NANOPARTICLE-MEDIATED GENE DELIVERY, GENOMIC EDITING AND LIGAND-TARGETED MODIFICATION IN VARIOUS CELL POPULATIONS
An improved nanoparticle for transfecting cells is provided. The nanoparticle includes a core polyplex and a silica coating on the core polyplex and, optionally, a polymer attached to an outer surface of the silica coating, where the polyplex includes an anionic polymer, a cationic polymer, a cationic polypeptide, and a polynucleotide. Also provided is an improved method of modifying intracellular polynucleotides. The method includes contacting a cell with a nanoparticle that includes a core polyplex and a silica coating on the core polyplex and, optionally, a polymer attached to an outer surface of the silica coating, where the polyplex includes an anionic polymer, a cationic polymer, a cationic polypeptide, and a polynucleotide.
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This application is a continuation application and claims priority benefit of commonly owned and co-pending U.S. patent application Ser. No. 15/024,264, filed Mar. 23, 2016, which is a National stage application of PCT/US2014/057000, filed on Sep. 23, 2014, which claims priority under 35 U.S.C. § 119 to U.S. Provisional Application No. 61/881,072, filed Sep. 23, 2013, the entire disclosures of each of the prior applications are hereby incorporated herein by reference.GOVERNMENT RIGHTS STATEMENT
This invention was made with U.S. Government support under R01 AG030637 awarded by the National Institutes of Health. The U.S. Government has certain rights in the invention.BACKGROUND OF THE INVENTION Technical Field
The present invention generally relates to use of nanoparticles to transfect cells. More particularly, the present invention relates to coated nanoparticles with a polyplex core for intracellular delivery of ploynucleotides to modify gene expression.Background Information
Introducing polynucleotides into cells to alter gene expression requires appropriate packaging of the polynucleotides to protect them from degradation before cell entry, to permit entry into cells, and to direct delivery to the appropriate subcellular compartment. Effectiveness in altering expression may also depend on time-frames of release of polynucleotides from packaging after cellular entry. Available nanoparticle-based technologies for modifying gene expression suffer from low levels of cellular transfection and limited effectiveness upon transfection, at least in part because of their limitations in satisfying the foregoing requirements. It is therefore desirable to obtain a nanoparticle-based transfection agent and method of use thereof that addresses all of these requirements to enhance effectiveness.SUMMARY OF THE INVENTION
The shortcomings of the prior art are overcome, and additional advantages are provided, through the provision, in one aspect, of a nanoparticle. The nanoparticle includes a core polyplex and a silica coating on the core polyplex, and the polyplex includes an anionic polymer, a cationic polymer, a cationic polypeptide, and a polynucleotide. In another aspect, the nanoparticle may also include a polymer attached to an outer surface of the silica coating.
A method of modifying intracellular polynucleotides is also provided. The method includes contacting a cell with a nanoparticle that includes a core polyplex and a silica coating on the core polyplex, and the polyplex includes an anionic polymer, a cationic polymer, a cationic polypeptide, and a polynucleotide. In another aspect, the nanoparticle may also include a polymer attached to an outer surface of the silica coating.
Additional features and advantages are realized through the techniques of the present invention. These, and other objects, features and advantages of this invention will become apparent from the following detailed description of the various aspects of the invention taken in conjunction with the accompanying drawings.
One or more aspects of the present invention are particularly pointed out and distinctly claimed as examples in the claims at the conclusion of the specification. The foregoing and other objects, features, and advantages of the invention are apparent from the following detailed description taken in conjunction with the accompanying drawings in which:
Aspects of the present invention and certain features, advantages, and details thereof, are explained more fully below with reference to the non-limiting embodiments illustrated in the accompanying drawings. Descriptions of well-known materials, fabrication tools, processing techniques, etc., are omitted so as to not unnecessarily obscure the invention in detail. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the invention, are given by way of illustration only, and are not by way of limitation. Various substitutions, modifications, additions and/or arrangements within the spirit and/or scope of the underlying inventive concepts will be apparent to those skilled in the art from this disclosure.
The present disclosure provides, in part, a multilayered nanoparticle for transfecting cells with agents to modify gene expression. Nanoparticles designed for improved serum stability, targetted delivery to specific cell types, greater nuclear specificity and compartment-specific unpackaging, improved ability to retain significant payload levels during initial stages of internalization, and ability to maintain release of payload for a various durations following internalization, and methods of use thereof, are provided.
In one aspect, complexes of polynucleotides with polymers, or polyplexes, created by condensation of cationic polymers and polynucleotides in the presence of anionic polymers may mediate increased transfection efficiency over polynucleotide-cationic polymer conjugates. Though this process may produce more particles and increase the net surface area of nanoparticles exposed for cellular uptake, an improved electrostatic repulsory element may also be at play in releasing nucleic acids through this technique. Surprisingly, in contrast to a more rapid disaggregation of nucleotides from nanoparticle polyplexes that include anionic polymers as would have been predicted on the basis of existing literature, in one aspect of the present invention, including an anionic polymer in a nanoparticle polyplex core may prolong the duration of intracellular residence of the nanoparticle and release of agents that affect gene expression or otherwise regulate cellular function, or payloads.
In another aspect, the presence of a cationic polypeptide in a nanoparticle may mediate stability, subcellular compartmentalization, and payload release. As one example, fragments of the N-terminus of histone peptides, referred to generally as histone tail peptides, within various polyplexes are not only capable of being deprotonated by various histone modifications, such as in the case of histone acetyltransferase-mediated acetylation, but may also mediate effective nuclear-specific unpackaging as components of polyplexes. Their trafficking may be reliant on alternative endocytotic pathways utilizing retrograde transport through the Golgi and endoplasmic reticulum, and the nature of histones existing inside of the nuclear envelope suggests an innate nuclear localization sequence on histone tail peptides. In one aspect of the present invention, including a histone tail peptide may promote nuclear localization of nanoparticles and result in enzyme-mediated release of polynucleotide payload therefrom.
In another aspect, silica coatings of polyplexes may seal their payloads before and during initial cellular uptake. Commonly used polyplexes consisting of poly(ethylenimine) and DNA have a tendency to shed the majority (˜90%) of their payloads during cellular internalization, with the remaining payload often remaining bound to its cationic nanocarrier's polymeric remains. With transiently stabilizing interlayers of silica, greater intracellular delivery efficiency may be observed despite decreased probability of cellular uptake. In another aspect of the present invention, coating a nanoparticle polyplex with a silica coating may seal the polyplex, stablizing it until its release upon processing in the intended subcellular compartment.
In another aspect of the present invention, transfection efficiency may be further increased by adding another layer of cationic polymer, making the delivery efficiency as much as two orders of magnitude greater than a bare or silica-coated polyplex, presumably due to the anionic nature of an oligomeric silica coating being cell repulsive. In a further aspect, silica-coated polyplexes and their further-layered derivatives are stable in serum and are suitable for in vivo experiments unlike cationic polymer/nucleic acid conjugates on their own.
In one example, a cationic polymer may comprise a poly(arginine), such as poly(L-arginine). A cationic polymer within the polyplex may have a molecular weight of between 1 kDa and 200 kDa. A cationic polymer within the polyplex may also have a molecular weight of between 10 kDa and 100 kDa. A cationic polymer within the polyplex may also have a molecular weight of between 15 kDa and 50 kDa. In one example, a cationic polymer comprises poly(L-arginine) with a molecular weight of approximately 29 kDa, as represented by SEQ ID NO: 1 (PLR). In another example, a cationic polymer may comprise linear poly(ethylenimine) with a molecular weight of 25 kDa (PEI). In another example, a cationic polymer may comprise branched poly(ethylenimine) with molecular weight of 10 kDa. In another example, a cationic polymer may comprise branched poly(ethylenimine) with a molecular weight of 70 kDa. In another example, a cationic polymer may comprise a D-isomer of poly(arginine) or of any of the foregoing polymers such as polypeptides, which may be particularly advantageous because polymers such as polypeptides containing a D-isomer may be less susceptible to degradation within a cell and therefore have a prolonged effect on influencing payload release and the rate thereof over time.
In another embodiment, a nanoparticle may include or contain, in addition to or in place of any of the foregoing cationic polypeptides, a nuclear localization sequence. A cationic polypeptide may comprise a nuclear localization sequence on its N- or C-terminus. A nuclear localization sequence may comprise an importin or karyopherin substrate, or may have or contain a sequence corresponding to SEQ ID NO: 8. In another embodiment, a nanoparticle may include, in addition to or in place of any of the foregoing cationic polypeptides, a mitochondrial localization signal or a peptide fragment of mtHSP70.
In another aspect, a nanoparticle may comprise a layer of polymers attached to or electrostatically bound with the external surface of coated polyplex, such as to or with the external surface of a silica coating. Such external polymers may serve to prevent cellular repulsion of the coated polyplex so as to promote contact with and uptake by a cell. An external polymer layer may also serve to promote internalization by specific cell types, such as if the externally attached polymer is or mimics a ligand to a receptor expressed by a cell type of which transfection is desired. A polymer in a polymer layer attached to the outer surface of coating on a polyplex may be from between 0.1 to 20 kDa in size, or may be up to 40 or 50 kDa in size.
Examples of polymer comprising a polymer layer attached to the external surface of the coated core polyplex include those represented by SEQ ID NO: 4, which is an approximately 10 kDa poly(arginine) polymer, and SEQ ID NO: 5, which is human vasoactive endothelial growth factor protein, in accordance with the present invention. In another example, a polymer comprising a layer attached to the external surface of the coated core polyplex may comprise an anchor substrate of from between 1 to 25 repeating anionic or cationic moieties at the N-terminus, C-terminus, 5′, or 3′ end of a polymer, polypeptide, or polynucleotide to provide electrostatic conjugation of a targeting motif contained in the polymer, polypeptide, or polynucleotide to the coated polyplex core. In another example, a polymer comprising a layer attached to the external surface of the coated core polyplex may comprise a polymer, polypeptide, or polynucleotide sequence that exhibits base pair complementarity or binding affinity for an amino acid sequence binding motif to bind additional layers that may be added thereupon.
In another aspect of the present invention, illustrated in
In a further aspect, the present invention includes optimized ratios of anionic and cationic polymers, cationic polypeptides, and polynucleotides for complexation of a polyplex core as part of a nanoparticle. In one example, plasmid DNA was fluorescently tagged with ethidium bromide (40 ng EtBr/ug DNA) before addition of various polymeric constituents in molar [1(positive)]:[1(negative)] ratios of [amine (n)]:[phosphate (p)+carboxylate (c)], or of c:p in the instance of poly(D-glutamic acid) (PDGA; SEQ ID NO: 2) addition. Addition of linear poly(ethylenimine) (PEI, 25 kDa) was compared to addition of poly(L-arginine) (PLR, 29 kDa; SEQ ID NO: 1) independently, as well as in conjunction with a H3K4(Me3) histone tail peptide (HTP; SEQ ID NO: 3), in order to quantify similar complexation behaviors between the two polymers as part of a binary complex (i.e., PEI+DNA or PEI+DNA) or ternary complexes (HTP+PEI+DNA or HTP+PLR+DNA). Where a cationic polymer and cationic polypeptide were both present, the relative molar ratio of each component was 60%:40%, respectively. A Zeiss filter and spectrophotometer were used to excite EtBr-tagged DNA at 510 nm for an emission at 595 nm, and results were compared amongst various formulations with unbound EtBr as a negative control.
After complexing PLR-HTP-DNA, PEI-HTP-DNA, PLR-DNA and PEI-DNA polyplexes and determining that PDGA possesses no ability to cause complexation of polynucleotides, PDGA's influence on formation kinetics was established by comparison of [5.5(positive)]:[1(negative)] and [10(positive)]:[1(negative)] molar ratios of [amine (n)]:[phosphate (p)] and [amine (n)]:[phosphate (p)+carboxylate (c)] on complexation efficiencies in order to determine effects of excess cationic and equalized charge ratios on nanoparticle complexation. Inclusion of carboxylate groups from PDGA was expected to have effects on overall formation kinetics comparable to inclusion of phosphate groups from DNA. Relative fluorescence was compared to DNA without addition of polymers or polypeptides or EtBr in the absence of DNA as controls.
Effects of including a cationic polymer and cationic polypeptide on polyplex destabilization were also determined, as shown in
Dynamic light scattering (BRAND) was used to determine the hydrodynamic radii of nanoparticles at various stages of formation. Nanoparticles containing core polyplexes with plasmid DNA, PLR, PDGA, and HTP, at a molar ratio of [amide]:[(phosphate)] of 5.5:1 were complexed as described. Some polyplex cores were further coated with silica as described. And some silica-coated polyplexes were further layered with cationic polymer (SEQ ID NO: 4) as described. 30-60 minutes of measurements were obtained following initial core formation of ternary complexes, silica coating of cores, and cationic polymer-coating of silica-coated cores.
Cellular uptake of nanoparticles was also determined. Fluorescein isothiocyanate (FITC) was covalently conjugated to amines of PEI (25 kDa linear) and PLR (29 kDa) such that the molar ratio of amines to FITC was 100:1. The reaction was performed in darkness at room temperature for four hours in equal volumes of water and DMSO. In order to establish conjugation, a 0.05% w/v 500 uL solution of each fluorescently modified polymer was centrifuged in a 10 kDa Nanosep® filter and the eluate's fluorescence intensity (485 ex./520 em.) was compared to the unfiltered polymer solution as well as water. mCherry plasmid (Addgene) was included in nanoparticles to permit fluorescent detection of plasmid-driven expression.
MC3T3 murine osteoblasts were cultured on polystyrene T-75 tissue culture plastic flasks (Corning, Calif., USA). Dulbecco's modified eagle medium supplemented with 10% Fetal Bovine Serum (Thermo Fisher Scientific, VA, USA) was used for osteoblasts along with 1% penicillin/streptomycin (Invitrogen, NY, USA). Xylenol orange was added to the cell culture media from day 15 to day 25 after initiation of cell culture. At day 25 cells were fixed and assayed for mineralization. For mCherry plasmid delivery using FITC-modified nanoparticles, osteoblasts were plated at 1000 cells/well in 96-well plates and allowed to adhere for 12-16 hours in antibiotic-free DMEM containing 10% FBS. Immediately before transfection, medium was replaced with equal volumes of OptiMEM-suspended nanoparticles and DMEM containing 10% FBS.
All complexes were FITC-labeled and subjected to qualitative observation of fluorescence intensity (488/520 ex./em.) before transfection. 96-well-plated osteoblasts (1000 cells/well) were transfected with 200 ng of plasmids in triplicates for each binary (plasmid and cationic polymer), ternary (plasmid and cationic polymer, plus anionic polymer or cationic polypeptide), and quaternary (plasmid, cationic polymer, anionic polymer, and cationic polypeptide) complex as well as its silica-coated counterpart, with 1 control and 8 experimental sets (n=3) in total. 5% serum was used in order to study effects of serum on extracellular properties of aggregation.
At 30-hours post-transfection, bimodal fluorescent imaging allowed for simultaneous observation of FITC-labeled nanoparticles (488 ex./520 em.) and the mCherry gene expression that they were responsible for (633 ex./680 em.). A minimum of 20 cells were observed at different locations in each well and representative images were obtained. ImageJ was used to process the overlaid images and combine phase-contrast, 488/520 and 633/680 channels.
Photomicrographs demonstrating cellular uptake are shown in
Further coating of silica-coated nanoparticles (DNA-HTP-PDGA-PLR+Si) with poly(arginine) (SEQ ID NO: 4) causes nanoparticles to be stable in serum and causes extended residence of nanoparticle payload within cells.
Layering silica-coated polyplex cores with polymers specifically directed to bind to particular cell types can further enhance uptake. Associating ligands for cellular receptors with the surface of a nanoparticle can enhance affinity of the nanoparticle for cells that express such receptors and increase transfection of such cells. As one example in accordance with the present invention, silica-coated polyplexes were coated with VEGF (SEQ ID NO: 5), a high-affinity ligand for VEGF receptors, which are expressed at high levels by human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with silica-coated FITC-conjugated polyplexes with poly(L-arginine) (SEQ ID NO: 4) or human VEGF (SEQ ID NO: 5) attached to the outer surface of the silica coating for 40 min before being washed twice with PBS then resuspended in DMEM (10% FBS). Cells were imaged 4 hrs later. After this short incubation period, only low levels of transfection with nanoparticles containing a poly(L-arginine) layer attached to the external silica surface (
In another aspect of the invention, a polynucleotide encoding a nuclease may be incorporated into the nanoparticle polyplex core. As one nonlimiting example, a polynucleotide that encodes and drives expression of a TALEN (Transcription Factor-Like Effector Nucleases) may be included in the nanoparticle. Like Zinc Finger Nucleases, TALENs utilize a modular DNA binding motif (TALE) that can be modified to introduce new DNA binding specificities and even nucleases (TALEN). TALEs consist of multiple repeat variable diresidues (RVDs) which each specify binding to a single nucleotide. TALE arrays are made by stringing together RVDs in a specific order to provide specificity and binding affinity to desired DNA sequences. Commonly, these genome-splicing tools are engineered by fusing non-specific cleavage domains, such as FokI nucleases, to TALEs. TALEN assembly protocols are available that allow assembly of these repetitive sequences, including an open source assembly method known as Golden Gate.
In another aspect of the present invention, nanoparticles may be designed and used in a manner to regulate expression of signaling molecules to alter cellular function. For example, sequences of chromosomal DNA may be deleted or altered to generate cellular or animal models of disease states or treatments therefor, or to treat disease states or otherwise enhance human health. One nonlimiting example of a protein whose expression may be modified in accordance with the present invention is sclerostin (SOST). SOST binding to the LRPS/6 receptor inhibits Wnt signaling, perhaps via feedback systems between Wnt3A, Wnt7B, Wnt10A, sclerostin, β-catenin, LEF1, and TCF1. Desuppressing these cascades via removal of sclerostin may result in significantly increased mineralization activity.
Osteoprogenitor (OPG) and RANKL are also expected to play a responsive role to SOST deletion, where RANKL expresses itself as a receptor for promoting osteoclastogenesis via osteoclast-linked RANK or ODF (osteoclast differentiation factor) binding, and OPG binds antagonistically to RANKL. Thus, the ratio between OPG and RANKL is a determinant of the relationship between bone formation and resorption. However, single cultures of osteoblasts will communicate through other forms of paracrine signaling and this ratio should be more reflective of behavior of altered cells in co-culture with osteoclasts or in vivo.
In another aspect of the present invention, a nanoparticle may be designed so as to allow transfection with a TALEN that may disrupt expression of SOST and consequently generate a high bone-mass phenotype. As one example, TALENS may be engineered to specifically bind to loci in the SOST gene and create double-stranded breaks in the genome to disrupt transcription or translation and reduce SOST expression. As a further example, a nanoparticle may contain plasmids that encode two TALENs that create double-stranded breaks on either side of the chromosomal locus of the start codon for SOST. Repair of endogenous genomic DNA following excision of the sequence encoding the start codon may result in transcription of sclerostin mRNA lacking the start codon that cannot be properly translated into SOST protein, thereby driving down SOST expression and activity. A diagrammatic representation of this model is shown in
A nanoparticle may also comprise other TALEN sequences, targeting SOST or any other gene of interest, and also may comprise other expression vectors, in accordance with the present invention. A nanoparticle may comprise other types of polynucleotides or analogs thereof, such as species of RNA or DNA including mRNA, siRNA, miRNA, aptamers, shRNA, AAV-derived nucleic acids, morpholeno RNA, peptoid and peptide nucleic acids, cDNA, DNA origami, DNA and RNA with synthetic nucleotides, DNA and RNA with predefined secondary structures, CRISPR sequences, and multimers and oligomers, and any combination of the foregoing, in accordance with the present invention. In another example, a nanoparticle may comprise polynucleotides whose sequence may encode other products such as any protein or polypeptide whose expression is desired. A skilled artisan would recognize that the foregoing examples are in accordance with the present invention and may be encompassed by claims thereto.
Following transfection of MC3T3 murine osteoblasts with nanoparticles designed to knock down SOST expression in accordance with the present invention, ELISA and quantitative real-time PCR (qPCR) assays were performed on cell lysate and supernatant fractions.
qPCR was also performed to determine whether down-regulation of SOST expression with nanoparticles in accordance with the present invention may have downstream effects on other components of the relevant signaling cascade. Cells were transfected as described above. Results on expression of numerous components of the signaling pathway (SOST, β-catenin, TCF1, LEF1, Wnt3A, Wnt7B, Wnt10b, OPG, and RANKL), at 5, 14, and 21 days after transfection with different amounts of nanoparticles as indicated, are shown in
TCF/LEF-1-mediated transcription may also be upregulated following knockdown of SOST expression in accordance with the present invention. MC3T3-E1 cells were transfected with TOPflash and control FOPflash luciferase reporter plasmid constructs (Addgene#12456 and 12457) that contain TCF/LEF-1 binding sites. The cells were plated at the density of 5000 cells/well of the 8-well labtek chamber slides and transfected with 1 ug of TOPflash and FOPflash plasmid separately. To control for the efficiency of transfection a control plasmid Renilla (Promega) was used.
Knockdown of SOST expression in accordance with the present invention may also increase mineralization in stromal bone marrow cells and osteoblasts. Mineralization was quantified by two separate methods, first based on image thresholding of xylenol-orange-labeled vital cultures using MATLAB (Mathworks, Natick, Mass.), and second by atomic absorption spectroscopy (AAS). For the xylenol orange threshold, images of both phase and fluorescence (with Texas Red Filter Set) were taken in five adjacent regions of wells, and then stitched into a larger 8-bit image (4×, Nikon Ti-100). The phase channel was subtracted from the fluorescence, and a threshold was set to half the level between the background and signal (−6 dB). The number of pixels above the threshold were counted and used to express the percentage of mineralized area in each well. The combination of phase and fluorescence allowed for unbound xylenol orange to be distinguished, whereas the use of decibel levels allowed for correction of the varied background levels in each image.
Mineralization was also quantified by atomic absorption with an atomic absorption spectrometer (AA-Perkin Elmer, Mass.). Each well was prepared by adding 0.5 mL of 10% nitric acid, and the resultant calcium content was measured relative to a standard curve and compared between groups. Care was taken to minimize interference due to ionized calcium precipitating with phosphate phases, so a large excess of potassium and lanthanum ions was added to each well.
While several aspects of the present invention have been described and depicted herein, alternative aspects may be effected by those skilled in the art to accomplish the same objectives. Accordingly, it is intended by the appended claims to cover all such alternative aspects as fall within the true spirit and scope of the invention.
1. A nanoparticle comprising:
- a core polyplex and a silica coating thereon;
- wherein said core polyplex comprises an anionic polymer, a cationic polymer, a cationic polypeptide, and a polynucleotide.
2. The nanoparticle of claim 1 wherein the anionic polymer is poly(D-glutamic acid).
3. The nanoparticle of claim 1 wherein the cationic polymer is selected from the group consisting of poly(ethylenimine) and poly(L-arginine).
4. The nanoparticle of claim 1 wherein the cationic polypeptide is a histone tail peptide.
5. The nanoparticle of claim 4 wherein the histone tail peptide is human H3 histone tail peptide.
6. The nanoparticle of claim 1 wherein the anionic polymer is poly(D-glutamic acid), the cationic polymer is selected from the group consisting of poly(ethylenimine) and poly(L-arginine), and the cationic polypeptide is a histone tail peptide.
7. The nanoparticle of claim 6 wherein the polynucleotide comprises a nucleotide sequence that encodes a nuclease.
8. The nanoparticle of claim 7 wherein the nuclease is a TALEN.
9. The nanoparticle of claim 8 wherein the TALEN is capable of inducing a break at a site-specific locus of DNA, wherein the break results in a change of expression of a protein encoded by a gene.
10. The nanoparticle of claim 9 wherein the change is a decrease and the gene encodes a sclerostin protein.
11. A nanoparticle of claim 6, further comprising a polymer attached to an outer surface of said silica coating.
12. A nanoparticle of claim 11, wherein said polymer attached to an outer surface of said silica coating comprises poly(L-arginine) or a vasoactive endothelial growth factor peptide.
13. A method of modifying intracellular polynucleotides comprising;
- contacting a cell with a nanoparticle, wherein said nanoparticle comprises a core polyplex and a silica coating thereon;
- wherein said core polyplex comprises an anionic polymer, a cationic polymer, a cationic polypeptide, and a polynucleotide.
14. The method of claim 13 wherein the anionic polymer is poly(D-glutamic acid).
15. The method of claim 13 wherein the cationic polymer is selected from the group consisting of poly(ethylenimine) and poly(L-arginine).
16. The method of claim 13 wherein the cationic polypeptide is a histone tail peptide.
17. The method of claim 16 wherein the histone tail peptide is human H3 histone tail peptide.
18. The method of claim 13 wherein the anionic polymer is poly(D-glutamic acid), the cationic polymer is selected from the group consisting of poly(ethylenimine) and poly(L-arginine), and the cationic polypeptide is a histone tail peptide.
19. The method of claim 18 wherein the polynucleotide comprises a nucleotide sequence that encodes a nuclease.
20. The method of claim 19 wherein the nuclease is a TALEN.
21. The method of claim 20 wherein the TALEN is capable of inducing a break at a site-specific locus of DNA, wherein the break results in a change of expression of a protein encoded by a gene.
22. The method of claim 21 wherein the change is a decrease and the gene encodes a sclerostin protein.
23. The method of claim 18, further comprising a polymer attached to an outer surface of said silica coating.
24. The method of claim 23, wherein said polymer attached to an outer surface of said silica coating comprises poly(L-arginine) or a vasoactive endothelial growth factor peptide.