PEPTIDE/PARTICLE DELIVERY SYSTEMS

Info

Publication number: 20160374949
Type: Application
Filed: Oct 12, 2011
Publication Date: Dec 29, 2016
Patent Grant number: 9717694
Applicant: THE JOHNS HOPKINS UNIVERSITY (Baltimore, MD)
Inventors: Jordan Jamieson Green (Nottingham, MD), Aleksander S. Popel (Lutherville, MD), Joel Chaim Sunshine (Pikesville, MD), Ron B. Shmueli (Baltimore, MD), Stephany Yi Tzeng (Baltimore, MD), Kristen Lynn Kozielski (Baltimore, MD)
Application Number: 13/272,042

Abstract

Polymeric nanoparticles, microparticles, and gels for delivering cargo, e.g., a therapeutic agent, such as a peptide, to a target, e.g., a cell, and their use for treating diseases, including angiogenesis-dependent diseases, such as age-related macular degeneration and cancer, are disclosed. Methods for formulating, stabilizing, and administering single peptides or combinations of peptides via polymeric particle and gel delivery systems also are disclosed.

Description

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos. 61/392,224, filed Oct. 12, 2010; 61/542,995, filed Oct. 4, 2011; and 61/543,046, filed Oct. 4, 2011, each which is incorporated herein by reference in its entirety.

BACKGROUND

Biomaterials have the potential to significantly impact medicine as delivery systems for imaging agents, biosensors, drugs, and genes. Farokhzad O C. Nanotechnology for drug delivery: the perfect partnership. Expert Opin Drug Deliv 2008; 5(9):927-9; Putnam D. Polymers for gene delivery across length scales. Nat Mater 2006; 5(6):439-51; Brigger I, Dubernet C, Couvreur P. Nanoparticles in cancer therapy and diagnosis. Adv Drug Deliv Rev 2002; 54(5):631-51. Challenges exist, however, in creating a delivery vehicle capable of effective, safe, and controlled release of sensitive biomolecules. Although rapid advances have been made for sustained delivery of small molecule drugs using biotechnology, similar advances have not been made for the delivery of peptides, siRNA, or combinations of biological agents.

SUMMARY

The presently disclosed subject matter provides polymeric nanoparticles, microparticles, and gels for delivering cargo, e.g., a therapeutic agent, such as a peptide, to a target, e.g., a cell, and their use for treating multiple diseases, including angiogenesis-dependent diseases, such as age-related macular degeneration and cancer. Methods for formulating, stabilizing, and administering single peptides or combinations of peptides via polymeric particle and gel delivery systems, for example, using a controlled release strategy, also are disclosed.

In some aspects, the presently disclosed subject matter provides a bioreducible, hydrolytically degradable polymer of formula (Ia):

wherein:

n is an integer from 1 to 10,000;

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino, alkylamino, dialkylamino, trialkylamino, aryl, ureido, heterocyclic, aromatic heterocyclic, cyclic, aromatic cyclic, halogen, hydroxyl, alkoxy, cyano, amide, carbamoyl, carboxylic acid, ester, carbonyl, carbonyldioxyl, alkylthioether, and thiohydroxyl groups;

wherein R₁can be present or absent and when present the compound of formula (I) further comprises a counter ion selected from the group consisting of chloride, fluoride, bromide, iodide, sulfate, nitrate, fumarate, acetate, carbonate, stearate, laurate, and oleate; and

wherein at least one R comprises a backbone of a diacrylate having the following structure:

wherein X₁and X₂are each independently substituted or unsubstituted C₂-C₂₀alkylene, and wherein each X₁and X₂can be the same or different.

In other aspects, the presently disclosed subject matter provides a nanoparticle, microparticle, or gel comprising a compound of formula (I):

wherein:

n is an integer from 1 to 10,000;

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino, alkylamino, dialkylamino, trialkylamino, aryl, ureido, heterocyclic, aromatic heterocyclic, cyclic, aromatic cyclic, halogen, hydroxyl, alkoxy, cyano, amide, carbamoyl, carboxylic acid, ester, carbonyl, carbonyldioxyl, alkylthioether, and thiohydroxyl groups;

wherein R₁can be present or absent and when present the compound of formula (I) further comprises a counter ion selected from the group consisting of chloride, fluoride, bromide, iodide, sulfate, nitrate, fumarate, acetate, carbonate, stearate, laurate, and oleate; and

at least one of R, R′, and R″ comprise a reducible or degradable linkage, and wherein each R, R′, or R″ can independently be the same or different;

under the proviso that when at least one R group comprises an ester linkage of the formula —C(═O)—O— and the compound of formula (I) comprises a poly(beta-amino ester), then the compound of formula (I) must also comprise one or more of the following characteristics:

(a) each R group is different;

(b) each R″ group is different;

(c) each R″ group is not the same as any of R′, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉;

(d) the R″ groups degrade through a different mechanism than the ester-containing R groups, wherein the degradation of the R″ group is selected from the group consisting of a bioreducible mechanism or an enzymatically degradable mechanism; and/or

(e) the compound of formula (I) comprises a substructure of a larger cross-linked polymer, wherein the larger cross-linked polymer comprises different properties from compound of formula (I);

and one or more peptides selected from the group consisting of an anti-angiogenic peptide, an anti-lymphangiogenic peptide, an anti-tumorigenic peptide, and an anti-permeability peptide.

In other aspects, the presently disclosed subject matter provides a multilayer particle comprising a core and one or more layers, wherein the core comprises a material selected from the group consisting of a compound of formula (I), a gold nanoparticle, an inorganic nanoparticle, an organic polymer, and the one or more layers comprise a material selected from the group consisting of a compound of formula (I), an organic polymer, one or more peptides, and one or more additional biological agents. In yet other aspects, the presently disclosed subject matter provides a microparticle comprising a compound of formula (I), poly(lactide-co-glycolide) (PLGA), or combinations thereof.

In other aspects, the presently disclosed subject matter provides a method for stabilizing a suspension of nanoparticles and/or microparticles of formula (I), the method comprising: (a) providing a suspension of nanoparticles and/or microparticles of formula (I); (b) admixing a lyroprotectant with the suspension; (c) freezing the suspension for a period of time; and (d) lyophilizing the suspension for a period of time.

In further aspects, the presently disclosed subject matter provides a pellet or scaffold comprising one or more lyophilized particle, wherein the one or more lyophilized particle comprises a compound of formula (I).

In yet further aspects, the presently disclosed subject matter provides a method of treating a disease or condition, the method comprising administering to a subject in need of treatment thereof a therapeutically effective amount of a nanoparticle, microparticle, gel, or multilayer particle comprising a compound of formula (I), wherein the nanoparticle, microparticle, gel, or multilayer particle further comprises a therapeutic agent specific for the disease or condition to be treated. In some aspects, the disease or condition comprises an angiogenesis-dependent disease or condition, including, but not limited to, cancer and age-related macular degeneration. In other aspects, the disease or condition is a non-angiogenic disease or condition. In certain aspects, the therapeutic agent encapsulated with the presently disclosed particles can be selected from the group consisting of gene, DNA, RNA, siRNA, miRNA, is RNA, agRNA, smRNA, a nucleic acid, a peptide, a protein, a chemotherapeutic agent, a hydrophobic drug, a small molecule drug, and combinations thereof.

Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

FIG. 1 is an illustration of the presently disclosed multilayer particles;

FIG. 2 is a scheme for producing hydrogels comprising the presently disclosed materials.

FIG. 3 shows a scheme for producing stable nanoparticle suspensions;

FIGS. 4A-4D show representative polymer structures tuned to peptide cargos;

FIGS. 5A and 5B show representative formation and sizing of polymer/peptide nanoparticles (by nanoparticle tracking analysis on a Nanosight LM10);

FIG. 6 shows DEAH peptide release by 336 nanoparticles at 4° C. (above) and 37° C. (below);

FIG. 7 shows HUVEC viability/proliferation assays with polymer/SP6001/DEAH peptide;

FIG. 8 shows HUVEC migration assays with 336 polymer/DEAH peptide;

FIG. 9 shows in vivo 336 polymer nanoparticle/SP6001 DEAH peptide;

FIG. 10 shows (top) Particle size and (bottom) cell viability effects of various polymer/SP2012 nanoparticles as compared to peptide only of non-cytotoxic polymers;

FIG. 11 shows polymer/peptide formulations for alternative peptides;

FIG. 12 shows data for FITC-tagged bovine serum albumin (BSA) mixed with a macromer solution containing 10% (w/v) PEGDA (Mn˜270 Da) with various amounts of B4S4, dissolved in a 1:1 (v/v) mixture of DMSO and PBS;

FIG. 13 shows an SEM of increasing B4S4 from top [0.2% w/w] to bottom [5% w/w]);

FIG. 14 shows the size distribution of appropriately freeze-dried particles (bottom left, right-most histogram) remains the same as freshly-prepared particles (bottom left, left-most histogram). Freeze-dried particles also remain more stable in serum-containing medium than freshly-prepared particles (upper left). Using DNA-loaded nanoparticles, transfection efficiency is comparable between fresh particles and particles lyophilized with sucrose (right) even after 3 months of storage;

FIG. 15 is Left: brightfield+GFP+DsRed, showing presence of cells (green) being transfected with DsRed (red) on a bone scaffold (brightfield). Right: GFP and DsRed shown only;

FIG. 16 demonstrates that DsRed expression was observed within 4 days and remained very robust even after 12 days: top=1 day, middle=4 days, bottom=12 days after transfection;

FIG. 17 demonstrates the incorporation of DNA-loaded nanoparticles into natural and synthetic scaffolds, disks, microparticles, and hydrogels;

FIG. 18 demonstrates transfection of GFP⁺ glioblastoma cells with scrambled (control) siRNA (top panels) or siRNA against GFP (bottom);

FIGS. 19A-19C show activity of R6-series polymers at delivering siRNA to knockdown GFP signal in GB cells; % Knockdown of GFP expression in GFP+ glioblastoma cells transfected with siRNA against GFP, normalized to cells transfected with scrambled siRNA, using various BR6 polymers as a transfection agent: (A) transfection with acrylate-terminated BR6 polymers with either S3, S4 or S5 as the side chain; (B) transfection with E10 end-capped versions of the polymers in Figure A; and (C) GFP fluorescence images of cells transfected with BR6-S4-Ac complexed scrambled RNA (top) vs. siRNA against GFP (bottom);

FIG. 20 shows gel retardation assay of siRNA with BR6-S5-E10 at varying ratios of polymer to RNA. The polymer effectively retards siRNA (top), but in the presence of 5 mM glutathione siRNA is released immediately (bottom). These data demonstrate the hypothesized intracellular release of siRNA and elucidates the mechanism by which nanoparticles formed using BR6 facilitate strong siRNA transfection and GFP knockdown;

FIG. 21 shows that an E10-endcapped polymer (top) retards siRNA efficiently, but upon addition of 5 mM glutathione, siRNA is immediately released (bottom). Numbers refer to the w/w ratio of polymer-to-siRNA in all cases;

FIG. 22 shows that the same base polymer as shown in FIG. 25 with a different endcap (E7, 1-(3-aminopropyl)-4-methylpiperazine) also retards siRNA (top) but is not affected by application of glutathione (bottom);

FIG. 23 provides gel permeation chromatography data of BR6 polymerized with S4 at a BR6:S4 ratio of 1.2:1 at 90° C. for 24 hours, before and after end-capping with E7;

FIG. 24 shows that knockdown efficiency also is affected by molecular weight of the polymer. 1.2:1, 1.1:1, and 1.05:1 refer to the ratio of reactants in the base polymer step growth reaction;

FIG. 25 demonstrates combined DNA (RFP) and siRNA delivery (against GFP) in GB;

FIG. 26 shows that siRNA knockdown is affected by the endcap (E), base polymer (increasing hydrophobicity from L to R within each E), and molecular weight (increasing L to R within each base polymer);

FIG. 27 shows 4410, 200 w/w (blue line on above graph), 8 days after transfection: Left: hMSCs treated with scrambled control; Right: hMSCs treated with siRNA;

FIG. 28 demonstrates that in variable molecular weight embodiments, polymer molecular weight is between 4.00-10.00 kDa for siRNA delivery;

FIG. 29 demonstrates the use of the presently disclosed materials for DNA delivery;

FIG. 30 shows GB Transfection;

FIG. 31 shows 551 GB cells cultured as neurospheres (undifferentiated);

FIG. 32 demonstrates that, for a DNA delivery application, in some embodiments, polymer molecular weight is between 3.00-10.0 kDa;

FIG. 33 provides representative characteristics exhibited by the presently disclosed biodegradable polymers;

FIG. 34 demonstrates the delivery of DNA to GB bulk tumor cells for representative biomaterials;

FIG. 35 demonstrates the transfection of genes to BCSC for representative presently disclosed biomaterials;

FIG. 36 demonstrates the delivery of DNA to fetal (healthy) cells;

FIG. 37 demonstrates the delivery of DNA to BCSCs;

FIGS. 38 and 39 demonstrate the delivery of apoptosis-inducing genes in BCSCs;

FIG. 40 shows that particles lyophilized with sucrose and used immediately are as effective in transfection as freshly prepared particles;

FIGS. 41 and 42 demonstrate the use of the presently disclosed materials and methods for long-term gene delivery;

FIG. 43 demonstrates siRNA delivery to GB cells;

FIGS. 44 and 45 provide a comparison of siRNA vs. DNA delivery in GB cells;

FIG. 46 depicts a strategy of combining nanoparticles within microparticles to extend release further. PLGA or blends of PLGA with the presently disclosed polymers are used to form microparticles by single or double emulsion;

FIG. 47 shows DEAH-FITC release from microparticles comprising a presently disclosed polymer and a peptide;

FIG. 48 shows slow extended release from microparticles containing nanoparticles that contain peptides; and

FIG. 49 shows in vivo effects of microparticle formulations in both the CNV and rho/VEGF model over time.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

I. Peptide/Particle Delivery Systems

The presently disclosed subject matter provides compositions of matter, methods of formulation, and methods of treatment utilizing drug delivery systems comprising one or more degradable polymers and one or more biological agents. The polymers described in these systems must be biodegradable. Mechanisms for this degradability include, but are not limited to, hydrolytic degradation, enzymatic degradation, and disulfide reduction. The biological agents described in these systems include, but are not limited to, therapeutic or diagnostic agents, such as small molecules, peptides, proteins, DNA, siRNA, miRNA, is RNA, contrast agents, and other agents one skilled in the field would wish to encapsulate. In particular embodiments, biological therapeutic agents that are sensitive to degradation and sized approximately 10,000-25,000 Da, including siRNA and peptides, are suitable for use with the presently disclosed materials.

Peptide drugs in polymeric delivery systems are useful for various therapeutic and diagnostic applications. Some embodiments of the presently disclosed subject matter are useful for treating angiogenesis-dependent diseases including, but not limited to, age-related macular degeneration (AMD) and cancer. One particular embodiment of the presently disclosed subject matter includes specific peptide sequences, as well as methods of formulating, stabilizing, and administering these peptides as single agents or as combinations of peptides via polymeric nanoparticle-based, microparticle-based, gel-based, or conjugate-based delivery systems.

The presently disclosed nanoparticles, microparticles, and gels can be used to deliver cargo, for example a therapeutic agent, such as a peptide or protein, to a target, for example, a cell. The cargo delivered by the presently disclosed nanoparticles, microparticles, and gels can act, in some embodiments, as a therapeutic agent or a biosensor agent. Combinations of polymeric materials and cargo, for example a single peptide or combination of peptides, can be formulated by the presently disclosed methods, which allows for the control, or tuning, of the time scale for delivery.

Further, the presently disclosed polymeric materials can be used to form self-assembled electrostatic complexes, micelles, polymersomes, emulsion-based particles, and other particle formulations known to one of ordinary skill in the art. Nanoparticles formed from the presently disclosed polymeric materials can be formulated into larger microparticles to further extend duration and timing of release. Lyophilized formulations that can maintain longer shelf life and stability also are described. The presently disclosed particles can be administered as a powder, cream, ointment, implant, or other reservoir device.

The presently disclosed nanoparticles, microparticles, and gels can be used to treat many diseases and conditions including, but not limited to, all types of cancers, ophthalmic diseases, cardiovascular diseases, and the like. In particular embodiments, the disease or condition treated by the presently disclosed nanoparticles, microparticles, and gels include breast cancer and age-related macular degeneration.

A. Bioreducible and Hydrolytically Degradable Two-Component Degradable Polymers

The presently disclosed materials offer several advantages for use in delivering cargo, e.g., a therapeutic agent, such as a peptide or siRNA, to a target, e.g., a cell. Such advantages include a slower degradation in the extracellular environment and a quicker degradation in the intracellular environment. Further, the method of synthesis allows for diversity of monomer starting materials and corresponding facile permutations of polymer structure. The presently disclosed materials can be used to form self-assembled nanoparticles, blended microparticles, gels, and bioconjugates. The presently disclosed polymers also have the following advantages compared to other drug delivery polymers known in the art: a higher polymerization than with disulfide acrylamides, which is important for various applications because it can be used to tune both binding/encapsulation and release; two time scales for degradation (hydrolytic degradation in water and disulfide reduction due to glutathione inside the cell), which facilitates drug release and reduces potential cytotoxicity; tunable structural diversity, with hydrophobic, hydrophilic, and charged moieties to aid in encapsulation of a target biological agent; and, usefulness for drug delivery, including high siRNA delivery, even without end-modification of the polymer.

Certain polyesters have been shown previously to form nanoparticles in the presence of biological agents, such as nucleic acids, and facilitate their entry into a cell. In such materials, release of the nucleic acid is modulated by hydrolytic degradation of the polyester polymer. The addition of a bioreducible disulfide moiety into the backbone of these polymers, however, can specifically target release to the reducing intracellular environment.

Accordingly, a library of bioreducible polyesters can be synthesized by oxidizing and acrylating various mercapto-alcohols (representative diacrylates formed from the presently disclosed synthetic process are shown in Scheme 1 below), then reacting with amine side chains. The structure of a representative bioreducible polyester, e.g., 2,2′-disulfanediylbis(ethane-2,1-diyl)diacrylate (BR6) polymerized with S4, also is shown in Scheme 1.

In other embodiments, amine-containing molecules can be reacted to terminal groups of the polymer. In particular embodiments, this amine-containing molecule also contains poly(ethylene glycol) (PEG) or a targeting ligand. In other embodiments, the disulfide acrylates are not reacted with amines, but are instead polymerized through other mechanisms including, but not limited to, free radical polymerization to form network polymers and gels. In other embodiments, oligomers are first formed and then the oligomers are polymerized to form block co-polymers or gels.

More particularly, the presently disclosed subject matter provides a bioreducible, hydrolytically degradable polymer of formula (Ia):

wherein:

n is an integer from 1 to 10,000;

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino, alkylamino, dialkylamino, trialkylamino, aryl, ureido, heterocyclic, aromatic heterocyclic, cyclic, aromatic cyclic, halogen, hydroxyl, alkoxy, cyano, amide, carbamoyl, carboxylic acid, ester, carbonyl, carbonyldioxyl, alkylthioether, and thiohydroxyl groups;

wherein R₁can be present or absent and when present the compound of formula (I) further comprises a counter ion selected from the group consisting of chloride, fluoride, bromide, iodide, sulfate, nitrate, fumarate, acetate, carbonate, stearate, laurate, and oleate; and

wherein at least one R comprises a backbone of a diacrylate having the following structure:

wherein X₁and X₂are each independently substituted or unsubstituted C₂-C₂₀alkylene, and wherein each X₁and X₂can be the same or different.

In some embodiments, the bioreducible, hydrolytically degradable polymer of claim 1, wherein at least one R comprises a backbone of a diacrylate selected from the group consisting of:

or co-oligomers comprising combinations thereof, wherein the diacrylate can be the same or different.

Additional R, R′, and R″ groups are defined immediately herein below as for compounds disclosed in International PCT Patent Application Publication No. WO/2010/132879 for “Multicomponent Degradable Cationic Polymers,” to Green et al., which is incorporated herein by reference in its entirety.

B. Hydrolytic and Bioreducible Polymeric Particle Formulations for Delivery of Peptides.

Multicomponent degradable cationic polymers suitable for the delivery of peptides to a target are disclosed in International PCT Patent Application Publication No. WO/2010/132879 for “Multicomponent Degradable Cationic Polymers,” to Green et al., which is incorporated herein by reference in its entirety. Such polymers, in addition to the presently disclosed polymers can be used to deliver cargo, e.g., a therapeutic agent, to a target, e.g., a cell.

In some embodiments, the presently disclosed subject matter generally provides multicomponent degradable cationic polymers. In some embodiments, the presently disclosed polymers have the property of biphasic degradation. Modifications to the polymer structure can result in a change in the release of therapeutic agents, which can occur over multiple time scales. In some embodiments, the presently disclosed polymers include a minority structure, e.g., an endcapping group, which differs from the majority structure comprising most of the polymer backbone. In other embodiments, the bioreducible oligomers form block copolymers with hydrolytically degradable oligomers. In yet other embodiments, the end group/minority structure comprises an amino acid or chain of amino acids, while the backbone degrades hydrolytically and/or is bioreducible.

As described in more detail herein below, small changes in the monomer ratio used during polymerization, in combination with modifications to the chemical structure of the end-capping groups used post-polymerization, can affect the efficacy of delivery of a therapeutic agent to a target. Further, changes in the chemical structure of the polymer, either in the backbone of the polymer or end-capping groups, or both, can change the efficacy of target delivery to a cell. In some embodiments, small changes to the molecular weight of the polymer or changes to the endcapping groups of the polymer, while leaving the main chain, i.e., backbone, of the polymer the same, can enhance or decrease the overall delivery of the target to a cell. Further, the “R” groups that comprise the backbone or main chain of the polymer can be selected to degrade via different biodegradation mechanisms within the same polymer molecule. Such mechanisms include, but are not limited to, hydrolytic, bioreducible, enzymatic, and/or other modes of degradation.

In some embodiments, the presently disclosed compositions can be prepared according to Scheme 2:

In some embodiments, at least one of the following groups R, R′, and R″ contain reducible linkages and, for many of the presently disclosed materials, additional modes of degradation also are present. More generally, R′ can be any group that facilitates solubility in water and/or hydrogen bonding, for example, OH, NH, and SH. Representative degradable linkages include, but are not limited to:

The end group structures, i.e., R″ groups in Scheme 2, for the presently disclosed cationic polymers are distinct and separate from the backbone structures (R) structures, the side chain structures (R′), and end group structures of the intermediate precursor molecule for a given polymeric material.

More particularly, in some embodiments, the presently disclosed subject matter includes a nanoparticle, microparticle, or gel comprising a compound of formula (I):

wherein:

n is an integer from 1 to 10,000;

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino, alkylamino, dialkylamino, trialkylamino, aryl, ureido, heterocyclic, aromatic heterocyclic, cyclic, aromatic cyclic, halogen, hydroxyl, alkoxy, cyano, amide, carbamoyl, carboxylic acid, ester, carbonyl, carbonyldioxyl, alkylthioether, and thiohydroxyl groups;

wherein R₁can be present or absent and when present the compound of formula (I) further comprises a counter ion selected from the group consisting of chloride, fluoride, bromide, iodide, sulfate, nitrate, fumarate, acetate, carbonate, stearate, laurate, and oleate; and

at least one of R, R′, and R″ comprise a reducible or degradable linkage, and wherein each R, R′, or R″ can independently be the same or different;

under the proviso that when at least one R group comprises an ester linkage of the formula —C(═O)—O— and the compound of formula (I) comprises a poly(beta-amino ester), then the compound of formula (I) must also comprise one or more of the following characteristics:

(a) each R group is different;

(b) each R″ group is different;

(c) each R″ group is not the same as any of R′, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₉, and R₉;

(d) the R″ groups degrade through a different mechanism than the ester-containing R groups, wherein the degradation of the R″ group is selected from the group consisting of a bioreducible mechanism or an enzymatically degradable mechanism; and/or

(e) the compound of formula (I) comprises a substructure of a larger cross-linked polymer, wherein the larger cross-linked polymer comprises different properties from compound of formula (I);

and one or more peptides selected from the group consisting of an anti-angiogenic peptide, an anti-lymphangiogenic peptide, an anti-tumorigenic peptide, and an anti-permeability peptide.

In some embodiments of the nanoparticle, microparticle, or gel n is an integer from 1 to 1,000; in some embodiments, n is an integer from 1 to 100; in some embodiments, n is an integer from 1 to 30; in some embodiments, n is an integer from 5 to 20; in some embodiments, n is an integer from 10 to 15; and in some embodiments, n is an integer from 1 to 10.

In particular embodiments, the reducible or degradable linkage comprising R, R′, and R″ is selected from the group consisting of an ester, a disulfide, an amide, an anhydride or a linkage susceptible to enzymatic degradation, subject to the proviso provided hereinabove.

In more particular embodiments, R comprises a backbone of a diacrylate selected from the group consisting of:

In some embodiments, wherein R′ comprises a side chain derived from compound selected from the group consisting of:

In some embodiments, R″ comprises an end group derived from a compound selected from the group consisting of

In other embodiments, the compound of formula (I) is subject to the further proviso that if at least one R group comprises an ester linkage, then the R″ groups impart one or more of the following characteristics to the compound of formula (I): independent control of cell-specific uptake and/or intracellular delivery of a particle; independent control of endosomal buffering and endosomal escape; independent control of DNA release; triggered release of an active agent; modification of a particle surface charge; increased diffusion through a cytoplasm of a cell; increased active transport through a cytoplasm of a cell; increased nuclear import within a cell; increased transcription of an associated DNA within a cell; increased translation of an associated DNA within a cell; increased persistence of an associated therapeutic agent within a cell, wherein the therapeutic agent is selected from the group consisting of DNA, RNA, a peptide or a protein.

More particularly, any poly(beta-amino ester) specifically disclosed or claimed in U.S. Pat. No. 6,998,115; U.S. Pat. No. 7,427,394; U.S. patent application publication no. US2005/0265961; and U.S. patent publication no. US2010/0036084, each of which is incorporated herein by reference in its entirety, is explicitly excluded from the presently disclosed compounds of formula (I). In particular, the poly(beta-amino ester)s disclosed in U.S. Pat. No. 6,998,115; U.S. Pat. No. 7,427,394; U.S. patent application publication no. US2005/0265961; and U.S. patent publication no. US2010/0036084 are symmetrical, i.e., both R groups as defined in formula (I) herein are the same. In certain embodiments of the presently disclosed compounds of formula (I), when at least one R comprises an ester linkage, the two R groups of formula (I) are not the same, i.e., in such embodiments, the compounds of formula (I) are not symmetrical.

In particular embodiments, the reducible or degradable linkage comprising R, R′, and R″ is selected from the group consisting of an ester, a disulfide, an amide, an anhydride or a linkage susceptible to enzymatic degradation, subject to the above-mentioned provisos.

Further, in some embodiments of the compound of formula (I), n is an integer from 1 to 1,000; in other embodiments, n is an integer from 1 to 100; in other embodiments, n is an integer from 1 to 30; in other embodiments, n is an integer from 5 to 20; in other embodiments, n is an integer from 10 to 15; and in other embodiments, n is an integer from 1 to 10.

In some embodiments, R″ can be an oligomer as described herein, e.g., one fully synthesized primary amine-terminated oligomer, and can be used as a reagent during the second reaction step of Scheme 2. This process can be repeated iteratively to synthesize increasingly complex molecules.

In other embodiments, R″ can comprise a larger biomolecule including, but not limited to, poly(ethyleneglycol) (PEG), a targeting ligand, including, but not limited to, a sugar, a small molecule, an antibody, an antibody fragment, a peptide sequence, or other targeting moiety known to one skilled in the art; a labeling molecule including, but not limited to, a small molecule, a quantum dot, a nanoparticle, a fluorescent molecule, a luminescent molecule, a contrast agent, and the like; and a branched or unbranched, substituted or unsubstituted alkyl chain.

In some embodiments, the branched or unbranched, substituted or unsubstituted alkyl chain is about 2 to about 5 carbons long; in some embodiments, the alkyl chain is about 6 to about 8 carbons long; in some embodiments, the alkyl chain is about 9 to about 12 carbons long; in some embodiments, the alkyl chain is about 13 to about 18 carbons long; in some embodiments, the alkyl chain is about 19 to about 30 carbons long; in some embodiments, the alkyl chain is greater than about 30 carbons long.

In certain embodiments, both R″ groups, i.e., the end groups of the polymer, comprise alkyl chains. In other embodiments, only one R″ group comprises an alkyl chain. In some embodiments, at least one alkyl chain is terminated with an amino (NH₂) group. In other embodiments, the at least one alkyl chain is terminated with a hydroxyl (OH) group.

In some embodiments, the PEG has a molecular weight of about 5 kDa or less; in some embodiments, the PEG has a molecular weight of about 5 kDa to about 10 kDa; in some embodiments, the PEG has a molecular weight of about 10 kDa to about 20 kDa; in some embodiments, the PEG has a molecular weight of about 20 kDa to about 30 kDa; in some embodiments, the PEG is greater than 30 kDa. In certain embodiments, both R″ groups comprise PEG. In other embodiments, only one R″ group comprises PEG.

Further, in some embodiments, one R″ group is PEG and the other R″ group is a targeting ligand and/or labeling molecule as defined herein above. In other embodiments, one R″ group is an alkyl chain and the other R″ group is a targeting ligand and/or labeling molecule.

Representative monomers used to synthesize the presently disclosed cationic polymers include, but are not limited to, those provided immediately herein below. The presently disclosed subject matter is not limited to the representative monomers disclosed herein, but also includes other structures that one skilled in the art could use to create similar biphasic degrading cationic polymers. For each type of cargo, a particular biodegradable polymer can be tuned through varying the constituent monomers used to form the backbone (designated as “B” groups), side-chains (designated as “S” groups), and end-groups (designated as “E” groups) of the polymer.

In particular embodiments, as depicted in Scheme 4, the presently disclosed cationic polymers comprise a polyalcohol structure, i.e., the side chain represented by R′ in Scheme 2 comprises an alcohol.

In such embodiments, the end group structures (R″) and the backbone structures (R) are defined as above and the side chain must contain at least one hydroxyl (OH) group.

In yet other embodiments, the presently disclosed cationic polymer comprises a specific poly(ester amine) structure with secondary non-hydrolytic modes of degradation. In such embodiments, the cationic polymer comprises a polyester that degrades through ester linkages (hydrolytic degradation) that is further modified to comprise bioreducible groups as end (R″) groups.

Representative bioreducible end groups in such embodiments include, but are not limited to:

In some embodiments, the presently disclosed cationic polymer comprises a specific poly(ester amine alcohol) structure with secondary non-hydrolytic modes of degradation. In such embodiments, the cationic polymer comprises a specific structure where a polyester that degrades through ester linkages (hydrolytic degradation) is modified to contain bioreducible groups as end groups.

In yet other embodiments, the presently disclosed cationic polymer comprises a specific poly(amido amine) structure having disulfide linking groups in the polymer backbone and an independent, non-reducible amine contacting group at the terminal ends of the polymer.

In such embodiments, R₁and R₂are alkyl chains. In some embodiments, the alkyl chain is 1-2 carbons long; in some embodiments, the alkyl chain is 3-5 carbons long; in some embodiments, the alkyl chain is 6-8 carbons long; in some embodiments, the alkyl chain is 9-12 carbons long; in some embodiments, the alkyl chain is 13-18 carbons long; in some embodiments, the alkyl chain is 19-30 carbons long; and in some embodiments, the alkyl chain is greater than 30 carbons long

Suitable non-reducible amino R″ groups for such embodiments include, but are not limited to:

In other embodiments, the presently disclosed cationic polymers comprise a specific poly(amido amine alcohol) structure having disulfide linking groups in the polymer backbone and an independent non-reducible amine contacting group at the terminal ends of the polymer.

In yet other embodiments, the presently disclosed cationic polymer comprises a copolymer of representative oligomers as described hereinabove. Such embodiments include, but are not limited to, a poly(amido amine) structure having disulfides in the polymer backbone and an independently degradable (non-reducible) group at least one end of the polymer. Such embodiments also include using a cross-linker to add bioreducible linkages to hydrolytically degradable materials and also provide for higher molecular weight materials. A representative example of this embodiment, along with suitable monomers is as follows:

In particular embodiments, the presently disclosed polymer is selected from the group consisting of:

Further aspects of the presently disclosed subject matter include: (a) the R substituent groups that make up the presently disclosed polymers degrade via different biodegradation mechanisms within the same polymer. These biodegradation mechanisms can include hydrolytic, bioreducible, enzymatic, and/or other modes of degradation; (b) the ends of the polymer include a minority structure that differs from the majority structure that comprises most of the polymer backbone; (c) in several embodiments, the side chain molecules contain hydroxyl (OH)/alcohol groups.

In some embodiments: (a) the backbone is bioreducible and the end groups of the polymer degrade hydrolytically; (b) the backbone degrades hydrolytically and the end groups are bioreducible; and (c) hydrolytically degradable oligomers are cross-linked with a bioreducible cross-linker; (d) bioreducible oligomers form block copolymers with hydrolytically degradable oligomers; and (e) the end group/minority structure comprises an amino acid or chain of amino acids, whereas the backbone degrades hydrolytically and/or is bioreducible.

One way to synthesize the presently disclosed materials is by the conjugate addition of amine-containing molecules to acrylates or acrylamides. This reaction can be done neat or in a solvent, such as DMSO or THF. Reactions can take place at a temperature ranging from about room temperature up to about 90° C. and can have a duration from about a few hours to about a few weeks. The presently disclosed methods can be used to create linear or branched polymers. In some embodiments, the molecular weight (MW) has a range from about 1 kDa to about 5 kDa, in other embodiments, the MW has a range from about 5 kDa to about 10 kDa, in other embodiments the MW has a range from about 10 kDa to about 15 kDa, in other embodiments, the MW has a range from about 15 kDa to about 25 kDa, in other embodiments, the MW has a range from about 25 kDa to about 50 kDa, and in other embodiments, the MW has a range from about 50 kDa to about 100 kDa. In other embodiments, the polymer forms a network, gel, and/or scaffold of apparent molecular weight greater than 100 kDa.

In particular embodiments, the presently disclosed subject matter provides hydrolytic and bioreducible polymeric particle formulations for the delivery of one or more peptides to a target. In some embodiments of the presently disclosed formulations, the particles are nanoparticles and, in other embodiments, they are microparticles. Some applications are to cancer and others are to ophthalmic diseases.

Accordingly, in some embodiments, the presently disclosed approach includes degradable nanoparticles, microparticles, and gels that release a peptide, which is capable of therapeutic activity through multiple modes of action. The presently disclosed peptides can simultaneously inhibit: (1) endothelial cell proliferation; (2) endothelial cell adhesion, (3) endothelial cell migration, (4) tumor cell proliferation, (5) tumor cell adhesion, and (6) tumor cell migration.

When combined with such peptides, the presently disclosed nanoparticles, microparticles, and gels: (1) protect and increase the persistence of the peptides that would otherwise be rapidly cleared in vivo; (2) allow passive targeting of tumor vasculature via nanoparticle biophysical properties to enable enhanced efficacy at the target site of action; (3) enable extended peptide release and minimized dosing schedules for affected patients; and (4) facilitate a continuous peptide concentration rather than a pulsatile profile that would be caused by bolus injections and fast clearance.

The presently disclosed microparticles have similar benefits to the nanoparticles except that they also persist longer and have an easier route for clinical administration. On the other hand, another advantage of the presently disclosed nanoparticles is that they are better able to passively target the peptides to tumor vasculature than are the microparticles. Representative embodiments of the presently disclosed microparticles are provided in Example 10, herein below.

Further, in some embodiments, one or more peptides, which can be the same or different, can be combined, e.g., encapsulated, directly or individually into different nanoparticles that then can be combined into the same microparticles.

C. Biodegradable Nanoparticles for Sustained Peptide Delivery

Selected polymers are able to encapsulate selected peptides possessing varied chemical properties. Changes to polymer structure, including small changes to the ends of the polymer only, can vary biophysical properties of these particles. These properties can be important to tune for effective in vivo peptide delivery. A small subset of the potential polymer library was screened to measure the effect of encapsulating the antiangiogenic peptides chemokinostatin-1 and pentastatin-1 within polymeric particles compared to unencapsulated, free peptides. Polymeric encapsulation of peptides enhanced the ability of the peptides to inhibit the proliferation of endothelial cells. An example of representative polymers encapsulating peptides is provided in Scheme 5.

Name Sequence Theor. pI MW DEAH box poly8 EIELVEEEPPF 3.51 1330.45 Wispostatin-1 SPWSPCSTSCGLGVS 7.80 1838.08 TRI Pentastatin LRRFSTMPFMFCNI 9.02 2454.93 NNVCNF Chemokinostatin NGRKACLNPASPIV 10.03 2625.19 KKIIEKMLNS

In other embodiments, particles synthesized and composed as described above are then used as a “core” inner particle for future coatings to create multi-component (also referred to herein as multi-layer) particles. For other embodiments, other nanoparticles are used as cores, such as an inorganic nanoparticles (like gold) or soft polymeric nanoparticles, for example, as disclosed in International PCT Patent Application Publication No. WO/2010/132879 for “Multicomponent Degradable Cationic Polymers,” to Green et al., which is incorporated herein by reference in its entirety. In each embodiment, the core particle is then coated with charged polymers as described above, peptides as described above, and other biological agents. Exemplary embodiments of multilayer particles are illustrated in FIG. 1.

Layering can be mediated by electrostatic forces and alternate cationic and anionic layers can be used to incorporate additional peptides and biological agents. Polyelectrolytes, including degradable polymers and peptides, also are used to provide structure to the multilayers. Multilayers can release drugs, peptides, and biological agents from the particle due to hydrolytic degradation, enzyme activity, disulfide reduction, and/or diffusion.

D. Polymeric Gels for Controlled Release of Biological Agents.

i. Hydrogels (or “Organogels”) for Protein/Peptide Release

In some embodiments, the presently disclosed subject matter provides photocrosslinked gels for controlled release of cargo, including, but not limited to peptides and proteins. Such gels can be tuned for release of other drugs. In some embodiments, for example, as illustrated in FIG. 2, a solution of acrylate-terminated polymers is made using either acrylate-terminated polymers, such as poly(β-amino esters) (PBAEs), poly(ethylene glycol)diacrylate (PEGDA), small crosslinkers including, but not limited to, 1,4-butanediol diacrylate, or a mixture of the above. Because many of these materials are amphiphilic, a variety of solvents can be used, including water, PBS, and DMSO, to encapsulate drugs within them. Addition of a small amount (0.05% w/v) of photoinitiator and exposure to long-wave UV light for a period of time, e.g., 5-15 min at 1-3 mW, causes formation of a drug-loaded gel.

The gel swelling properties can vary with pH by taking advantage of the PBAE portions, which can be reversibly protonated. Changing ratios of PBAE to PEGDA and the addition of crosslinkers changes swelling properties by changing pore size or overall hydrophobicity. For example, doping in increasing amounts of a more hydrophobic PBAE (B4S4) into a network of hydrophilic PEGDA causes the release kinetics to slow when measuring protein release.

E. Stable Formulations

To increase stability of nanoparticles in suspension, especially with hydrolytically-degradable polymers, the presently disclosed subject matter provides a method of keeping DNA or other cargo stable and functional after storage. For example, freeze-drying often causes denaturation of biological molecules or irreversible aggregation and inactivation of nanoparticles. Referring now to FIG. 3, by adding sucrose as a lyoprotectant at a final concentration of, for example, 7.5-45 mg/mL, the presently disclosed subject matter demonstrates that particles can be freeze dried and stored, for example, at 4° C. or −20° C. for extended periods, e.g., months, without significant change in physicochemical or biological properties. Certain formulations, when stored dry, also might be stable at ambient temperatures up to 40° C. Furthermore, the presently disclosed process allows particles to be prepared in advance and used much more easily in a clinical setting. The presently disclosed subject matter also demonstrates that particles can be concentrated in this way much more highly than would be possible with free polymer, which may be advantageous for dose adjustment in clinical or pre-clinical models.

F. Inclusion of Lyophilized Nanoparticles into Pellets/Scaffolds for Long-Term Delivery

The presently disclosed nanoparticles can be stored in a dry form and can be used in gene delivery via three-dimensional (3D) constructs. While DNA is used as a cargo in this example, other cargos of interest to one skilled in the art including, but not limited to, siRNA, peptides, protein, imaging agents, and the like, can be used, as well. In other embodiments, DNA-loaded nanoparticles were incorporated into natural and synthetic scaffolds, disks, microparticles, and hydrogels for various potential applications.

G. Methods of Treating Angiogenesis-Dependent Diseases

Although significant progress has been made in treating angiogenesis-dependent diseases, such as cancers, major challenges remain in terms of development of drug resistance, metastasis and overall survival rates. Studies designed to decipher the modes of drug resistance have revealed that tumors are very versatile and use multiple pathways to continue to survive and metastasize. See Chiang A C, Massague J. Molecular basis of metastasis. N Engl J Med 2008; 359(26):2814-23; Gupta G P, Massague J. Cancer metastasis: building a framework. Cell 2006; 127(4):679-95. Resistance has been observed for both cytotoxic and antiangiogenic agents. Thus, multimodal therapeutic design emerges as a promising, and perhaps even a mandatory strategy for treatment of cancer. See Sawyers C L. Cancer: mixing cocktails. Nature 2007; 449(7165):993-6; Dorrell M I, Aguilar E, Scheppke L, Barnett F H, Friedlander M. Combination angiostatic therapy completely inhibits ocular and tumor angiogenesis. Proc Natl Acad Sci USA 2007; 104(3):967-72.

The key attributes of tumor growth and metastasis are: angiogenesis, which facilitates the supply of the growing tumor with oxygen and nutrients; lymphangiogenesis, which facilitates the spreading of cancer cells through the lymphatics; and cancer cell proliferation. Angiogenesis, in particular, plays a critical role in the growth of tumors and antiangiogenic therapies have the potential to treat cancer, either alone or in combination with conventional chemotherapies, by starving tumors of oxygen and nutrients. There is a need, however, to find more potent anti-cancer therapeutics, including antiangiogenic therapeutics, as well as delivery systems for these therapeutics. The presently disclosed subject matter can address all of these attributes in a combined system.

Many forms of cancer, including breast cancer, are dependent on angiogenesis, the growth of blood vessels. There is a great medical need for the development of a safe, effective, and inexpensive means of antiangiogenic therapy. One promising approach is the use of antiangiogenic peptides as the active agents. In some embodiments, the presently disclose'd subject matter provides peptides derived from several classes of proteins that are effective at preventing angiogenesis. In other embodiments, the presently disclosed subject matter provides other peptides that are able to inhibit cancer through additional mechanisms including, but not limited to, antilymphangiogenesis and apoptosis. In their current form, however, all of these peptides have a short in vivo half-life and they are not suitable for systemic administration or for long-term action. Thus, there is a need to package, protect, and deliver these peptides in a more stable, sustained fashion.

Accordingly, the presently disclosed biomaterials facilitate delivery of combinations of these peptides in an engineered fashion to synergistically kill cancer or treat other diseases, in particular, other angiogenesis-dependent diseases. More particularly, the presently disclosed subject matter provides an effective array of safe, biodegradable polymers for use in forming peptide-containing nanoparticles, microparticles, gels, and conjugates. The presently disclosed biomaterials can be used to construct particles, gels, and conjugates that vary in their biophysical properties and in biological properties, such as tumor accumulation and peptide release.

The presently disclosed formulations work through one or more of the following mechanisms: antiangiogenesis; inhibition of human endothelial cell proliferation and migration; inhibition of lymphatic endothelial cell proliferation and migration; and promotion of cancer apoptosis, as well as other mechanisms. The presently disclosed materials and methods can safely, effectively, and relatively inexpensively treat age-related macular degeneration (AMD), cancer, and other diseases.

Further, siRNA is a promising technology to silence the activity of many biological targets in many diseases including cancer, cardiovascular diseases, infectious diseases, neurological diseases, ophthalmic diseases, and others. In some cases, siRNA can be used to reach previously undruggable targets. The method of delivery and examples described herein for siRNA delivery apply equally to other similar RNA molecules including, but not limited to is RNA, agRNA, saRNA, and miRNA.

H. Nanoparticle-Mediated Multimodal Peptide Delivery

Conventional anti-angiogenesis treatments have proven to be very expensive with limited clinical success, particularly in breast cancer. The presently disclosed strategy combines more effective and multimodal therapeutic agents with nanomedicine to provide a delivery system to enhance their therapeutic effect. More particularly, the presently disclosed subject matter provides a single system that incorporates multimodal therapeutic activity, including, but not limited to, antiangiogenic activity, antilymphangiogenic activity, and apoptotic activity, and can be effective in limiting both tumor growth and metastasis.

Generally, small peptides possess many advantageous characteristics as therapeutic agents, including high specificity and low toxicity. Reichert J. Development trends for peptide therapeutics. Tufts Center for the Study of Drug Development 2008 [cited 2010; Available from: http://www.peptidetherapeutics.org/PTF_Summary_2008.pdf]. The main disadvantage of small peptides as therapeutic agents, however, is their short half-life. The presently disclosed subject matter capitalizes on the advantages of peptide agents by developing novel antiangiogenic, antilymphangiogenic, and apoptotic peptides targeting multiple pathways, and overcoming the disadvantages by designing a multi-agent nanocarrier system.

Approximately 25 peptides have been approved by the FDA, however, to date none of these approved peptides are antiangiogenic. Rosca E V, Koskimaki J E, Rivera C G, Pandey N B, Tamiz A P, Popel A S. Anti-angiogenic peptides for cancer therapeutics. Curr Pharm Biotechnol, 12(8):1101-1116 (2011). Several endogenous proteins/polypeptides, including angiostatin, endostatin, proteolytic fragments of collagen IV, pigment epithelium-derived factor, and thrombospondin, have antiangiogenic properties and can induce apoptosis in endothelial cells. Lucas R, Holmgren L, Garcia I, Jimenez B, Mandriota S J, Borlat F, et al. Multiple forms of angiostatin induce apoptosis in endothelial cells. Blood 1998; 92(12):4730-41. These proteins/polypeptides are large, however, and are not ideal for use as therapeutic agents. Further, full length human proteins, although theoretically not foreign to an individual's body, induce an immune response in some individuals.

More recently, a bioinformatics approach has allowed identification of candidate antiangiogenic regions of several proteins and synthetic peptides corresponding to those short sequences that possess the ability to suppress proliferation and migration of vascular endothelial cells in vitro and angiogenesis in vivo. Delivering such peptides to a cell and prolonging the duration of their activity, however, remains a challenge.

Although peptides are much easier to produce and are more scalable and less immunogenic than full-length proteins, they are eliminated from the body more quickly. The presently disclosed subject matter can increase and sustain residence time, increase accumulation in tumor vasculature, and maximize the therapeutic effects of such peptides. The presently disclosed subject matter combines biomaterial synthesis, sustained drug delivery, and anti-cancer peptide creation to provide nanoparticle-, microparticle-, and gel-based systems for sustained peptide delivery. The presently disclosed biodegradable biomaterials can be tuned for the encapsulation, protection, and sustained release of each type of peptide.

The use of the presently disclosed nanoparticles, microparticles, and gels limits toxicity because they can extravasate from the leaky neovasculature of the tumors and be trapped in the interstitium of the tumor once the anti-angiogenic compounds kill or normalize the vasculature. Further, the presently disclosed subject matter demonstrates that effective biomaterials for anti-cancer peptide nanoparticles, microparticles, and gels can be fabricated. Multiple anti-cancer peptides and other peptides can be combined within the same particle for multimodal peptide delivery, as well as multimodal therapy with other active agents including, but not limited to, other peptides, nucleic acids, proteins, small molecules, and the like.

More particularly, in some embodiments, the presently disclosed subject matter provides peptides that work through multiple biological mechanisms in combination with the presently disclosed biomaterials, including multilayer and multi-peptide nanoparticle formulations. An array of biodegradable polymers can be used to encapsulate peptides to create nanoparticles having varied biophysical properties and release kinetics. Each peptide can have a specialized subset of materials employed for its encapsulation. Referring now to FIG. 4, differing chemical structures can be synthesized by the conjugate addition of amines to acrylates or acrylamides of differing structure. The polymer structure can be tuned through variation to the backbone, side chain, end-group, hydrophobicity, and degradability. Unlike the polymeric materials disclosed in International PCT Patent Application Publication No. WO/2010/132879 for “Multicomponent Degradable Cationic Polymers,” to Green et al., which are cationic, i.e., positively charged, the presently disclosed polymers can be positively charged, whereas others can be negatively charged, others neutral and hydrophobic, and still others amphiphilic. The diversity of the presently disclosed biomaterials comes from the chemical diversity of the R groups (R, R′, R″) in the biomaterial array and from parameter tuning during particle fabrication.

For example, to create the presently disclosed multi-peptide particles, hydrophobic core particles first are constructed by self-assembly, for example between the somatotropin-derived peptide, the collagen IV-derived peptide, and a hydrophobic polymer. These nanoparticles are then coated by charged biodegradable polymers and peptides following a particle coating and layer-by-layer technique that modifies techniques previously described. Green J J, Chiu E, Leshchiner E S, Shi J, Langer R, Anderson D G. Electrostatic ligand coatings of nanoparticles enable ligand-specific gene delivery to human primary cells. Nano Lett 2007; 7(4):874-9; Shmueli R B, Anderson D G, Green J J. Electrostatic surface modifications to improve gene delivery. Expert Opin Drug Deliv 7(4):535-50. Through this process, the charged peptides (serpin-derived and chemokine-derived) can be incorporated into these multilayers. Charged biological agents, such as peptides and nucleic acids, can serve as both the therapeutic agent and the support polyelectrolyte in the presently disclosed systems.

In one embodiment, peptides can self-assemble with the presently disclosed polymers in an aqueous buffer due to physical, hydrophobic, and electrostatic forces. Zhang S, Uludag H. Nanoparticulate systems for growth factor delivery. Pharm Res 2009; 26(7):1561-80. In other embodiments, peptide-containing micelles can be formed by synthetic polymer-mPEG (e.g., E15 from FIG. 4) block copolymers. Depending on formulation parameters, polymer/peptide particle sizes can be tuned from approximately 50 nm to approximately 500 nm.

As an alternative strategy for polymers in the library that are more hydrophobic or have higher glass transition temperatures, peptides can be encapsulated by a double emulsion procedure. In this method, droplets of aqueous buffer containing peptide are dispersed in the hydrophobic polymer phase and then the polymer phase is itself dispersed in another aqueous phase to form the polymeric particles. Jain R A. The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials 2000; 21(23):2475-90. As an alternative technique, blends of novel hydrophobic polymers and poly(lactic-co-glycolic acid) also can be made to form particles with unique degradation properties. Little S R, Lynn D M, Ge Q, Anderson D G, Puram S V, Chen J Z, et al. Poly-beta amino ester-containing microparticles enhance the activity of nonviral genetic vaccines. Proc Natl Acad Sci USA 2004; 101(26):9534-9.

I. Peptides for Anti-Angiogenesis, Anti-Lymphangiogenesis, Anti-Tumor, and Anti-Permeability Activity.

Several classes of peptides have been developed that show either anti-proliferative or anti-migratory activity or both on endothelial cells. These peptides appear to function through distinct mechanisms of action and have been tested both in vitro and in vivo in tumor xenografts and in ocular mouse models. These peptides include a 24-mer peptide NGRKACLNPASPIVKKIIEKMLNS derived from the CXC chemokine protein GRO-α/CXCL1 and a collagen IV derived and modified 20-mer peptide LRRFSTMPFMF-Abu-NINNV-Abu-NF as a highly potent anti-proliferative and anti-migratory peptide targeting α_vβ1 integrins on both endothelial and tumor cells; here Abu is the 2-Aminobutyric acid introduced in the sequence to facilitate translation to human.

An 11-mer anti-angiogenic peptide EIELVEEEPPF derived from the serpin domain of DEAH box polypeptide also has been identified that shows significant inhibition of MDA-MB-231 tumor xenograft growth. A somatotropin family peptide LLRISLLLIESWLE (SP5031) derived from transmembrane 45 protein that also has been identified and has anti-proliferative and anti-migratory activity on both endothelial cells and lymphatic endothelial cells. It is believed that this peptide is the first antilymphangiogenic peptide agent. Combining these peptides together can result in a peptide-based system that inhibits angiogenesis by several different mechanisms and also inhibits lymphangiogenesis that has been shown to promote tumor metastasis.

Representative peptides suitable for encapsulation with the presently disclosed biomaterials include those disclosed in International PCT Patent Application Publication Number WO2007/033215 A2 for “Compositions Having Antiangiogenic Activity and Uses Thereof,” to Popel et al., published Mar. 22, 2007; International PCT Patent Application Publication Number WO2008/085828 A2 for “Peptide Modulators of Angiogenesis and Use Thereof,” to Popel, published Jul. 17, 2008; U.S. Provisional Patent Application No. 61/421,706, filed Dec. 12, 2010, which is commonly owned; and U.S. Provisional Patent Application No. 61/489,500, filed May 24, 2011, which also is commonly owned, each of which is incorporated herein by reference in its entirety.

Accordingly, in some embodiments, peptide suitable for use in the presently disclosed subject matter are disclosed in Tables 1-10 of International PCT Patent Application Publication Number WO2008/085828 A2 for “Peptide Modulators of Angiogenesis and Use Thereof,” to Popel, published Jul. 17, 2008, which is incorporated herein by reference in its entirety.

Accordingly, in some embodiments, the presently disclosed subject matter provides a nanoparticle, microparticle, or gel comprising one or more peptides, wherein the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof comprising one of the following amino acid sequences:

TSP Motif: W-X(2)-C-X(3)-C-X(2)-G, CXC Motif: G-X(3)-C-L Collagen Motif: C-N-X(3)-V-C Collagen Motif: P-F-X(2)-C Somatotropin Motif: L-X(3)-L-L-X(3)-S-X-L Serpin Motif: L-X(2)-E-E-X-P

wherein X denotes a variable amino acid and the number in parentheses denotes the number of variable amino acids; W denotes tryptophan; C denotes cysteine, G denotes glycine, V denotes valine; L denotes leucine, P is proline, and wherein the peptide reduces blood vessel formation in a cell, tissue or organ.

In other embodiments, the one or more peptide comprises an amino acid sequence shown in Table 1-6, 8 and 9.

In other embodiments, the one or more peptide comprises an isolated peptide or analog thereof having at least 85% identity to an amino acid sequence shown in Table 1-10.

In other embodiments, the one or more peptide comprises an amino acid sequence shown in Table 1-10. In yet other embodiments, the one or more peptide consists essentially of an amino acid sequence shown in Table 1-10.

In particular embodiments, the one or more peptide comprises an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of:

Placental Lactogen LLRISLLLIESWLE hGH-V LLRISLLLTQSWLE GH2 LLHISLLLIQSWLE Chorionic somatomammotropin LLRLLLLIESWLE Chorionic somatomammotropin LLHISLLLIESRLE hormone-like 1 Transmembrane protein 45A LLRSSLILLQGSWF IL-17 receptor C RLRLLTLQSWLL Neuropeptide FF receptor 2 LLIVALLFILSWL Brush border myosin-I LMRKSQILISSWF

wherein the peptide reduces blood vessel formation in a cell, tissue or organ.

In yet more particular embodiments, the one or more peptide comprises an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of:

DEAH box polypeptide 8 EIELVEEEPPF Caspase 10 AEDLLSEEDPF CKIP-1 TLDLIQEEDPS

wherein the peptide reduces blood vessel formation in a cell, tissue or organ.

In further embodiments, the one or more peptide comprises an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of:

Collagen type IV, alpha6 LPRFSTMPFIYCNINEVCHY fibril

wherein the peptide reduces blood vessel formation in a cell, tissue or organ.

TABLE 1 The TSP-1 containing 20-mer with all the possible amino acid substitutions AA#1 AA#2 AA#3 AA#4 AA#5 AA#6 AA#7 AA#8 AA#9 AA#10 S (9) P (13) W (29) S (14) P (9) C (29) S (26) V (7) T (15) C (29) T (9) E (5) T (5) A (5) N (2) A (6) S (10) G (6) S (3) G (5) Q (4) T (1) R (5) R (3) Q (2) A (2) E (2) D (3) K (4) N (1) A (1) Q (1) D (1) E (3) G (2) K (1) R (1) K (1) S (2) A (1) R (1) T (2) V (1) E (1) AA#11 AA#12 AA#13 AA#14 AA#15 AA#16 AA#17 AA#18 AA#19 AA#20 G (26) G (10) G (29) V (8) Q (11) T (10) R (26) S (5) R (15) R (1) S (2) K (4) I (4) S (7) F (4) S (2) T (5) V (1) N (1) R (4) M (3) R (6) K (3) Q (1) V (5) M (4) T (3) K (2) Q (3) R (3) T (2) H (2) Y (2) S (3) H (3) L (2) A (1) A (1) L (2) E (2) D (1) E (1) E (1) Q (2) S (1) F (1) M (1) A (1) P (1) K (1) N (1) I (1) R (1) V (1) S (1) Q (1) W (1) Y (1)

TABLE 2 TSPs Motif: W-X(2)-C-X(3)-C-X(2)-G Number of Locations: 166 Number of Different Proteins: 54 Accession First Number|Protein Amino Last Amino # Name acid acid Sequence 1 O00622|CYR61_HUMAN 236 246 WsqCsktCgtG 2 O14514|BAI1_HUMAN 270 280 WgeCtrdCggG 3 O14514|BAI1_HUMAN 363 373 WsvCsstCgeG 4 O14514|BAI1_HUMAN 418 428 WslCsstCgrG 5 O14514|BAI1_HUMAN 476 486 WsaCsasCsqG 6 O14514|BAI1_HUMAN 531 541 WgsCsvtCgaG 7 O15072|ATS3_HUMAN 975 985 WseCsvtCgeG 8 O60241|BAI2_HUMAN 306 316 WsvCsltCgqG 9 O60241|BAI2_HUMAN 361 371 WslCsrsCgrG 10 O60241|BAI2_HUMAN 416 426 WgpCstsCanG 11 O60241|BAI2_HUMAN 472 482 WslCsktCdtG 12 O60242|BAI3_HUMAN 300 310 WstCsvtCgqG 13 O60242|BAI3_HUMAN 354 364 WslCsftCgrG 14 O60242|BAI3_HUMAN 409 419 WsqCsvtCsnG 15 O60242|BAI3_HUMAN 464 474 WsgCsksCdgG 16 O75173|ATS4_HUMAN 529 539 WgdCsrtCggG 17 O76076|WISP2_HUMAN 201 211 WgpCsttCglG 18 O95185|UNC5C_HUMAN 269 279 WsvCnsrCgrG 19 O95388|WISP1_HUMAN 223 233 WspCstsCglG 20 O95389|WISP3_HUMAN 216 226 WtpCsrtCgmG 21 O95450|ATS2_HUMAN 863 873 WspCskpCggG 22 O95450|ATS2_HUMAN 984 994 WsqCsvtCgnG 23 P07996|TSP1_HUMAN 388 398 WtsCstsCgnG 24 P07996|TSP1_HUMAN 444 454 WssCsvtCgdG 25 P07996|TSP1_HUMAN 501 511 WdiCsvtCggG 26 P13671|CO6_HUMAN 32 42 WtsCsktCnsG 27 P13671|CO6_HUMAN 75 85 WqrCpinCllG 28 P14222|PERF_HUMAN 374 384 WrdCsrpCppG 29 P27918|PROP_HUMAN 86 96 WapCsvtCseG 30 P27918|PROP_HUMAN 145 155 WepCsvtCskG 31 P27918|PROP_HUMAN 202 212 WtpCsasChgG 32 P29279|CTGF_HUMAN 206 216 WsaCsktCgmG 33 P35442|TSP2_HUMAN 390 400 WtqCsvtCgsG 34 P35442|TSP2_HUMAN 446 456 WssCsvtCgvG 35 P35442|TSP2_HUMAN 503 513 WsaCtvtCagG 36 P48745|NOV_HUMAN 213 223 WtaCsksCgmG 37 P49327|FAS_HUMAN 627 637 WeeCkqrCppG 38 P58397|ATS12_HUMAN 551 561 WshCsrtCgaG 39 P58397|ATS12_HUMAN 832 842 WteCsvtCgtG 40 P58397|ATS12_HUMAN 952 962 WseCsvsCggG 41 P58397|ATS12_HUMAN 1321 1331 WseCsttCglG 42 P58397|ATS12_HUMAN 1372 1382 WskCsrnCsgG 43 P58397|ATS12_HUMAN 1431 1441 WsqCsrsCggG 44 P58397|ATS12_HUMAN 1479 1489 WdlCstsCggG 45 P59510|ATS20_HUMAN 976 986 WsqCsrsCggG 46 P59510|ATS20_HUMAN 1031 1041 WseClvtCgkG 47 P59510|ATS20_HUMAN 1086 1096 WgpCtttCghG 48 P59510|ATS20_HUMAN 1162 1172 WtpCsvsCgrG 49 P59510|ATS20_HUMAN 1217 1227 WspCsasCghG 50 P59510|ATS20_HUMAN 1314 1324 WgsCsssCsgG 51 P59510|ATS20_HUMAN 1368 1378 WgeCsqtCggG 52 P59510|ATS20_HUMAN 1427 1437 WtsCsasCgkG 53 P59510|ATS20_HUMAN 1483 1493 WneCsvtCgsG 54 P59510|ATS20_HUMAN 1664 1674 WskCsvtCgiG 55 P82987|ATL3_HUMAN 84 94 WsdCsrtCggG 56 P82987|ATL3_HUMAN 427 437 WtaCsvsCggG 57 P82987|ATL3_HUMAN 487 497 WsqCtvtCgrG 58 P82987|ATL3_HUMAN 573 583 WsaCsttCgpG 59 P82987|ATL3_HUMAN 712 722 WgpCsatCgvG 60 P82987|ATL3_HUMAN 768 778 WqqCsrtCggG 61 P82987|ATL3_HUMAN 828 838 WskCsvsCgvG 62 P82987|ATL3_HUMAN 1492 1502 WsqCsvsCgeG 63 P82987|ATL3_HUMAN 1606 1616 WkpCtaaCgrG 64 Q13591|SEM5A_HUMAN 604 614 WspCsttCgiG 65 Q13591|SEM5A_HUMAN 662 672 WerCtaqCggG 66 Q13591|SEM5A_HUMAN 793 803 WsqCsrdCsrG 67 Q13591|SEM5A_HUMAN 850 860 WtkCsatCggG 68 Q496M8|CI094_HUMAN 259 269 WsaCtrsCggG 69 Q6S8J7|POTE8_HUMAN 27 37 WccCcfpCcrG 70 Q6UXZ4|UNC5D_HUMAN 261 271 WsaCnvrCgrG 71 Q6UY14|ATL4_HUMAN 53 63 WasCsqpCgvG 72 Q6UY14|ATL4_HUMAN 732 742 WtsCsrsCgpG 73 Q6UY14|ATL4_HUMAN 792 802 WsqCsvrCgrG 74 Q6UY14|ATL4_HUMAN 919 929 WgeCsseCgsG 75 Q6UY14|ATL4_HUMAN 979 989 WspCsrsCqgG 76 Q6ZMM2|ATL5_HUMAN 44 54 WtrCsssCgrG 77 Q76LX8|ATS13_HUMAN 1081 1091 WmeCsvsCgdG 78 Q86TH1|ATL2_HUMAN 56 66 WtaCsrsCggG 79 Q86TH1|ATL2_HUMAN 631 641 WseCsrtCgeG 80 Q86TH1|ATL2_HUMAN 746 756 WgpCsgsCgqG 81 Q86TH1|ATL2_HUMAN 803 813 WerCnttCgrG 82 Q86TH1|ATL2_HUMAN 862 872 WseCtktCgvG 83 Q8IUL8|CILP2_HUMAN 155 165 WgpCsgsCgpG 84 Q8IZJ1|UNC5B_HUMAN 255 265 WspCsnrCgrG 85 Q8N6G6|ATL1_HUMAN 42 52 WseCsrtCggG 86 Q8N6G6|ATL1_HUMAN 385 395 WtaCsssCggG 87 Q8N6G6|ATL1_HUMAN 445 455 WspCtvtCgqG 88 Q8TE56|ATS17_HUMAN 552 562 WsmCsrtCgtG 89 Q8TE56|ATS17_HUMAN 809 819 WegCsvqCggG 90 Q8TE56|ATS17_HUMAN 870 880 WspCsatCekG 91 Q8TE56|ATS17_HUMAN 930 940 WsqCsasCgkG 92 Q8TE56|ATS17_HUMAN 981 991 WstCsstCgkG 93 Q8TE57|ATS16_HUMAN 595 605 WspCsrtCggG 94 Q8TE57|ATS16_HUMAN 936 946 WsaCsrtCggG 95 Q8TE57|ATS16_HUMAN 995 1005 WaeCshtCgkG 96 Q8TE57|ATS16_HUMAN 1060 1070 WsqCsvtCerG 97 Q8TE57|ATS16_HUMAN 1135 1145 WsqCtasCggG 98 Q8TE58|ATS15_HUMAN 848 858 WgpCsasCgsG 99 Q8TE58|ATS15_HUMAN 902 912 WspCsksCgrG 100 Q8TE59|ATS19_HUMAN 642 652 WspCsrtCsaG 101 Q8TE59|ATS19_HUMAN 924 934 WedCdatCggG 102 Q8TE59|ATS19_HUMAN 985 995 WtpCsrtCgkG 103 Q8TE59|ATS19_HUMAN 1096 1106 WskCsitCgkG 104 Q8TE60|ATS18_HUMAN 598 608 WseCsrtCggG 105 Q8TE60|ATS18_HUMAN 940 950 WstCskaCagG 106 Q8TE60|ATS18_HUMAN 1000 1010 WsqCsktCgrG 107 Q8TE60|ATS18_HUMAN 1061 1071 WseCsatCglG 108 Q8TE60|ATS18_HUMAN 1132 1142 WqqCtvtCggG 109 Q8WXS8|ATS14_HUMAN 856 866 WapCskaCggG 110 Q8WXS8|ATS14_HUMAN 977 987 WsqCsatCgeG 111 Q92947|GCDH_HUMAN 225 235 WarCedgCirG 112 Q96RW7|HMCN1_HUMAN 4538 4548 WraCsvtCgkG 113 Q96RW7|HMCN1_HUMAN 4595 4605 WeeCtrsCgrG 114 Q96RW7|HMCN1_HUMAN 4652 4662 WgtCsesCgkG 115 Q96RW7|HMCN1_HUMAN 4709 4719 WsaCsvsCggG 116 Q96RW7|HMCN1_HUMAN 4766 4776 WgtCsrtCngG 117 Q96RW7|HMCN1_HUMAN 4823 4833 WsqCsasCggG 118 Q99732|LITAF_HUMAN 116 126 WlsCgslCllG 119 Q9C0I4|THS7B_HUMAN 49 59 WgrCtgdCgpG 120 Q9C0I4|THS7B_HUMAN 345 355 WspCsktCrsG 121 Q9C0I4|THS7B_HUMAN 746 756 WtpCprmCqaG 122 Q9C0I4|THS7B_HUMAN 1009 1019 WgsCsssCgiG 123 Q9C0I4|THS7B_HUMAN 1258 1268 WteCsqtCghG 124 Q9C0I4|THS7B_HUMAN 1381 1391 WstCeltCidG 125 Q9H324|ATS10_HUMAN 530 540 WgdCsrtCggG 126 Q9H324|ATS10_HUMAN 808 818 WtkCsaqCagG 127 Q9H324|ATS10_HUMAN 867 877 WslCsrsCdaG 128 Q9H324|ATS10_HUMAN 927 937 WseCtpsCgpG 129 Q9H324|ATS10_HUMAN 986 996 WgeCsaqCgvG 130 Q9HCB6|SPON1_HUMAN 510 520 WspCsisCgmG 131 Q9HCB6|SPON1_HUMAN 567 577 WdeCsatCgmG 132 Q9HCB6|SPON1_HUMAN 623 633 WsdCsvtCgkG 133 Q9HCB6|SPON1_HUMAN 677 687 WseCnksCgkG 134 Q9HCB6|SPON1_HUMAN 763 773 WseCtklCggG 135 Q9NS62|THSD1_HUMAN 349 359 WsqCsatCgdG 136 Q9P283|SEM5B_HUMAN 615 625 WalCstsCgiG 137 Q9P283|SEM5B_HUMAN 673 683 WskCssnCggG 138 Q9P283|SEM5B_HUMAN 804 814 WssCsrdCelG 139 Q9P283|SEM5B_HUMAN 861 871 WspCsasCggG 140 Q9P2N4|ATS9_HUMAN 1006 1016 WteCsksCdgG 141 Q9P2N4|ATS9_HUMAN 1061 1071 WseClvtCgkG 142 Q9P2N4|ATS9_HUMAN 1116 1126 WvqCsvtCgqG 143 Q9P2N4|ATS9_HUMAN 1191 1201 WtpCsatCgkG 144 Q9P2N4|ATS9_HUMAN 1247 1257 WssCsvtCgqG 145 Q9P2N4|ATS9_HUMAN 1337 1347 WgaCsstCagG 146 Q9P2N4|ATS9_HUMAN 1391 1401 WgeCtklCggG 147 Q9P2N4|ATS9_HUMAN 1450 1460 WssCsvsCgrG 148 Q9P2N4|ATS9_HUMAN 1506 1516 WsqCsvsCgrG 149 Q9P2N4|ATS9_HUMAN 1564 1574 WqeCtktCgeG 150 Q9P2N4|ATS9_HUMAN 1621 1631 WseCsvtCgkG 151 Q9P2N4|ATS9_HUMAN 1686 1696 WgsCsvsCgvG 152 Q9UHI8|ATS1_HUMAN 568 578 WgdCsrtCggG 153 Q9UHI8|ATS1_HUMAN 863 873 WgeCsksCelG 154 Q9UHI8|ATS1_HUMAN 917 927 WssCsktCgkG 155 Q9UKP4|ATS7_HUMAN 547 557 WsiCsrsCgmG 156 Q9UKP4|ATS7_HUMAN 924 934 WtkCtvtCgrG 157 Q9UKP5|ATS6_HUMAN 519 529 WgeCsrtCggG 158 Q9UKP5|ATS6_HUMAN 801 811 WseCsatCagG 159 Q9UNA0|ATS5_HUMAN 576 586 WgqCsrsCggG 160 Q9UNA0|ATS5_HUMAN 884 894 WlaCsrtCdtG 161 Q9UP79|ATS8_HUMAN 536 546 WgeCsrtCggG 162 Q9UP79|ATS8_HUMAN 842 852 WseCsstCgaG 163 Q9UPZ6|THS7A_HUMAN 203 213 WseCsktCgsG 164 Q9UPZ6|THS7A_HUMAN 780 790 WtsCpssCkeG 165 Q9UPZ6|THS7A_HUMAN 1044 1054 WsrCsksCgsG 166 Q9UPZ6|THS7A_HUMAN 1423 1433 WslCqltCvnG

TABLE 3 The C-X-C chemokine 22-mer with all the possible amino acid substitutions AA#1 AA#2 AA#3 AA#4 AA#5 AA#6 AA#7 AA#8 AA#9 AA#10 AA#11 N (4) G (6) R (3) K (3) A (2) C (6) L (6) D (4) P (6) A (2) A (3) D (2) K (3) E (2) I (2) N (2) E (2) S (2) Q (1) L (1) D (1) E (1) V (1) K (1) AA#12 AA#13 AA#14 AA#15 AA#16 AA#17 AA#18 AA#19 AA#20 AA#21 AA#22 P (6) F (2) V (3) K (4) K (5) I (3) I (4) E (3) K (6) I (3) L (6) I (1) L (2) Q (2) R (1) V (3) V (2) Q (3) F (1) M (1) I (1) K (1) R (1) M (1) W (1)

TABLE 5 Collagens Motif: C-N-X(3)-V-C Number of Locations: 24 Number of Different Proteins: 24 Accession First Number|Protein Amino Last Amino # Name acid acid Sequence 1504 O14514|BAI1_HUMAN 400 406 CNnsaVC 1505 O75093|SLIT1_HUMAN 507 513 CNsdvVC 1506 O75534|CSDE1_HUMAN 733 739 CNvwrVC 1507 P02462|CO4A1_HUMAN 1505 1511 CNinnVC 1508 P08572|CO4A2_HUMAN 1549 1555 CNpgdVC 1509 P09758|TACD2_HUMAN 119 125 CNqtsVC 1510 P25391|LAMA1_HUMAN 751 757 CNvhgVC 1511 P29400|CO4A5_HUMAN 1521 1527 CNinnVC 1512 P53420|CO4A4_HUMAN 1525 1531 CNihqVC 1513 P83110|HTRA3_HUMAN 48 54 CNcclVC 1514 Q01955|CO4A3_HUMAN 1505 1511 CNvndVC 1515 Q13625|ASPP2_HUMAN 1002 1008 CNnvqVC 1516 Q13751|LAMB3_HUMAN 572 578 CNrypVC 1517 Q14031|CO4A6_HUMAN 1527 1533 CNineVC 1518 Q8WWQ8|STAB2_HUMAN 1970 1976 CNnrgVC 1519 Q96GX1|TECT2_HUMAN 642 648 CNrneVC 1520 Q99965|ADAM2_HUMAN 621 627 CNdrgVC 1521 Q9BX93|PG12B_HUMAN 112 118 CNqldVC 1522 Q9BYD5|CNFN_HUMAN 32 38 CNdmpVC 1523 Q9H013|ADA19_HUMAN 659 665 CNghgVC 1524 Q9HBG6|IF122_HUMAN 436 442 CNllvVC 1525 Q9P2R7|SUCB1_HUMAN 152 158 CNqvlVC 1526 Q9UBX1|CATF_HUMAN 89 95 CNdpmVC 1527 Q9UKF2|ADA30_HUMAN 638 644 CNtrgVC

TABLE 7A Table of the amino acid sequences of the peptides predicted similar to Growth Hormone Peptide Protein Name Peptide Location sequence Placental Lactogen AAA98621(101-114) LLRISLLLIESWLE hGH-V AAB59548(101-114) LLRISLLLTQSWLE GH2 CAG46722(101-114) LLHISLLLIQSWLE Chorionic AAA52116(101-113) LLRLLLLIESWLE somatomammotropin Chorionic AAI19748(12-25) LLHISLLLIESRLE somatomammotropin hormone-like 1 Transmembrane NP_060474(181-194) LLRSSLILLQGSWF protein 45A IL-17 receptor C Q8NAC3(376-387) RLRLLTLQSWLL Neuropeptide Q9Y5X5(378-390) LLIVALLFILSWL FF receptor 2 Brush border AAC27437(719-731) LMRKSQILISSWF myosin-I

TABLE 7B Table of the amino acid sequences of the peptides predicted similar to PEDF. Peptide Protein Name Peptide Location sequence DEAH box polypeptide AAH47327(438-448) EIELVEEEPPF 8 Caspase 10 CAD32371(67-77) AEDLLSEEDPF CKIP-1 CAI14263(66-76) TLDLIQEEDPS

TABLE 8 Amino acid sequences of peptides that contain the somatotropin motif. Somatotropins Motif: L-X(3)-L-L-X(3)-S-X-L Number of Locations: 139 Number of Different Proteins: 139 Accession # Number|Protein Name First Aminoacid Last Aminoacid Sequence 1834 O14569|C56D2_HUMAN 164 175 LvgyLLgsaSlL 1835 O15287|FANCG_HUMAN 416 427 LceeLLsrtSsL 1836 O15482|TEX28_HUMAN 338 349 LatvLLvfvStL 1837 O43914|TYOBP_HUMAN 11 22 LllpLLlavSgL 1838 O60609|GFRA3_HUMAN 15 26 LmllLLlppSpL 1839 O75844|FACE1_HUMAN 279 290 LfdtLLeeySvL 1840 O95747|OXSR1_HUMAN 90 101 LvmkLLsggSvL 1841 P01241|SOMA_HUMAN 102 113 LrisLLliqSwL 1842 P01242|SOM2_HUMAN 102 113 LrisLLliqSwL 1843 P01243|CSH_HUMAN 102 113 LrisLLlieSwL 1844 P02750|A2GL_HUMAN 83 94 LpanLLqgaSkL 1845 P03891|NU2M_HUMAN 149 160 LnvsLLltlSiL 1846 P04201|MAS_HUMAN 151 162 LvcaLLwalScL 1847 P05783|K1C18_HUMAN 338 349 LngiLLhleSeL 1848 P07359|GP1BA_HUMAN 3 14 LlllLLllpSpL 1849 P09848|LPH_HUMAN 35 46 LtndLLhnlSgL 1850 P11168|GTR2_HUMAN 136 147 LvgaLLmgfSkL 1851 P12034|FGF5_HUMAN 3 14 LsflLLlffShL 1852 P13489|RINI_HUMAN 247 258 LcpgLLhpsSrL 1853 P14902|I23O_HUMAN 196 207 LlkaLLeiaScL 1854 P16278|BGAL_HUMAN 135 146 LpawLLekeSiL 1855 P19838|NFKB1_HUMAN 558 569 LvrdLLevtSgL 1856 P22079|PERL_HUMAN 512 523 LvrgLLakkSkL 1857 P23276|KELL_HUMAN 53 64 LilgLLlcfSvL 1858 P24394|IL4RA_HUMAN 4 15 LcsgLLfpvScL 1859 P29320|EPHA3_HUMAN 5 16 LsilLLlscSvL 1860 P31512|FMO4_HUMAN 524 535 LaslLLickSsL 1861 P35270|SPRE_HUMAN 26 37 LlasLLspgSvL 1862 P41250|SYG_HUMAN 20 31 LpprLLarpSlL 1863 P42575|CASP2_HUMAN 114 125 LedmLLttlSgL 1864 P46721|SO1A2_HUMAN 396 407 LleyLLyflSfL 1865 P51665|PSD7_HUMAN 201 212 LnskLLdirSyL 1866 P59531|T2R12_HUMAN 188 199 LisfLLsliSlL 1867 P69849|NOMO3_HUMAN 1180 1191 LiplLLqltSrL 1868 P98161|PKD1_HUMAN 82 93 LdvgLLanlSaL 1869 P98171|RHG04_HUMAN 153 164 LqdeLLevvSeL 1870 P98196|AT11A_HUMAN 1077 1088 LaivLLvtiSlL 1871 Q08431|MFGM_HUMAN 10 21 LcgaLLcapSlL 1872 Q08AF3|SLFN5_HUMAN 533 544 LvivLLgfkSfL 1873 Q12952|FOXL1_HUMAN 293 304 LgasLLaasSsL 1874 Q13275|SEM3F_HUMAN 2 13 LvagLLlwaSlL 1875 Q13394|MB211_HUMAN 300 311 LngiLLqliScL 1876 Q13609|DNSL3_HUMAN 8 19 LlllLLsihSaL 1877 Q13619|CUL4A_HUMAN 213 224 LlrsLLgmlSdL 1878 Q13620|CUL4B_HUMAN 349 360 LlrsLLsmlSdL 1879 Q14406|CSHL_HUMAN 84 95 LhisLLlieSrL 1880 Q14667|K0100_HUMAN 8 19 LlvlLLvalSaL 1881 Q15155|NOMO1_HUMAN 1180 1191 LiplLLqltSrL 1882 Q15760|GPR19_HUMAN 279 290 LilnLLfllSwL 1883 Q53RE8|ANR39_HUMAN 166 177 LacdLLpcnSdL 1884 Q5FWE3|PRRT3_HUMAN 586 597 LatdLLstwSvL 1885 Q5GH73|XKR6_HUMAN 630 641 LlyeLLqyeSsL 1886 Q5GH77|XKR3_HUMAN 194 205 LnraLLmtfSlL 1887 Q5JPE7|NOMO2_HUMAN 1180 1191 LiplLLqltSrL 1888 Q5JWR5|DOP1_HUMAN 506 517 LpqlLLrmiSaL 1889 Q5UIP0|RIF1_HUMAN 2413 2424 LsknLLaqiSaL 1890 Q5VTE6|ANGE2_HUMAN 175 186 LsqdLLednShL 1891 Q5VU43|MYOME_HUMAN 1932 1943 LreaLLssrShL 1892 Q5VYK3|ECM29_HUMAN 1296 1307 LipaLLeslSvL 1893 Q68D06|SLN13_HUMAN 554 565 LvivLLgfrSlL 1894 Q6GYQ0|GRIPE_HUMAN 641 652 LwddLLsvlSsL 1895 Q6NTF9|RHBD2_HUMAN 166 177 LvpwLLlgaSwL 1896 Q6ZMH5|S39A5_HUMAN 217 228 LavlLLslpSpL 1897 Q6ZMZ3|SYNE3_HUMAN 532 543 LhnsLLqrkSkL 1898 Q6ZVD8|PHLPL_HUMAN 313 324 LfpiLLceiStL 1899 Q6ZVE7|GOT1A_HUMAN 23 34 LfgtLLyfdSvL 1900 Q70J99|UN13D_HUMAN 927 938 LrveLLsasSlL 1901 Q7Z3Z4|PIWL4_HUMAN 139 150 LriaLLyshSeL 1902 Q7Z6Z7|HUWE1_HUMAN 841 852 LqegLLqldSiL 1903 Q7Z7L1|SLN11_HUMAN 554 565 LvivLLgfrSlL 1904 Q86SM5|MRGRG_HUMAN 223 234 LlnfLLpvfSpL 1905 Q86U44|MTA70_HUMAN 78 89 LekkLLhhlSdL 1906 Q86UQ4|ABCAD_HUMAN 3182 3193 LlnsLLdivSsL 1907 Q86WI3|NLRC5_HUMAN 1485 1496 LlqsLLlslSeL 1908 Q86YC3|LRC33_HUMAN 263 274 LffpLLpqySkL 1909 Q8IYK4|GT252_HUMAN 9 20 LawsLLllsSaL 1910 Q8IYS0|GRM1C_HUMAN 485 496 LesdLLieeSvL 1911 Q8IZL8|PELP1_HUMAN 33 44 LrllLLesvSgL 1912 Q8IZY2|ABCA7_HUMAN 1746 1757 LftlLLqhrSqL 1913 Q8N0X7|SPG20_HUMAN 322 333 LfedLLrqmSdL 1914 Q8N6M3|CT142_HUMAN 33 44 LagsLLkelSpL 1915 Q8N816|TMM99_HUMAN 96 107 LlpcLLgvgSwL 1916 Q8NBM4|PDHL1_HUMAN 15 26 LsksLLlvpSaL 1917 Q8NCG7|DGLB_HUMAN 555 566 LtqpLLgeqSlL 1918 Q8NFR9|I17RE_HUMAN 80 91 LcqhLLsggSgL 1919 Q8NGE3|O10P1_HUMAN 9 20 LpefLLlgfSdL 1920 Q8TCV5|WFDC5_HUMAN 8 19 LlgaLLavgSqL 1921 Q8TDL5|LPLC1_HUMAN 165 176 LriqLLhklSfL 1922 Q8TE82|S3TC1_HUMAN 1025 1036 LegqLLetiSqL 1923 Q8TEQ8|PIGO_HUMAN 857 868 LvflLLflqSfL 1924 Q8TEZ7|MPRB_HUMAN 127 138 LlahLLqskSeL 1925 Q8WWN8|CEND3_HUMAN 1481 1492 LeeqLLqelSsL 1926 Q8WZ84|OR8D1_HUMAN 43 54 LgmiLLiavSpL 1927 Q92535|PIGC_HUMAN 253 264 LfalLLmsiScL 1928 Q92538|GBF1_HUMAN 1224 1235 LrilLLmkpSvL 1929 Q92743|HTRA1_HUMAN 262 273 LpvlLLgrsSeL 1930 Q92935|EXTL1_HUMAN 19 30 LllvLLggfSlL 1931 Q93074|MED12_HUMAN 401 412 LqtiLLccpSaL 1932 Q96DN6|MBD6_HUMAN 740 751 LgasLLgdlSsL 1933 Q96GR4|ZDH12_HUMAN 48 59 LtflLLvlgSlL 1934 Q96HP8|T176A_HUMAN 29 40 LaklLLtccSaL 1935 Q96K12|FACR2_HUMAN 380 391 LmnrLLrtvSmL 1936 Q96KP1|EXOC2_HUMAN 339 350 LldkLLetpStL 1937 Q96MX0|CKLF3_HUMAN 40 51 LkgrLLlaeSgL 1938 Q96Q45|AL2S4_HUMAN 387 398 LvvaLLvglSwL 1939 Q96QZ0|PANX3_HUMAN 136 147 LssdLLfiiSeL 1940 Q96RQ9|OXLA_HUMAN 269 280 LpraLLsslSgL 1941 Q9BY08|EBPL_HUMAN 178 189 LipgLLlwqSwL 1942 Q9BZ97|TTY13_HUMAN 30 41 LclmLLlagScL 1943 Q9H1Y0|ATG5_HUMAN 85 96 LlfdLLassSaL 1944 Q9H254|SPTN4_HUMAN 1422 1433 LdkkLLhmeSqL 1945 Q9H330|CI005_HUMAN 430 441 LgkfLLkvdSkL 1946 Q9H4I8|SEHL2_HUMAN 175 186 LlqrLLksnShL 1947 Q9HCN3|TMEM8_HUMAN 200 211 LpqtLLshpSyL 1948 Q9NQ34|TMM9B_HUMAN 4 15 LwggLLrlgSlL 1949 Q9NR09|BIRC6_HUMAN 1400 1411 LlkaLLdnmSfL 1950 Q9NRA0|SPHK2_HUMAN 296 307 LgldLLlncSlL 1951 Q9NRU3|CNNM1_HUMAN 156 167 LgalLLlalSaL 1952 Q9NTT1|U2D3L_HUMAN 99 110 LskvLLsicSlL 1953 Q9NVH2|INT7_HUMAN 623 634 LridLLqafSqL 1954 Q9NVM9|CL011_HUMAN 350 361 LtnfLLngrSvL 1955 Q9NZD1|GPC5D_HUMAN 60 71 LptqLLfllSvL 1956 Q9P2E9|RRBP1_HUMAN 1226 1237 LrqlLLesqSqL 1957 Q9P2G4|K1383_HUMAN 397 408 LlnaLLvelSlL 1958 Q9P2V4|LRIT1_HUMAN 541 552 LpltLLvccSaL 1959 Q9UDY8|MALT1_HUMAN 33 44 LrepLLrrlSeL 1960 Q9UEW8|STK39_HUMAN 138 149 LvmkLLsggSmL 1961 Q9UGN4|CM35H_HUMAN 188 199 LlllLLvgaSlL 1962 Q9UHD4|CIDEB_HUMAN 189 200 LghmLLgisStL 1963 Q9UIG8|SO3A1_HUMAN 270 281 LcgaLLffsSlL 1964 Q9UPA5|BSN_HUMAN 353 364 LgasLLtqaStL 1965 Q9UPX8|SHAN2_HUMAN 609 620 LtgrLLdpsSpL 1966 Q9Y239|NOD1_HUMAN 318 329 LsgkLLkgaSkL 1967 Q9Y2I2|NTNG1_HUMAN 526 537 LlttLLgtaSpL 1968 Q9Y2U2|KCNK7_HUMAN 92 103 LpsaLLfaaSiL 1969 Q9Y2Y8|PRG3_HUMAN 7 18 LpflLLgtvSaL 1970 Q9Y586|MB212_HUMAN 300 311 LngiLLqliScL 1971 Q9Y5X0|SNX10_HUMAN 106 117 LqnaLLlsdSsL 1972 Q9Y5X5|NPFF2_HUMAN 379 390 LivaLLfilSwL

TABLE 10 Table containing the amino acid sequence of the peptide predicted similar to Tumstatin/Tum4 Protein Name Peptide Location Peptide sequence Collagen CAI40758.1: LPRFSTMPFIYCNINEVCHY type IV, 1630-1648 alpha6 fibril

In other embodiments, the following peptides suitable for use with the presently disclosed subject matter are disclosed in Table 1 of International PCT Patent Application Publication Number WO2007/033215 A2 for “Compositions Having Antiangiogenic Activity and Uses Thereof,” to Popel et al., published Mar. 22, 2007, which is incorporated herein by reference in its entirety.

TABLE 11 Anti-Angiogenic Peptide sequences SEQ ID NO. Thrombospondin Containing Proteins 2287 ADAM-9 Q13443: 649-661 KCHGHGVCNSNKN 2288 ADAM-12 O43184: 662-675 MQCHGRGVCNNRKN 2289 ADAMTS-1 Q9UHI8: 566-584 GPWGDCSRTCGGGVQYTMR 2290 ADAMTS-2 CAA05880.1: 982-998 GPWSQCSVTCGNGTQER 2291 ADAMTS-3 NP_055058.1: 973-989 GPWSECSVTCGEGTEVR 2292 ADAMTS-4 CAH72146.1: 527-540 GPWGDCSRTCGGGV 2293 ADAMTS-4 CAH72146.1: 527-545 GPWGDCSRTCGGGVQFSSR 2294 ADAMTS-5 NP_008969.1: 882-898 GPWLACSRTCDTGWHTR 2295 ADAMTS-6 NP_922932.2: 847-860 QPWSECSATCAGGV 2296 ADAMTS-6 NP_922932.2: 847-863 QPWSECSATCAGGVQRQ 2297 ADAMTS-7 AAH61631.1: 1576-1592 GPWGQCSGPCGGGVQRR 2298 ADAMTS-7 AAH61631.1: 828-841 GPWTKCTVTCGRGV 2299 ADAMTS-8 Q9UP79: 534-547 GPWGECSRTCGGGV 2300 ADAMTS-8 Q9UP79: 534-552 GPWGECSRTCGGGVQFSHR 2301 ADAMTS-9 Q9P2N4: 1247-1261 WSSCSVTCGQGRATR 2302 ADAMTS-9 Q9P2N4: 1335-1351 GPWGACSSTCAGGSQRR 2303 ADAMTS-9 Q9P2N4: 595-613 SPFGTCSRTCGGGIKTAIR 2304 ADAMTS-10 Q9H324: 528-546 TPWGDCSRTCGGGVSSSSR 2305 ADAMTS-12 P58397: 1479-1493 WDLCSTSCGGGFQKR 2306 ADAMTS-12 P58397: 549-562 SPWSHCSRTCGAGV 2307 ADAMTS-13 AAQ88485.1: 751-765 WMECSVSCGDGIQRR 2308 ADAMTS-14 CAI13857.1: 980-994 WSQCSATCGEGIQQR 2309 ADAMTS-15 CAC86014.1: 900-916 SAWSPCSKSCGRGFQRR 2310 ADAMTS-16 Q8TE57: 1133-1149 SPWSQCTASCGGGVQTR 2311 ADAMTS-16 Q8TE57: 1133-1150 SPWSQCTASCGGGVQTRS 2312 ADAMTS-18 Q8TE60: 1131-1146 PWQQCTVTCGGGVQTR 2313 ADAMTS-18 Q8TE60: 1131-1147 PWQQCTVTCGGGVQTRS 2314 ADAMTS-18 Q8TE60: 998-1014 GPWSQCSKTCGRGVRKR 2315 ADAMTS-18 Q8TE60: 596-614 SKWSECSRTCGGGVKFQER 2316 ADAMTS-19 CAC84565.1: 1096-1111 WSKCSITCGKGMQSRV 2317 ADAMTS-20 CAD56159.3: 1478-1494 NSWNECSVTCGSGVQQR 2318 ADAMTS-20 CAD56159.3: 1309-1326 GPWGQCSSSCSGGLQHRA 2319 ADAMTS-20 CAD56159.3: 1661-1675 WSKCSVTCGIGIMKR 2320 ADAMTS-20 CAD56160.2: 564-581 PYSSCSRTCGGGIESATR 2321 BAI-1 O14514: 361-379 SPWSVCSSTCGEGWQTRTR 2322 BAI-2 O60241: 304-322 SPWSVCSLTCGQGLQVRTR 2323 BAI-3 CAI21673.1: 352-370 SPWSLCSFTCGRGQRTRTR 2324 C6 AAB59433.1: 30-48 TQWTSCSKTCNSGTQSRHR 2325 CILP AAQ89263.1: 156-175 SPWSKCSAACGQTGVQTRTR 2326 CILP-2 AAN17826.1: 153-171 GPWGPCSGSCGPGRRLRRR 2327 CTGF CAC44023.1: 204-221 TEWSACSKTCGMGISTRV 2328 CYR61 AAR05446.1: 234-251 TSWSQCSKTCGTGISTRV 2329 Fibulin-6 CAC37630.1: 1574-1592 SAWRACSVTCGKGIQKRSR 2330 Fibulin-6 CAC37630.1: 1688-1706 QPWGTCSESCGKGTQTRAR 2331 Fibulin-6 CAC37630.1: 1745-1763 ASWSACSVSCGGGARQRTR 2332 NOVH AAL92490.1: 211-228 TEWTACSKSCGMGFSTRV 2333 Papilin NP_775733.2: 33-51 SQWSPCSRTCGGGVSFRER 2334 Papilin NP_775733.2: 342-359 GPWAPCSASCGGGSQSRS 2335 Properdin AAP43692.1: 143-161 GPWEPCSVTCSKGTRTRRR 2336 ROR-1 CAH71706.1: 313-391 CYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPEL NGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPAC 2337 ROR-1 CAH71706.1: 310-391 NHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRF PELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPAC 2338 ROR-1 CAH71706.1: 311-388 HKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFP ELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDI 2339 ROR-1 CAH71706.1: 311-391 HKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFP ELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPAC 2340 ROR-1 CAH71706.1: 312-392 KCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPE LNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACD 2341 ROR-2 Q01974: 315-395 QCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPE LGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSCS 2342 ROR-2 Q01974: 314-391 HQCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFP ELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDV 2343 ROR-2 Q01974: 314-394 HQCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFP ELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSC 2344 ROR-2 Q01974: 314-395 HQCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFP ELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSCS 2345 ROR-2 Q01974: 315-394 QCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPE LGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSC 2346 Semaphorin 5A NP_003957.1: 660-678 GPWERCTAQCGGGIQARRR 2347 Semaphorin 5A NP_003957.1: 848-866 SPWTKCSATCGGGHYMRTR 2348 Semaphorin 5B AAQ88491.1: 916-934 TSWSPCSASCGGGHYQRTR 2349 SCO-spondin XP_379967.2: 3781-3799 GPWEDCSVSCGGGEQLRSR 2350 THSD1 AAQ88516.1: 347-365 QPWSQCSATCGDGVRERRR 2351 THSD3 AAH33140.1: 280-298 SPWSPCSGNCSTGKQQRTR 2352 THSD6 AAH40620.1: 44-60 WTRCSSSCGRGVSVRSR 2353 TSP-2 CAI23645.1: 444-462 SPWSSCSVTCGVGNITRIR 2354 TSP-2 CAI23645.1: 501-519 SPWSACTVTCAGGIRERTR 2355 TSRC1 AAH27478.1: 140-159 SPWSQCSVRCGRGQRSRQVR 2356 UNC5C AAH41156.1: 267-285 TEWSVCNSRCGRGYQKRTR 2357 UNC5D AAQ88514.1: 259-277 TEWSACNVRCGRGWQKRSR 2358 VSGP/F-spondin BAB18461.1: 567-583 WDECSATCGMGMKKRHR 2359 VSGP/F-spondin BAB18461.1: 621-639 SEWSDCSVTCGKGMRTRQR 2360 WISP-1 AAH74841.1: 221-238 SPWSPCSTSCGLGVSTRI 2361 WISP-2 AAQ89274.1: 199-216 TAWGPCSTTCGLGMATRV 2362 WISP-3 CAB16556.1: 191-208 TKWTPCSRTCGMGISNRV Collagens 2363 α1CIV CAH74130.1: 1479-1556 NERAHGQDLGTAGSCLRKFSTMPFLFCNINNVCNFASRNDYSY WLSTPEPMPMSMAPITGENIRPFISRCAVCEAPAM 2364 α1CIV CAH74130.1: 1494-1513 LRKFSTMPFLFCNINNVCNF 2365 α1CIV CAH74130.1: 1504-1523 FCNINNVCNFASRNDYSYWL 2366 α1CIV CAH74130.1: 1610-1628 SAPFIECHGRGTCNYYANA 2367 α2CIV CAH71366.1: 1517-1593 QEKAHNQDLGLAGSCLARFSTMPFLYCNPGDVCYYASRNDKSY WLSTTAPLPMMPVAEDEIKPYISRCSVCEAPAIA 2368 α2CIV CAH71366.1: 1542-1561 YCNPGDVCYYASRNDKSYWL 2369 α2CIV CAH71366.1: 1646-1664 ATPFIECNGGRGTCHYYAN 2370 α4CIV CAA56943.1: 1499-1575 QEKAHNQDLGLAGSCLPVFSTLPFAYCNIHQVCHYAQRNDRSY WLASAAPLPMMPLSEEAIRPYVSRCAVCEAPAQA 2371 α4CIV CAA56943.1: 1514-1533 LPVFSTLPFAYCNIHQVCHY 2372 α4CIV CAA56943.1: 1524-1543 YCNIHQVCHYAQRNDRSYWL 2373 α4CIV CAA56943.1: 1628-1646 AAPFLECQGRQGTCHFFAN 2374 α5CIV AAC27816.1: 1495-1572 NKRAHGQDLGTAGSCLRRFSTMPFMFCNINNVCNFASRNDYSY WLSTPEPMPMSMQPLKGQSIQPFISRCAVCEAPAV 2375 α5CIV AAC27816.1: 1510-1529 LRRFSTMPFMFCNINNVCNF 2376 α5CIV AAC27816.1: 1520-1539 FCNINNVCNFASRNDYSYWL 2377 α5CIV AAC27816.1: 1626-1644 SAPFIECHGRGTCNYYANS 2378 α6CIV CAI40758.1: 1501-1577 QEKAHNQDLGFAGSCLPRFSTMPFIYCNINEVCHYARRNDKSY WLSTTAPIPMMPVSQTQIPQYISRCSVCEAPSQA 2379 α6CIV CAI40758.1: 1526-1545 YCNINEVCHYARRNDKSYWL 2380 α6CIV CAI40758.1: 1630-1648 ATPFIECSGARGTCHYFAN CXC Chemokines 2381 ENA-78/CXCL5 AAP35453.1: 86-108 NGKEICLDPEAPFLKKVIQKILD 2382 ENA-78/CXCL5 AAP35453.1: 48-103 RCVCLQTTQGVHPKMISNLQVFAIGPQCSKVEVVASLKNGKEIC LDPEAPFLKKVI 2383 ENA-78/CXCL5 AAP35453.1: 51-107 CLQTTQGVHPKMISNLQVFAIGPQCSKVEVVASLKNGKEICLDP EAPFLKKVIQKIL 2384 GCP-2/CXCL6 AAH13744.1: 86-109 NGKQVCLDPEAPFLKKVIQKILDS 2385 GCP-2/CXCL6 AAH13744.1: 47-106 LRCTCLRVTLRVNPKTIGKLQVFPAGPQCSKVEVVASLKNGKQV CLDPEAPFLKKVIQKI 2386 GCP-2/CXCL6 AAH13744.1: 48-103 RCTCLRVTLRVNPKTIGKLQVFPAGPQCSKVEVVASLKNGKQV CLDPEAPFLKKVI 2387 GCP-2/CXCL6 AAH13744.1: 51-107 CLRVTLRVNPKTIGKLQVFPAGPQCSKVEVVASLKNGKQVCLDP EAPFLKKVIQKIL 2388 GRO-α/CXCL1 AAP35526.1: 80-103 NGRKACLNPASPIVKKIIEKMLNS 2389 GRO-α/CXCL1 AAP35526.1: 42-97 RCQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKAC LNPASPIVKKII 2390 GRO-α/CXCL1 AAP35526.1: 44-101 QCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKACLN PASPIVKKIIEKML 2391 Gro-β/CXCL2 AAH15753.1: 42-97 RCQCLQTLQGIHLKNIQSVKVKSPGPHCAQTEVIATLKNGQKAC LNPASPMVKKII 2392 GRO-γ/MIP-2β/CXCL3 AAA63184.1: 79-100 NGKKACLNPASPMVQKIIEKIL 2393 GRO-γ/MIP-2β/CXCL3 AAA63184.1: 43-100 QCLQTLQGIHLKNIQSVNVRSPGPHCAQTEVIATLKNGKKACLN PASPMVQKIIEKIL 2394 GRO-γ/MIP-2β/CXCL3 AAA63184.1: 41-96 RCQCLQTLQGIHLKNIQSVNVRSPGPHCAQTEVIATLKNGKKAC LNPASPMVQKII 2395 IL-8/CXCL8 AAP35730.1: 35-94 QCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDP KENWVQRVVEKFLK 2396 IL-8/CXCL8 AAP35730.1: 72-94 DGRELCLDPKENWVQRVVEKFLK 2397 IP-10/CXCL10 AAH10954.1: 29-86 RCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCL NPESKAIKNLL 2398 MIG/CXCL9 Q07325: 32-91 SCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLKNGVQTCLNP DSADVKELIKKWEK 2399 PF-4/CXCL4 AAK29643.1: 43-100 CVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQ APLYKKIIKKLLE 2400 THBG-β/CXCL7 AAB46877.1: 100-121 DGRKICLDPDAPRIKKIVQKKL 2401 THBG-β/CXCL7 AAB46877.1: 62-117 RCMCIKTTSGIHPKNIQSLEVIGKGTHCNQVEVIATLKDGRKICL DPDAPRIKKIV 2402 THBG-β/CXCL7 AAB46877.1: 64-121 MCIKTTSGIHPKNIQSLEVIGKGTHCNQVEVIATLKDGRKICLDP DAPRIKKIVQKKL Kringle Containing Proteins 2403 AK-38 protein AAK74187.1: 14-93 DCMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTFIPGTNK WAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLCA 2404 AK-38 protein AAK74187.1: 12-94 QDCMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTFIPGTN KWAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLCA 2405 AK-38 protein AAK74187.1: 13-90 DCMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTFIPGTNK WAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDI 2406 AK-38 protein AAK74187.1: 14-93 CMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTFIPGTNK WAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLC 2407 Hageman fct/cf XII AAM97932.1: 216-292 SCYDGRGLSYRGLARTTLSGAPCQPWASEATYRNVTAEQARN WGLGGHAFCRNPDNDIRPWCFVLNRDRLSWEYCDL 2408 Hageman fct/cf XII AAM97932.1: 214-295 KASCYDGRGLSYRGLARTTLSGAPCQPWASEATYRNVTAEQAR NWGLGGHAFCRNPDNDIRPWCFVLNRDRLSWEYCDLAQC 2409 Hageman fct/cf XII AAM97932.1: 215-296 ASCYDGRGLSYRGLARTTLSGAPCQPWASEATYRNVTAEQARN WGLGGHAFCRNPDNDIRPWCFVLNRDRLSWEYCDLAQCQ 2410 HGF P14210: 127-206 NCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDL QENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQC 2411 HGF P14210: 127-207 NCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDL QENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCS 2412 HGF P14210: 304-377 ECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKC KDLRENYCRNPDGSESPWCFTTDPNIRVGYC 2413 HGF P14210: 210-289 ECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERYPD KGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCA 2414 HGF P14210: 304-383 ECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKC KDLRENYCRNPDGSESPWCFTTDPNIRVGYCSQIPNC 2415 Hyaluronan binding NP_004123.1: 192-277 DDCYVGDGYSYRGKMNRTVNQHACLYWNSHLLLQENYNMFM EDAETHGIGEHNFCRNPDADEKPWCFIKVTNDKVKWEYCDVSA CS 2416 Hyaluronan binding NP_004123.1: 192-276 DDCYVGDGYSYRGKMNRTVNQHACLYWNSHLLLQENYNMFM EDAETHGIGEHNFCRNPDADEKPWCFIKVTNDKVKWEYCDVSAC 2417 KREMEN-1 BAB40969.1: 31-114 ECFTANGADYRGTQNWTALQGGKPCLFWNETFQHPYNTLKYP NGEGGLGEHNYCRNPDGDVS- PWCYVAEHEDGVYWKYCEIPAC 2418 KREMEN-1 BAB40969.1: 31-115 ECFTANGADYRGTQNWTALQGGKPCLFWNETFQHPYNTLKYP NGEGGLGEHNYCRNPDGDVSPWCYVAEHEDGVYWKYCEIPACQ 2419 KREMEN-2 BAD97142.1: 35-119 ECFQVNGADYRGHQNRTGPRGAGRPCLFWDQTQQHSYSSASDP HGRWGLGAHNFCRNPDGDVQ-PWCYVAETEEGIYWRYCDIPSC 2420 KREMEN-2 BAD97142.1: 34-119 SECFQVNGADYRGHQNRTGPRGAGRPCLFWDQTQQHSYSSASD PHGRWGLGAHNFCRNPDGDVQPWCYVAETEEGIYWRYCDIPSC 2421 Lp(a) NP_005568.1: 1615-1690 TEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHS RTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPG 2422 Lp(a) NP_005568.1: 3560-3639 QDCYYHYGQSYRGTYSTTVTGRTCQAWSSMTPHQHSRTPENYP NAGLTRNYCRNPDAEIRPWCYTMDPSVRWEYCNLTQC 2423 Lp(a) NP_005568.1: 4123-4201 QCYHGNGQSYRGTFSTTVTGRTCQSWSSMTPHRHQRTPENYPN DGLTMNYCRNPDADTGPWCFTMDPSIRWEYCNLTRC 2424 Lp(a) NP_005568.1: 4225-4308 EQDCMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTFIPGT NKWAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLCA 2425 Macrophage stim. 1 AAH48330.1: 188-268 EAACVWCNGEEYRGAVDRTESGRECQRWDLQHPHQHPFEPGK FLDQGLDDNYCRNPDGSERPWCYTTDPQIEREFCDLPRC 2426 Macrophage stim. 1 AAH48330.1: 368-448 QDCYHGAGEQYRGTVSKTRKGVQCQRWSAETPHKPQFTFTSEP HAQLEENFCRNPDGDSHGPWCYTMDPRTPFDYCALRRC 2427 Macrophage stim. 1 AAH48330.1: 368-449 QDCYHGAGEQYRGTVSKTRKGVQCQRWSAETPHKPQFTFTSEP HAQLEENFCRNPDGDSHGPWCYTMDPRTPFDYCALRRCA 2428 Macrophage stim. 1 AAH48330.1: 370-448 CYHGAGEQYRGTVSKTRKGVQCQRWSAETPHKPQFTFTSEPHA QLEENFCRNPDGDSHGPWCYTMDPRTPFDYCALRRC 2429 Thrombin/cf II AAL77436.1: 105-186 EGNCAEGLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHP GADLQENFCRNPDSSTTGPWCYTTDPTVRRQECSIPVC 2430 Thrombin/cf II AAL77436.1: 106-186 GNCAEGLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHPG ADLQENFCRNPDSSTTGPWCYTTDPTVRRQECSIPVC 2431 Thrombin/cf II AAL77436.1: 107-183 NCAEGLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHPGA DLQENFCRNPDSSTTGPWCYTTDPTVRRQECSI 2432 Thrombin/cf II AAL77436.1: 107-186 NCAEGLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHPGA DLQENFCRNPDSSTTGPWCYTTDPTVRRQECSIPVC 2433 tPA AAH95403.1: 214-293 DCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQ ALGLGKHNYCRNPDGDAKPWCHVLKSRRLTWEYCDV 2434 tPA AAH95403.1: 213-296 SDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSA QALGLGKHNYCRNPDGDAKPWCHVLKSRRLTWEYCDVPSC 2435 tPA AAH95403.1: 213-297 SDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSA QALGLGKHNYCRNPDGDAKPWCHVLKSRRLTWEYCDVPSCS 2436 tPA AAH95403.1: 214-296 DCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQ ALGLGKHNYCRNPDGDAKPWCHVLKSRRLTWEYCDVPSC Somatotropins 2437 GH-1 NP_000506.2: 26-160 AFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSF LQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQ FLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPR 2438 GH-2 CAG46700.1: 26-160 AFPTIPLSRLFDNAMLRARRLYQLAYDTYQEFEEAYILKEQKYSF LQNPQTSLCFSESIPTPSNRAKTQQKSNLELLRISLLLIQSWLEPV QLLRSVFANSLVYGASDSNVYRHLKDLEEGIQTLMWRLEDGSPR 2439 Placental lactogen AAP35572.1: 26-160 AVQTVPLSRLFDHAMLQAHRAHQLAIDTYQEFEETYIPKDQKYS FLHDSQTSFCFSDSIPTPSNMEETQQKSNLELLRISLLLIESWLEPV RFLRSMFANNLVYDTSDSDDYHLLKDLEEGIQTLMGRLEDGSRR 2440 Somatoliberin AAH62475.1: 26-145 AFPTIPLSRLFDNASLRAHRLHQLAFDTYQEFNPQTSLCFSESIPT PSMREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGA SDSNVYDLLKDLEEGIQTLMGRLEDGSPR TIMPs 2441 TIMP 3 AAA21815.1: 148-171 ECLWTDMLSNFGYPGYQSKHYACI 2442 TIMP 4 AAV38433.1: 175-198 ECLWTDWLLERKLYGYQAQHYVCM

In particular embodiments, the presently disclosed subject matter provides a nanoparticle, microparticle, or gel comprising a compound of Formula (I), wherein the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof comprising the amino acid sequence W-X₂-C-X₃-C-X₂-G, wherein X denotes a variable amino acid; W is tryptophan; C is cysteine, G is glycine; and wherein the peptide reduces blood vessel formation in a cell, tissue or organ.

In some embodiments, the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of:

THSD-1: QPWSQCSATCGDGVRERRR; THSD-3: SPWSPCSGNCSTGKQQRTR; THSD-6: WTRCSSSCGRGVSVRSR; CILP: SPWSKCSAACGQTGVQTRTR; WISP-1: SPWSPCSTSCGLGVSTRI; WISP-2: TAWGPCSTTCGLGMATRV; WISP-3: TKWTPCSRTCGMGISNRV; F-spondin: SEWSDCSVTCGKGMRTRQR; F-spondin: WDECSATCGMGMKKRHR; CTGF: TEWSACSKTCGMGISTRV; fibulin-6: ASWSACSVSCGGGARQRTR; fibulin-6: QPWGTCSESCGKGTQTRAR; fibulin-6: SAWRACSVTCGKGIQKRSR; CYR61: TSWSQCSKTCGTGISTRV; NOVH: TEWTACSKSCGMGFSTRV; UNC5-C: TEWSVCNSRCGRGYQKRTR; UNC5-D: TEWSACNVRCGRGWQKRSR; SCO-spondin: GPWEDCSVSCGGGEQLRSR; Properdin: GPWEPCSVTCSKGTRTRRR; C6: TQWTSCSKTCNSGTQSRHR; ADAMTS-like-4: SPWSQCSVRCGRGQRSRQVR; ADAMTS-4: GPWGDCSRTCGGGVQFSSR; ADAMTS-8: GPWGECSRTCGGGVQFSHR; ADAMTS-16: SPWSQCTASCGGGVQTR; ADAMTS-18: SKWSECSRTCGGGVKFQER; semaphorin 5A: GPWERCTAQCGGGIQARRR; semaphorin 5A: SPWTKCSATCGGGHYMRTR; semaphoring 5B: TSWSPCSASCGGGHYQRTR; papilin: GPWAPCSASCGGGSQSRS; papilin: SQWSPCSRTCGGGVSFRER; ADAM-9: KCHGHGVCNS; and ADAM-12: MQCHGRGVCNNRKN,

wherein A is alanine; I is isoleucine; M is methionine; H is histidine; Y is tyrosine; K is lysine; W is tryptophan; C is cysteine, T is threonine, S is serine; N is asparagine; G is glycine; R is arginine; V is valine, P is proline, and Q is glutamine wherein the peptide reduces blood vessel formation in a cell, tissue or organ.

In other embodiments, the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof having at least 85% identity to an amino acid sequence selected from the group consisting of:

ENA-78: NGKEICLDPEAPFLKKVIQKILD; CXCL6: NGKQVCLDPEAPFLKKVIQKILDS; CXCL1: NGRKACLNPASPIVKKIIEKMLNS; Gro-γ: NGKKACLNPASPMVQKIIEKIL; Beta thromboglobulin/CXCL7: DGRKICLDPDAPRIKKIVQK KL, Interleukin 8 (IL-8)/CXCL8: DGRELCLDPKENWVQRVVEKF LK, GCP-2: NGKQVCLDPEAPFLKKVIQKILDS,

wherein A is alanine; I is isoleucine; F is phenylalanine; D is aspartic acid; M is methionine; H is histidine; Y is tyrosine; K is lysine; W is tryptophan; C is cysteine, T is threonine, S is serine; N is asparagine; G is glycine; R is arginine; V is valine, P is proline, and Q is glutamine; and wherein the peptide reduces blood vessel formation in a cell, tissue or organ.

In yet other embodiments, the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of

Alpha 6 fibril of type 4 collagen: YCNINEVCHYARRND KSYWL; Alpha 5 fibril of type 4 collagen: LRRFSTMPFMFCNIN NVCNF; Alpha 4 fibril of type 4 collagen: AAPFLECQGRQGTCH FFAN; Alpha 4 fibril of type 4 collagen: LPVFSTLPFAYCNIH QVCHY; Alpha 4 fibril of type 4 collagen: YCNIHQVCHYAQRND RSYWL, and Collagen type IV, alpha6 fibril LPRFSTMPFIYCNINE VCHY;

wherein A is alanine; I is isoleucine; F is phenylalanine; D is aspartic acid; M is methionine; H is histidine; Y is tyrosine; K is lysine; W is tryptophan; C is cysteine, T is threonine, S is serine; N is asparagine; G is glycine; R is arginine; V is valine, P is proline, and Q is glutamine wherein the peptide reduces blood vessel formation in a cell, tissue or organ.

In other embodiments, peptides suitable for use in the presently disclosed subject matter are disclosed in U.S. Provisional Patent Application No. 61/421,706, filed Dec. 12, 2010, which is commonly owned, and is incorporated herein by reference in its entirety.

SEQ ID NO. ID Sequence 2443 SP2000 LRRFSTMPFMFCNINNVCNF 2444 SP2002 LRRFSTMPFMFGNINNVGNF 2445 SP2004 LRRFSTMPFMF 2446 SP2006 LRRFSTMPFMF-Abu-NINV 2447 SP2007 LRRFSTMPFMF-Abu 2448 SP2008 LRRFSTMP 2449 SP2009 NINNV-Abu-NF 2450 SP2010 FMF-Abu-NINNV-Abu-NF 2451 SP2011 STMPFMF-Abu-NINNV-Abu-NF 2452 SP2012 LRRFSTMPFMF-Abu-NINNV-Abu-NF 2453 SP2013 LNRFSTMPF 2454 SP2014 LRRFST-Nle-PF-Nle-F 2455 SP2015 LRRFSTMPAMF-Abu-NINNV-Abu-NF 2456 SP2016 LRRFSTMPFAF-Abu-NINNV-Abu-NF 2457 SP2017 LRRFSTMPFMA-Abu-NINNV-Abu-NF 2458 SP2018 LRRFSTMPF-Nle-F-Abu-NINNV-Abu-NF 2459 SP2019 LRRFSTMPFM(4-ClPhen)-Abu-CNINNV- Abu-NF 2460 SP2020 F-Abu-NINNV-Abu-N 2461 SP2021 F-Abu-NIN 2462 SP2022 LRRFSTMPFMFSNINNVSNF 2463 SP2023 LRRFSTMPFMFANINNVANF 2464 SP2024 LRRFSTMPFMFININNVINF 2465 SP2025 LRRFSTMPFMFTNINNVTNF 2466 SP2026 LRRFSTMPFMFC(AllyGly)NINNV (AllyGly)NF 2467 SP2027 LRRFSTMPFMFVNINNVVNF 2468 SP2028 LRRFSTMPFMF-Abu-NINN 2469 SP2029 LRRFSTMPFMFTNINV 2470 SP2030 F-Abu-NINV 2471 SP2031 FTNINNVTN 2472 SP2032 LRRFSTMPFMFTNINN 2473 SP2033 LRRFSTMPFMFININN 2474 SP2034 LRRFSTMPF-Da-FININNVINF 2475 SP2035 LRRFSTAPFAFININNVINF 2476 SP2036 LRRFSTMPFAFININNVINF;

wherein Abu is 2-aminobutyric acid; Nle is Norleucine; and AllyGly is allyglycine.

In other embodiments, peptides suitable for use in the presently disclosed subject matter are disclosed in U.S. Provisional Patent Application No. 61/489,500, filed Way 24, 2011, which also is commonly owned, and is incorporated herein by reference in its entirety.

SEQ ID NO. ID Sequence 2477 SP5001 RLRLLTLQSWLL 2478 SP5028 LMRKSQILISSWF 2479 SP5029 LLIVALLFILSWL 2480 SP5030 LLRLLLLIESWLE 2481 SP5031 LLRSSLILLQGSWF 2482 SP5032 LLHISLLLIESRLE 2483 SP5033 LLRISLLLIESWLE

III. Definitions

Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs.

While the following terms in relation to compounds of Formulae I-X are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter. These definitions are intended to supplement and illustrate, not preclude, the definitions that would be apparent to one of ordinary skill in the art upon review of the present disclosure.

The terms substituted, whether preceded by the term “optionally” or not, and substituent, as used herein, refer to the ability, as appreciated by one skilled in this art, to change one functional group for another functional group provided that the valency of all atoms is maintained. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. The substituents also may be further substituted (e.g., an aryl group substituent may have another substituent off it, such as another aryl group, which is further substituted, for example, with fluorine at one or more positions).

When the term “independently selected” is used, the substituents being referred to (e.g., R groups, such as groups R₁, R₂, and the like, or variables, such as “m” and “n”), can be identical or different. For example, both R₁and R₂can be substituted alkyls, or R₁can be hydrogen and R₂can be a substituted alkyl, and the like.

A named “R” or group will generally have the structure that is recognized in the art as corresponding to a group having that name, unless specified otherwise herein. For the purposes of illustration, certain representative “R” groups as set forth above are defined below.

The term hydrocarbon, as used herein, refers to any chemical group comprising hydrogen and carbon. The hydrocarbon may be substituted or unsubstituted. As would be known to one skilled in this art, all valencies must be satisfied in making any substitutions. The hydrocarbon may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic. Illustrative hydrocarbons are further defined herein below and include, for example, methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl, methoxy, diethylamino, and the like.

As used herein the term “alkyl” refers to C_1-20inclusive, linear (i.e., “straight-chain”), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl)hydrocarbon radicals derived from a hydrocarbon moiety containing between one and twenty carbon atoms by removal of a single hydrogen atom. Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, iso-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C_1-8alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, “alkyl” refers, in particular, to C_1-8straight-chain alkyls. In other embodiments, “alkyl” refers, in particular, to C_1-8branched-chain alkyls.

Alkyl groups can optionally be substituted (a “substituted alkyl”) with one or more alkyl group substituents, which can be the same or different. The term “alkyl group substituent” includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), or aryl.

Thus, as used herein, the term “substituted alkyl” includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

“Cyclic” and “cycloalkyl” refer to a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The cycloalkyl group can be optionally partially unsaturated. The cycloalkyl group also can be optionally substituted with an alkyl group substituent as defined herein, oxo, and/or alkylene. There can be optionally inserted along the cyclic alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms,

wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl, thus providing a heterocyclic group. Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl.

The term “cycloalkylalkyl,” as used herein, refers to a cycloalkyl group as defined hereinabove, which is attached to the parent molecular moiety through an alkyl group, also as defined above. Examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.

The terms “cycloheteroalkyl” or “heterocycloalkyl” refer to a non-aromatic ring system, unsaturated or partially unsaturated ring system, such as a 3- to 10-member substituted or unsubstituted cycloalkyl ring system, including one or more heteroatoms, which can be the same or different, and are selected from the group consisting of N, O, and S, and optionally can include one or more double bonds. The cycloheteroalkyl ring can be optionally fused to or otherwise attached to other cycloheteroalkyl rings and/or non-aromatic hydrocarbon rings. Heterocyclic rings include those having from one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. In certain embodiments, the term heterocylic refers to a non-aromatic 5-, 6-, or 7-membered ring or a polycyclic group wherein at least one ring atom is a heteroatom selected from O, S, and N (wherein the nitrogen and sulfur heteroatoms may be optionally oxidized), including, but not limited to, a bi- or tri-cyclic group, comprising fused six-membered rings having between one and three heteroatoms independently selected from the oxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each 7-membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to an aryl or heteroaryl ring. Representative cycloheteroalkyl ring systems include, but are not limited to pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidyl, piperazinyl, indolinyl, quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and the like.

The term “alkenyl” as used herein refers to a monovalent group derived from a C_1-20inclusive straight or branched hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom. Alkenyl groups include, for example, ethenyl (i.e., vinyl), propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.

The term “cycloalkenyl” as used herein refers to a cyclic hydrocarbon containing at least one carbon-carbon double bond. Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.

The term “alkynyl” as used herein refers to a monovalent group derived from a straight or branched C_1-20hydrocarbon of a designed number of carbon atoms containing at least one carbon-carbon triple bond. Examples of “alkynyl” include ethynyl, 2-propynyl(propargyl), 1-propyne, 3-hexyne, and the like.

“Alkylene” refers to a straight or branched bivalent aliphatic hydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group also can be optionally unsaturated and/or substituted with one or more “alkyl group substituents.” There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as “alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described. Exemplary alkylene groups include methylene (—CH₂—); ethylene (—CH₂—CH₂-); propylene (—(CH₂)₃—); cyclohexylene (—C₆H₁₀—); —CH═CH—CH═CH—; —CH═CH—CH₂—; —(CH₂)_q—N(R)—(CH₂)_r—, wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl (—O—CH₂—O—); and ethylenedioxyl (—O—(CH₂)₂—O—). An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons.

The term “aryl” is used herein to refer to an aromatic substituent that can be a single aromatic ring, or multiple aromatic rings that are fused together, linked covalently, or linked to a common group, such as, but not limited to, a methylene or ethylene moiety. The common linking group also can be a carbonyl, as in benzophenone, or oxygen, as in diphenylether, or nitrogen, as in diphenylamine. The term “aryl” specifically encompasses heterocyclic aromatic compounds. The aromatic ring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether, diphenylamine and benzophenone, among others. In particular embodiments, the term “aryl” means a cyclic aromatic comprising about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5- and 6-membered hydrocarbon and heterocyclic aromatic rings.

The aryl group can be optionally substituted (a “substituted aryl”) with one or more aryl group substituents, which can be the same or different, wherein “aryl group substituent” includes alkyl, substituted alkyl, alkenyl, alkynyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, acyl, halo, haloalkyl, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, amino, alkylamino, dialkylamino, trialkylamino, acylamino, aroylamino, carbamoyl, cyano, alkylcarbamoyl, dialkylcarbamoyl, carboxyaldehyde, carboxyl, alkoxycarbonyl, carboxamide, arylthio, alkylthio, alkylene, thioalkoxyl, and mercapto.

Thus, as used herein, the term “substituted aryl” includes aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

Specific examples of aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, and the like.

The terms “heteroaryl” and “aromatic heterocycle” and “aromatic heterocyclic” are used interchangeably herein and refer to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from sulfur, oxygen, and nitrogen; zero, one, or two ring atoms are additional heteroatoms independently selected from sulfur, oxygen, and nitrogen; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like. Aromatic heterocyclic groups can be unsubstituted or substituted with substituents selected from the group consisting of branched and unbranched alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, thioalkoxy, amino, alkylamino, dialkylamino, trialkylamino, acylamino, cyano, hydroxy, halo, mercapto, nitro, carboxyaldehyde, carboxy, alkoxycarbonyl, and carboxamide. Specific heterocyclic and aromatic heterocyclic groups that may be included in the compounds of the invention include: 3-methyl-4-(3-methylphenyl)piperazine, 3 methylpiperidine, 4-(bis-(4-fluorophenyl)methyl)piperazine, 4-(diphenylmethyl)piperazine, 4(ethoxycarbonyl)piperazine, 4-(ethoxycarbonylmethyl)piperazine, 4-(phenylmethyl)piperazine, 4-(1-phenylethyl)piperazine, 4-(1,1-dimethylethoxycarbonyl)piperazine, 4-(2-(bis-(2-propenyl)amino)ethyl)piperazine, 4-(2-(diethylamino)ethyl)piperazine, 4-(2-chlorophenyl)piperazine, 4(2-cyanophenyl)piperazine, 4-(2-ethoxyphenyl)piperazine, 4-(2-ethylphenyl)piperazine, 4-(2-fluorophenyl)piperazine, 4-(2-hydroxyethyl)piperazine, 4-(2-methoxyethyl)piperazine, 4-(2-methoxyphenyl)piperazine, 4-(2-methylphenyl)piperazine, 4-(2-methylthiophenyl)piperazine, 4(2-nitrophenyl)piperazine, 4-(2-nitrophenyl)piperazine, 4-(2-phenylethyl)piperazine, 4-(2-pyridyl)piperazine, 4-(2-pyrimidinyl)piperazine, 4-(2,3-dimethylphenyl)piperazine, 4-(2,4-difluorophenyl)piperazine, 4-(2,4-dimethoxyphenyl)piperazine, 4-(2,4-dimethylphenyl)piperazine, 4-(2,5-dimethylphenyl)piperazine, 4-(2,6-dimethylphenyl)piperazine, 4-(3-chlorophenyl)piperazine, 4-(3-methylphenyl)piperazine, 4-(3-trifluoromethylphenyl)piperazine, 4-(3,4-dichlorophenyl)piperazine, 4-(3,4-dimethoxyphenyl)piperazine, 4-(3,4-dimethylphenyl)piperazine, 4-(3,4-methylenedioxyphenyl)piperazine, 4-(3,4,5-trimethoxyphenyl)piperazine, 4-(3,5-dichlorophenyl)piperazine, 4-(3,5-dimethoxyphenyl)piperazine, 4-(4-(phenylmethoxy)phenyl)piperazine, 4-(4-(3,1-dimethylethyl)phenylmethyl)piperazine, 4-(4-chloro-3-trifluoromethylphenyl)piperazine, 4-(4-chlorophenyl)-3-methylpiperazine, 4-(4-chlorophenyl)piperazine, 4-(4-chlorophenyl)piperazine, 4-(4-chlorophenylmethyl)piperazine, 4-(4-fluorophenyl)piperazine, 4-(4-methoxyphenyl)piperazine, 4-(4-methylphenyl)piperazine, 4-(4-nitrophenyl)piperazine, 4-(4-trifluoromethylphenyl)piperazine, 4-cyclohexylpiperazine, 4-ethyl piperazine, 4-hydroxy-4-(4-chlorophenyl)methylpiperidine, 4-hydroxy-4-phenylpiperidine, 4-hydroxypyrrolidine, 4-methylpiperazine, 4-phenylpiperazine, 4-piperidinylpiperazine, 4-(2-furanyl)carbonyl)piperazine, 4-((1,3-dioxolan-5-yl)methyl)piperazine, 6-fluoro-1,2,3,4-tetrahydro-2-methylquinoline, 1,4-diazacylcloheptane, 2,3-dihydroindolyl, 3,3-dimethylpiperidine, 4,4-ethylenedioxypiperidine, 1,2,3,4-tetrahydroisoquinoline, 1,2,3,4-tetrahydroquinoline, azacyclooctane, decahydroquinoline, piperazine, piperidine, pyrrolidine, thiomorpholine, and triazole. The heteroaryl ring can be fused or otherwise attached to one or more heteroaryl rings, aromatic or non-aromatic hydrocarbon rings, or heterocycloalkyl rings. A structure represented generally by the formula:

as used herein refers to a ring structure, for example, but not limited to a 3-carbon, a 4-carbon, a 5-carbon, a 6-carbon, a 7-carbon, and the like, aliphatic and/or aromatic cyclic compound, including a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure, comprising a substituent R group, wherein the R group can be present or absent, and when present, one or more R groups can each be substituted on one or more available carbon atoms of the ring structure. The presence or absence of the R group and number of R groups is determined by the value of the variable “n,” which is an integer generally having a value ranging from 0 to the number of carbon atoms on the ring available for substitution. Each R group, if more than one, is substituted on an available carbon of the ring structure rather than on another R group. For example, the structure above where n is 0 to 2 would comprise compound groups including, but not limited to:

and the like.

A dashed line representing a bond in a cyclic ring structure indicates that the bond can be either present or absent in the ring. That is, a dashed line representing a bond in a cyclic ring structure indicates that the ring structure is selected from the group consisting of a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure.

When a named atom of an aromatic ring or a heterocyclic aromatic ring is defined as being “absent,” the named atom is replaced by a direct bond.

As used herein, the term “acyl” refers to an organic acid group wherein the —OH of the carboxyl group has been replaced with another substituent and has the general formula RC(═O)—, wherein R is an alkyl, alkenyl, alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic group as defined herein). As such, the term “acyl” specifically includes arylacyl groups, such as an acetylfuran and a phenacyl group. Specific examples of acyl groups include acetyl and benzoyl.

The terms “alkoxyl” or “alkoxy” are used interchangeably herein and refer to a saturated (i.e., alkyl-O—) or unsaturated (i.e., alkenyl-O— and alkynyl-O—) group attached to the parent molecular moiety through an oxygen atom, wherein the terms “alkyl,” “alkenyl,” and “alkynyl” are as previously described and can include C₁-C₂₀inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl, sec-butoxyl, t-butoxyl, and n-pentoxyl, neopentoxy, n-hexoxy, and the like.

The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether, for example, a methoxyethyl or an ethoxymethyl group.

“Aryloxyl” refers to an aryl-O— group wherein the aryl group is as previously described, including a substituted aryl. The term “aryloxyl” as used herein can refer to phenyloxyl or hexyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.

“Aralkyl” refers to an aryl-alkyl-group wherein aryl and alkyl are as previously described, and included substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.

“Aralkyloxyl” refers to an aralkyl-O— group wherein the aralkyl group is as previously described. An exemplary aralkyloxyl group is benzyloxyl.

“Alkoxycarbonyl” refers to an alkyl-O—CO— group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl.

“Aryloxycarbonyl” refers to an aryl-O—CO— group. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

“Aralkoxycarbonyl” refers to an aralkyl-O—CO— group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.

“Carbamoyl” refers to an amide group of the formula —CONH₂. “Alkylcarbamoyl” refers to a R′RN—CO— group wherein one of R and R′ is hydrogen and the other of R and R′ is alkyl and/or substituted alkyl as previously described. “Dialkylcarbamoyl” refers to a R′RN—CO— group wherein each of R and R′ is independently alkyl and/or substituted alkyl as previously described.

The term carbonyldioxyl, as used herein, refers to a carbonate group of the formula —O—CO—OR.

“Acyloxyl” refers to an acyl-O— group wherein acyl is as previously described.

The term “amino” refers to the —NH₂group and also refers to a nitrogen containing group as is known in the art derived from ammonia by the replacement of one or more hydrogen radicals by organic radicals. For example, the terms “acylamino” and “alkylamino” refer to specific N-substituted organic radicals with acyl and alkyl substituent groups respectively.

The terms alkylamino, dialkylamino, and trialkylamino as used herein refer to one, two, or three, respectively, alkyl groups, as previously defined, attached to the parent molecular moiety through a nitrogen atom. The term alkylamino refers to a group having the structure —NHR′ wherein R′ is an alkyl group, as previously defined; whereas the term dialkylamino refers to a group having the structure —NR′R″, wherein R′ and R″ are each independently selected from the group consisting of alkyl groups. The term trialkylamino refers to a group having the structure —NR′R″R′″, wherein R′, R″, and R′″ are each independently selected from the group consisting of alkyl groups. Additionally, R′, R″, and/or R′″ taken together may optionally be —(CH₂)_k— where k is an integer from 2 to 6. Examples include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino, iso-propylamino, piperidino, trimethylamino, and propylamino.

The terms alkylthioether and thioalkoxyl refer to a saturated (i.e., alkyl-S—) or unsaturated (i.e., alkenyl-S— and alkynyl-S—) group attached to the parent molecular moiety through a sulfur atom. Examples of thioalkoxyl moieties include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

“Acylamino” refers to an acyl-NH— group wherein acyl is as previously described. “Aroylamino” refers to an aroyl-NH— group wherein aroyl is as previously described.

The term “carbonyl” refers to the —(C═O)— group. The term “carboxyl” refers to the —COOH group. Such groups also are referred to herein as a “carboxylic acid” moiety.

The terms “halo,” “halide,” or “halogen” as used herein refer to fluoro, chloro, bromo, and iodo groups.

The term “hydroxyl” refers to the —OH group.

The term “hydroxyalkyl” refers to an alkyl group substituted with an —OH group.

The term “mercapto” refers to the —SH group.

The term “oxo” refers to a compound described previously herein wherein a carbon atom is replaced by an oxygen atom.

The term “nitro” refers to the —NO₂group.

The term “thio” refers to a compound described previously herein wherein a carbon or oxygen atom is replaced by a sulfur atom.

The term “sulfate” refers to the —SO₄group.

The term thiohydroxyl or thiol, as used herein, refers to a group of the formula —SH.

The term ureido refers to a urea group of the formula —NH—CO—NH₂.

Throughout the specification and claims, a given chemical formula or name shall encompass all tautomers, congeners, and optical- and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist.

As used herein the term “monomer” refers to a molecule that can undergo polymerization, thereby contributing constitutional units to the essential structure of a macromolecule or polymer.

A “polymer” is a molecule of high relative molecule mass, the structure of which essentially comprises the multiple repetition of unit derived from molecules of low relative molecular mass, i.e., a monomer.

As used herein, an “oligomer” includes a few monomer units, for example, in contrast to a polymer that potentially can comprise an unlimited number of monomers. Dimers, trimers, and tetramers are non-limiting examples of oligomers.

Further, as used herein, the term “nanoparticle,” refers to a particle having at least one dimension in the range of about 1 nm to about 1000 nm, including any integer value between 1 nm and 1000 nm (including about 1, 2, 5, 10, 20, 50, 60, 70, 80, 90, 100, 200, 500, and 1000 nm and all integers and fractional integers in between). In some embodiments, the nanoparticle has at least one dimension, e.g., a diameter, of about 100 nm. In some embodiments, the nanoparticle has a diameter of about 200 nm. In other embodiments, the nanoparticle has a diameter of about 500 nm. In yet other embodiments, the nanoparticle has a diameter of about 1000 nm (1 μm). In such embodiments, the particle also can be referred to as a “microparticle. Thus, the term “microparticle” includes particles having at least one dimension in the range of about one micrometer (μm), i.e., 1×10⁻⁶meters, to about 1000 μm. The term “particle” as used herein is meant to include nanoparticles and microparticles.

It will be appreciated by one of ordinary skill in the art that nanoparticles suitable for use with the presently disclosed methods can exist in a variety of shapes, including, but not limited to, spheroids, rods, disks, pyramids, cubes, cylinders, nanohelixes, nanosprings, nanorings, rod-shaped nanoparticles, arrow-shaped nanoparticles, teardrop-shaped nanoparticles, tetrapod-shaped nanoparticles, prism-shaped nanoparticles, and a plurality of other geometric and non-geometric shapes. In particular embodiments, the presently disclosed nanoparticles have a spherical shape.

The subject treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein.

“Associated with”: When two entities are “associated with” one another as described herein, they are linked by a direct or indirect covalent or non-covalent interaction. Preferably, the association is covalent. Desirable non-covalent interactions include hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, etc.

“Biocompatible”: The term “biocompatible”, as used herein is intended to describe compounds that are not toxic to cells. Compounds are “biocompatible” if their addition to cells in vitro results in less than or equal to 20% cell death, and their administration in vivo does not induce inflammation or other such adverse effects.

“Biodegradable”: As used herein, “biodegradable” compounds are those that, when introduced into cells, are broken down by the cellular machinery or by hydrolysis into components that the cells can either reuse or dispose of without significant toxic effect on the cells (i.e., fewer than about 20% of the cells are killed when the components are added to cells in vitro). The components preferably do not induce inflammation or other adverse effects in vivo. In certain preferred embodiments, the chemical reactions relied upon to break down the biodegradable compounds are uncatalyzed.

“Effective amount”: In general, the “effective amount” of an active agent or drug delivery device refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the encapsulating matrix, the target tissue, and the like.

“Peptide” or “protein”: A “peptide” or “protein” comprises a string of at least three amino acids linked together by peptide bonds. The terms “protein” and “peptide” may be used interchangeably. Peptide may refer to an individual peptide or a collection of peptides. Inventive peptides preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in an inventive peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. In a preferred embodiment, the modifications of the peptide lead to a more stable peptide (e.g., greater half-life in vivo). These modifications may include cyclization of the peptide, the incorporation of D-amino acids, etc. None of the modifications should substantially interfere with the desired biological activity of the peptide.

“Polynucleotide” or “oligonucleotide”: Polynucleotide or oligonucleotide refers to a polymer of nucleotides. Typically, a polynucleotide comprises at least three nucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

“Small molecule”: As used herein, the term “small molecule” refers to organic compounds, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have relatively low molecular weight and that are not proteins, polypeptides, or nucleic acids. Typically, small molecules have a molecular weight of less than about 1500 g/mol. Also, small molecules typically have multiple carbon-carbon bonds. Known naturally-occurring small molecules include, but are not limited to, penicillin, erythromycin, taxol, cyclosporin, and rapamycin. Known synthetic small molecules include, but are not'limited to, ampicillin, methicillin, sulfamethoxazole, and sulfonamides.

By “analog” is meant a chemical compounds having a structure that is different from the general structure of a reference agent, but that functions in a manner similar to the reference agent. For example, a peptide analog having a variation in sequence or having a modified amino acid.

By “thrombospondin (TSP) derived peptide” is meant a peptide comprising a TSP motif: W-X(2)-C-X(3)-C-X(2)-G. Exemplary TSP derived peptides are shown in Tables 1 and 2. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence of the peptide. TSP1 derived peptides include, for example, those derived from proteins WISP-1

(SPWSPCSTSCGLGVSTRI), NOVH(TEWTACSKSCGMGFSTRV) and UNC5C (TEWSVCNSRCGRGYQKRTR).

By “CXC derived peptide” is meant a peptide comprising a CXC Motif: G-X(3)-C-L. Exemplary CXC derived peptides are shown in Table 3. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence. CXC derived peptides include, for example, those derived from proteins GRO-α/CXCL1 (NGRKACLNPASPIVKKIIEKMLNS), GRO-γ/MIP-21β/CXCL3 (NGKKACLNPASPMVQKEEKIL), and ENA-78/CXCL5 (NGKEICLDPEAPFLKKVIQKILD).

By “Collagen IV derived peptide” is meant a peptide comprising a C—N—X(3)-V-C or P—F-X(2)-C collagen motif. Exemplary collagen IV derived peptides are shown in Table 5. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence. Type IV collagen derived peptides include, for example, LRRFSTMPFMFCNINNVCNF and FCNINNVCNFASRNDYSYWL, and LPRFSTMPFIYCNINEVCHY.

By “Somatotropin derived peptide” is meant a peptide comprising a Somatotropin Motif: L-X(3)-L-L-X(3)-S—X-L. Exemplary somatotropin derived peptides are shown in Table 8. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence.

By “Serpin derived peptide” is meant a peptide comprising a Serpin Motif: L-X(2)-E-E-X—P. Exemplary serpin derived peptides are shown in Table 9. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence.

By “Beta 1 integrin” is meant a polypeptide that binds a collagen IV derived peptide or that has at least about 85% identity to NP_596867 or a fragment thereof.

By “Beta 3 integrin” is meant a polypeptide that binds a collagen IV derived peptide or that has at least about 85% identity to P05106 or a fragment thereof.

By “CD36” is meant a CD36 glycoprotein that binds to a thrombospondin-derived peptide or that has at least about 85% identity to NP_001001548 or a fragment thereof. CD36 is described, for example, by Oquendo et al., “CD36 directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes,” Cell 58: 95-101, 1989.

By “CD47” is meant a CD47 glycoprotein that binds to a thrombospondin-derived peptides or that has at least about 85% identity to NP_000315 or a fragment thereof. CD47 is described, for example, by Han et al., “CD47, a ligand for the macrophage fusion receptor, participates in macrophage multinucleation.” J. Biol. Chem. 275: 37984-37992, 2000.

By “CXCR3” is meant a G protein coupled receptor or fragment thereof having at least about 85% identity to NP_001495. CXCR3 is described, for example, by Trentin et al., “The chemokine receptor CXCR3 is expressed on malignant B cells and mediates chemotaxis.” J. Clin. Invest. 104: 115-121, 1999.

By “blood vessel formation” is meant the dynamic process that includes one or more steps of blood vessel development and/or maturation, such as angiogenesis, vasculogenesis, formation of an immature blood vessel network, blood vessel remodeling, blood vessel stabilization, blood vessel maturation, blood vessel differentiation, or establishment of a functional blood vessel network.

By “angiogenesis” is meant the growth of new blood vessels originating from existing blood vessels. Angiogenesis can be assayed by measuring the total length of blood vessel segments per unit area, the functional vascular density (total length of perfused blood vessel per unit area), or the vessel volume density (total of blood vessel volume per unit volume of tissue).

By “vasculogenesis” is meant the development of new blood vessels originating from stem cells, angioblasts, or other precursor cells.

By “blood vessel stability” is meant the maintenance of a blood vessel network.

By “alteration” is meant a change in the sequence or in a modification (e.g., a post-translational modification) of a gene or polypeptide relative to an endogeneous wild-type reference sequence.

By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

By “antibody” is meant any immunoglobulin polypeptide, or fragment thereof, having immunogen binding ability.

In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

A “cancer” in an animal refers to the presence of cells possessing characteristics typical of cancer-causing cells, for example, uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cellular markers. In some circumstances, cancer cells will be in the form of a tumor; such cells may exist locally within an animal, or circulate in the blood stream as independent cells, for example, leukemic cells.

By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

By “isolated nucleic acid molecule” is meant a nucleic acid (e.g., a DNA) that is free of the genes, which, in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule which is transcribed from a DNA molecule, as well as a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

“By “neoplasia” is meant a disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Solid tumors, hematological disorders, and cancers are examples of neoplasias.

By “operably linked” is meant that a first polynucleotide is positioned adjacent to a second polynucleotide that directs transcription of the first polynucleotide when appropriate molecules (e.g., transcriptional activator proteins) are bound to the second polynucleotide.

By “peptide” is meant any fragment of a polypeptide. Typically peptide lengths vary between 5 and 1000 amino acids (e.g., 5, 10, 15, 20, 25, 50, 100, 200, 250, 500, 750, and 1000).

By “polypeptide” is meant any chain of amino acids, regardless of length or post-translational modification.

By “promoter” is meant a polynucleotide sufficient to direct transcription. By “reduce” is meant a decrease in a parameter (e.g., blood vessel formation) as detected by standard art known methods, such as those described herein. As used herein, reduce includes a 10% change, preferably a 25% change, more preferably a 40% change, and even more preferably a 50% or greater change.

By “reference” is meant a standard or control condition.

By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and even more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e⁻³and e⁻¹⁰⁰indicating a closely related sequence.

“Sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window, and can take into consideration additions, deletions and substitutions. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (for example, charge or hydrophobicity) and therefore do not deleteriously change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have sequence similarity. Approaches for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, for example, according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17, 1988, for example, as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).

“Percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions, substitutions, or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions, substitutions, or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

The term “substantial identity” or “homologous” in their various grammatical forms in the context of polynucleotides means that a polynucleotide comprises a sequence that has a desired identity, for example, at least 60% identity, preferably at least 70% sequence identity, more preferably at least 80%, still more preferably at least 90% and even more preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, more preferably at least 70%, 80%, 85%, 90%, and even more preferably at least 95%.

Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. However, nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This may occur, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, although such cross-reactivity is not required for two polypeptides to be deemed substantially identical.

An “expression vector” is a nucleic acid construct, generated recombinantly or synthetically, bearing a series of specified nucleic acid elements that enable transcription of a particular gene in a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-preferred regulatory elements, and enhancers.

A “recombinant host” may be any prokaryotic or eukaryotic cell that contains either a cloning vector or expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.

The term “operably linked” is used to describe the connection between regulatory elements and a gene or its coding region. That is, gene expression is typically placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. Such a gene or coding region is said to be “operably linked to” or “operatively linked to” or “operably associated with” the regulatory elements, meaning that the gene or coding region is controlled or influenced by the regulatory element.

A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 5, 10, or 15 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, about 100 amino acids, or about 150 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides about 300 nucleotides or about 450 nucleotides or any integer thereabout or therebetween.

Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2: 482, 1981; by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48: 443, 1970; by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 8: 2444, 1988; by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 7 Science Dr., Madison, Wis., USA; the CLUSTAL program is well described by Higgins and Sharp, Gene, 73: 237-244, 1988; Corpet, et al., Nucleic Acids Research, 16:10881-10890, 1988; Huang, et al., Computer Applications in the Biosciences, 8:1-6, 1992; and Pearson, et al., Methods in Molecular Biology, 24:7-331, 1994. The BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York, 1995. New versions of the above programs or new programs altogether will undoubtedly become available in the future, and can be used with the present invention.

Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs, or their successors, using default parameters (Altschul et al., Nucleic Acids Res, 2:3389-3402, 1997). It is to be understood that default settings of these parameters can be readily changed as needed in the future.

As those ordinary skilled in the art will understand, BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 17:149-163, 1993) and XNU (Clayerie and States, Comput. Chem., 17:191-1, 1993) low-complexity filters can be employed alone or in combination.

As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

A “tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a subject” includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments, ±100% in some embodiments ±50%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The following Examples are offered by way of illustration and not by way of limitation.

Example 1 Methods Synthesis of BR6

All chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, Mo., USA) and used without further purification. Bis(2-hydroxyethyl)disulfide (15.4 g, 10 mmol) and triethylamine (TEA, 37.5 mL, 300 mmol) were dissolved in 450 mL of tetrahydrofuran previously dried with NaSO₄in a 1000 ml round bottom flask. The flask was flushed with N₂for 10 min and then maintained under a N₂environment. Acryloyl chloride (24.4 mL, 300 mmol) was dissolved in 50 mL tetrahydrofuran then added to the flask dropwise over 2 hrs while stirring. The reaction was carried out for 24 hrs, then the TEA HCl precipitate was removed by filtration, and the solvent was removed by rotary evaporation. The product was dissolved in 100 mL dichloromethane and washed five times with 200 mL of an aqueous solution of 0.2 M Na₂CO₃and three times with distilled water. The solution was dried with NaSO₄and the solvent was removed by rotary evaporation.

Polymer Synthesis

Base monomer BR6 was polymerized with side chain monomers S3, S4, and S5 at a base:side chain ratio of 1.2:1 by weight without solvent at 90° C. for 24 hrs while stirring. For end-capping with E10, base polymer was dissolved in anhydrous dimethyl sulfoxide at 100 mg/mL with 0.2 mM end-cap. The reaction was allowed to proceed for 1 hr at room temperature while shaking.

Example 2 Characteristics of Representative Polymer/Peptide Nanoparticles

Referring now to FIG. 5 are shown representative formation and sizing of polymer/peptide nanoparticles (by nanoparticle tracking analysis on a Nanosight LM10). Selected peptides and PBAEs were diluted in 25 mM sodium acetate buffer and then together in different weight-to-weight ratios. In some embodiments w/w is unity, 1:1, in other embodiments there is an excess of polymer to peptide. In some embodiments this ratio is 5:1, in other embodiments between 1:1-10:1, in other embodiments it is 10:1 to 20:1. In FIG. 6, both a 5:1 and 1:1 ratio is shown. The mixtures were incubated at room temperature for up to 10 minutes to allow for self-assembly and then loaded into the NanoSight laser cell. Using NanoSight nanoparticle tracking software and analysis, individual particles were tracked in order to determine the average size distribution of the particles. In the case of more hydrophilic peptides (DEAH Box poly8) and PBAEs (336), there were very few background particles, that is very few particles of peptide or PBAE only. However, when self-assembled, a noticeable nanoparticle distribution was observed, with an average size ranging from 100-150 nm. In the case of hydrophobic peptides and PBAEs, they have the possibility of aggregating with themselves. In the case of the peptide-PBAE mixture, a shift in the mean can be observed as a way to detect difference in nanoparticle formation.

Referring now to FIG. 6, is shown DEAH peptide release by 336 nanoparticles at 4° C. (above) and 37° C. (below). Changing polymer to peptide formulation ratios and concentrations are key to tune release. Slowing the reaction rate of degradation of the liable polymer bonds extends release from nanoparticles. This is shown by change in temperature, but could also be accomplished by increasing hydrophobicity of the polymer, increasing the molecular weight between liable ester groups, or other modifications known by someone in the art; FITC labeled DEAH peptide and 336 polymer were mixed and incubated for up to 10 minutes in sodium acetate buffer. Mixtures at different peptide concentrations, but constant polymer to peptide ratios, as well as peptide only were added to a 96-well plate. Fluorescence measurements were obtained using a plate reader and measured over time. The plates were kept either at 4° C. or 37° C.

Referring now to FIG. 7, is shown HUVEC viability/proliferation assays with polymer/SP6001/DEAH peptide; the CellTiter 96® AQ_ueousOne Solution Cell Proliferation assay was used to see the effect of both peptide and polymer on cell proliferation and viability. Polymers at the right concentrations have minimal cytotoxic effect on the cells, such as 336 below 100 uM. The individual peptides and Polymers were diluted in sodium acetate buffer and added to HUVECs in a 96-well plate. After incubating for a few days, the assay substrate was added and then incubated for a few hours at 37° C. Absorbance measurements were performed using a plate reader.

Referring now to FIG. 8 is shown HUVEC migration assays with 336 polymer/DEAH peptide. These nanoparticles inhibit endothelial migration in addition to proliferation and viability. Peptide-polymer nanoparticles were made as described previously. Samples were added to HUVEC cells and migration was measured using the ACEA time course cell migration system. Nanoparticle formulations at a total peptide concentration of 20 uM were able to inhibit migration more than any peptide only at 20 μM.

Referring now to FIG. 9 is shown in vivo 336 polymer nanoparticle/SP6001 DEAH peptide; Peptide-336 polymer nanoparticles were formulated as previously described and intravitreously injected to test in vivo efficacy. ACNV laser mouse model was used on C57 BL/6 female mice. The mice receive laser eye treatments on day zero, followed by the intravitreous injections. Mice are then perfused with fluorescein labeled dextran on day 14 and choroidal flat mounts (bottom) were analyzed via fluorescence microscopy. On day 14, both the peptide only and nanoparticles formulations significantly reduced angiogenesis in the eye (top) and did so to a similar extent. This suggests that all peptide was released from nanoparticles by day 14.

Referring now to FIG. 10 is shown (top) Particle size and (bottom) cell viability effects of various polymer/SP2012 nanoparticles as compared to peptide only of non-cytotoxic polymers; a range of polymer structures were mixed with SP2000 series peptides, in a similar manner as described above. Similar sizing is found with peptides from the same class with similar structural properties. For example, SP2000, SP2012, SP2024, SP2034, and SP2036 can be encapsulated similarly to each other with the same polymers, but different from peptides from other classes such as SP6001. Sizing was performed using the Malzern Zetasizer. Size strongly depends on polymer choice. Using the same cell viability assay as described previously, effects of nanoparticle vs. peptide only on HUVECs in a 96-well plate. Non-cytotoxic polymers are shown here. Referring once again to FIG. 10, the (top) panel suggests that some of these peptide-polymer formulations have an increased effect on HUVEC cell proliferation and viability (y-axis ratio less than one) as compared to peptide only. (Data also are normalized to any polymer-only effects. Pep-pol/pol/SP2012 refers to the change in cell proliferation/viability due to the peptide/polymer nanoparticle formulation divided by any change in cell proliferation/viability from the same dose of polymer by itself and this quantity divided by the change in cell proliferation/viability by delivering the same amount of peptide SP2012 as a bolus);

FIG. 11 shows polymer/peptide formulations for alternative peptides. Peptide-polymer formulations made as described previously. Here two different classes of peptides are used. Experiments performed in a 96-well plate, with final results obtained using the same cell viability/proliferation assay as described previously. An increased effect (decreased metabolic activity) is observed for the nanoparticle formulations over the free peptide.

Example 3 Hydrogels for Protein/Peptide Release

As shown in FIG. 12, FITC-tagged bovine serum albumin (BSA) was mixed with a macromer solution containing 10% (w/v) PEGDA (Mn-270 Da) with various amounts of B4S4, dissolved in a 1:1 (v/v) mixture of DMSO and PBS. Irgacure 2959 was added at 0.05% (w/v), and the solution was briefly vortexed and immediately polymerized to form gels. The gels were incubated at 37° C. in 1×PBS with shaking. PBS was removed at each time point to measure fluorescence.

The observed slowed release is due to two factors: first, increased overall hydrophobicity can decrease the movement of water in and out of the gel, reducing degradation rate and protein release. Furthermore, this method of mixing relatively hydrophobic diacrylates with hydrophilic diacrylates in a co-solvent (mixture of water and DMSO) that can dissolve both types of polymer causes the spontaneous formation of micro-emulsions within the gel (see SEM in FIG. 13; increasing B4S4 from top [0.2% w/w] to bottom [5% w/w]). Similar to traditionally studied controlled-release microparticles, these microparticles within photopolymerized gels could serve as another way to tune the release of an encapsulated peptide, protein, or drug.

Example 4 Stable Formulations

In this formulation nanoparticles were formed by mixing PBAE and DNA in 25 mM sodium acetate buffer (pH 5) at a 30:1 polymer:DNA ratio (w/w). After 10 min of incubation, sucrose solution was added at various concentrations. The particles were mixed, then frozen at −80° C. for 1 hr and lyophilized for 48 hr. They then were used for transfection or sizing or were stored at either room temperature, 4° C. or −20° C. and tested at various timepoints.

Referring now to FIG. 14, the size distribution of appropriately freeze-dried particles (bottom left, right-most histogram) remains the same as freshly-prepared particles (bottom left, left-most histogram). Freeze-dried particles also remain more stable in serum-containing medium than freshly-prepared particles (upper left). Using DNA-loaded nanoparticles, transfection efficiency is comparable between fresh particles and particles lyophilized with sucrose (right) even after 3 months of storage. Modifying type of sugar and concentration of sugar modulates the stability of the degradable nanoparticles.

Example 5 Inclusion of Lyophilized Nanoparticles into Pellets/Scaffolds

For coating of natural or pre-made synthetic scaffolds, DNA nanoparticles were prepared by mixing DNA and polymer in a sodium acetate buffer. Sucrose was added for a final concentration of 15 mg/mL, and the solution was used to coat the surface of a trabecular bone construct. This construct was then lyophilized for 2 days before being seeded with primary human cells (−50% GFP⁺ for ease of visualization). Referring now to FIG. 15, DsRed expression was observed within 24 hr, indicating that the nanoparticles remained functional and able to transfect cells in this new system.

Lyophilized nanoparticles also can be mixed with PLGA microparticles to form a larger construct that can be more easily manipulated and also can tune controlled release properties. In this embodiment, DsRed DNA-containing nanoparticles were compressed into a pellet with PLGA microparticles. This pellet was then placed within a well containing primary human glioblastoma cells (−20% GFP⁺ for ease of visualization through the opaque pellet). Referring now to FIG. 16, DsRed expression was observed within 4 days and remained very robust even after 12 days. Referring once again to FIG. 16, top=1 day, middle=4 days, bottom=12 days after transfection.

Further, as demonstrated in FIG. 17, DNA-loaded nanoparticles have been incorporated into natural and synthetic scaffolds, disks, microparticles, and hydrogels.

Example 6 Bioreducible Polymeric Particle Formulations for Delivery of siRNA

Reducible functional groups mediate successful siRNA-delivery, including transfection. In this example, GFP⁺ primary human glioblastoma cells were seeded in 96-well plates at a density of 10⁴cells/well in complete culture medium (DMEM/F-12 with 10% FBS and 1% antibiotic-antimycotic) and allowed to adhere overnight. Just before transfection, the culture medium was changed to serum-free medium. Particles were prepared by diluting polymer and siRNA both in 25 mM sodium acetate buffer (pH 5), then mixing them at a 100:1 polymer:siRNA ratio (w/w). Nanoparticles formed spontaneously after 10 min of incubation and were added to the cells in medium at a 1:5 ratio (v/v) and a final concentration of 60 nM. Each polymer/siRNA treatment group was paired with a control group using a scrambled siRNA sequence (scrRNA). Cells were incubated with the particles for 4 hr. The medium and particles were then aspirated and replaced with complete medium. On each of the following days, GFP expression was measured using a Synergy 2 multiplate fluorescence reader (Biotek). Background fluorescence was measured from GFP cells in medium and was subtracted from all other readings. Knockdown was calculated by normalizing GFP fluorescence (excitation 485 nm, emission 528 nm) from the siRNA-treated cells to the scrRNA-treated cells. Medium was changed every 3 days.

The reducible disulfide bond in the endgroup E10 (cystamine dihydrochloride) drastically improves siRNA delivery and gene knockdown. Referring now to FIG. 18, GFP⁺ glioblastoma cells were transfected with scrambled (control) siRNA (top panels) or siRNA against GFP (bottom). The polymers used as transfection agents consisted of B3-S5 at a 1.1:1 molar ratio, endcapped with (from left to right) E10, E3 (1,3=diaminopentane), or E6 (2-(3-aminopropylamino)ethanol). With the endgroups tested, the base polymer B3-S5 was able to achieve up to 8% knockdown; with E10 as the endgroup, over 80% knockdown was observed.

Referring now to FIGS. 1A-1C, the activity of R6-series polymers at delivering siRNA to knockdown GFP signal is GB cells is further demonstrated. % Knockdown of GFP expression in GFP+ glioblastoma cells transfected with siRNA against GFP, normalized to cells transfected with scrambled siRNA, using various BR6 polymers as a transfection agent. (A) Transfection with acrylate-terminated BR6 polymers with either S3, S4 or S5 as the side chain; (B) Transfection with E10 end-capped versions of the polymers in Figure A; and (C) GFP fluorescence images of cells transfected with BR6-S4-Ac complexed scrambled RNA (top) vs. siRNA against GFP (bottom);

Without wishing to be bound to any one particular theory, it is likely that E10 facilitates siRNA delivery by augmenting intracellular release because it degrades in the reducing intracellular environment. Results from gel retardation assay supports this hypothesis. Gel retardation assays were carried out by adding polymer of varying concentrations in sodium acetate buffer to a constant concentration of siRNA in sodium acetate. After 10 min of incubation, a solution of 30% glycerol in water was added at a 1:5 volumetric ratio as a loading buffer. Bromophenol blue or other dyes were not added, as they were found to interfere with binding. Samples were loaded into a 1% agarose gel with 1 μg/mL ethidium bromide at 125 ng siRNA per well. Samples were run for 15 min under 100 V, then visualized using UV exposure.

Referring now to FIG. 20, a gel retardation assay of siRNA with BR6-S5-E10 at varying ratios of polymer to RNA is shown. The polymer effectively retards siRNA (top), but in the presence of 5 mM glutathione siRNA is released immediately (bottom). These data demonstrate the hypothesized intracellular release of siRNA and elucidates the mechanism by which nanoparticles formed using BR6 facilitate strong siRNA transfection and GFP knockdown.

Referring also to FIG. 21, an E10-endcapped polymer (top) retards siRNA efficiently, but upon addition of 5 mM glutathione, siRNA is immediately released (bottom). Numbers refer to the w/w ratio of polymer-to-siRNA in all cases.

Referring now to FIG. 22, the same polymer as in FIG. 21, but with a different endcap (E7, 1-(3-aminopropyl)-4-methylpiperazine) also retards siRNA (top), but is not affected by application of glutathione (bottom).

Referring now to FIG. 23, gel permeation chromatography data of BR6 polymerized with S4 at a BR6:S4 ratio of 1.2:1 at 90° C. for 24 hours, before and after end-capping with E7, are provided.

Referring now to FIG. 24, knockdown efficiency also is affected by molecular weight of the polymer. In FIG. 24, 1.2:1, 1.1:1, and 1.05:1 refer to the ratio of reactants in the base polymer step growth reaction, which affects the ultimate molecular weight. Top 4310 formulations were able to achieve greater knockdown over time compared to commercially available reagents like Lipofectamine 2000 (Lipo).

Referring now to FIG. 25, combined DNA (RFP) and siRNA delivery (against GFP) in GB; GFP+GB cells were treated with scrambled siRNA (top) or siRNA against GFP (bottom), causing visible knockdown. Interestingly, different polymer structures seem ideal for siRNA versus DNA delivery or for both. One polymer effective in both was used to deliver both siRNA against GFP and plasmid DsRed DNA to GFP+ hMSCs, resulting in the ability to turn green cells red.

Referring now to FIG. 26, siRNA knockdown is affected by the endcap (E), base polymer (increasing hydrophobicity from L to R within each E), and molecular weight (increasing L to R within each base polymer). One endcap that shows high knockdown even at lower molecular weights is E10, which is strikingly more effective than the other endcaps tested for the same base polymers. Other PBAEs were also highly effective when synthesized at high molecular weight.

Referring now to FIG. 27, is shown 4410, 200 w/w (blue line on above graph), 8 days after transfection: Left: hMSCs treated with scrambled control; Right: hMSCs treated with siRNA.

Referring now to FIG. 28, in some embodiments, polymer molecular weight is between 4.00-10.00 kDa for siRNA delivery.

Example 7 DNA Delivery

Referring now to FIG. 29, the presently disclosed biomaterial can be used for other forms of delivery, for example DNA delivery. DNA transfection shows some similar trends compared with siRNA, but with different optimal endcaps. Specific polymer structure is critical to determine which polymers are effective for DNA delivery or siRNA delivery or both. Both DNA and siRNA transfection depend less on MW with high polymer hydrophobicity. High GFP DNA delivery was achieved using PBAEs, with transfection in 10% serum and at 5 μg DNA/mL. Referring now to FIG. 30, several formulations with up to 90% transfection and high (>90%) viability are shown.

Referring now to FIG. 31, GB transfection is demonstrated. More particularly, 551 GB cells cultured as neurospheres (undifferentiated). They were plated in monolayer on laminin 24 hr before transfection with DsRed DNA using 447 LG (red). 48 hr after transfection, they were stained for nestin (blue). Red and blue overlaid (left) show that transfection occurred in nestin+ cells (nestin only: right).

Referring now to FIG. 32, for a DNA delivery application, in some embodiments, polymer molecular weight is between 3.00-10.0 kDa.

Example 8 In Vivo Activity for Selected Peptides

In some embodiments, the presently disclosed subject matter demonstrates in vivo activity for selected peptides in DIVAA angioreactors and a lung cancer xenograft model, Koskimaki J E, Karagiannis E D, Tang B C, Hammers H, Watkins D N, Pili R, et al. Pentastatin-1, a collagen IV derived 20-mer peptide, suppresses tumor growth in a small cell lung cancer xenograft model. BMC Cancer 2010; 10:29, and in a breast cancer xenograft model using MDA-MB-231 cells. Koskimaki J E, Karagiannis E D, Rosca E V, Vesuna F, Winnard P T, Jr., Raman V, et al. Peptides derived from type IV collagen, CXC chemokines, and thrombospondin-1 domain-containing proteins inhibit neovascularization and suppress tumor growth in MDA-MB-231 breast cancer xenografts. Neoplasia 2009; 11(12):1285-91.

Following orthotopic inoculation of SCID mice in the mammary fat pad area using 2×10⁶cells, tumors grew to approximately 100 mm³in 2 weeks; at that time 100 μL of peptide solution was injected i.p. once a day at peptide doses 10-20 mg/kg. PBS solution was injected as control. Several peptides have been found to inhibit tumor growth. See FIG. 33A. The microvessel density was determined by screening the immunohistologically stained CD31 sections. Inhibition of LEC migration in the ACEA migration assay also was determined (see FIG. 33B).

Representative data showing the activity of free peptide and peptide encapsulated in the presently disclosed polymeric particles are shown in FIG. 33D, which shows the metabolic activity of free peptides and peptides in polymeric particles.

Example 9 Non-Viral Gene Delivery for Treatment of Glioblastoma and Brain Cancer Stem Cells

Glioblastoma (GB) is a grade IV brain cancer as defined by the WHO and is the most common primary CNS tumor in the United States. Current treatment includes surgical resection, radiotherapy, and chemotherapy. The median survival with treatment is approximately 14 months.

Brain cancer stem cells (BCSCs) possess genetic and morphological features similar to neural stem cells. Small numbers of BCSCs can initiate gliomas. BCSCs are refactory to conventional anti-cancer treatments.

Gene delivery typically is accomplished by either vaccine-mediated or polymer mediates techniques. Virus-mediated gene delivery is highly efficient, insertional mutagenesis, and toxicity/immunogenicity. Polymer-mediated gene delivery is chemically versatile, potentially safer than vaccine-mediated gene delivery, but typically is less efficient. See Green et al., 2008. Acc. Chem. Res. 41(6):749-59; Putnam 2006. Nat. Mater. 5(6):439-51.

Non-viral, e.g., polymer-mediated gene delivery, can be accomplished, in some embodiments, by using poly(beta-amino esters) (PBAEs). In particular embodiments, PBAEs suitable for use in target delivery can be synthesized in a two-step reaction provided herein below in Scheme 6 and can form nanocomplexes with negatively-charged cargo (e.g., DNA, siRNA) via electrostatic interactions as disclosed, for example, in some embodiments described in International PCT Patent Application Publication No. WO/2010/132879 for “Multicomponent Degradable Cationic Polymers,” to Green et al., which is incorporated herein by reference in its entirety.

In some embodiments, the presently disclosed subject matter demonstrates the delivery of DNA to GB cells, i.e., bulk tumor (non-stem cells; verifies the efficacy of the presently disclosed methods in BCSCs; demonstrates the delivery of apoptosis-inducing genes in BCSCs; provides practical considerations for translation of the presently disclosed methods; and discusses how the presently disclosed methods can be used in conjunction with other methods for treating GB.

The delivery of DNA to GB cells, bulk tumor (non-stem cells) and the efficacy of the presently disclosed methods to deliver DNA to BCSCs is demonstrated in FIGS. 36-39.

Referring now to FIG. 34, the delivery of DNA to GB bulk tumor cells is demonstrated for representative biomaterials. Referring now to FIG. 35, the transfection of genes to BCSC is demonstrated for representative presently disclosed biomaterials. FIG. 36 demonstrates the delivery of DNA to fetal (healthy) cells. FIG. 37 also demonstrates the delivery of DNA to BCSCs. The delivery of apoptosis-inducing genes in BCSCs is demonstrated in FIGS. 38 to 39.

These data demonstrate that PBAEs can be used for highly effective DNA delivery to GB cells, including tumor-initiating stem cells; transfection occurs even in 3D neurospheres in suspension; transfection is much less efficient in non-cancer cells (F34 fetal cells) as compared to GB cells; and transfection with secreted TRAIL causes more death in BCSCs with not significant effect on healthy cells.

In practical considerations for translation, for lyophilized nanoparticles, the presently disclosed methods provide an ease of preparation, e.g., only water needs to be added to the lyophilized nanoparticles, long-term storage, large, consistent batches, manipulation for uses in other devices, and stability in suspension. See scheme in FIG. 3.

As shown in FIG. 40, particles lyophilized with sucrose and used immediately are as effective in transfection as freshly prepared particles. Further, no loss in efficiency is observed within three months; and approximately 50% efficiency is retained after six months. The use of the presently disclosed materials and methods for long-term gene delivery is demonstrated in FIGS. 41 and 42. Other methods for treatment of GB include siRNA delivery to GB cells (FIG. 43).

A comparison of siRNA vs. DNA delivery in GB cells is shown in FIGS. 44 and 45. More particularly, as shown in FIG. 45, both 4410 and 447 can form complexes with DNA and siRNA; a higher weight ratio of polymer-to-nucleic acid is needed for siRNA than for DNA; E10 polymers release siRNA immediately, but not DNA, upon addition of glutathione (GSH).

In summary, PBAE/nucleic acid nanoparticles can be fabricated in a form that remains stable over time and allow flexibility for clinical use; PBAEs can be used for effective DNA or siRNA delivery to GB-derived BCSCs; and efficient release of cargo is necessary for effective nucleic acid delivery, especially with siRNA.

Example 10 Microparticles for Peptide Delivery In some embodiments, microparticles for controlled release of nanoparticles, which themselves encapsulate biological agents, are illustrated in FIGS. 14-17.

More particularly, FIG. 46 depicts a strategy of combining nanoparticles within microparticles to extend release further. PLGA or blends of PLGA can be combined with the presently disclosed polymers to form microparticles by double emulsion. FIG. 47 shows release of a representative peptide, DEAH-FITC, from a presently disclosed microparticle. FIG. 48 shows slow extended release from microparticles containing nanoparticles that contain peptides; FITC-DEAH peptide was first mixed with the 336 PBAE to allow for self-assembly into nanoparticles and was then mixed with BSA (middle) or not (bottom) to form an aqueous mixture. This mixture was added to a DCM-PLGA phase and sonicated to form a w/o suspension. This suspension was then added to a PVA solution and homogenized to form the final w/o/w suspension. This mixture was finally added to another PVA solution to allow for the DCM to evaporate and harden the formed microparticle. Different release profiles can potentially be obtained as seen above for the different microparticle formulations. In all cases, there is a long-term release of the peptide. Forming nanoparticles that encapsulate the peptide within the microparticles, extends the release compared to encapsulating peptide directly into microparticles (middle figure). The particles can be designed to have different release depending on the local environment (top figure). In some embodiments, release is constant over time and zero-order with respect to time (bottom figure).

Referring now to FIG. 49 in shown the in vivo effects of microparticle formulations in both the CNV and rhoNEGF model over time. DEAH (SP6001)-336 PBAE nanoparticle formulation made as described previously. (Top) Intravitreal injections into CNV model mice as described previously show comparable effects after 14 days, even though only small fraction of peptide is released over that time from microparticles. (Middle) and (Bottom) A genetic model of wet form of age-related macular degeneration in mice used to test long-term effect of microparticles. After 1 week (middle) comparable effects seen in reduction of angiogenesis. After 8 weeks (bottom), however, while peptide only no longer inhibits angiogenesis, the microparticle still does, as it is still releasing peptide over this time. While PLGA is used to form the microparticles used above, other polymers may be used including the synthetic polyesters and polyamides described above. In certain embodiments, blends of these polymer are combined with PLGA to form microparticles with differing environmental sensitivity and release properties; (a) the effect of microparticle (SP-6001) in CNV model mouse; (b) the effect of microparticle (SP-6001) in rhoNEGF (V6) mouse, 1 week after injection; and (c) the effect of microparticle (SP-6001) in rhoNEGF (V6) mouse, 8 weeks after injection.

REFERENCES

All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

Chiang A C, Massague J. Molecular basis of metastasis. N Engl J Med 2008; 359(26):2814-23.
Gupta G P, Massague J. Cancer metastasis: building a framework. Cell 2006; 127(4):679-95.
Sawyers C L. Cancer: mixing cocktails. Nature 2007; 449(7165):993-6.
Dorrell M I, Aguilar E, Scheppke L, Barnett F H, Friedlander M. Combination angiostatic therapy completely inhibits ocular and tumor angiogenesis. Proc Natl Acad Sci USA 2007; 104(3):967-72.
Farokhzad O C. Nanotechnology for drug delivery: the perfect partnership. Expert Opin Drug Deliv 2008; 5(9):927-9.
Putnam D. Polymers for gene delivery across length scales. Nat Mater 2006; 5(6):439-51.
Brigger I, Dubernet C, Couvreur P. Nanoparticles in cancer therapy and diagnosis. Adv Drug Deliv Rev 2002; 54(5):631-51.
Green J J, Shi J, Chiu E, Leshchiner E S, Langer R, Anderson D G. Biodegradable polymeric vectors for gene delivery to human endothelial cells. Bioconjug Chem 2006; 17:1162-9.
Green J J, Chiu E, Leshchiner E S, Shi J, Langer R, Anderson D G. Electrostatic ligand coatings of nanoparticles enable ligand-specific gene delivery to human primary cells. Nano Lett 2007; 7(4):874-9.
Harris T J, Green J J, Fung P W, Langer R, Anderson D G, Bhatia S N. Tissue-specific gene delivery via nanoparticle coating. Biomaterials 2010; 31(5):998-1006.
Green J J, Zugates G T, Tedford N C, Huang Y, Griffith L G, Lauffenburger D A, et al. Combinatorial modification of degradable polymers enables transfection of human cells comparable to adenovirus. Adv Mater 2007; 19(19):2836-42.
Lee J S, Green J J, Love K T, Sunshine J, Langer R, Anderson D G. Gold, poly(beta-amino ester) nanoparticles for small interfering RNA delivery. Nano Lett 2009; 9(6):2402-6.
Reichert J. Development trends for peptide therapeutics. Tufts Center for the Study of Drug Development 2008 [cited 2010; Available from: http://www.peptidetherapeutics.org/PTF_Summary_2008.pdf
Rosca E V, Koskimaki J E, Rivera C G, Pandey N B, Tamiz A P, Popel A S. Anti-angiogenic peptides for cancer therapeutics. Curr Pharm Biotechnol 12(8):1101-1116 (2011).
Lucas R, Holmgren L, Garcia I, Jimenez B, Mandriota S J, Borlat F, et al. Multiple forms of angiostatin induce apoptosis in endothelial cells. Blood 1998; 92(12):4730-41.
Green J J, Langer R, Anderson D G. A combinatorial polymer library approach yields insight into nonviral gene delivery. Acc Chem Res 2008; 41(6):749-59.
Shmueli R B, Anderson D G, Green J J. Electrostatic surface modifications to improve gene delivery. Expert Opin Drug Deliv 7(4):535-50.
Zhang S, Uludag H. Nanoparticulate systems for growth factor delivery. Pharm Res 2009; 26(7):1561-80.
Jain R A. The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials 2000; 21(23):2475-90.
Little S R, Lynn D M, Ge Q, Anderson D G, Puram S V, Chen J Z, et al. Poly-beta amino ester-containing microparticles enhance the activity of nonviral genetic vaccines. Proc Natl Acad Sci USA 2004; 101(26):9534-9.
Koskimaki J E, Karagiannis E D, Tang B C, Hammers H, Watkins D N, Pili R, et al. Pentastatin-1, a collagen IV derived 20-mer peptide, suppresses tumor growth in a small cell lung cancer xenograft model. BMC Cancer 2010; 10:29.
Koskimaki J E, Karagiannis E D, Rosca E V, Vesuna F, Winnard P T, Jr., Raman V, et al. Peptides derived from type IV collagen, CXC chemokines, and thrombospondin-1 domain-containing proteins inhibit neovascularization and suppress tumor growth in MDA-MB-231 breast cancer xenografts. Neoplasia 2009; 11(12):1285-91.

Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

1. A bioreducible, hydrolytically degradable polymer of formula (Ia): wherein:

n is an integer from 1 to 10,000;

R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino, alkylamino, dialkylamino, trialkylamino, aryl, ureido, heterocyclic, aromatic heterocyclic, cyclic, aromatic cyclic, halogen, hydroxyl, alkoxy, cyano, amide, carbamoyl, carboxylic acid, ester, carbonyl, carbonyldioxyl, alkylthioether, and thiohydroxyl groups;

wherein R1 can be present or absent and when present the compound of formula (I) further comprises a counter ion selected from the group consisting of chloride, fluoride, bromide, iodide, sulfate, nitrate, fumarate, acetate, carbonate, stearate, laurate, and oleate; and

wherein at least one R comprises a backbone of a diacrylate having the following structure:

wherein X1 and X2 are each independently substituted or unsubstituted C2-C20 alkylene, and wherein each X1 and X2 can be the same or different.

2. The bioreducible, hydrolytically degradable polymer of claim 1, wherein at least one R comprises a backbone of a diacrylate selected from the group consisting of: or co-oligomers comprising combinations thereof, wherein the diacrylate can be the same or different.

3. The bioreducible, hydrolytically degradable polymer of claim 1, wherein R′ comprises a side chain derived from compound selected from the group consisting of:

4. The bioreducible, hydrolytically degradable polymer of claim 1, wherein R″ comprises an end group comprising an acrylate functional group or an end group derived from a compound selected from the group consisting of:

5. The bioreducible, hydrolytically degradable polymer of claim 1, wherein n is an integer from 1 to 100.

6. The bioreducible, hydrolytically degradable polymer of claim 1, wherein n is an integer from 1 to 30.

7. The bioreducible, hydrolytically degradable polymer of claim 1, wherein n is an integer from 5 to 20.

8. The bioreducible, hydrolytically degradable polymer of claim 1, wherein n is an integer from 10 to 15.

9. The bioreducible, hydrolytically degradable polymer of claim 1, wherein n is an integer from 1 to 10.

10. The bioreducible, hydrolytically degradable polymer of claim 1, wherein at least one R″ comprises a biomolecule selected from the group consisting of poly(ethyleneglycol) (PEG), a targetinaligand, and a labeling molecule.

11. A nanoparticle, microparticle, or gel comprising the bioreducible, hydrolytically degradable polymer of claim 1.

12. The nanoparticle, microparticle, or gel of claim 11 further comprising a therapeutic agent.

13. The nanoparticle, microparticle, or gel of claim 12, wherein the therapeutic agent is a peptide or a combination of peptides.

14. The nanoparticle, microparticle, or gel of claim 12, wherein the therapeutic agent is an siRNA or a combination of siRNA.

15. The nanoparticle, microparticle, or gel of claim 12, wherein the therapeutic agent is selected from the group consisting of a gene, DNA, RNA, siRNA, miRNA, is RNA, agRNA, smRNA, a nucleic acid, a peptide, a protein, a chemotherapeutic agent, a hydrophobic drug, a small molecule drug, and combinations thereof.

16. A nanoparticle, microparticle, or gel comprising a compound of formula (I): wherein:

n is an integer from 1 to 10,000;

R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino, alkylamino, dialkylamino, trialkylamino, aryl, ureido, heterocyclic, aromatic heterocyclic, cyclic, aromatic cyclic, halogen, hydroxyl, alkoxy, cyano, amide, carbamoyl, carboxylic acid, ester, carbonyl, carbonyldioxyl, alkylthioether, and thiohydroxyl groups;

wherein R1 can be present or absent and when present the compound of formula (I) further comprises a counter ion selected from the group consisting of chloride, fluoride, bromide, iodide, sulfate, nitrate, fumarate, acetate, carbonate, stearate, laurate, and oleate; and

at least one of R, R′, and R″ comprise a reducible or degradable linkage, and wherein each R, R′, or R″ can independently be the same or different;

under the proviso that when at least one R group comprises an ester linkage of the formula —C(═O)—O— and the compound of formula (I) comprises a poly(beta-amino ester), then the compound of formula (I) must also comprise one or more of the following characteristics:

(a) each R group is different;

(b) each R″ group is different;

(c) each R″ group is not the same as any of R′, R1, R2, R3, R4, R5, R6, R7, R8, and R9;

(d) the R″ groups degrade through a different mechanism than the ester-containing R groups, wherein the degradation of the R″ group is selected from the group consisting of a bioreducible mechanism or an enzymatically degradable mechanism; and/or

(e) the compound of formula (I) comprises a substructure of a larger cross-linked polymer, wherein the larger cross-linked polymer comprises different properties from compound of formula (I);

and one or more peptides selected from the group consisting of an anti-angiogenic peptide, an anti-lymphangiogenic peptide, an anti-tumorigenic peptide, and an anti-permeability peptide.

17. The nanoparticle, microparticle, or gel of claim 16, wherein n is an integer from 1 to 1,000.

18. The nanoparticle, microparticle, or gel of claim 16, wherein n is an integer from 1 to 100.

19. The nanoparticle, microparticle, or gel of claim 16, wherein n is an integer from 1 to 30.

20. The nanoparticle, microparticle, or gel of claim 16, wherein n is an integer from 5 to 20.

21. The nanoparticle, microparticle, or gel of claim 16, wherein n is an integer from 10 to 15.

22. The nanoparticle, microparticle, or gel of claim 16, wherein n is an integer from 1 to 10.

23. The nanoparticle, microparticle, or gel of claim 16, wherein the reducible or degradable linkage comprising R, R′, and R″ is selected from the group consisting of an ester, a disulfide, an amide, an anhydride or a linkage susceptible to enzymatic degradation, subject to the proviso of claim 16.

24. The nanoparticle, microparticle, or gel of claim 16, wherein R comprises a backbone of a diacrylate selected from the group consisting of:

25. The nanoparticle, microparticle, or gel of claim 16, wherein R′ comprises a side chain derived from compound selected from the group consisting of:

26. The nanoparticle, microparticle, or gel of claim 16, wherein R″ comprises an end group derived from a compound selected from the group consisting of

27. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide is a collagen IV peptide.

28. The nanoparticle, microparticle, or gel of claim 27, wherein the collagen IV peptide is selected from the group consisting of: (SEQ ID NO: 2443) LRRFSTMPFMFCNINNVCNF (SEQ ID NO: 2444) LRRFSTMPFMFGNINNVGNF (SEQ ID NO: 2445) LRRFSTMPFMF (SEQ ID NO: 2446) LRRFSTMPFMF-Abu-NINV (SEQ ID NO: 2447) LRRFSTMPFMF-Abu (SEQ ID NO: 2448) LRRFSTMP (SEQ ID NO: 2449) NINNV-Abu-NF (SEQ ID NO: 2450) FMF-Abu-NINNV-Abu-NF (SEQ ID NO: 2451) STMPFMF-Abu-NINNV-Abu-NF (SEQ ID NO: 2452) LRRFSTMPFMF-Abu-NINNV-Abu-NF (SEQ ID NO: 2453) LNRFSTMPF (SEQ ID NO: 2454) LRRFST-Nle-PF-Nle-F (SEQ ID NO: 2455) LRRFSTMPAMF-Abu-NINNV-Abu-NF (SEQ ID NO: 2456) LRRFSTMPFAF-Abu-NINNV-Abu-NF (SEQ ID NO: 2457) LRRFSTMPFMA-Abu-NINNV-Abu-NF (SEQ ID NO: 2458) LRRFSTMPF-Nle-F-Abu-NINNV-Abu-NF (SEQ ID NO: 2459) LRRFSTMPFM(4-ClPhen)-Abu-CNINNV-Abu-NF (SEQ ID NO: 2460) F-Abu-NINNV-Abu-N (SEQ ID NO: 2461) F-Abu-NIN (SEQ ID NO: 2462) LRRFSTMPFMFSNINNVSNF (SEQ ID NO: 2463) LRRFSTMPFMFANINNVANF (SEQ ID NO: 2464) LRRFSTMPFMFININNVINF (SEQ ID NO: 2465) LRRFSTMPFMFTNINNVTNF (SEQ ID NO: 2466) LRRFSTMPFMFC(AllyGly)NINNV(AllyGly)NF (SEQ ID NO: 2467) LRRFSTMPFMFVNINNVVNF (SEQ ID NO: 2468) LRRFSTMPFMF-Abu-NINN (SEQ ID NO: 2469) LRRFSTMPFMFTNINV (SEQ ID NO: 2470) F-Abu-NINV (SEQ ID NO: 2471) FTNINNVTN (SEQ ID NO: 2472) LRRFSTMPFMFTNINN (SEQ ID NO: 2473) LRRFSTMPFMFININN (SEQ ID NO: 2474) LRRFSTMPF-dA-FININNVINF (SEQ ID NO: 2475) LRRFSTAPFAFININNVINF (SEQ ID NO: 2476) LRRFSTMPFAFININNVINF;

wherein Abu is 2-aminobutyric acid; Nle is Norleucine; and AllyGly is allyglycine.

29. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide is selected from the group consisting of: RLRLLTLQSWLL (SEQ ID NO: 2477) LMRKSQILISSWF (SEQ ID NO: 2478) LLIVALLFILSWL (SEQ ID NO: 2479) LLRLLLLIESWLE (SEQ ID NO: 2480) LLRSSLILLQGSWF (SEQ ID NO: 2481) LLHISLLLIESRLE (SEQ ID NO: 2482) LLRISLLLIESWLE (SEQ ID NO: 2483)

30. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof comprising the amino acid sequence W-X2-C-X3-C-X2-G (SEQ ID NO: 2486), wherein X denotes a variable amino acid; W is tryptophan; C is cysteine, G is glycine; and wherein the peptide reduces blood vessel formation in a cell, tissue or organ.

31. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of: THSD-1: QPWSQCSATCGDGVRERRR; (SEQ ID NO: 2350) THSD-3: SPWSPCSGNCSTGKQQRTR; (SEQ ID NO: 2351) THSD-6: WTRCSSSCGRGVSVRSR; (SEQ ID NO: 2352) CILP: SPWSKCSAACGQTGVQTRTR; (SEQ ID NO: 2325) WISP-1: SPWSPCSTSCGLGVSTRI; (SEQ ID NO: 2360) WISP-2: TAWGPCSTTCGLGMATRV; (SEQ ID NO: 2361) WISP-3: TKWTPCSRTCGMGISNRV; (SEQ ID NO: 2362) F-spondin: SEWSDCSVTCGKGMRTRQR; (SEQ ID NO: 2359) F-spondin: WDECSATCGMGMKKRHR; (SEQ ID NO: 2358) CTGF: TEWSACSKTCGMGISTRV; (SEQ ID NO: 2327) fibulin-6: ASWSACSVSCGGGARQRTR; (SEQ ID NO: 2331) fibulin-6: QPWGTCSESCGKGTQTRAR; (SEQ ID NO: 2330) fibulin-6: SAWRACSVTCGKGIQKRSR; (SEQ ID NO: 2329) CYR61: TSWSQCSKTCGTGISTRV; (SEQ ID NO: 2328) NOVH: TEWTACSKSCGMGFSTRV; (SEQ ID NO: 2332) UNC5-C: TEWSVCNSRCGRGYQKRTR; (SEQ ID NO: 2356) UNC5-D: TEWSACNVRCGRGWQKRSR; (SEQ ID NO: 2357) SCO-spondin: GPWEDCSVSCGGGEQLRSR; (SEQ ID NO: 2349) Properdin: GPWEPCSVTCSKGTRTRRR; (SEQ ID NO: 2335) C6: TQWTSCSKTCNSGTQSRHR; (SEQ ID NO: 2324) ADAMTS-like-4: SPWSQCSVRCGRGQRSRQVR; (SEQ ID NO: 2355) ADAMTS-4: GPWGDCSRTCGGGVQFSSR; (SEQ ID NO: 2293) ADAMTS-8: GPWGECSRTCGGGVQFSHR; ADAMTS-16: SPWSQCTASCGGGVQTR; (SEQ ID NO: 2310) ADAMTS-18: SKWSECSRTCGGGVKFQER; (SEQ ID NO: 2315) semaphorin 5A: GPWERCTAQCGGGIQARRR; (SEQ ID NO: 2346) semaphorin 5A: SPWTKCSATCGGGHYMRTR; (SEQ ID NO: 2347) semaphoring 5B: TSWSPCSASCGGGHYQRTR; (SEQ ID NO: 2348) papilin: GPWAPCSASCGGGSQSRS; (SEQ ID NO: 2334) papilin: SQWSPCSRTCGGGVSFRER; (SEQ ID NO: 2333) ADAM-9: KCHGHGVCNS; (SEQ ID NO: 2497) and ADAM-12: MQCHGRGVCNNRKN, (SEQ ID NO: 2288) wherein A is alanine; I is isoleucine; M is methionine; H is histidine; Y is tyrosine; K is lysine; W is tryptophan; C is cysteine, T is threonine, S is serine; N is asparagine; G is glycine; R is arginine; V is valine, P is proline, and Q is glutamine wherein the peptide reduces blood vessel formation in a cell, tissue or organ.

32. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof having at least 85% identity to an amino acid sequence selected from the group consisting of: ENA-78: NGKEICLDPEAPFLKKVIQKILD; (SEQ ID NO: 2381) CXCL6: NGKQVCLDPEAPFLKKVIQKILDS; (SEQ ID NO: 2384) CXCL1: NGRKACLNPASPIVKKIIEKMLNS; (SEQ ID NO: 2388) Gro-γ: NGKKACLNPASPMVQKIIEKIL; (SEQ ID NO: 2392) Beta thromboglobulin/CXCL7: DGRKICLDPDAPRIKKIVQKKL, (SEQ ID NO: 2400) Interleukin 8 (IL-8)/CXCL8: DGRELCLDPKENWVQRVVEKFLK, (SEQ ID NO: 2396) GCP-2: NGKQVCLDPEAPFLKKVIQKILDS, (SEQ ID NO: 2384) wherein A is alanine; I is isoleucine; F is phenylalanine; D is aspartic acid; M is methionine; H is histidine; Y is tyrosine; K is lysine; W is tryptophan; C is cysteine, T is threonine, S is serine; N is asparagine; G is glycine; R is arginine; V is valine, P is proline, and Q is glutamine; and wherein the peptide reduces blood vessel formation in a cell, tissue or organ.

33. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of Alpha 6 fibril of type 4 collagen: YCNINEVCHYARRNDKSYWL; (SEQ ID NO: 2379) Alpha 5 fibril of type 4 collagen: LRRFSTMPFMFCNINNVCNF; (SEQ ID NO: 2443) Alpha 4 fibril of type 4 collagen: AAPFLECQGRQGTCHFFAN; (SEQ ID NO: 2373) Alpha 4 fibril of type 4 collagen: LPVFSTLPFAYCNIHQVCHY; (SEQ ID NO: 2371) Alpha 4 fibril of type 4 collagen: YCNIHQVCHYAQRNDRSYWL, (SEQ ID NO: 2372) and Collagen type IV, alpha6 fibril LPRFSTMPFIYCNINEVCHY (SEQ ID NO: 2494) wherein A is alanine; I is isoleucine; F is phenylalanine; D is aspartic acid; M is methionine; H is histidine; Y is tyrosine; K is lysine; W is tryptophan; C is cysteine, T is threonine, S is serine; N is asparagine; G is glycine; R is arginine; V is valine, P is proline, and Q is glutamine wherein the peptide reduces blood vessel formation in a cell, tissue or organ.

34. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof comprising one of the following amino acid sequences: TSP Motif: W-X(2)-C-X(3)-C-X(2)-G, (SEQ ID NO: 2486) CXC Motif: G-X(3)-C-L Collagen Motif: C-N-X(3)-V-C (SEQ ID NO: 2487) Collagen Motif: P-F-X(2)-C Somatotropin Motif: L-X(3)-L-L-X(3)-S-X-L (SEQ ID NO: 2488) Serpin Motif: L-X(2)-E-E-X-P (SEQ ID NO: 2489) wherein X denotes a variable amino acid and the number in parentheses denotes the number of variable amino acids; W denotes tryptophan; C denotes cysteine, G denotes glycine, V denotes valine; L denotes leucine, P is proline, and wherein the peptide reduces blood vessel formation in a cell, tissue or organ.

35. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide comprises an amino acid sequence shown in Table 1-6, 8 and 9.

36. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide comprises an isolated peptide or analog thereof having at least 85% identity to an amino acid sequence shown in Table 1-10.

37. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide comprises an amino acid sequence shown in Table 1-10.

38. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide consists essentially of an amino acid sequence shown in Table 1-10.

39. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide comprises an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of: Placental Lactogen LLRISLLLIESWLE (SEQ ID NO: 2483) hGH-V LLRISLLLTQSWLE (SEQ ID NO: 2490) GH2 LLHISLLLIQSWLE (SEQ ID NO: 2491) Chorionic LLRLLLLIESWLE (SEQ ID NO: 2480) somatomammotropin Chorionic LLHISLLLIESRLE (SEQ ID NO: 2482) somatomammotropin hormone-like 1 Transmembrane LLRSSLILLQGSWF (SEQ ID NO: 2481) protein 45A IL-17 receptor C RLRLLTLQSWLL (SEQ ID NO: 2477) Neuropeptide FF LLIVALLFILSWL (SEQ ID NO: 2479) receptor 2 Brush border LMRKSQILISSWF (SEQ ID NO: 2478) myosin-I wherein the peptide reduces blood vessel formation in a cell, tissue or organ.

40. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide comprises an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of: DEAH box polypeptide EIELVEEEPPF (SEQ ID NO: 2485) 8 (“DEAH” disclosed as SEQ ID NO: 2484) Caspase 10 AEDLLSEEDPF (SEQ ID NO: 2492) CKIP-1 TLDLIQEEDPS (SEQ ID NO: 2493) wherein the peptide reduces blood vessel formation in a cell, tissue or organ.

41. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide comprises an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of: Collagen LPRFSTMPFIYCNINEVCHY (SEQ ID NO: 2494) type IV, alpha6 fibril wherein the peptide reduces blood vessel formation in a cell, tissue or organ.

42. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide comprises a collagen peptide in combination with a somatotropin peptide.

43. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide has the sequence EIELVEEEPPF (SEQ ID NO: 2485) and the polymer has the following structure: wherein:

R is selected from the group consisting of:

R′ is selected from the group consisting of:

R″ is selected from the group consisting of:

44. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide has the sequence NGRKACLNPASPIVKKIIEKMLNS (SEQ ID NO: 2388) and the polymer has the following structure: wherein:

R is selected from the group consisting of:

R′ is selected from the group consisting of:

R″ is selected from the group consisting of:

45. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide has the sequence LLRISLLLIESWLE (SEQ ID NO: 2483) and the polymer has the following structure: wherein:

R is selected from the group consisting of:

R′ is selected from the group consisting of:

R″ is selected from the group consisting of:

46. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide has the sequence LRRFSTMPFMF-Abu-NINNV-Abu-NF (SEQ ID NO: 2452) and the polymer has the following structure: wherein: and

R is selected from the group consisting of:

R′ is selected from the group consisting of:

R″ is selected from the group consisting of:

47. The nanoparticle, microparticle, or gel of claim 16, wherein the polymer has the following structure: and the one or more peptide is selected from the group consisting of: EIELVEEEPPF; (SEQ ID NO: 2485) SPWSPCSTSCGLGVSTRI; (SEQ ID NO: 2360) LRRFSTMPFMFCNINNVCNF; (SEQ ID NO: 2375) and NGRKACLNPASPIVKKIIEKMLNS. (SEQ ID NO: 2388)

48. The nanoparticle, microparticle, or gel of claim 16, further comprising one or more peptides encapsulated therein.

49. A multilayer particle comprising a core and one or more layers, wherein the core comprises a material selected from the group consisting of a compound of formula (I), a gold nanoparticle, an inorganic nanoparticle, an organic polymer, and the one or more layers comprise a material selected from the group consisting of a compound of formula (I), an organic polymer, one or more peptides, and one or more additional biological agents.

50. The multilayer particle of claim 49, further comprising alternating cationic and anionic layers.

51. A microparticle comprising a compound of formula (I), poly(lactide-co-glycolide) (PLGA), or combinations thereof.

52. The microparticle of claim 51, further comprising a nanoparticle comprising a compound of formula (I).

53. The microparticle of claim 51, further comprising one or more peptides encapsulated therein.

54. A method for stabilizing a suspension of nanoparticles and/or microparticles of formula (I), the method comprising:

(a) providing a suspension of nanoparticles and/or microparticles of formula (I);

(b) admixing a lyroprotectant with the suspension;

(c) freezing the suspension for a period of time; and

(d) lyophilizing the suspension for a period of time.

55. The method of claim 54, wherein the lyroprotectant is sucrose.

56. A pellet or scaffold comprising one or more lyophilized particle, wherein the one or more lyophilized particle comprises a compound of formula (I).

57. The pellet or scaffold of claim 56, wherein the one or more lyophilized particle comprises cargo.

58. The pellet or scaffold of claim 57, wherein the cargo is selected from the group consisting of a peptide, a protein, an siRNA, DNA, an imaging agent, and combinations thereof.

59. The pellet or scaffold of claim 56, wherein the scaffold comprises a bone construct.

60. The pellet or scaffold of claim 56, further comprising one or more microparticle, which has the same or different composition as the one or more lyophilized particle.

61. The pellet or scaffold of claim 56, wherein the one or more lyophilized particle has a size of about 20 nm to about 100 nm.

62. The pellet or scaffold of claim 56, wherein the one or more lyophilized particle has a size of about 100 nm to about 300 nm.

63. The pellet or scaffold of claim 56, wherein the one or more lyophilized particle has a size of about 300 nm to about 1000 nm.

64. The pellet or scaffold of claim 56, wherein the one or more lyophilized particle has a size of about 1 micron to about 10 microns.

65. The pellet or scaffold of claim 56, wherein the one or more lyophilized particle has a size of about 10 microns to about 30 microns.

66. A method of treating a disease or condition, the method comprising administering to a subject in need of treatment thereof a therapeutically effective amount of a nanoparticle, microparticle, gel, or multilayer particle comprising a compound of formula (I), wherein the nanoparticle, microparticle, gel, or multilayer particle further comprises a therapeutic agent specific for the disease or condition to be treated.

67. The method of claim 66, wherein the disease or condition comprises an angiogenesis-dependent disease or condition.

68. The method of claim 67, wherein the angiogenesis disease or condition is selected from the group consisting of a cancer and age-related macular degeneration.

69. The method of claim 66, wherein the disease or condition is a non-angiogenic disease or condition.

70. The method of claim 66, wherein the therapeutic agent is selected from the group consisting of gene, DNA, RNA, siRNA, miRNA, is RNA, agRNA, smRNA, a nucleic acid, a peptide, a protein, a chemotherapeutic agent, a hydrophobic drug, a small molecule drug, and combinations thereof.