Patent Application
20180250328
This present disclosure relates to copolymers having antibacterial properties. More specifically, the present disclosure relates to metallocene-boronic acid-containing compounds and copolymers as antimicrobial agents.
Bacterial infections have become one of the most urgent global health threats, leading to increased healthcare costs, destruction of local tissues, patient disability, morbidity, and even death. If no effective strategies are taken to prevent and treat bacterial infections, it is estimated that by 2050 they could claim 10 million lives and cost up to 100 trillion dollars globally. However, commonly used antibiotics, such as penicillin and methicillin, have shown diminished antimicrobial efficacy, and numerous bacterial pathogens have accumulated multidrug resistance (MDR).
Multidrug-resistant Gram-negative bacteria are posing an increasingly alarming threat, making many last-resort antibiotics ineffective. Compared to therapies for Gram-positive strains, a recent analysis showed very few antibiotics in development could be promising for fighting these life-threatening Gram-negative bacterial infections. Due to having double cell membranes as an intrinsic defense, it is difficult for antibiotics to not only inhibit critical bacterial processes, but also penetrate two membrane barriers and escape efflux pumps. In many cases, these agents can cross the outer membrane, but stop short of penetrating the cytoplasmic membrane. Meanwhile, the antimicrobial agent must overcome efflux pumps even after the penetration of two membranes. With the frequency of MDR increasing at an alarming rate, there is an urgent need to develop new antimicrobial agents. It would be most desirable to have multiple pathogen-specific therapeutics that can target various types of bacteria, but especially Gram-negative bacteria. As such, the development of compounds that are effective against broad-spectrum bacterial strains including those with multidrug resistance would be useful.
Aspects and advantages of embodiments of the present disclosure will be set forth in part in the following description, or may be learned from the description, or may be learned through practice of the embodiments.
Polymeric compounds for targeting broad-spectrum bacterial strains are provided. The polymeric compounds can include at least one metallocene monomeric unit and at least one boronic acid monomeric unit. The metallocene monomeric unit can include a cationic metallocene moiety paired to an anion, and the anion can be an anionic antibiotic compound. The metallocene-boronic acid-containing copolymers can enhance interactions with bacterial cells and therefore improve antimicrobial effectiveness of antibiotics against not only Gram-positive bacterial strains but also Gram-negative bacterial strains. The cationic metallocene moiety can be attracted to negatively-charged bacterial membranes via electrostatic interaction. The boronic acid group can bind with peptidoglycan and polysaccharides on bacterial cell walls/membranes through the formation of reversible boronic esters. Thus, bacteria can be rapidly captured and cell membranes disrupted while the antibiotic targets and kills the bacteria. The metallocene-boronic acid-containing compounds and polymers demonstrate synergistic antimicrobial effects and excellent bactericidal function with broad spectrum activity against various strains of bacteria. Further, the compounds exhibit minimal toxicity and non-hemolytic activity in vitro and in vivo on mammalian cells.
A full and enabling disclosure of the present invention, including the best mode thereof to one skilled in the art, is set forth more particularly in the remainder of the specification, which includes reference to the accompanying figures.
Reference now will be made to the embodiments of the invention, one or more examples of which are set forth below. Each example is provided by way of an explanation of the invention, not as a limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as one embodiment can be used on another embodiment to yield still a further embodiment. Thus, it is intended that the present invention cover such modifications and variations as come within the scope of the appended claims and their equivalents. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention, which broader aspects are embodied exemplary constructions.
Chemical elements are discussed in the present disclosure using their common chemical abbreviation, such as that commonly found on a periodic table of elements. For example, hydrogen is represented by its common chemical abbreviation H, helium is represented by its common chemical abbreviation IIe, and so forth.
As used herein, the term “polymer” generally includes, but is not limited to, homopolymers; copolymers, such as, for example, block, graft, random and alternating copolymers; and terpolymers; and blends and modifications thereof. Furthermore, unless otherwise specifically limited, the term “polymer” shall include all possible geometrical configurations of the material. These configurations include, but are not limited to isotactic, syndiotactic, and random symmetries.
The term “organic” is used herein to refer to a class of chemical compounds that are comprised of carbon atoms. For example, an “organic polymer” is a polymer that includes carbon atoms in the polymer backbone, but may also include other atoms either in the polymer backbone and/or in side chains extending from the polymer backbone (e.g., oxygen, nitrogen, sulfur, etc.).
The term “pharmaceutically effective amount” refers to an amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought by a researcher or clinician. This amount can be a therapeutically effective amount.
Embodiments of the present disclosure include metallocene-boronic acid-containing copolymers. The metallocene-boronic acid-containing copolymers can be used in biomedical applications such as drugs and antimicrobial agents. The metallocene-boronic acid-containing copolymers can act as effective antimicrobial agents against a broad spectrum of bacterial pathogens, including Gram-positive bacteria, Gram-negative bacteria, and bacteria that has shown resistance to conventional antibiotics.
A metallocene is a compound having two cyclopentadienyl anions (Cp, which is C5H5−) bound to a metal center (M) in the oxidation state II, with the resulting general formula (C5H5)2M. Closely related to the metallocenes are the metallocene derivatives, (e.g. titanocene dichloride, vanadocene dichloride). However, a metallocene-containing cationic compound generally has a positive charge due to the metal center (M) being in the oxidation state I. Thus, the overall charge of the metallocene-containing cationic compound is +1, such that the metallocene-containing cationic compound is paired to an anion having a negative charge, such as hexafluorophosphate (PF6−), tetraphenylborate (BPh4−), tetrafluoroborate (BFr−), trifluoromethanesulfonate (OTf), F−, Cl−, Br−, I−, NO3−, acetate (Ac−), sulfate (SO42−), hydrogen sulfate (HSO4−), perchlorate (ClO4−), bromate (BrO3−), cyanide (CN−), thiocyanate (SCN−), hydroxide (OH−), dihydrogen phosphate (H2PO4−), or formate (HCOO−).
Referring to
I. Anion-Paired Metallocene-Containing Monomers
Generally, each anion-paired metallocene-containing monomer includes an anion-paired metallocene group covalently attached to a polymerizable group via an organic linker group (U.S. Pat. No. 9,402,394 of Tang, et al. teaches metallocene-containing compounds, the disclosure of which is incorporated by reference herein). Referring to
A cationic metallocene group includes two cyclopentadienyl rings bound to a metal center (M) in an oxidation state that leaves the cationic metallocene group with a positive charge (such as +1 or +2). Thus, the cationic metallocene group is generally paired with a counter ion. For example, an anion (X) can be present such that the charge of the resulting anion-paired cationic metallocene group is zero. The metals can include, for example, iron (Fe), cobalt (Co), rhodium (Rh), ruthenium (Ru), and mixtures thereof.
The anion (“X”) paired with the cationic metallocene-containing compound can be any suitable anion, including, but not limited to, hexafluorophosphate (PF6−), tetraphenylborate (BPh4−), tetrafluoroborate (BF4−), trifluoromethanesulfonate (OTf), F−, Cl−, Br−, I−, NO3−, acetate (Ac−), sulfate (SO42−), hydrogen sulfate (HSO4−), perchlorate (ClO4−), bromate (BrO3−), cyanide (CN−), thiocyanate (SCN−), hydroxide (OH−), dihydrogen phosphate (H2PO4−), or formate (HCOO−).
Various organic linker groups can be positioned between the polymerizable group (e.g., containing a vinyl group) and the anion-paired metallocene group. In one embodiment, the organic linker group includes a simple alkyl chain having a number (m) of repeating carbon atoms (e.g., —CH2—), with m being an integer of from 1 to about 50, such as 2 to about 40, and such as from 3 to about 20. In one particular embodiment, m is from 2 to about 12. As shown in the embodiment of
The organic linker group can also include any covalent linkage to one of the cyclopentadienyl rings of the anion-paired metallocene group, such as an amide linkage as shown in
The polymerizable group of the anion-paired metallocene-containing monomer can include a vinyl group, such as an acrylic group, a methacrylic group, a styrenic group, an acrylamide group, or a norbornene group, etc. For example,
II. Boronic Acid-Containing Monomers
The metallocene-containing monomers can be copolymerized with boronic acid containing monomers. The boronic acids are trivalent boron-containing organic compounds that possess one alkyl substituent (i.e., a C-B bond) and two hydroxyl groups (i.e. two B-OH bonds) to fill the remaining valences on the boron atom. Referring to
Diverse organic linker groups can be positioned between the polymerizable group (i.e., containing the vinyl group) and the boronic acid group. In the embodiment of
The polymerizable group of the boronic acid-containing monomer can include a vinyl group, such as an acrylamide group, methacrylamide group, acrylic group, a methacrylic group, a styrenic group, an acrylamide group, or a norbornene group. For example,
III. Metallocene-Boronic Acid-Containing Compounds and Polymers
The metallocene-containing monomers and boronic acid-containing monomers can be polymerized to form metallocene-boronic acid-containing copolymers (including block copolymers, random copolymers, graft copolymers, star copolymers and/or organic/inorganic hybrids) that contain at least one unit derived from metallocene moiety and one unit derived from boronic acid moiety (i.e., at least one metallocene monomer and at least one boronic acid monomer). In one embodiment, a metallocene-boronic acid-containing copolymer can be prepared by free radical and controlled/living radical copolymerization of a vinyl-boronic acid-containing monomer and a vinyl-metallocene-containing monomer. The copolymers can have anion-paired metallocene moieties arid boronic acid moieties on the side-chains.
For example, referring to
The properties of the metallocene-boronic acid-containing copolymers can be tuned by changing the comonomer structures (the polymerizable vinyl moiety, the linker, metallocene-containing moiety or boronic acid-containing moiety), the molecular weight of the polymer, and/or the relative amounts of any comonomers present. For example, the amount of metallocene monomer can range from about 50 wt. % to about 95 wt. %, such as from about 60 wt. % to about 80 wt. %, and such as from about 65 wt. % to about 75 wt. %. Further, the amount of boronic acid monomer can range from about 5 wt. % to about 50 wt. %, such as from about 15 wt. % to about 40 wt. %, and such as from about 25 wt. % to about 35 wt. %.
The molar ratio of metallocene monomer to boronic acid monomer (mols metallocene monomer/mols boronic acid monomer) can range from about 20 to about 1, such as from about 5 to about 15, and such as from about 8 to about 12. The average number of monomers in the copolymer chain can range from about 5 to about 300, such as from about 10 to about 200, and such as from about 20 to about 100. Additionally, in some embodiments the average molecular weight of metallocene-boronic acid copolymers can range from about 2,000 g/mol to about 100,000 g/mol, such as from about 10,000 g/mol to about 75,000 g/mol, and such as from about 20,000 g/mol to about 50,000 g/mol.
IV. Metallocene-Boronic Acid-Containing Copolymers as Antimicrobial Agents
Metallocene-boronic acid-containing compounds and polymers of the present disclosure can be used as antimicrobial agents. For example, cobaltocenium-boronic acid-containing copolymers of the present disclosure have shown antimicrobial activity against a broad spectrum of bacteria, including Gram positive bacteria (S. aureus and E. faecalis), Gram negative bacteria (E. coli, K. pneumoniae, P. vulgaris, and P. aeruginosa) and drug resistant bacteria (methicillin-resistant Staphylococcus aureus, MRSA).
Such copolymers can be administered in a pharmaceutically effective amount as an antibiotic to a subject (e.g., a living subject such as a human or animal) infected with such bacteria (i.e., a metallocene-boronic acid-containing antibiotic). The metallocene-boronic acid-containing antibiotic may be administered to the subject via any suitable routes of administration, such as oral, rectal, transmucosal, transnasal, intestinal, or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
Pharmaceutical compositions that include the metallocene-boronic acid-containing antibiotic may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Thus, such pharmaceutical compositions comprising the metallocene-boronic acid-containing antibiotic may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, artificial cerebrospinal fluid (CSF) or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated can used in the formulation. Such penetrants are generally known in the art. For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol. Cellulose preparations can also be utilized such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose, and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. Toxicity and therapeutic efficacy of the metallocene-boronic acid-containing antibiotic described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage and formulation may vary depending upon the dosage form employed and the route of administration utilized.
Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with the course of treatment lasting from several days to several weeks or until a cure is effected or diminution of the infected state is achieved. The amount of a metallocene-boronic acid containing antibiotic to be administered will, of course, be dependent on factors including the subject being treated, the severity of the affliction, and the manner of administration.
V. Antibiotic-Loaded Metallocene-Boronic Acid Copolymers
In one embodiment, the metallocene-boronic acid copolymers can be used to promote the effects of traditional antibiotics against a broad spectrum of bacterial pathogens including Gram positive bacteria (S. aureus and E. faecalis) and Gram negative bacteria (E. coli, K. pneumoniae, P. vulgaris, and P. aeruginosa). Metallocene-boronic acid-containing copolymers have at least one metallocene unit and one boronic acid unit (i.e., at least one metallocene monomer and at least one boronic acid monomer). The metallocene unit can have a positive charge. In such cationic metallocene-boronic acid-containing copolymers, the anion (X) as shown in
All β-lactam type antibiotics, such as penicillins, carbapenems, and cephalosporins (including the first, second, third, fourth and fifth generation), can be loaded in the metallocene-boronic acid-containing copolymers. These antibiotic-loaded metallocene-boronic acid-containing copolymers can produce excellent effects against Gram-negative and Gram-positive bacteria, and especially drug resistant bacteria. For example,
Thus, traditional antibiotics can be loaded in metallocene-boronic acid-containing materials and can improve antimicrobial ability against a broad spectrum of drug resistant bacterial pathogens. Exemplary antibiotics that can be paired with the cationic metallocene moiety in the metallocene-boronic acid-containing copolymer include, but are not limited to, penicillins (Penams): Amoxicillin, Ampicillin (Pivampicillin, Hetacillin, Bacampicillin, Metampicillin, Talampicillin), Epicillin, Carbenicillin (Carindacillin), Ticarcillin, Temocillin, Azlocillin, Piperacillin, Mezlocillin, Mecillinam Sulbenicillin, Clometocillin, Benzathine, benzylpenicillin, Procaine benzylpenicillin, Azidocillin, Penamecillin, Phenoxymethylpenicillin (V), Propicillin, Benzathine phenoxymethylpenicillin and Pheneticillin; cephalosporins, including the first, second, third, fourth, and fifth generations; carbapenems, including Biapenem, Ertapenem, Doripenem, Imipenem, Meropenem and Panipenem.
The properties of the antibiotic-loaded metallocene-boronic acid copolymers can be tuned by changing the comonomer structures (the polymerizable vinyl moiety, the linker, metallocene-containing moiety or boronic acid-containing moiety), the molecular weight of the polymer, the relative amounts of any comonomers, and/or the amount and type of antibiotic. For example, the antibiotic can be present in the antibiotic-loaded metallocene-boronic acid copolymer in an amount of from about 10 wt. % to about 50 wt. %, such from about 20 wt. % to about 40 wt. %, and such as from about 25 wt. % to about 35 wt. %. Further, the molar ratio of antibiotic to metallocene monomer (mols antibiotic / mols metallocene monomer) can range from about 0.30 to about 1.0, such as from about 0.45 to about 0.85, and such as from about 0.55 to about 0.75.
The amount of metallocene monomer can range from about 50 wt. % to about 95 wt. %, such as from about 60 wt. % to about 80 wt. %, and such as from about 65 wt. % to about 75 wt. %. Further, the amount of boronic acid monomer can range from about 5 wt. % to about 50 wt. %, such as from about 15 wt. % to about 40 wt. %, and such as from about 25 wt. % to about 35 wt. %.
The molar ratio of metallocene monomer to boronic acid monomer (mols metallocene monomer/mols boronic acid monomer) can range from about 20 to about 1, such as from about 5 to about 15, and such as from about 8 to about 12. The average number of monomers in the copolymer chain can range from about 5 to about 300, such as from about 10 to about 200, and such as from about 20 to about 100. Additionally, in some embodiments, the average molecular weight of antibiotic-loaded metallocene-boronic acid copolymers can range from about 2,000 g/mol to about 100,000 g/mol, such as from about 10,000 g/mol to about 75,000 g/mol, and such as from about 20,000 g/mol to about 50,000 g/mol.
The antimicrobial efficacy of various metallocene-boronic acid-containing copolymers including random copolymers, block copolymers, graft copolymers, star copolymers, and organic/inorganic hybrids was demonstrated was demonstrated through various experiments which are discussed in the Examples, below.
This example demonstrates the antimicrobial efficacy of metallocene-boronic acid-containing copolymers against Gram-positive S. aureus and Gram-negative E. coli.
The cobaltocenium-boronic acid-containing copolymers (PCo-PPB) were synthesized via reversible-addition fragmentation chain transfer (RAFT) polymerization using cobaltocenium-containing monomer (2-cobaltocenium amidoethyl methacrylate) and boronic acid-containing monomer (3-acrylamidophenylboronic acid) as co-monomers. Three cobaltocenium-boronic acid-containing copolymers with different weight fractions of boronic acid were synthesized by changing molar ratios of comonomers, while keeping their molecular weight similar (Mn 14,500 g/mol). The proportion of boronic acid in the copolymers was about 20 wt. % (PCo-PPB-1), 15 wt. % (PCo-PPB-2), and 8 wt. % (PCo-PPB-3), respectively.
For the bacteria, a single colony was inoculated in 30 mL tryptic soy broth (TSB) at 37° C. for 24 hours, shaking at 190 rpm/min. All bacteria were grown to an optical density of about 1.00 (OD600=1.00) for further use. To conduct the agar disk-diffusion assays, actively-growing cultures of each bacterial strain on mannitol salt agar (MSA) were inoculated on tryptic soy broth (TSB) agar plates. The bacterial growth culture (cell concentrations were 1.0×106 CFU/mL) was diluted from 10 μL to 1 mL in tryptic soy broth (TSB) and 100 μL was spread on TSB agar plates to form a bacterial lawn covering the plate surface. Then, 6 mm (diameter) filter discs were added to the plate surface, aqueous cobaltocenium-boronic acid-containing copolymers (PCo-PPB) at different concentrations were added to disks, and the plates were incubated at 28° C. for 18 h. The development of a clear zone around the disk was indicative of the ability of the compounds to kill bacteria.
As shown in
In order to verify the interaction between metallocene-boronic acid-containing copolymer and peptidoglycan as well as lipopolysaccharide, peptidoglycan, and lipopolysaccharide, extractions from cell membranes of S. aureus and E.coli were selected to model macromolecules. Firstly, the cobaltocenium-boronic acid-containing copolymer (PCo-PPB-1) and peptidoglycan (weight ratio 3:1) were mixed in dimethyl sulfoxide/water (DMSO/H2O) solvent for 6 h at room temperature, and then employed for Fourier-transform infrared spectroscopy (FTIR) analysis after freeze-drying. Compared with the spectra of cobaltocenium-boronic acid-containing copolymer and peptidoglycan alone, the characteristic peak of boronate ester (B-O-C stretching vibration) at 1050 cm−1 appeared in the spectrum of cobaltocenium-boronic acid-containing copolymer-peptidoglycan conjugates (
The utilization of metallocene-boronic acid-containing compounds, random copolymers, block copolymers, graft copolymers, star copolymers, and organic/inorganic hybrids as drug delivery materials for traditional antibiotics was demonstrated. Different commercially available antibiotics (including all β-lactam type antibiotics, such as penicillins, carbapenems and cephalosporins, including the first, second, third, fourth, and fifth generations) were loaded with cationic metallocene-containing polymers.
The ability of cationic metallocene-containing compounds and polymers to activate conventional antibiotics against drug-resistant bacterial pathogens was demonstrated. For example, cobaltocenium-boronic acid-containing copolymers loaded with penicillin G showed antimicrobial activity against a broad spectrum of bacteria, including Gram-positive bacteria (S. aureus and E. faecalis) and Gram-negative bacteria (E. coli, P. vulgaris, P. aeruginosa and K. pneumoniae).
Penicillin-G was loaded into cobaltocenium-boronic acid-containing-copolymers to form bioconjugates (labeled as PCo-PPB-Peni) via ionic complexation between cationic cobaltocenium and anionic antibiotic. High antibiotic loading capacity (31 wt %, the molar ratio of cobaltocenium moiety to penicillin is 1:0.6) was easily obtained due to the strong electrostatic interactions.
Disk-diffusion assays were used to evaluate the antimicrobial activity of penicillin loaded-cobaltocenium-boronic acid-containing copolymers against six strains of bacteria including Gram-positive bacteria (S. aureus and E. faecalis) and Gram-negative bacteria (E. coli, P. vulgaris, P. aeruginosa and K. pneumoniae). To compare bactericidal efficiency, a penicillin loaded-cobaltocenium-containing homopolymer (named as PCo-Peni) was prepared as a control.
As shown in
The inhibition effect of penicillin-loaded cobaltocenium-boronic acid-containing copolymers against six types of bacteria was further investigated by confocal scanning laser microscopy (CSLM). One (1) mL of active bacterial stock of various strains was introduced to 5 penicillin-G, 11 μg cobaltocenium-boronic acid-containing copolymer, and 16 μg penicillin-loaded cobaltocenium-boronic acid-containing copolymers (penicillin-G weight: 5 m), respectively. An untreated cell suspension was used as the control. Following 18-hour incubation at 37° C., 1 μL LIVE/DEAD BacLight (Bacterial Viability Kit; INVITROGEN INC.®) was added to the incubation solution. After incubation for 15 minutes, cells were imaged using a LEICA TCS SP5® Laser Scanning Confocal Microscope with a 63X oil immersion lens. When excited at 488 nm with Argon and Helium/Neon lasers, bacteria with intact membranes displayed green fluorescence (Emission=500 nm) and bacteria with disrupted membranes fluoresced red (Emission=635 nm). LIVE/DEAD bacteria viability assay by CSLM suggested penicillin-G and cobaltocenium-boronic acid-containing copolymers alone were not effective at killing bacteria at relatively low concentrations (
The morphologies of different bacteria after incubation with penicillin-loaded cobaltocenium-boronic acid-containing copolymers were examined by field-emission scanning electron microscopy (FESEM). Ten (10) μL of bacteria cell solution were grown overnight on one glass slide in a 12-well plate containing 1 mL of TSB medium at 37° C. Cell suspensions were diluted to OD600=1.0. Penicillin loaded cobaltocenium-boronic acid-containing copolymer (PCo-PPB-Peni) bioconjugates (16 μg, with penicillin-G weight 5 μg) were added to the 1 mL cell stock solution and incubated at 37° C. overnight. An unadulterated cell suspension was used as a control. The samples were then fixed in cacodylate buffered with 2.5% glutaraldehyde solution (pH=7.2) for 2-3 h at 4° C. and post-fixed with 1% osmium tetraoxide at 4° C. for 1 h. The samples were dried under their critical point, then coated with gold using DENTON DESK II SPUTTER COATER® for 120 s and observed by FESEM. An untreated cell suspension was used as the control. From the FESEM images in
The strong bactericidal efficacy of penicillin-loaded cobaltocenium-boronic acid-containing copolymers was believed to be attributed to the synergistic effects originating from the building blocks of cobaltocenium and phenylboronic acid.
Metallocene-boronic acid-containing copolymers demonstrated high efficacy in lysing bacterial cells as well as reducing β-lactamase activity. Furthermore, the cobaltocenium-boronic acid-containing copolymers possessed excellent biocompatibility, exhibiting non-hemolytic activity and minimal in vitro and in vivo toxicity.
To determine the toxicity of cobaltocenium-boronic acid-containing copolymers (PCo-PPB-1, about 20 wt. % boronic acid; PCo-PPB-2, about 15 wt. % boronic acid; and PCo-PPB-3, about 8 wt. % boronic acid), both in vitro and in vivo experiments were performed to determine their ability to induce programmed cell death (known as apoptosis) in immune cells. For this purpose, the cells were cultured with phosphate-buffered saline (PBS) solution and 10 and 50 μg/mL of cobaltocenium-boronic acid-containing copolymers for 24 h, and then fluorochrome-labeled with Annexin V (a member of the annexin family of intracellular proteins that can bind to phosphatidylserine in a calcium-dependent manner), which was employed to specifically target and identify apoptotic cells. It was found that the percentages of apoptotic cells after treatment with cobaltocenium-boronic acid-containing copolymers were very similar to that of the PBS control (
The immune cells were phenotyped for detection of cell subpopulations by targeting their unique markers with a specific antibody followed by detection using flow cytometry, which is a very sensitive technique to quantify large numbers of cells. Splenocytes (1×106) from PBS-treated groups or copolymer-treated groups of mice were washed with PBS (INVITROGEN®) and incubated in the dark for 30 min on ice with 0.5 μg of the following anti-mouse primary monoclonal antibodies (mAb): fluorescein isothiocyanate (FITC)-conjugated anti-CD3, phycoerythrin (PE)-anti-CD8 and allophycocyanin (APC)-anti-CD4 (all from BIOLEGEND®, Calif., USA), or FITC-anti-CD19 (BD PHARMINGEN®, San Diego, Calif., USA). For triple-staining studies, directly-conjugated monoclonal antibodies were simultaneously added to the sample. In the current example, flow cytometry was employed to detect whether the cobaltocenium-boronic acid-containing copolymers influenced the different populations of T and B cell lineages in the splenocytes after intraperitoneal injection of copolymers for 48 h. It was observed that the treatment of mice with any of the cobaltocenium-boronic acid-containing copolymers did not alter the percentages of the immune cells when compared to PBS-treated groups. The percentages of all cell types, including CD3+ T cells, CD4+ T helper/regulatory cells, the CD8+ cytotoxic T cells, as well as the CD19+ B cells from mice injected with the copolymers were similar to those of the PBS-injected mice (
Finally, the toxicity of cobaltocenium-boronic acid-containing copolymers was analyzed on red blood cells (RBCs) by evaluating whether they could lead to hemolysis of red blood cells (RBCs). Blood was collected from mice in heparinized tubes and diluted by mixing 800 μL of blood with 1000 μL PBS. Cobaltocenium-boronic acid-containing copolymer samples were prepared in PBS at concentrations of 10, 50, 100, and 500 μg/mL. Sixty (60) μL of the diluted blood samples were added to 3 mL of each polymer, PBS, and 0.1% Triton-X100 in PBS. Supernatants were then used to measure their optical density (OD) and the hemolysis percentage (%) was calculated. It was found that, even at concentrations of cobaltocenium-boronic acid-containing copolymers as high as 500 μg/mL, all showed lysis of RBCs to be extremely low (<10%) when compared to the negative control group (
In conclusion, the antimicrobial cobaltocenium-boronic acid-containing copolymers exhibited robust, synergistic antibacterial activity through electrostatic absorption onto bacterial membranes/cell walls via the cationic cobaltocenium moiety and the binding of boronic acid to peptidoglycan or lipopolysaccharides on the bacterial surface. Furthermore, these cobaltocenium-boronic acid-containing copolymers possessed excellent biocompatibility. After binding β-lactam antibiotic penicillin-G, the copolymer-antibiotic bioconjugates improved the vitality of antibiotics by protecting the antibiotics from β-lactamase hydrolysis and exhibited excellent antibacterial efficacy against six different strains of Gram-positive and Gram-negative bacteria. This new macromolecular design could open a promising paradigm for improving the vitality of conventional antibiotics against various strains of bacteria while exerting minimal toxicity to mammalian cells.
These and other modifications and variations to the present invention may be practiced by those of ordinary skill in the art, without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. In addition, it should be understood the aspects of the various embodiments may be interchanged both in whole or in part. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention so further described in the appended claims.
The present application claims priority to U.S. Provisional Patent Application Ser. No. 62/467,438 titled “METALLOCENE-BORONIC ACID-CONTAINING COMPOUNDS AND COPOLYMERS AS ANTIMICROBIAL AGENTS” of Tang, et al. filed on Mar. 6, 2017, the disclosure of which is incorporated by reference herein.
This invention was made with government support under R01A1120987 awarded by National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62467438 | Mar 2017 | US |
