Controlled Release of Usnic Acid from Biodegradable Polyesters to

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Controlled Release of Usnic Acid from Biodegradable Polyesters to Inhibit Biofilm Formation Queeny Dasgupta, Giridhar Madras, and Kaushik Chatterjee ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00680 • Publication Date (Web): 05 Dec 2016 Downloaded from http://pubs.acs.org on December 7, 2016

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ACS Biomaterials Science & Engineering

Controlled Release of Usnic Acid from Biodegradable Polyesters to Inhibit Biofilm Formation

Queeny Dasgupta1, Giridhar Madras1,2 , Kaushik Chatterjee 1,3* 1

Centre for Biosystems Science and Engineering, 2Department of Chemical Engineering and 3

Department of Materials Engineering Indian Institute of Science Bangalore 560012, India

*

Corresponding author +91-80-2293 3408; [email protected]

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ABSTRACT Controlled and sustained release of antibacterial drugs is a promising approach to address challenges related to bacterial infections in biomedical implants. Biocompatible polyols like xylitol and mannitol are frequently used to synthesize crosslinked, biodegradable polyesters. Xylitol based adipoyl and sebacoyl polyesters were synthesized by a catalyst free melt polyesterification technique. Unlike traditional drug delivery systems, the objective of this work was to develop biodegradable polymers with usnic acid (UA), a known antibacterial agent, entrapped in the polymer network. Apart from offering a wider control of the release kinetics and improved processability, the hydrolytic degradation results in the concomitant resorption of the polymer. Polymer properties such as degradation, modulus, and drug release were tuned through a subtle change in the chain length of the diacid. In one week, the xylitol based adipoyl ester degrades 41 % and releases 25 % of its initial drug loading whereas the sebacoyl ester degrades 23 % in and releases 9 % of the loaded drug. A kinetic model has been used to understand the UA release profiles and determine degradation and release parameters that influence release from the polymers. These polyesters are cytocompatible and exhibit excellent bactericidal activity against Staphyloccus aureus by inducing oxidative stress. This work enables a strategy to synthesize biodegradable polymers for potential to inhibit biofilm formation in vivo with tunable mechanical and degradation properties, and variable controlled release.

Keywords: Usnic acid; crosslinked polyesters; biodegradable polymers; controlled release; antibacterial.

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1. INTRODUCTION Nosocomial infections comprise a major healthcare burden and are a cause of alarming concern due to its widespread prevalence in the medical industry. Nearly 7 % of patients in developed countries and 10 % in developing countries acquire hospital acquired infections

1 2

.

Most countries lack a proper surveillance system to monitor these infections, thereby, making prevention the only remedy. The burden is particularly high for patients admitted to intensive care units 1. Apart from urinary tract infection, the second major cause of these infections is from surgical site implantations. These implants are extremely prone to hospital acquired infections and may have serious implications on implantation. A major concern of the biomaterials industry is that of biofilm formation on its surface

3, 4

. Various microorganisms may cause the

development of biofilms and, therefore, limit the use of certain potential materials for biomedical applications. These infections are mainly caused by Gram positive bacteria and S. aureus is the chief causative agent in such cases 5-7. The control and inhibition of S. aureus growth, therefore, deserves enormous attention to avert hospital acquired infections. Usnic acid (UA), a natural lichen extract with a dibenzofuran structure, has excellent antimicrobial properties anticancer

12, 13

8, 9

. It is used as an antibacterial, antimycobacterial 10, antifungal

11

and

agent that has led to increased interest in this compound. Due to its antioxidant,

anti-inflammatory properties and activity against Gram positive bacteria, it has also been recognized in wound healing

14, 15

. Nevertheless, its efficiency against biofilm formation

8

has

made it of primary potential interest for biomaterials research. In spite of all these remarkable properties and advantages, the use of UA remains limited due to its low aqueous solubility16 and reported cytotoxicity

17

. The first drawback is overcome by synthesizing UA derivatives that

have more favorable solubility 16 yet maintain the antimicrobial properties of UA. The latter is a 2 ACS Paragon Plus Environment


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more serious concern and is usually dealt by using low drug levels that do not disrupt hepatocyte function 18. Several drug delivery systems have been devised for delivering UA. These drug delivery systems include microcapsules19, magnetic nanoparticles

15, 23

20

and nanocapsules,18 polymer colloids,21 nanofluids 22,

etc. Encapsulation of UA in these polymeric vehicles enables the

drug to take advantage of their controlled release properties. UA has been encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres for use as a chemotherapeutic agent

24

.

Poly(vinylbenzyl chloride) based antimicrobial polymer colloids have been synthesized to enable uniform dispersion of UA and, thereby, increase bactericidal activity 21. Core/shell nanoparticle systems with Fe3O4/ Oleic acid/ UA have been reported to prevent biofilm formation by S. aureus 22. The low solubility of the drug typically makes high drug loading a challenge. It is, therefore, critical to engineer systems for controlled and targeted delivery of UA. As explained in a recent review,25 the incorporation of a bioactive moiety in the polymer backbone improves processability, stability and provides controlled release of the drug. In the present study, we have entrapped UA into a crosslinked polymer network. This is the first study that investigates the entrapping of UA in a network structure and its subsequent release. The polymeric carriers are xylitol based crosslinked polyesters, which are promising candidates for use as resorbable biomaterials

26

and can be biofunctionalized

27

. Xylitol is a

pentafunctional alcohol that can be easily metabolized and the diacid precursors of the polyesters are non-toxic. Combinatorial techniques have been devised earlier to control several properties like cell-material interaction,28 polymer degradation and release 26.


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The objectives of this study are three fold: (a) to synthesize crosslinked polyesters with UA entrapped in its network; (b) to attain a controlled release UA delivery system and (c) to check its pharmacological stability and antimicrobial activity when released from the crosslinked polyester. Two UA incorporated polyesters were synthesized using two different diacids, namely adipic and sebacic acid. This variation in the diacid precursor allowed us to understand the effect of the monomer chemistry on the physicochemical properties of polymers. The incorporation of UA into polyesters ensures that upon hydrolysis, the ester bonds would break and free the drug entrapped in its network. This hydrolysis would simultaneously degrade the polymer and make it a useful resorbable biomaterial. The cytocompatibility and antibacterial activity of this drug was also assessed for potential biomedical applications. 2. EXPERIMENTAL 2.1 Materials The chemicals, (+)-usnic acid (UA); adipoyl chloride (AC), sebacoyl chloride (SC); and xylitol were all procured from Sigma Aldrich. Solvents used at various stages of the work include pyridine, ethyl acetate (EtoAc), chloroform, dimethylsulfoxide (DMSO) and N,N-dimethylformamide (DMF), tetrahydrofuran (THF) and acetone (all from Merck, India). 2.2 Synthesis Step I: Synthesis of UA based adipoyl/sebacoyl diester UA (3.0 g, 8.7 mmol) was dissolved in anhydrous chloroform (35 mL). AC (2.37 g, 13 mmol) was added drop-wise to the stirred solution of UA in CHCl3 at 0 ˚C (over ice). UA and AC are taken in a molar ratio of 2:3. The actual reaction stoichiometry (UA: AC) is 2:1, wherein



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2 units of UA react with 1 unit of AC, and thus AC is taken in excess. Immediately, after the addition of AC, pyridine (6.3 mL, 78 mmol) was added to the reaction mixture. The reaction was conducted for 5 h. The reaction mixture was washed with water to remove unreacted diacid and pyridine. The product was obtained by partitioning the mixture into chloroform and water. The extracted organic layer was washed with brine and dried over anhydrous Na2SO4. The product (UA4_diester) obtained was dried in a rotary evaporator at 40 ˚C. The yield of UA4_diester was 62 %. A similar procedure was used wherein SC (3.11 g, 13 mmol) was added instead of AC to yield the corresponding UA8_diester (yield: 51 %). The molecular weight of the diester was verified by mass spectrometry based on direct injection with water as eluent as discussed in section 2.3.3. Step II: Transesterification of diester with xylitol The reagents, diester (UA4_diester or UA8_diester) and xylitol were mixed in equimolar concentrations in a round bottomed (RB) flask. Melt esterification polymerization was performed by melting the two precursors at 120 ˚C under nitrogen atmosphere and with continuous stirring for 2 h (Schematic 1). In the post-polymerization step, the prepolymer was cured at 120 ˚C under vacuum (60 mm Hg) for 6 days to obtain UA entrapped crosslinked polyester networks. The polyesters are named as PX4UA and PX8UA where PX4 and PX8 indicate poly(xylitol adipate) and poly(xylitol sebacate), respectively. The numbers represent the number of methylene (−CH2) units between the carboxylic acid groups in AC and SC, respectively.


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2.3 Materials characterization 2.3.1 FTIR spectroscopy FTIR spectra were recorded for chemical characterization of synthesized polyesters. Perkin-Elmer Frontier FT-NIR/MIR spectrometer was used to record FTIR spectra of samples. The final spectrum was taken as an average of 32 scans with a resolution of 4 cm−1 over the range 4000-650 cm−1. Universal attenuated total reflectance (uATR-FTIR) mode was used and the samples were placed on the uATR solid sample holder. 2.3.2 NMR spectroscopy Proton and 13C nuclear magnetic resonance (NMR) spectroscopy were used for chemical characterization. The spectra were recorded on a 400 MHz Bruker Avance NMR spectrometer between a range of 0−20 and 0−200 ppm for 1H and

13

C, respectively. NMR spectroscopy was

performed only on the soluble prepolymers, since the cured polyesters were insoluble in any solvent. 2 mg of the prepolymer was dissolved in 500 µL of CDCl3 with 0.03 % (v/v) tetramethylsilane as internal standard (Deutero, Germany). 2.3.3 LC-ESI Mass spectroscopy Mass spectra were obtained on a MicroMass ESI-TOF mass spectrometer (Waters Inc.). The samples were introduced into the column by direct injection using an autosampler with water as the mobile phase. A cone voltage of 140 V was used during the analysis.



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2.3.4 MALDI spectroscopy The prepolymer molecular weights were obtained by MALDI spectroscopy on an UltrafleXtreme MALDI TOF/TOF (Bruker Daltonics). 2 mg of the prepolymer samples were dissolved in 2 mL of 1:3 DMF: acetonitrile and the molecular weights were estimated. 2.3.5 Measurement of swelling ratio and gel content Crosslinking density was calculated by determining the swelling ratio. Swellingdeswelling studies of these polyesters were performed to enumerate swelling ratio (S). Swelling ratio was obtained by immersing polymer discs (diameter 5 mm and thickness 1 mm) in nonsolvent (hexane) at 37 ˚C and allowed to swell to attain constant weight. The samples were then dried at 37 ˚C until no further weight loss occurred. The swelling ratio (S in %) is: % =

×

(1)

In equation (1), Ws is the sample weight after swelling in hexane and Wd is the polymer weight after drying. The gel content indicates the fraction of the polymer that cannot be extracted by a solvent while the sol content represents the remaining fraction. The degree of crosslinking is directly proportional to the % gel content and is calculated using the standard protocol (as per ASTM standard D-2765). The polymer discs were initially dried in a vacuum desiccator and the weight of the polyester discs was measured (Wd1). The discs were then immersed and allowed to swell in N,N-DMF for 24 h until constant weight was achieved. The polymers were subsequently removed from DMF and dried to constant weight under vacuum (Wd2). The sol content was evaluated as: 7 ACS Paragon Plus Environment

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% = (

× )

(2)

2.3.6 Dynamic mechanical Analysis Dynamic mechanical analysis (DMA) was used for mechanical characterization of the samples. The mechanical properties were analyzed on a TA Instruments Q800 DMA. An isothermal (37 ˚C) frequency sweep ranging from 1 to 100 Hz with fixed amplitude/strain of 15 µm was applied to the polyester samples of rectangular geometry (dimensions: 30 mm × 5 mm× 1 mm) using the film tension clamp. 2.3.7 Thermogravimetric analysis Thermal degradation was performed with 10 mg of the sample at a constant heating rate of 5 ˚C/ min on a TG/DTA instrument (Perkin Elmer S11 Pyris Diamond) in an inert (nitrogen) environment up to 700 ˚C. 2.3.8 Differential scanning calorimetry The glass transition of the crosslinked polyesters was evaluated by a differential scanning calorimeter (DSC, TA Instruments, Q2000). Polyester samples weighing 3-5 mg were crimped in a copper pan. The samples were then subjected to a uniform temperature program ranging from −50 to 150 ˚C at a temperature ramp of 5 ˚C/ min. The thermal transitions were noted over the stated temperature range. 2.3.9 Hydrolytic degradation studies These studies are typically polymer weight loss studies to account for ester hydrolysis of the polymer under physiological conditions. Polymer films (1 mm thickness) were fabricated by



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drop casting the melted prepolymer and curing at 130 ˚C for six days. Polymeric discs (4.5 mm diameter) were punched from these films. These discs were placed in nylon mesh pouches (prepared by closing three sides and leaving a side open) and immersed in tubes containing 20 mL of phosphate buffered saline (PBS) to replicate physiological conditions (pH 7.4 and 37 ˚C). These tubes were placed in an incubator shaker at 100 rpm. The polymer pouches were removed from the buffer at specific time intervals (t), dried and weighed to measure weight loss. The following equation was used for measuring weight loss % =

×

(3)

In equation (3), Mo and Mt denote initial and final weights of the polymers, respectively. The effect of pH on hydrolytic degradation was also studied to evaluate polymer properties under varying acidic (e.g. stomach and GI tract) or basic (e.g. wounds) conditions in the body. Similar degradation studies were performed in phosphate buffered saline of pH of 3.2 and 9.0, respectively. The behavior of the polyesters were also studied in the absence of buffer (in deionized water) to account for the effect of degradation mediated pH change on subsequent degradation. 2.3.10 UA release studies Release studies of UA were performed to understand its release kinetics from the polyesters. Polymeric discs for release studies of UA were punched from 1 mm thick cured polymer films, as described above for hydrolytic degradation. Punched discs (4.5 mm diameter) were placed in nylon mesh bags, submerged in PBS (pH 7.4) and maintained at 37 ˚C. The release concentration of UA was determined by measuring its absorbance in a UV-vis


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spectrophotometer (Shimadzu, UV-1700 PharmaSpec). The characteristic absorption peaks of UA include a sharp peak at 244 nm, a broad peak at 278 nm and a prominent shoulder at 337 nm. The UA concentration calculations were made with respect to the calibration curve based on the absorbance at 337 nm. The total loading concentration was evaluated by dissolving discs in PBS of pH 9.0 and measuring the absorbance. The products of hydrolytic degradation of PX4 and PX8 have been characterized previously26 in detail to check for their absorbance at 337 nm. It has been observed that these products do not exhibit any absorbance at 337 nm, confirming that the absorbance is only due to the UA released. The UV spectrum was verified at every time point to ensure that the released drug is UA. The release of UA was also confirmed by mass spectrometry as discussed in section 2.3.3. The mass spectrometry showed the presence of UA (344 Da) and other short oligomers. 2.3.11 Cytocompatibility studies Three different types of cells were used to assess the cytocompatibility of the synthesized polyesters. Two cell lines, namely, NIH3T3 mouse embryonic fibroblasts (ATCC, USA) and HeLa cervical epithelial carcinoma cells (ATCC, USA) were used. These are robust cells commonly used for cytotoxicity experiments. Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Life Technologies) augmented with 10 % (v/v) fetal bovine serum (FBS, Gibco, Life Technologies) and antibiotics (1 % (v/v) penicillin-streptomycin) was used as growth medium and cells were grown at 37 ˚C in a 5% CO2 incubator. In addition, cytotoxicity was also evaluated against primary human mesenchymal stem cells (hMSCs, Stempeutics, India) derived from bone marrow of a 25 year old male donor. hMSCs were cultured in Knockout Dulbecco’s modified Eagle’s medium (koDMEM, Gibco, Life Technologies) augmented with 15 % (v/v)



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MSC-qualified fetal bovine serum (MSC-FBS, Gibco, Life Technologies) having 1 % (v/v) antibiotic-antimycotic supplemented 1 % (v/v) fibroblast growth factor (FGF). Cytocompatibility was evaluated using an indirect method by treating cells with conditioned medium. For conditioning of medium, 100 mg discs of PX4, PX8, PX4UA and PX8UA (ethanol/ UV sterilized discs) were degraded for 24 h in 5 mL complete culture medium (designated as Set I). 5×103 cells were seeded per well in a 48-well plate in fresh medium for 6 h. After initial cell attachment was achieved, the medium was replaced with 500 µL of preconditioned media in each well. Fresh medium was used as the control. Cells were cultured in this pre-conditioned medium for 1 day and 3 days to test cytocompatibility. The medium, fresh or conditioned, was renewed after every 24 h with the same fresh conditioned medium. The polymers PX4 and PX8 (without UA) were also used as controls to assess cytocompatibility. Independently, we allowed the polyester discs to degrade for 48 h in complete medium. This experiment was designed to assess the effect of higher UA concentration on the cells. Two parallel sets were set up. In one set the cells were treated with this 48 h conditioned medium (designated as Set II) and the second, where the cells were first exposed to the 24 h conditioned medium on day 1 and then treated with the 48 h conditioned medium on day 2 (designated as Set III). The latter was designed to mimic physiological conditions wherein minimal accumulation of the degradation products is expected as they will likely be continuously removed. After treatment in conditioned media, cell viability was evaluated after 1 day and 3 days using the WST-1 (water soluble tetrazolium salts, Roche) kit. WST-1 reagent indicates cell viability by producing a water soluble formazan product by the action of oxidoreductases present only in viable cells. For the cell proliferation assay, 20 µL of WST-1 was diluted in 200 µL


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DMEM complete medium. This solution (220 µL) was added to each of the wells and kept undisturbed at 37 ˚C in 5% CO2 for 4 h. The formation of water soluble formazan crystals was quantified by absorbance readouts at 440 nm in a well plate reader (Synergy HT, Biotek). The absorbance value corresponds directly to the number of cells in the well. The absorbance values from six independent wells were averaged to obtain the final absorbance. The data are presented as mean ± S.D. for n= 6. Analysis of variance (ANOVA) with Tukey’s test for p

Controlled Release of Usnic Acid from Biodegradable Polyesters to

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