Phosphorylated Tri-Block Copolymers with Anti-Microbial Properties
The disclosure provides products and methods of treating diseases and disorders involving microbial pathogens, such as intestinal microbial pathogens, e.g., Pseudomonas aeruginosa, by administering an effective amount of a phosphorylated polyethylene glycol compound of defined structural organization. Those diseases and disorders characterized by an epithelium attacked by a microbial pathogen are contemplated, including gastrointestinal infections and inflammation, e.g., treatment of intestinal or esophageal anastomosis or treatment or suppression of anastomotic leakage.
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This invention was made with government support under Grant No. R01GM062344-15 awarded by the National Institutes of Health and under Contract No. DE-AC002-06CH11357 awarded by the U.S. Department of Energy. The government has certain rights in the invention.INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
This application contains, as a separate part of the disclosure, a Sequence Listing in computer-readable form which is incorporated by reference in its entirety and identified as follows: Filename: 51283A_Seqlisting.txt; 781 bytes, created Sep. 25, 2017.FIELD
The disclosure relates generally to the fields of medical treatment, prevention or suppression of epithelial diseases and disorders including infections and inflammation.BACKGROUND
The promiscuous use of antibiotics had led to the emergence of antibiotic resistance at an unprecedented pace and continues to place patients at risk for life-threatening infections following major surgery. Many if not most of the pathogens that cause these infections use the intestinal tract as their primary site of colonization. Although surgeons routinely decontaminate the intestinal tract with antibiotics prior to surgery to prevent infection, this practice carries the unintended consequence of causing antibiotic resistance. Furthermore, overuse of antibiotics destroys the microbiome that normally protects against high risk pathogens. A more evolutionarily stable strategy to this problem would be to develop compounds that suppress pathogen virulence without affecting the growth of the pathogenic organisms, thereby preserving the microbiome. In this manner, bacterial pathogenicity could be contained and the colonization resistance of the normally protective microbiota preserved.
Phosphate is a key universal cue for bacteria to either enhance their virulence, for example when local phosphate is scarce, or to suppress virulence when phosphate is abundant. Phosphate can become depleted in the mammalian gut following physiologic stress and serves as a major trigger for colonizing bacteria to express virulence and invasiveness. This process cannot be reversed with oral or intravenous inorganic phosphate, as phosphate is nearly completely absorbed in the proximal small intestine.
One obstacle to using commercially available PEG 15-20 (Sigma) is that PEG 15-20 is not a pure tri-block polymer but rather a mixture of polymers of varying molecular weights. This situation limits the ability to further interrogate the molecular mechanisms by which the polymers protect both in vitro and in vivo, and the limited content of phosphate inhibits the opportunity to further improve the efficacy of the polymers.
In view of the foregoing observations, it is apparent that needs continue to exist in the art for products and methods useful in treating, preventing or suppressing diseases and disorders characterized by a pathogenic microbial attack on an epithelium.SUMMARY
The disclosure provides materials and methods for treating, preventing or suppressing diseases and conditions associated with pathogenic microbe-mediated epithelial diseases or disorders such as gastrointestinal infections or inflammation, or gastrointestinal anastomoses or anastomotic leaks, such as esophageal or intestinal anastomoses or anastomotic leaks. The materials for use in such circumstances are phosphorylated polyethylene glycol compounds of a defined structure, such as an A-B-A triblock copolymer structure (ABA-PEG-Pi). Notably, the ABA-PEG-Pi materials of the disclosure comprise a hydrophobic core such as a diphenylmethyl moiety and the materials exhibit a substantially similar molecular weight wherein about 80%, 90%, 95%, 96%, 97% 98%, 99%, 99.5% or 99.9% of the ABA-PEG-Pi molecules have the same molecular weight (plus or minus 5% or 10%). The particular structure of the ABA-PEG-Pi and the relatively constant structure result in effects on epithelial cell diseases and disorders mediated by obligate or opportunistic microbial pathogens.
One aspect of the disclosure is drawn to a triblock copolymer comprising: (a) a hydrophobic core; and (b) at least two polyethylene glycol chains wherein at least one polyethylene glycol chain is a phosphorylated polyethylene glycol comprising more than two phosphate groups. The hydrophobic core is a “B” block copolymer and the PEG chains are generally considered “A” block copolymers using the triblock copolymer terminology of the disclosure. It is recognized that triblock copolymers can be indicated as having an A-B-A or an A-B-A′ triblock copolymer structure depending on whether the at least two PEG chains are the same or not. In some embodiments, at least two polyethylene glycol chains are phosphorylated polyethylene glycol chains comprising more than two phosphate groups. In some embodiments, the hydrophobic core is a carbocyclic or heterocyclic ring, including embodiments wherein the ring is aromatic, such as a single ring or a plurality of rings. In some embodiments, the hydrophobic core is a diphenylmethyl moiety. In some embodiments, the hydrophobic core is a 4,4′-(propane-2,2-diyl)diphenolate salt. In some embodiments, the copolymer has a molecular weight of at least 8,000 daltons, at least 12,000 daltons, at least 15,000 daltons, at least 16,000 daltons, at least 20,000 daltons, or is between 15,000-20,000 daltons. In some embodiments, the copolymer is in solution. In some embodiments, the dispersity (Ð) of the triblock copolymer disclosed herein is less than or equal to 1.10. In some embodiments, the triblock copolymer is a phosphorylated form of ABA-PEG-PGly or ABA-PEG-PEEGE.
Another aspect of the disclosure is directed to a method of producing the triblock copolymer comprising (a) covalently attaching at least two polyethylene glycol chains to a hydrophobic core comprising a carbocyclic or heterocyclic ring; and (b) covalently attaching at least two phosphate groups to at least one polyethylene glycol chain. In some embodiments, at least two polyethylene glycol chains are each covalently attached to at least two phosphate groups.
Yet another aspect of the disclosure is a method of treating anastomosis comprising administering a therapeutically effective amount of a composition comprising a triblock copolymer disclosed herein to a subject in need. In some embodiments, the triblock copolymer has a molecular weight of at least 8,000 daltons, 12,000 daltons, 15,000 daltons, 16,000 daltons, 20,000 daltons, or between 15,000-20,000 daltons.
Still another aspect of the disclosure is a method of treating anastomotic leakage comprising administering a therapeutically effective amount of a composition comprising a triblock copolymer disclosed herein to a subject in need. In some embodiments, the triblock copolymer has a molecular weight of at least 8,000 daltons, 12,000 daltons, 15,000 daltons, 16,000 daltons, 20,000 daltons, or between 15,000-20,000 daltons.
In another aspect, the disclosure provides a method of preventing anastomotic leakage comprising administering an effective amount of a composition comprising a triblock copolymer disclosed herein to a subject at risk of anastomotic leakage. In some embodiments, the triblock copolymer has a molecular weight of at least 8,000 daltons, 12,000 daltons, 15,000 daltons, 16,000 daltons, 20,000 daltons, or between 15,000-20,000 daltons.
Yet another aspect of the disclosure is directed to a method of suppressing anastomotic leakage comprising administering an effective amount of a composition comprising a triblock copolymer disclosed herein to a subject at risk of anastomotic leakage. In some embodiments, the triblock copolymer has a molecular weight of at least 8,000 daltons, 12,000 daltons, 15,000 daltons, 16,000 daltons, 20,000 daltons, or between 15,000-20,000 daltons.
Another aspect of the disclosure is drawn to a method of inhibiting, e.g., preventing, microbial virulence, such as the virulence of Pseudomonas aeruginosa or the virulence of Enterococcus faecalis. Virulence is herein defined as the relative ability of a microorganism, such as P. aeruginosa or E. faecalis, to cause disease, which is a measure of the degree of pathogenicity. Stated alternatively, virulence reflects the capability of a microorganism to cause disease. Virulence in P. aeruginosa can be characterized by the P2 (or virulent) phenotype, wherein P. aeruginosa one or more of the following features: produces toxin(s), exhibits swarming motility, activates expression of the quorum-sensing system (QS), has elevated expression of PstS, and/or increases production of pyocyanin and/or pyoverdin. Virulence in E. faecalis can be characterized by increased collagenase production. Accordingly, the disclosure provides a method of inhibiting microbial virulence in the gastrointestinal tract comprising administering an effective amount of a composition comprising a triblock copolymer as disclosed herein to a subject at risk of inducing microbial virulence. In some embodiments, the triblock copolymer has a molecular weight of at least 8,000 daltons, 12,000 daltons, 15,000 daltons, 16,000 daltons, 20,000 daltons, or between 15,000-20,000 daltons. In some embodiments, the microbe is Pseudomonas aeruginosa. In some embodiments, the microbe is Enterococcus faecalis.
The methods of inhibiting microbial virulence are related to another aspect of the disclosure providing a method of treating a gastrointestinal microbe capable of developing a virulent phenotype comprising administering an effective amount of a composition comprising a triblock copolymer as disclosed herein to a subject comprising the gastrointestinal microbe. In some embodiments, the triblock copolymer has a molecular weight of at least 8,000 daltons, 12,000 daltons, 15,000 daltons, 16,000 daltons, 20,000 daltons, or between 15,000-20,000 daltons. In some embodiments, the gastrointestinal microbe capable of developing a virulent phenotype is Pseudomonas aeruginosa. In some embodiments, the gastrointestinal microbe capable of developing a virulent phenotype is Enterococcus faecalis.
Other features and advantages of the disclosure will become apparent from the following detailed description, including the drawing. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments, are provided for illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from the detailed description.
A phosphorylated polyethylene glycol compound of high molecular weight allows phosphate to be distributed along the entire gut and into the distal intestine where microbes such as bacteria are most abundant. The phosphorylated polyethylene glycol compounds of the disclosure have a triblock copolymer structure of ABA, with “A” referring to any polyethylene glycol, or derivative thereof, that is at least 8,000 daltons, 12,000 daltons, 15,000 daltons, 16,000 daltons, 20,000 daltons, or is between 15,000-20,000 daltons. The “B” component of the triblock structure is a hydrophobic compound capable of covalent linkage to two PEG molecules of the disclosure, or derivatives thereof. Exemplary hydrophobic cores are bisphenol A (BPA) and bisphenol E (BPE).
In discussing the compounds of the disclosure, and compositions comprising such compounds, the following terminology is used. “ABA” refers to the triblock structural organization of the compounds, with two like polymers, e.g., PEG, bracketing a “B” structure that is a hydrophobic core such as any aliphatic, carbocyclic, heterocyclic, or aromatic structure that is hydrophobic, e.g. any of the bisphenols. “PEG” refers to polyethylene glycol, and “PEG-Pi” refers to a phosphorylated polyethylene glycol. “EEGE” is ethoxyethyl glycidyl ether and “PEEGE” is polyethoxyethyl glycidyl ether. As described below, EEGE is de-protected and, once de-protected, EEGE groups become hydroxy groups and the structure is referred to as a polyglycidol, such as ABA-PEG-PGly. Compounds identified as ABA-PEG10k-E8, ABA-PEG16k-E12, and ABA-PEG20k-E18 refer to triblock copolymers having the ABA structure with 8 EEGE groups (E8) and PEG groups of 10 k in ABA-PEG8k-E8. For ABA-PEG16k-E12, the compound has the ABA structure with 12 EEGE groups (E12) and PEG groups of 16 k. In like manner, ABA-PEG20k-E18 has an ABA structure with 18 EEGE groups and PEG groups of 20 k. For compounds identified as ABA-PEG10k-G10, ABA-PEG16k-G14 and ABA-PEG20k-G20, “G10” refers to 10 hydroxyl groups created by de-protection of EEGE (the “G” is a reference to the compound as a polyglycidol), while “G14” and “G20” refer to 14 and 20 hydroxyl groups, respectively. Compounds defined as ABA-PEG10k-Pi10, ABA-PEG16k-Pi14, and ABA-PEG20k-Pi20 refer to compounds having the ABA triblock copolymer structure with 10, 14, or 20 phosphoryl groups (e.g., phosphate groups), respectively, resulting from phosphorylation of a polyglycidol. Consistent with the naming convention explained above, PEG10k, PEG16k, and PEG20k refer to PEG groups of 10 k, 16 k and 20 k, respectively. It is apparent that the number of functional groups (e.g., EEGE) ultimately rendered amenable to phosphorylation can vary in the compounds according to the disclosure, and the size of PEG molecules bearing those functional groups can vary, including PEG molecules in a compound totaling at least 8,000 daltons, at least 12,000 daltons, at least 15,000 daltons, at least 16,000 daltons, at least 20,000 daltons or between 15,000-20,000 daltons.
The phosphate content of compounds delivered to the intestine is particularly important for any protective effect because local phosphate concentrations support bacterial growth while at the same time suppressing bacterial virulence . The mechanism underlying this effect involves phosphosensory/phosphoregulatory circuits that are a universal feature of most bacteria and play a key role in virulence .
Analysis of PEG15-20 (Sigma) showed that it was not a pure tri-block polymer but rather a mixture of polymers of varying molecular weights including ABA triblock, AB diblock and homopolymer poly(ethylene glycol) structures (
The disclosure will be more fully understood by reference to the following examples, which detail exemplary embodiments of the disclosure. The examples should not, however, be construed as limiting the scope of the disclosure.EXAMPLES Example 1
Materials and Methods.
Materials. Bisphenol A (BPA, >99%, Aldrich), naphthalene (99%, Aldrich), diphenylmethane (99%, Aldrich), and phosphorus oxychloride (POCl3, 99%, Aldrich) were used as received. Ethylene oxide (EO, >99%, lecture bottle, Praxair) and Ethoxyethyl glycidyl ether (EEGE, 97%, Synthonix) were treated with di-n-butylmagnesium for 20 minutes, and distilled into Schlenk flasks before use. Tetrahydrofuran (THF, HPLC, inhibitor free, Aldrich) was purified with solvent purification system (Mbraun SPS-800) and distilled from a sodium naphthalenide solution directly before use. Diphenylmethylpotassium (DPMK) was prepared as described. Initially, a potassium naphthalenide solution was prepared in dry THF with 1:4 molar ratio of naphthalene to potassium. After stirring for 12 hours, 0.66 mole equivalents of diphenylmethane were introduced into the solution via syringe, and the solution was allowed to stir at room temperature for at least 12 hours prior to use.
Synthesis of phosphorylated PEG-based block copolymers with a hydrophobic core. Sequential Anionic Polymerization of ABA-PEG-PEEGE. A series of ABA-PEG-PEEGE were synthesized by the sequential anionic polymerization of EO and EEGE in a custom heavy-wall glass reaction flask on a Schlenk line. In a typical reaction, BPA (251 mg, 1.1 mmol) dissolved in 120 mL anhydrous THF at 0° C. under dry nitrogen atmosphere, was titrated with DPMK to form the initiator, followed by addition of the first monomer EO (22.0 g, 500 mmol). After stirring for 1 hour, the mixture was heated to 50° C. and reacted for 3 days to attain complete conversion of the EO monomer. Then, the second monomer EEGE (2.9 g, 19.8 mmol) was injected into the flask and allowed to react for another 3 days. The polymerization was terminated with methanol and the polymer was recovered by precipitation in cold diethyl ether. Different chain lengths of EO and EEGE are adjusted by the feed ratio of [EO]/[Initiator] and [EEGE]/[Initiator].
Hydrolysis of ABA-PEG-PEEGE. Hydrolysis of EEGE segments of block copolymer was carried out in THF with 4 wt % of HCl and stirred at room temperature for 30 minutes. The polymers were then purified by precipitating in cold hexane and finally dried under vacuum at 60° C. to get a yellowy wax-like product, i.e., ABA-PEG-PGly (PGly: Poly glycerol). The disappearance of peaks at 4.70 ppm (q, 1H), 1.29 ppm (d, 3H), 1.19 ppm (t, 3H) in 1H-NMR confirmed the success of de-protection.
Phosphorylation of ABA-PEG-PGly. ABA-PEG-Pis (Pi: Poly phosphoric acid) were prepared by phosphorylation of ABA-PEG-PGly, which was performed in a flame-dried flask under dry nitrogen atmosphere. ABA-PEG-PGly was dissolved in anhydrous THF at 50° C., and a ten-fold equivalent molar amount of POCl3 was added at once via gas-tight syringe. The solution was stirred under nitrogen pressure for 3 hours, and then quenched by the addition of a small amount of water. After evaporation of THF and dialysis against Milli-Q water, the sample was lyophilized to give a white flocculent product. 31P-NMR (D20): 6-0.18 ppm.
Synthesis of phosphorylated PEG-based block copolymers without a hydrophobic core (PEG-Pi). The synthetic strategy of PEG-Pi is quite similar to that of ABA-PEG-Pi, with the exception that the polymerization started with hydrophilic ethylene glycol instead of hydrophobic BPA, as shown in Scheme 2. First, PEG-PEEGE was prepared by starting with ethylene glycol. Next, polymerizing through the sequential adding of EO and EEGE and then using the same hydrolysis process (as used to obtain PEG-PGly) and the same phosphorylation process, PEG-Pi was obtained.
Characterization of block copolymers. 1H and 31P-NMR spectra were obtained at a Bruker Ultrashield Plus 500 MHz spectrometer and referenced internally to solvent proton signal. Apparent molecular weights and dispersity (D) were characterized with a gel permeation chromatography (GPC) system equipped with a Waters 1515 pump, a Wyatt Optilab T-rEX differential refractive index (RI) detector, and a Waters 2998 photodiode array (PDA) detector. For ABA-PEG-PEEGEs, ABA-PEG-PGlys, PEG-PEEGEs and PEG-PGlys, THF was used for elution at 35° C. with an elution rate of 0.8 mL/minute. Three Waters Styragel columns were used and calibrated by polystyrene standards (Aldrich). ABA-PEG-Pis and PEG-Pis were measured in 0.1 M NaNO3 (aq) at 25° C. with an elution rate of 1.0 mL/minute on the same setup, except three Waters Ultrahydrogel columns in series were used and calibrated by PEO standards (Aldrich). Regarding dispersity of a polymer, monodispersed means the polymer itself, meaning all the polymer chains have nearly identical chain lengths. For amphiphilic block copolymers in selective solvent, for example ABA-PEG-Pi in aqueous solutions, water is a good solvent for a hydrophilic block, but a poor solvent for a hydrophobic block. In this situation, a block copolymer can self-assemble into aggregated structures, such as micelles, vesicles, and the like. Sometimes, micelles/vesicles and much larger structures further aggregated from micelles/vesicles can co-exist in the same solution.
Even for block copolymers with very narrow dispersity, under some circumstances, the block copolymers can form non-uniform structures in solutions. Even when a block copolymer forms uniform aggregated structures in solution, these structures can be monodispersed, meaning all the aggregates have nearly identical size and shape.
Biological tests. Bacterial strains. Pseudomonas aeruginosa strains MPAO1-P1 and MPAO1-P2  were used in all experiments. The MPAO1-P1 strain and its derivative mutant ΔPvdD were used to create the reporter constructs, MPAO1-P1/pstS-EGFP and ΔPvdD/pstS-EGFP.
Construction of pSensor-PstS-EGFP. The promoter region of the pstS gene (P. aeruginosa MPAO1) was cloned in a pSensor vector. As noted in Example 4, PstS is the phosphate-binding component of the ABC-type transporter complex pstSACB involved in phosphate transport into the bacterial cytoplasm. The pSensor consists of a pUCP24 vector backbone and Gateway C.1 cassette (Invitrogen) in frame with the EGFP reporter gene (derived from pBI-EGFP) cloned into the Sma1 and Pst1/Hind III sites of the pUCP24 MCS region, respectively. The region upstream of pstS was amplified by PCR (Platinum PCR SuperMix (Invitrogen) using primers PstS_F: CACCTATCCCAAAACCCCTGGTCA (SEQ ID NO:1) and PstS_R: CAAACGCTTGAGTTTCATGCCTTG (SEQ ID NO:2), and cloned into the Gateway entry vector (pCR8/GW/Topo kit (Invitrogen)). Nucleotide sequence and orientation of the inserts were confirmed by sequencing, inserts were transferred into pSensor vector via LR reaction using Gateway LR Clonase II Enzyme Mix (Invitrogen). Throughout the study, vector constructs were propagated in One Shot TOP10 Chemically Competent E. coli cells. Gentamycin (100 μg/ml) selection was used for pUCP24 and pSensor and Ampicillin (100 μg/ml) for pBI-EGFP vectors. The QIAGEN Plasmid Mini Kit (Qiagen) was used for plasmid DNA extraction.
PstS expression. P. aeruginosa MPAO1-P1/pstS-EGFP or ΔPvdD/pstS-EGFP were grown on tryptic soy agar plates supplemented with 100 μg/ml gentamicin (Gm100) overnight. A few colonies from the overnight plates were used to inoculate liquid TSB+Gm100 for overnight growth. The overnight culture was used to inoculate fresh TSB+Gm100 at 1:100 dilution and grown to OD600 nm=0.5. Cells were pelleted by centrifugation at 3300×g for 5 minutes, and washed twice with defined citrate media (DCM: sodium citrate, 4.0 g/L (Sigma, S4641), (NH4)2SO4, 1.0 g/L (Sigma, A4915), MgSO4·7H2O, 0.2 g/L (Fisher, M63-50). DCM medium is limited in both phosphate and iron. Potassium phosphate buffer, pH 6.0 (PPB), was used for phosphate supplementation. The supplementation of DCM with PPB 0.1 mM was defined for phosphate limitation (DCM-Pi0.1), and with PPB 25 mM for phosphate abundance (DCM-Pi25). Washed cells were resuspended in DCM-Pi0.1+Gm100 or DCM-Pi25+Gm100, respectively, and grown overnight. In experiments carried out to test the phosphorylated polymers, bacterial cells were washed in DCM-Pi0.1 and resuspended in DCM-Pi0.1+Gm100 supplemented with 2 mM ABA-PEG-Pis or ABA-PEG-PGlys and adjusted to pH 6.0 with KOH. After overnight growth, fluorescence (excitation 485/10, emission 528/20) and absorbance (600 nm) were measured with a FLx800 fluorescent reader (Biotek Instruments). Fluorescence readings were normalized to absorbance. Culture conditions were 37° C. with shaking at 180 rpm (C25 Incubator Shaker, New Brunswick Scientific, Edison, N.J.).
Pyocyanin production during low-phosphate conditions. P. aeruginosa MPAO1-P2, which is known to produce higher amounts of pyocyanin than MPAO1-P1 , was used in this set of experiments. The design of the experiments was similar to the experiments described above for PstS expression except they were performed in the absence of Gm in the DCM media. 2 μM Fe3+ (1 μM Fe2(SO4)3) was added to the media in order to enhance the production of pyocyanin. Pyocyanin was extracted by chloroform followed by re-extraction in the 0.2N HCl and measured at OD520 nm as previously described . Before extraction, cell density was measured by the absorbance at 600 nm, and pyocyanin values were normalized to bacterial cell density.
Pyocyanin production following exposure to virulence activating factor U-50,488, kappa opioid agonist. P. aeruginosa was known to be triggered to express enhanced virulence when exposed to kappa opioid, host factors known to be released into the gut during physiologic stress . P. aeruginosa MPAO1-P1, which is highly sensitive to U-50,488, was used in these experiments.
MPAO1-P1 was grown on tryptic soy agar plates overnight, and a few colonies were used to inoculate liquid TSB for overnight growth. Overnight cultures were used to inoculate fresh TSB at 1:100 dilution and allowed to grow out for 1 hour. Next, 200 μM U-50,488 (Sigma) was added, and growth was continued for 10 hours. Pyocyanin was extracted and measured as described above.
Pyoverdin production. P. aeruginosa MPAO1-P1 was used in these experiments. The design of the experiments was similar to the experiments described above for PstS expression except they were performed without Gm in the DCM media. Pyoverdin was measured by fluorescence (400/10 excitation, 460/40 emission) using FLx800 fluorescent reader (Biotek Instruments). Data were normalized to cell density measured as absorbance at 600 nm.
Caenorhabditis elegans killing assays. C. elegans N2 nematodes provided by the Caenorhabditis Genetic Center (CGC), University of Minnesota, were used in these experiments. Synchronization and pre-fasting of worms was performed by transferring them onto plain plates with kanamycin as previously described . P. aeruginosa MPAO1-P1 was grown overnight in tryptone/yeast extract medium (TY, tryptone, 10 g/L; yeast extract, 5 g/L) and diluted at 1:100 in 0.1×TY (TY diluted 10-fold with water). Potassium phosphate buffer, pH 6.0, was included in the 0.1×TY to a final concentration of 0.1 mM. After 1 hour of growth, the kappa-opioid receptor agonist U-50,488 was added to a final concentration of 50 μM followed by 2 hours growth as previously described [18, 19]. Two ml of the microbial culture was adjusted to room temperature and poured in the 30-mm-diameter dishes into which pre-fasting nematodes (10 nematodes per plate) were transferred. P. aeruginosa was grown overnight in TY media diluted 1:100 in either 0.1×TY or 0.1×TY containing polymers at 2 mM (or 5% in selected experiments, as indicated) final concentration and adjusted to pH 5.2 with KOH. Plates were incubated at room temperature, without shaking, and mortality was defined if worms did not respond to the touch of a platinum picker.
Statistical analyses. All data are from 3 or more replicates and presented as the mean with standard deviation presented as error bars. Statistical analysis was performed using SigmaPlot software. In C. elegans experiments, Long-rank (Mantiel-Cox) test (GraphPad Prizm 7) was used with significance accepted as a p-value<0.05. In in vitro experiments, Student t-tests were used and significance determined to be p-value<0.05.
Scanning electron microscopy (SEM). P. aeruginosa MPAO1 was grown in tryptic soy broth (TSB) overnight. Overnight cultures (2 ml) were centrifuged at 6,000 rpm, 5 minutes, room temperature, and pellets were gently washed (3 times) with DCM-Pi0.1 (see description of PstS expression, above). Washed pellets were suspended in 1 ml of DCM-Pi0.1 or 2 mM ABA-PEG20k-Pi20 or 2 mM PEG20k-Pi20. ABA-PEG20k-Pi20 and PEG20k-Pi20 solutions were prepared in DCM-Pi0.1. The pH of these phosphorylated polymers is around 2-3, therefore, the pH was adjusted by KOH to DCM-Pi0.1 (pH5.5). Bacteria were grown for 4 hours, then cells were pelleted by centrifugation at 6,000 rpm, 5 minutes, room temperature, and gently washed (3 times) with phosphate-buffered saline (PBS). Bacterial cells were then dropped onto glass coverslips coated with poly-L-lysine. Cells were fixed in 3% glutaraldehyde buffered with 0.1 M phosphate buffer, pH 7.2, washed with 0.1 M phosphate buffer, and dehydrated in a graded ethanol solution in water (30% increased gradually to 100%; 20 minutes each). The samples were dried with a Leica CPD300 critical point dryer and coated with Pt(80)/Pd(20) to a thickness of 2 nm using a Cressington sputter coater, model 208HR. SEM images were obtained using a Zeiss Merlin FE-SEM with an accelerating voltage of 1 kV and a working distance of 3 mm.Example 2
Design and Synthesis of Phosphorylated PEG-Based Block Copolymer with a Hydrophobic Core (ABA-PEG-Pi).
The purpose of this study was to develop phosphate-containing PEG-based block copolymers with a defined ABA structure and molecular weight and to identify their effectiveness in suppressing microbial virulence using biological tests. The ABA structure and phosphate were shown to be involved in providing the biologic function of Pi-PEG 15-20. However, molecular weight measurements (
PEG chains contain only one or two terminal hydroxyl groups suitable for further functionalization. To incorporate more hydroxyl groups per polymer chain, sequential anionic copolymerization of ethylene oxide (EO) with a functional epoxide monomer, ethoxy ethyl glycidyl ether (EEGE), an ethoxyl ethylacetal protected glycidyl was used to acquire block copolymers with polyethylene oxide as backbone, along with controllable hydroxyl groups [20-22]. As depicted in
This strategy allowed access to a series of block copolymers with defined ABA architecture, which consisted of three distinctive segments: (i) B group represents the small, yet very hydrophobic bis-phenol A moiety at the polymer center, (ii) PEG blocks adjacent to the bi-aromatic center formed the inert spacer and the inner part of hydrophilic A groups. As an integral part of the architecture, the chain length of the PEG block played a key role in the hydrophobicity/hydrophilicity balance of the whole polymer, (iii) phosphorylated polyglycidol block acts as the outer part of hydrophilic A groups, offering biological functionality and defined phosphate content.
Three ABA-PEG-Pis, ABA-PEG10k-Pi10, ABA-PEG16k-Pi14 and ABA-PEG20k-Pi20 were synthesized. 10 k, 16 k and 20 k corresponded to the different molecular weight of PEG block. By incorporating more repeating units of phosphate (10, 14 to 20 repeating units in the above phosphorylated HMW PEGs, respectively), almost identical molar concentration of phosphate can be maintained for each block copolymer (e.g., for 1 g of each block copolymer, the molar concentration of phosphate were 0.78, 0.77 and 0.80 mmol, respectively, for ABA-PEG10k-Pi10, ABA-PEG16k-Pi14 and ABA-PEG20k-Pi20). Initiated from bis-phenoxide, the sequential anionic ring-opening polymerization of EO and EEGE was successful. This can be confirmed by the chemical shifts seen in 1H-NMR spectra (
where Ii and Ib are the integration of Peak i and b in
The degree of polymerization of PEG block (NEG) was given by
where Ic˜h is the integration of Peak c˜h in
Nomenclature of the polymers: Take ABA-PEG10k-E8/ABA-PEG10k-G10/ABA-PEG10k-Pi10 as examples. 10 k is the designed molecular weight of PEG block; E8 means the designed repeating units of EEGE block is 8; G10 means the designed repeating units of Glycerol is 10. Because hydrolysis of EEGE block released 8 alcohol groups plus 2 primary alcohol groups at the chain ends, the total is 10 repeating units for Glycerol; Pi10 indicates that the designed repeating units of the phosphorylated Glycerol block is 10. Other polymer designations disclosed herein follow this nomenclature scheme.
a: ABA-PEG-PEEGE and ABA-PEG-PGly samples were measured in THF against PS standards; ABA-PEG-Pi samples were measured in 0.1 M NaNO3 against PEO standards.b: Measured by GPC.
c: Calculated from NMR.
d: Nhydroxyl=NEEGE+2 primary alcohol groups at chain ends, NMR confirmed the complete de-protection of EEGE repeat units.
e: Nphosphate of ABA-PEG-Pi samples were determined by phosphoric acid titration determinations.
EEGE was chosen to be the outer block, due to the advantages that: (i) it has a similar main chain to PEG and can be co-polymerized with EO through an anionic mechanism, (ii) this structural similarity also indicates that PEG-PEEGE should be non-toxic and safe, which is relevant to the use of ABA-PEG-PEEGE in biomedical applications, and (iii) the protective ethoxy ethylacetal groups can be easily removed by acidic hydrolysis, yielding pendant hydroxyl group in each repeating unit, offering perfect functionalization sites for phosphorylation. Complete hydrolysis could be verified by the disappearance of specific EEGE signals i, j and k, comparing
In order to further identify the degree of phosphorylation, phosphoric acid titration experiments were performed to identify the average number of phosphate groups per polymer chain. Briefly, 0.1M of sodium hydroxide (NaOH) solution was titrated into the ABA-PEG-Pi/PEG-Pi solution, and the pH changes were monitored using a pH meter with automatic temperature compensation.
where [NaOH] is the concentration of sodium hydroxide solution, V1 and V2 are the volume of sodium hydroxide solution consumed at firsts titration end and second titration end, respectively. m is the mass of ABA-PEG-Pi polymer used in the titration, and Mn is the number average molecular weight of ABA-PEG-Pi polymer. Theoretically, the volume of NaOH solution consumed at the second titration end (V2) should be twice that found at the first titration end (V1). In the experiment disclosed herein, V2 is a little bit lower than 2V1. Without wishing to be bound by theory, this may be due to the dissociation constant difference between the phosphoric acid units at the chain ends and those far from the chain ends.
Through the above method, the average number of phosphate groups per polymer chain Nphosphate for ABA-PEG10K-Pi10, ABA-PEG16K-Pi14 and ABA-PEG20k-Pi20 were determined to be 9.8±0.2, 13.0±0.8, and 19.5±0.5, respectively. These results confirm that phosphorylation of available hydroxyl groups was complete.Example 3
Synthesis of Phosphorylated PEG-Based Block Copolymer without Hydrophobic Core (PEG-Pi).
In order to demonstrate the structural importance of the hydrophobic moiety, another phosphate-containing PEG-based block copolymer without a hydrophobic core, namely PEG-Pi, was synthesized for comparison. The only structural difference between PEG-Pi and ABA-PEG-Pi is the center moiety (
It is also important to note that, due to the use of a living anionic polymerization technique, the dispersity (D) of all these PEG-based block copolymers were kept narrow (<1.10). Significant broadening of the corresponding GPC traces was not observed even after de-protection and phosphorylation (
ABA-PEG-Pis Inhibit Phosphate Signaling in P. aeruginosa Under Phosphate-Limiting Conditions.
Multiple biological tests were performed to assess the functionality of the synthesized polymers as anti-virulence compounds. Expression of the phosphate transport protein PstS in P. aeruginosa was used as a biomarker to determine phosphate availability of the various polymers. If PstS expression was increased, it served as a proxy indicating that extracellular phosphate was depleted and unavailable within the phosphorylated compound. On the other hand, if PstS was observed to be decreased, it indicated that P. aeruginosa detected sufficient phosphate availability in the test compound. PstS is the phosphate-binding component of the ABC-type transporter complex pstSACB involved in phosphate transport into the bacterial cytoplasm. PstS is known to be induced by phosphate limitation and suppressed in a phosphate-rich extracellular environment. In order to track the expression of PstS, the pSensor-PstS-EGFP plasmid (see Example 1) was electroporated in the P. aeruginosa MPAO1-P1 strain to yield the MPAO1-P1/pstS-EGFP reporter strain. The expression of PstS was detected by fluorescence (excitation 485/10, emission 528/20) normalized to cell density measured by the absorbance at 600 nm. As a control, PstS expression in P. aeruginosa grown in low phosphate- and high phosphate-defined citrated media (DCM) was used. Data indicated, as expected, that PstS expression was increased in low-phosphate medium and was nearly completely suppressed in medium containing 25 mM inorganic phosphate. All three phosphorylated polymers (ABA-PEG10k-Pi10, ABA-PEG16k-Pi14, and ABA-PEG20k-Pi20, 2 mM) (
ABA-PEG-Pis Significantly Decrease Pyocyanin Production by P. aeruginosa Under Phosphate Limited Conditions and During Exposure to Opioids.
One of the most distinguishing features of strains of P. aeruginosa is their production of pyocyanin, a water-soluble blue-green phenazine compound. Pyocyanin is one of the major toxins of P. aeruginosa that induces rapid apoptosis of human neutrophils, and thus defines the virulence of this highly lethal opportunistic pathogen. The production of pyocyanin is controlled by the quorum sensing system (QS), a central virulence circuit in P. aeruginosa and other pathogens. The PstS-PhoB phosphate regulon, a two component membrane regulator, is activated during phosphate limitation and is involved in the transcriptional activation of QS. Thus, enrichment of media with phosphate leads to suppression of pyocyanin production [23, 24]. Therefore, ABA-PEG-Pi was examined to determine whether Pi can suppress pyocyanin production in P. aeruginosa in phosphate-limited medium using DCM-Pi0.1. In this set of experiments, a MPAO1-P2 strain was used that produces a higher amount of pyocyanin compared to the MPAO1-P1 strain . In experiments, it was found that supplementation of media with iron increased pyocyanin production in this nutrient-limited DCM media. Therefore, DCM media was supplemented with 2 μM Fe3+ (1 μM Fe2(SO4)3). Results demonstrated that both ABA-PEG10k-Pi10 and ABA-PEG20k-Pi20 significantly decreased pyocyanin production in P. aeruginosa MPAO1-P2 (
It has been demonstrated that endogenous opioid compounds are released into the intestine during physiologic stress and induce pyocyanin production via the quorum sensing (QS) system of virulence activation [17, 25]. The MPAO1-P1 strain was shown to be highly responsive to the synthetic kappa opioid U-50,488 in terms of pyocyanin production . Consistent with previous results, pyocyanin production was demonstrated to be significantly increased in MPAO1-P1 when exposed to 200 μM of the kappa-opioid receptor agonist U-50,488 (
ABA-PEG-Pis Attenuated Animal Mortality Caused by P. aeruginosa Exposed to Opioids.
Two animal models (i.e., small animal model of Caenorhabditis elegans and mouse model) were developed to assess local phosphate depletion at sites of colonization of P. aeruginosa, and their use validated the fidelity between these models [23, 26]. The C. elegans model was used in the experiments disclosed herein in which the opioid-induced lethality of P. aeruginosa was shown to be suppressed by the delivery of inorganic phosphate. In order to test the in vivo efficacy of the de novo synthesis of APA-PEG-Pi compounds, conditions of both opioid exposure and phosphate limitation were created. Results indicated that all three ABA-PEG-Pi polymers, at equal concentrations of 2 mM, effectively decreased C. elegans mortality (
The hydrophobic core BPA in ABA-PEG20k-Pi20 significantly contributed to bacterial coating and its in vivo protection against lethality. In order to confirm that it is the unique ABA structure of ABA-PEG20k-Pi20 that plays a significant role in its protective capacity, PEG20k-Pi20 was synthesized. This polymer has a similar structure to ABA-PEG20k-Pi20, but lacks the hydrophobic core (
Oral Administration of Phosphorylated Polymers is Effective to Prevent Sepsis and Modulate Intestinal Homeostasis.
An experiment was conducted to assess the effects of phosphorylated polymers, i.e., PPi-6 and ABA-PEG20k-Pi20, delivered orally as drinking solutions to mice subjected to 30% hepatectomy and intestinal infection with the human pathogen community. More particularly, the mice were divided into the following groups. Group 1 contained starvation-positive, antibiotic-positive mice receiving no phosphorylated PEG but exposed to the pathogen community. Data from three groups of mice were analyzed. Absolute (
The results shown in
ABA-PEG-Pis Attenuated Collagenase Activity of Collagenolytic Enterococcus faecalis and Reduced Anastomotic Leakage Rates.
Overnight incubation of two different Enterococcus faecalis collagenolytic strains (E2 and E27) with 2 mM of ABA-PEG20k-Pi20 led to near complete inhibition of collagenase production in both strains (from 18,000 to 1,000 and from 68,000 to 5,000 collagenase units (n=6, p<0.001)) without suppressing bacterial growth. Next, the compound was tested in a mouse model of E. faecalis—mediated anastomotic leak by providing 1% ABA-PEG20k-Pi20 in drinking water. At post-operative day (POD) 7, anastomotic healing was assessed and the total amount of E. faecalis present at the anastomotic site and the percentage of E. faecalis expressing the collagenolytic phenotype were determined. Based on calculated anastomotic healing scores, leak rates were decreased from 80% to 20% in mice drinking 1% ABA-PEG20k-Pi20 (n=10, p<0.01). In ABA-PEG20k-Pi20 drinking mice, the concentration of phosphate in distal colon mucus was increased two-fold, the mean population of E. faecalis at the site of anastomosis was decreased 12-fold with the percentage of collagenolytic colonies in entire populations decreased from 20% (no ABA-PEG20k-Pi20) to 1% (with ABA-PEG20k-Pi20) (n=5, p<0.05).
Sodium hexametaphosphate (PPi-6) profoundly suppresses the collagenase production in gram negative strains S. marcescens and P. aeruginosa and PPi-6 supplementation in the drinking water protects mice from colonic anastomosis leakage induced by these strains. However, PPi-6 only slightly attenuated collagenase production in E. faecalis. As described herein, ABA-PEG20k-Pi20 suppresses collagenase activity in E. faecalis and promotes anastomotic healing in a mouse model of anastomotic leak caused by intestinal injection of E. faecalis.
Two groups of mice were involved in this study. All mice were subjected to colon anastomosis followed by an enema of E. faecalis E2 at POD1. The first group received water, and the second group received 1% ABA-PEG20k-Pi20 dissolved in water. At the time of sacrifice at POD7, the tissues and luminal contents were collected at the site of anastomosis, homogenized, and used for culturing. Total Enterococcus colonization at the anastomosis site and luminal content were determined by culture analysis using Enterococcal selective plates (BD Difco) on which all Enterococcus species produced black-pigmented colonies. The CFU count was normalized to the sample weight. The total Enterococcus population was not significantly different between the two groups in both the lumen and the tissue at the anastomotic site. The collagenolytic population was evaluated on Enterococcal plates covered with skim milk on which collagenolytic species produced black colonies with a clearing halo surrounding the colony. The amount of collagenolytic colonies of E. faecalis was significantly reduced by ABA-PEG20k-Pi20 in both tissue and luminal contents.
Mucus layers at anastomotic sites are enriched with phosphate in mice treated with ABA-PEG20k-Pi20. Phosphate concentration was measured in mucus layer scrapped off from 1 cm2 area at the site of anastomosis at the time of sacrifice at POD7. The concentration of phosphate was significantly increased in ABA-PEG20k-Pi20 group (E. faecalis group 23.22±5.73 versus E. faecalis+ABA-PEG20k-Pi20, 48.42±7.633 μM, p=0.0297, n=5).
The non-phosphorylated parent polymer ABA-PEG20 k is less effective compared to ABA-PEG20k-Pi20. The phosphorylated polymer ABA-PEG20k-Pi20 is more potent compared to its parent non-phosphorylated compound ABA-PEG20 k for suppression of collagenolytic activity of E. faecalis. A comparative analysis was performed for ABA-PEG20k-Pi20 versus ABA-PEG20 k in our mouse model and found that ABA-PEG20 k is less effective compared to ABA-PEG20k-Pi20. Both anastomotic leak scores and number of collagenolytic colonies of E. faecalis were significantly higher in ABA-PEG20 k group.
The inability of PPi-6 to suppress the collagenolytic activity of E. faecalis in vitro relates to its inability in vivo to improve anastomotic healing. Orally delivered 6-mer polyphosphate (PPi-6) has been recently proved as a potent protective compound in anastomotic leak induced by gram-negative collagenolytic strains of S. marcescens and P. aeruginosa. The efficacy of this compound in vivo correlated to its ability to profoundly suppress collagenase activity in these strains in vivo. However, PPi-6 does not affect the collagenolytic activity in E. faecalis, and does not promote a growth. Actually, PPi-6 at concentrations of >2 mM even suppressed the E. faecalis growth. Respectively, in in vivo experiments, the oral PPi-6 did not improve the anastomotic healing complications caused by E. faecalis.
Preparing a bowel is a critical step in colonic surgery. In most cases, it includes mechanical cleansing and a treatment with oral non-absorbable antibiotics. However, as it becomes evident, this procedure cannot guaranty the bacterial clearance, and there are multiple evidences that bacterial pathogens such E. faecalis and P. aeruginosa are often cultured from leaking anastomosis. These bacteria were shown to complicate anastomotic healing by degradation of collagen and thereby preventing normal extracellular matrix rearrangement that is required for wound healing. Given the emergence of multi-drug resistant bacteria, novel formulations are required for bowel preparation. Among components of a bowel preparation, those responsible for decrease of bacterial collagenolytic activities should be considered. Such components are to be directed to specific bacterial phenotype rather than to antibiotic-like effect. For this, scientific-based approaches are required. It was recently demonstrated a potency of a phosphate-based therapy using PPi-6 to improve anastomotic healing by suppressing collagenase activity of gram-negative pathogens. In the present study, it was demonstrated that a potential of yet another phosphate-based therapeutic compound, a phosphorylated triblock copolymer ABA-PEG20k-Pi20 to suppress the collagenolytic activity in gram-positive bacterium Enterococcus faecalis, a pathogen tightly associated with anastomotic leak. The polymer ABA-PEG20k-Pi20 was synthesized de novo and verified for its ability to coat bacteria and make its phosphate available for delivery and virulence suppression. In the present study, ABA-PEG20k-Pi20 delivers phosphate to the site of anastomosis, significantly attenuates collagenolytic populations of E. faecalis at anastomotic sites and enhances anastomotic healing. The ability of ABA-PEG20k-Pi20 to suppress collagenolytic activity is critical for its in vivo protective effect as its non-phosphorylated parent compound ABA-PEG20 k and inorganic polyphosphate PPi-6 that were less effective for prevention of collagenolytic activity of E. faecalis were also less protective to prevent anastomotic leak caused by this pathogen. In some embodiments, ABA-PEG20k-Pi20 could be combined with PPi-6 to formulate a bowel preparation against broad range collagenolytic pathogens.
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From the disclosure herein it will be appreciated that, although specific embodiments of the disclosure have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the disclosure.
13. A method of producing the triblock copolymer comprising
- (a) covalently attaching at least two polyethylene glycol chains to a hydrophobic core comprising a carbocyclic or heterocyclic ring;
- (b) covalently attaching at least two phosphate groups to at least one polyethylene glycol chain.
14. The method of claim 13 wherein at least two polyethylene glycol chains are each covalently attached to at least two phosphate groups.
Filed: Jan 18, 2023
Publication Date: Sep 14, 2023
Applicants: The University of Chicago (Chicago, IL), UChicago Argonne, LLC (Chicago, IL)
Inventors: John C. Alverdy (Glenview, IL), Matthew Tirrell (Chicago, IL), Olga Y. Zaborina (Brookfield, IL), Jun Mao (Chicago, IL), Wei Chen (Chicago, IL)
Application Number: 18/156,058