Article Cite This: Macromolecules XXXX, XXX, XXX−XXX
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Cyclopropenium-Based Biodegradable Polymers Noam Y. Steinman,† Rachel L. Starr,‡ Spencer D. Brucks,‡ Chen Belay,† Rinat Meir,‡ Jacob Golenser,§ Luis M. Campos,*,‡ and Abraham J. Domb*,† Institute of Drug Research, School of Pharmacy-Faculty of Medicine, and §Department of Microbiology and Molecular Genetics, The Kuvin Centre for the Study of Infectious and Tropical Diseases, The Hebrew University of Jerusalem, Jerusalem 91120, Israel ‡ Department of Chemistry, Columbia University, New York, New York 10027, United States Macromolecules Downloaded from pubs.acs.org by UNIV AUTONOMA DE COAHUILA on 05/02/19. For personal use only.
†
S Supporting Information *
ABSTRACT: Cationic polymers offer a wide range of potential biomedical applications. Often, these materials suffer from a lack of degradability under biological conditions, preventing their translation in vivo. We present herein the synthesis and characterization of a series of novel biodegradable polymers bearing cationic cyclopropenium along a polyester backbone, either linear or cross-linked. The polymers are synthesized stepwise via the reaction between diol-functionalized tris(amino)-cyclopropenium (TAC) monomers and diacyl chlorides. Incorporation of the TAC moiety with a permanent, pH-independent charge, and hydrophobic groups with sufficient bulkiness causes the polyelectrolyte to form an aqueous dispersion of nanoparticles with a positive surface charge. Smaller hydrophobic TAC substituents inhibit nanoparticle formation because of a lack of hydrophobic bonds within the core of the nanoparticle. The polymers undergo hydrolytic degradation and swell significantly, displaying an important framework for the drug-delivery capabilities of a hydrolytically degradable cationic polyester. One polymer displayed potent antimicrobial activity against Staphylococcus epidermidis. These polymers may have use for the delivery of anionic bioactive agents.
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INTRODUCTION Biodegradable polymers offer many advantages to the biomedical and pharmaceutical industries, particularly in controlled drug delivery, long-term implants, tissue engineering, and regenerative medicine.1 Synthetic biodegradable polymers are highly tunable, allowing for optimization of functionality. The rate of biodegradation may also be tailored to fit a desired application, vastly enhancing the potential for in vivo application of polymers. By installing a specific functionality onto a biodegradable polymeric backbone, certain properties like degradability and structural integrity can be enhanced. A particularly important class of polymers frequently used in biomedicine for many different purposes are polycations. They can act as antimicrobial agents by adsorbing to the negatively charged bacterial cell surface, disrupting the cell wall and promoting cell death.2,3 Alternatively, they can form electrostatic complexes with negatively charged nucleic acids,4 enabling the transfection of genetic material. Other applications such as regenerative medicine, tissue engineering, and protein delivery systems can also benefit by utilizing polycations.5−13 Therefore, there is a compelling need for developing potent cationic moieties.14 The cyclopropenium cation has great potential to be used as a cationic moiety in pharmaceutical applications. First synthesized by Breslow in 1957,15 it is the smallest Hückel aromatic ring, possessing an unusually high degree of ionic and thermodynamic stability for a carbocation.16 Its carbon© XXXX American Chemical Society
centered cation is stable across a wide pH range, including in strongly alkaline environments.17 This cationic stability is important when using cyclopropenium in medicine, as its cation remains robust at most pH levels18 and at high temperatures,19 allowing variability and flexibility in a variety of environments. This is unique compared to most other polycations used in medicine, which have pH-dependent, nitrogen-centered cations. We previously reported the first incorporation of cyclopropenium into polymeric structures through reversible deactivation radical polymerization and via post polymerization functionalization.20,21 However, in these systems, cyclopropenium was either appended to polystyrene or poly(ethylene imine) backbones with limited translation to in vivo biomedical contexts due to a lack of biodegradability. Here, we describe for the first time the incorporation of cyclopropenium into a biodegradable polymer backbone, thus improving its applicability for biomedical applications. We demonstrate the synthesis and characterization of both linear and crosslinked biodegradable cyclopropenium polymers. In addition to synthesis, the polyelectrolytes’ rates of hydrolytic degradation as well as their swelling behavior and nanoparticle formation are described. Both linear and crosslinked polymers degrade in pharmaceutically relevant time periods (>50% degradation within one week). Those linear Received: March 4, 2019 Revised: April 18, 2019
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DOI: 10.1021/acs.macromol.9b00430 Macromolecules XXXX, XXX, XXX−XXX
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Poly(CY-co-terephthalate) (p(CY-Te)), 4. A solution of terephthaloyl chloride (81 mg, 0.40 mmol) in DCM (5 mL) was added to a solution of CY-OH (2, 122.8 mg, 0.23 mmol) and pyridine (0.6 mL) in DCM (20 mL). TEA (0.9 mL) was added after 20 h of stirring at rt. After 4 more days of stirring at rt, the mixture was washed with water (3 × 10 mL) and brine (1 × 20 mL), dried over Na2SO4, and evaporated to dryness. The crude product was taken up in DCM and precipitated into hexane. Decanting of solvents followed by drying under vacuum afforded p(Cy-Te) (4, 27.3 mg, 25%). 1H NMR (300 MHz, CDCl3): δ 8.11−7.73 (m, 4H), 4.66−4.57 (m, 4H), 4.22−3.95 (m, 4H), 3.11−2.96 (m, 4H), 1.81−1.05 (m, 40H). 13C NMR (75 MHz, CDCl3): δ 178, 173, 130, 110, 61, 53, 32, 29, 26, 25. IR: ν 2929 (s), 2855 (s), 1720 (s), 1605 (w), 1501 (s), 1448 (w), 1368 (w), 1264 (s), 1249 (s), 1103 (s), 1018 (w), 989 (w), 894 (s), 731 (s). Poly(IP-co-adipate) (p(IP-Ad)), 5. Diisopropylamine (2.7 mL, 19 mmol) was added slowly to a solution of TCC (0.3 mL, 2.4 mmol) in DCM (25 mL) and the mixture was stirred overnight at rt. The mixture was then washed with 1 M HCl (15 mL) and brine (15 mL), dried over Na2SO4, and evaporated to dryness. The crude product was dissolved in CHCl3 (12 mL) and charged with TEA (1.0 mL). A solution of diethanolamine (606 mg, 5.8 mmol) in CHCl3 (3 mL) was then added. After stirring the mixture overnight at rt, TEA (4.2 mL), adipoyl chloride (1.9 mL, 13 mmol), and CHCl3 (5 mL) were added to the solution. TEA (1.0 mL) was added after 21 h of stirring at rt, and stirring continued for another 3 days. The mixture was then washed with DDW (3 × 10 mL), dried over Na2SO4 and evaporated to dryness. The crude mixture was taken up in DCM and precipitated into diethyl ether. Decanting of solvents followed by drying under vacuum afforded p(IP-Ad) (5, 92 mg, 9%). 1H NMR (300 MHz, CDCl3): δ 4.28−4.13 (m, 4H), 3.83 (dd, J = 12, 6, 4H), 3.64−3.53 (m, 4H), 2.36−2.23 (m, 4H), 1.68−1.61 (m, 4H), 1.31 (d, J = 9, 12H). 13C NMR (75 MHz, CDCl3): δ 173, 168, 119, 117, 63, 62, 55, 52, 47, 45, 38, 34, 33, 27, 25, 22. IR: ν 2953 (s), 1723 (s), 1642 (s), 1507 (s), 1436 (w), 1349 (w), 1183 (w), 1110 (w), 1023 (w), 833 (w), 770 (w). 1,2,3-Tris(diethanolamino)cyclopropenium Chloride (DEA-Cl), 6, and Poly(DEA-Cl-co-adipate) (p(DEA-Ad)), 7. A solution of diethanolamine (520 mg, 4.95 mmol) in DCM (15 mL) and CHCl3 (3 mL) was added to a cooled solution of TCC (0.20 mL, 1.6 mmol) in DCM (2 mL). TEA (0.70 mL, 5.0 mmol) was added and the solution was left to stir at rt for 4 days to afford monomer 6. Pyridine (2.5 mL) was added to the solution followed by adipoyl chloride (0.73 mL, 5.0 mmol), and the resulting mixture was stirred at rt for 24 h. The murky mixture was filtered and the residue was washed with DCM (1 × 50 mL) and DDW (2 × 50 mL). The cake was dried under high vacuum to afford crosslinked p(DEA-Ad) (7, 551 mg, 55%). IR: ν 2950 (s), 1809 (w), 1728 (s), 1600 (s), 1533 (s), 1281 (w), 1168 (w), 1142 (w), 1059 (w), 972 (w), 735 (w), 684 (w). Degradation Studies. The polymers were pressed into tablets of diameter 1.3 cm and shaken in 10 mL of 0.1 M phosphate-buffered saline (PBS) solution (pH 7.4) at 37 °C for 4 weeks. Media were exchanged for fresh PBS at regular intervals (one day, one week, three weeks, and four weeks) to avoid solution saturation, and checked for small molecules by MS-ESI. When the medium was exchanged, the tablets were weighed before and after drying to determine weight loss and swelling capacity. Nanoparticle Analysis. Hydrodynamic diameter and zeta potential were measured on a Malvern Zetasizer Nano ZS. The polymers were dispersed in H2O and diluted to a concentration of 0.1 mg/mL. The reported diameters are the average of at least three measurements, where each measurement comprises 10 acquisitions. The zeta potentials were calculated according to the Smoluchowski approximation. The nanoparticles were analyzed on a Jeol (Jem-1400Plus equipped with Gatan Orius SC 600 camera) transmission electron microscope using negative staining. Briefly, 5 μL of the sample in water was placed on formvar/carbon coated copper grids (200 mesh, EMS), mixed with 5 μL of uranyl acetate (2%) for ∼10 s, excess mix of the sample and stain was gently absorbed, and the grids were air-dried.
polymers with sufficiently hydrophobic components spontaneously form nanoparticles of diameters 260−500 nm with a zeta potential ranging from +9 to 12 mV upon dispersion in aqueous media. Staphylococcus epidermidis growth was inhibited at 25.6 μg/mL polymer concentration.
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EXPERIMENTAL SECTION
Materials. Tetrachlorocyclopropene (TCC), dicyclohexylamine, diethanolamine, triethylamine (TEA), diisopropylamine, terephthaloyl chloride, and adipoyl chloride were all purchased from SigmaAldrich (Rehovot, Israel). Organic solvents were obtained from BioLab Ltd. (Jerusalem, Israel). General. Chemical reactions were performed in oven-dried glassware under Ar gas. 1H and 13C NMR spectra were obtained on a Varian 300 MHz spectrometer with CDCl3 as the solvent and tetramethylsilane as shift reference. Mass spectrometry electrospray ionization (MS-ESI) was recorded on a ThermoQuest, Finnigan LCQ-Duo instrument in the positive ionization mode. Fourier transform infrared (FTIR) analysis was performed using a Smart iTR ATR sampling accessory for a Nicolet iS10 spectrometer with a diamond crystal. Viscosity measurements were performed on a Cannon SimpleVIS portable viscometer, and dilute polymer solutions were compared to the pure solvent as reference. Synthesis. 1-Chloro-2,3-bis(dicyclohexylamino)cyclopropenium Chloride (BACCy), 1. Dicyclohexylamine (9.8 mL, 49 mmol) was added slowly to an ice-bath cooled solution of TCC (1.0 mL, 8.2 mmol) in dichloromethane (DCM, 200 mL). The reaction mixture was stirred overnight and rise to room temperature (rt). The solids were filtered off and the filtrate was washed with 1 M HCl (3 × 50 mL) and brine (1 × 50 mL). The organic layer was dried over Na2SO4 and evaporated to dryness. The crude product was triturated 4× with hot ethyl acetate, and the off-white solid was dried under high vacuum to afford 1-chloro-2,3-bis(dicyclohexylamino)cyclopropenium chloride (BACCy) (1, 3.8 g, 8.2 mmol, quantitative yield). Spectral data were consistent with the literature.20 1-Diethanolamino-2,3-bis(dicyclohexylamino)cyclopropenium Chloride (CY-OH), 2. TEA (3.4 mL, 24 mmol) and diethanolamine (1.8 g, 17 mmol) in chloroform (CHCl3, 10 mL) were added to a solution of BACCy (1, 3.8 g, 8.2 mmol) in CHCl3 (40 mL) and the reaction mixture was left to stir at rt overnight. The solution was then washed with brine (3 × 10 mL), dried over Na2SO4, and evaporated to dryness to afford CY-OH (2, 3.0 g, 5.6 mmol, 69%). Spectral data were consistent with the literature.22 Poly(CY-co-adipate) (p(CY-Ad)), 3. The polymer was synthesized via a multistep process. TEA (2.3 mL, 16 mmol) and adipoyl chloride (0.20 mL, 1.4 mmol) were added to a solution of CY-OH (2, 1.5 g, 2.8 mmol) in CHCl3 (15 mL). After overnight stirring at rt, additional TEA (1.2 mL, 8.3 mmol) and adipoyl chloride (0.1 mL, 0.7 mmol) were added. After overnight stirring at rt, additional CHCl3 (2 mL), TEA (0.6 mL, 4.2 mmol), and adipoyl chloride (0.05 mL, 0.3 mmol) were added. After 72 h of stirring at rt, CHCl3 (5 mL), TEA (0.3 mL, 2.2 mmol), and adipoyl chloride (0.5 mL of 5% solution in CHCl3, 0.2 mmol) were added. After overnight stirring at rt, TEA (0.15 mL, 1.1 mmol) and adipoyl chloride (0.25 mL of 5% solution in CHCl3, 0.09 mmol) were added. After overnight stirring at rt, TEA (0.3 mL, 2.2 mmol) and adipoyl chloride (0.1 mL, 0.7 mmol) were added. The reaction was finally left to stir for 5 days at rt, filtered, diluted with CHCl3, washed with H2O (3 × 10 mL), and brine (1 × 10 mL). The organic layer was dried over Na2SO4 and evaporated to dryness. The crude product was dissolved in CHCl3 and precipitated into n-hexane. Decantation of solvents and drying under high vacuum afforded p(CY-Ad) (3, 0.86 g, 48%).1H NMR (300 MHz, CDCl3): δ 4.33− 4.22 (m, 4 H), 3.89 (br s, 4 H), 3.68−3.60 (m, 4 H), 3.33 (br s, 4 H), 2.25 (br s, 8 H), 1.91−1.03 (m, 40 H). 13C NMR (75 MHz, CDCl3): δ 178, 173, 172, 120, 71, 66, 61, 60, 58, 54, 51, 32, 26, 25. IR: ν 3369 (w), 2926 (s), 2854 (s), 1733 (s), 1500 (s), 1448 (m), 1370 (w), 1346 (w), 1249 (w), 1227 (w), 1145 (w), 1112 (w), 1075 (w), 1017 (w), 894 (m), 748 (s), 658 (w). B
DOI: 10.1021/acs.macromol.9b00430 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules Scheme 1. Synthesis of p(CY-Ad) (3)a
(a) Dicyclohexylamine, DCM, 0 °C → rt, overnight; (b) diethanolamine, TEA, CHCl3, rt, overnight; (c) adipoyl chloride, TEA, rt, 12 days stepwise.
a
Scheme 2. Synthesis of p(CY-Te) (4)
Antibacterial Studies. S. epidermidis and E. coli 8957 were grown in Lambda Broth (LB). Working suspensions were then prepared by dilution with LB in order to reach to the initial optical density (O.D.) of 0.1 at 600 nm. Next, three solutions of p(CY-Ad) (3) were prepared from solutions of 3.2 mg/mL in dimethyl sulfoxide and dilution in LB growth medium: 25.6, 8.5, and 2.8 μg/mL. Using an automated dispenser, 50 μL of the working bacteria suspension was added to flat bottom wells of a microtiter plate (96-well flat bottom plate, Nunc.). Then, 200 μL of the appropriate polymer solution was added. Solutions of doxycycline (3.2 μg/mL) as the positive control and LB medium as the negative control were tested in parallel, and each test was performed in triplicate. The remaining wells of the plate were filled with 250 μL of the LB medium only. The microtiter plate was incubated at 37 °C for 22 h. O.D. was measured at 600 nm every 2 h using a spectrophotometer (UV−vis detector, Tecan Spark 10M instrument).
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To vary the polyester backbone, we exchanged the aliphatic adipoyl chloride for the aromatic terephthaloyl chloride, forming poly(CY-co-terephthalate), (p(CY-Te), 4) (Scheme 2). To exchange the amino substituents on the cyclopropenium ring, we synthesized an analogous bifunctional monomer to compound 2 by substituting cyclopropenium with diisopropylamine,20 instead of dicyclohexylamine. Poly(IP-co-adipate) (p(IP-Ad), 5) was then synthesized in an analogous pathway to that of p(CY-Ad), 3 (Scheme 3). Scheme 3. Synthesis of p(IP-Ad) (5)
RESULTS AND DISCUSSION
Synthesis of Cyclopropenium Polyesters. A series of hydrolytically degradable cyclopropenium polymers was synthesized via a stepwise polymerization between a bifunctional cyclopropenium monomer and a diacyl chloride to obtain a polyester. To achieve the first bifunctional cyclopropenium monomer, we substituted two of the three positions of the cyclopropenium ring with dicyclohexylamine to give bisaminocyclopropenium chloride BACCy (1).23 Dicyclohexylamine is sterically bulky, which prevented trissubstitution of the cyclopropenium ring and left one position available for further modification. We then used excess diethanolamine to substitute the third position of the cyclopropenium ring, yielding 1-diethanolamine-2,3(bisdicyclohexylamino)cyclopropenium chloride (CY-OH, 2) as the primary diol monomer.22 The diol monomer was reacted with adipoyl chloride to form poly(CY-co-adipate) (p(CY-Ad), 3) via stepwise polymerization (Scheme 1). Several analogues of polymer 3 were obtained by modifying either (i) the polyester backbone, (ii) the bisamino cyclopropenium substituents, or (iii) the connectivity of the polymer via crosslinking.
In a recently published article, we described using cyclopropenium as a chemical crosslinker of poly(ethylenimine) (PEI), thereby introducing a permanent, pH-independent cationic component to the polymer.21 Integration of a permanent cation throughout crosslinked PEI may afford advantages in biomedicine,24,25 but applicability is limited due to a lack of biodegradability of the PEI backbone. Here, we incorporated cyclopropenium at the monomer level to allow for flexibility in choosing the polymer backbone, in this case a crosslinked network of biodegradable ester bonds. A hexafunctional cyclopropenium monomer was synthesized by reacting TCC with excess diethanolamine, affording tris(diethanolamine)cyclopropenium chloride (DEA-Cl, 6). This monomer reacted at all six hydroxyl positions with adipoyl C
DOI: 10.1021/acs.macromol.9b00430 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules Scheme 4. Synthesis of p(DEA-Ad) (7)a
a
(a) Diethanolamine, TEA, DCM/CHCl3, 4 days, rt; (b) pyridine, adipoyl chloride (3:1 adipoyl chloride/6), rt, 24 h.
Figure 1. 1H NMR spectra of CY-OH (2) and p(CY-Ad) (3) with peak assignments. Downfield shift and broadening of protons C and D were evidence of ester formation on the diethanolamine backbone.
Figure 2. 13C NMR spectra of CY-OH (2) and p(CY-Ad) (3) with peak assignments. A new carbonyl shift (C9) was observed for the polymer (3), and C2 shifted downfield, confirming formation of ester linkage.
chloride to afford a polymer with a high degree of crosslinking, poly(DEA-co-adipate), (p(DEA-Ad), 7) (Scheme 4). Polymer Characterization. Formation of linear polyesters 3−5 was confirmed spectroscopically by 1H and 13C NMR. IR spectroscopy was used to visualize the ester linkages along the polymer backbone by characteristic strong ester bends ranging between 1733 and 1720 cm−1. Linear polymer molecular weights (MWs) were calculated by measuring the intrinsic viscosity as an alternative to standard tools such as size-
exclusion chromatography, because of the drawback of charged polymers adhering to the chromatography columns, rendering them unsuitable to our means.20 Below is an example of the spectroscopic characterization of p(CY-Ad) (3). Spectra for compounds 4, 5, and 7 are available in the Supporting Information. 1 H NMR. Polyester formation was confirmed by 1H NMR (Figure 1) by observing a downfield chemical shift of the CH2 groups of diethanolamine (Hc) in CY-OH (2) upon D
DOI: 10.1021/acs.macromol.9b00430 Macromolecules XXXX, XXX, XXX−XXX
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Figure 3. FT-IR spectra of CY-OH (2) and p(CY-Ad) (3) confirmed ester bond formation. A characteristic ester peak at 1733 cm−1 was observed for polyester 3, as well as the disappearance of the characteristic alcohol peak at 3316 cm−1.
Table 1. Chemical Properties of Cyclopropenium Polyesters Polymer 3 4 5 7
Architecture linear linear linear crosslink
Backbone adipate terephthalate adipate adipate
Cyclopropenium Amine Substituent cyclohexyl cyclohexyl isopropyl N/A
MW (kDa)a 10.4 11.5 16.6 N/A
DH (nm)b 260 ± 20 510 ± 20 N/A N/A
ζ potential (mV)b 12 ± 1 9±3 N/A N/A
a
Viscosity average molecular weight was estimated by measuring polymers’ intrinsic viscosity by dilute solution viscometry and calculated using Mark−Houwink parameters for similar compounds. bHydrodynamic diameter and zeta potential were measured at a concentration of 0.1 mg/mL in aqueous media.
The reaction of a diol with a diacyl acid is useful for forming a biodegradable backbone,26,27 but step-growth polymerizations are not conducive to synthesizing high-molecular weight polymers.28 Polymers 3−5 represent the first hydrolytically degradable cyclopropenium polymers with a MW above 10 kDa. Degradation Studies. We compared the degradation profiles of linear (p(CY-Ad) (3)) and crosslinked (p(DEA-Ad) (7)) cyclopropenium polyesters under physiological conditions to explore their applicability to medicine and healing.1 Biodegradation of a polymer eliminates the need for the polymer to be removed from the body, so it is a crucial characteristic for in vivo applications such as drug delivery. A polyester powder was pressed into a tablet of diameter 1.3 cm and exposed to the representative physiological conditions, which include maintaining physiological pH (with PBS at pH 7.4) and temperature (37 °C) in aqueous solution. In just over one week, 50% of linear polymer 3 mass degraded to a constituent monomer (Figure 4A), and after four weeks about 75% of the polymer mass was degraded (Figure 4B). Weight loss of crosslinked polymer 7 was slightly more rapid than linear polymer 3, with over 50% of the polymer mass lost to the aqueous medium after only four days and 78% lost after four weeks at the representative physiological conditions. Detection of degradation products by MS-ESI showed that both polymers degraded via hydrolysis of the ester bonds, affording the corresponding monomeric cyclopropenium. The increased rate of weight loss of crosslinked polymer 7 may be due to the relative hydrophilicity of its corresponding monomer 6 (Figure 4A). While the linear polymer degraded into the relatively hydrophobic monomer 2, monomer 6 is more hydrophilic, hastening polymer weight loss to the aqueous medium. To broaden the applicability of the linear and crosslinked cyclopropenium polyesters as drug delivery systems, we tested
polymerization to form p(CY-Ad) (3), demonstrating the formation of ester bonds. The broadening of these peaks was also indicative of polymer formation. 13 C NMR. In the 13C NMR spectra (Figure 2), the diethanolamine peaks (C1 and C2) were shifted downfield upon polymerization. C2, adjacent to the hydroxyl group in 2, shifted noticeably downfield when the ester bonds were formed as a result of polymerization to 3. Additionally, the appearance of a characteristic carbonyl peak (C9) was consistent with ester bond formation. Infrared spectroscopy. IR spectroscopy (Figure 3) showed the decreasing intensity of the OH band at 3316 cm−1 and the simultaneous appearance of a characteristic strong ester CO band at 1733 cm−1 in 2 upon polymerization to 3. This was consistent with the terminal OH groups of diol monomer 2 having been converted to ester bonds in polyester 3. Crosslinked p(DEA-Ad) (7) was insoluble in all solvents due to its high degree of crosslinking, therefore NMR and viscosity studies could not be performed. A lack of solubility implied a high degree of crosslinking, and ester bond formation was confirmed via IR by a strong ester band at 1728 cm−1. Molecular Weight Determination. The MW of linear polymers 3−5 were estimated using dilute solution viscometry. Viscosity average molecular weight was calculated based on the parameters of two known polymers that model each new polymer. For polymers based on adipate (3, 5), the parameters for poly(styrene sulfonate) (an aromatic polyelectrolyte to model polycyclopropenium) and poly(ethylene adipate) (to model the polyester chain) were used. The estimated viscosity average MW of polymers 3 and 5 were then taken as an average of these values and were determined to be 10.4 and 11.5 kDa, respectively. For polymer 4 based on terephthalate, the parameters of poly(ethylene terephthalate) were used instead and the estimated viscosity average molecular weight was 16.6 kDa (Table 1). E
DOI: 10.1021/acs.macromol.9b00430 Macromolecules XXXX, XXX, XXX−XXX
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Figure 4. Biodegradation and swelling of linear (3) and crosslinked (7) cyclopropenium polyesters. Both polymers degraded into constituent monomers (A). Over the course of the degradation, the polymers’ swelling showed their ability to absorb water as their structures degraded (B), indicating crucial hydrophilicity. Degradation is represented as % polymer remaining (w/w), and swelling as % water absorption (water/polymer wt %).
drug-delivery candidates.31−33 Current drawbacks include a lack of biodegradability and pH-dependent charge,34 both of which are addressed by the cyclopropenium polyester system presented here. At a concentration of 0.1 mg/mL, linear polymer 3 spontaneously formed cationic surface-charged nanoparticles of diameter 260 ± 20 nm and zeta potential +12 ± 1 mV (Table 1). Transmission electron microscopy revealed the spherical morphology of the nanoparticles (Figure 5). High charge density on stable spherical biodegradable nanoparticles offers the potential to overcome major difficulties in drug delivery. Under the same conditions, linear polymer 4 spontaneously formed cationic surface-charged nanoparticles of diameter 510 ± 20 nm and zeta potential +9 ± 3 mV (Table 1). The increased nanoparticle size was likely due to the added bulkiness of the terephthalate rings, as the surface charge remained almost unchanged from polymer 3. Polymer 5, however, did not form nanoparticles in aqueous media, likely due to the loss of hydrophobicity by exchanging cyclohexyl rings for diisopropyl groups. Lacking the large aliphatic substituents, polymer 5 was stable enough in aqueous media that it did not collapse into nanoparticles in order to shield interactions with water. Crosslinked polymer 7 also did not form nanoparticles in water. We attribute this to the high
their swelling under physiological conditions. Swelling indicates hydrophilicity, a critical element of drug delivery systems, as the absorbed water could dissolve an incorporated drug and then release it slowly by diffusion.29 The swelling of the polymers was tested by weighing the tablet throughout the degradation study before drying. The linear polymer showed increased swelling capacity over time (Figure 4B). Once the parent polymer degraded by 75%, it absorbed over 200% of its remaining weight in water. Crosslinked polymer 7 also displayed increased swelling capacity over time, absorbing over 700% of its weight in water after four weeks of hydrolytic degradation due to the nonrigid nature of its crosslinked polymer network and the hydrophilicity of its corresponding monomer, 6.30 As degradation proceeded, the deforming of the crosslinked network and exposure of free hydroxyl groups increased the swelling capacity of the polymer. Indeed, the crosslinked polymer (7) maintained a higher swelling capacity throughout the degradation process compared to linear polyester 3. The polymers’ ability to hydrolytically degrade in pharmaceutically relevant time periods, in addition to their ability to absorb large amounts of water, promotes their potential as drug delivery systems. Nanoparticle Dispersion in Water. Water-dispersible polyelectrolyte nanoparticles have been studied extensively as F
DOI: 10.1021/acs.macromol.9b00430 Macromolecules XXXX, XXX, XXX−XXX
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CONCLUSIONS We report the facile synthesis of the first biodegradable cyclopropenium-containing polymer, and take advantage of the modularity of the synthetic route. A library of polyesters was synthesized by modifying the backbone, side groups, and architecture of the polymers. Some of the polymers form surface-charged cationic nanoparticles, and all polymers degrade hydrolytically in pharmaceutically relevant time periods. The polymers’ swelling capacity further contributes to their potential to be used in drug delivery systems. Furthermore, initial antimicrobial studies showed promising results, opening the door for the development of a new class of potent antimicrobials. This new class of polymers offers potential use as carriers for anionic agents that electrostatically bind to the cationic sites in the polymer. Bioactive agents, including plasmid DNA, siRNA, anionic proteins and small molecules are of particular interest. The polymer carrier is biodegradable and should be eliminated from the body after the drug has been depleted.
Figure 5. Transmission electron microscopy image of p(CY-Ad) (3) in aqueous media shows the fully formed, stable, surface-charged nanoparticle.
degree of crosslinking, preventing collapse into orderly nanoparticles. Antimicrobial Studies. Cationic polymers bearing hydrophobic moieties tend to exhibit strong antimicrobial activity by disrupting the cell membrane and thereby causing cell death.2,13,35 In order to test the potential applicability of our biodegradable cyclopropenium polymers as antibacterial materials, we tested p(CY-Ad) (3) nanoparticles for antimicrobial activity against S. epidermidis and E. coli 8957. S. epidermidis was chosen as a representative species due to its role in causing nosocomial infections and by forming biofilms on medical devices.36 The E. coli strain is not pathogenic. The antibacterial activity of 3 on Gram-positive S. epidermidis was compared to the Gram-negative E. coli 8957 in order to determine selectivity of the antimicrobial activity of the polymer. Indeed, p(CY-Ad) displayed partial bacterial growth inhibition of S. epidermidis at a concentration of 8.5 μg/mL, and completely prevented growth when used at 25.6 μg/mL. No effect was observed at 2.8 μg/mL (Figure 6). p(CY-Ad)
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.9b00430.
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NMR and IR spectra and antimicrobial controls (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (L.M.C.). *E-mail:
[email protected] (A.J.D.). ORCID
Noam Y. Steinman: 0000-0001-6938-6894 Rinat Meir: 0000-0001-6615-0288 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was funded in part by the National Science Foundation (NSF CAREER DMR-1351293), ACS Petroleum Research Fund, and 3 M Non-Tenured Faculty Award.
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REFERENCES
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Figure 6. p(CY-Ad) displayed effective inhibition of bacteria growth (quantified by O.D.) over a period of 22 h. At 2.8 μg/mL, no effect was observed compared to the negative control of bacteria in LB medium. At 8.5 μg/mL, partial inhibition was observed, and at 25.6 μg/mL, total inhibition was observed.
displayed no antibacterial effect on E. coli 8957, likely due to structural differences between the Gram-positive S. epidermidis and Gram-negative E. coli 8957. Positive and negative controls confirmed the polymer’s antibacterial activity as equivalent to commercial doxycycline when the polymer was introduced at 25.6 μg/mL (Figure S7). These results demonstrate a marked improvement on previously reported cationic polymers, which required a concentration of over 100 μg/mL to achieve antibacterial activity on S. epidermidis.37,38 G
DOI: 10.1021/acs.macromol.9b00430 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules
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