Modulated in Vitro Biocompatibility of a Unique Cross-Linked Salicylic

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Modulated in vitro biocompatibility of unique crosslinked salicyclic acid-poly (#-caprolactone) based biodegradable polymer Nitu Bhaskar, Nagarajan Padmavathy, Shubham Jain, Suryasarathi Bose, and Bikramjit Basu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10711 • Publication Date (Web): 11 Oct 2016 Downloaded from http://pubs.acs.org on October 12, 2016

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ACS Applied Materials & Interfaces

Modulated In Vitro Biocompatibility of a Unique Crosslinked Salicylic Acid-Poly (Ɛ-Caprolactone) Based Biodegradable Polymer

Nitu Bhaskara#, Nagarajan Padmavathyb#, Shubham Jaina, Suryasarathi Boseb*, Bikramjit Basua, c,*

Author Address a

Laboratory for Biomaterials, Materials Research Centre, Indian Institute of Science, Bangalore-560012, India b Department of Materials Engineering Indian Institute of Science, Bangalore-560012, India c Center for Biosystems Science and Engineering, Indian Institute of Science, Bangalore560012, India

# *

Nitu Bhaskar and Nagarajaan Padmavathy have contributed equally for the present work. Corresponding author: E-mail: [email protected], E-mail: [email protected].

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Abstract Herein, we report the development of a unique architecture by chemically crosslinking the salicylic acid (SA) based poly (anhydride-ester) onto a biodegradable amine functionalized polycaprolactone (PCL), using lactic acid as a spacer. The ester and amide linkages in the SA-PCL polymer, synthesized through melt condensation, were confirmed by NMR and FTIR spectroscopic techniques. The enzymatic and non-enzymatic hydrolytic degradation profile exhibited linear degradation kinetics over extended time period (>5 weeks). The compatibility and growth of C2C12 myoblast cells were found to be significantly improved on the fast degrading SA-PCL substrates as compared to that over neat PCL and amine functionalized PCL. Further, the decreased RBC (red blood cell) damage, illustrated by 0.4% haemolysis activity and minimal number of platelet adhesion on SA-PCL polymeric surface confirmed good hemocompatibility of as-synthesized polymer. Together with moderate bactericidal property, the spectrum of properties of this novel polymer can be attributed to the synergistic effect of the presence of chemical moieties of salicylic acid and amine groups in PCL. To sum up, it is considered that SA-PCL based crosslinked composite can be utilized as a new biodegradable polymer. Keywords: Biocompatibility; Degradation; Polycaprolactone; Polymer, Salicylic Acid


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1. Introduction In recent years, biodegradable synthetic polymers have made significant impact in various fields of biomedical applications, especially in tissue engineering, gene therapy and also as controlled drug delivery systems 1-3 4 5 . The synthetic polymers offer greater advantages than natural polymers, because their chemistry can be tailored to give a wider range of physicochemical properties. Together with tunable biodegradation rate, such properties are useful to minimize the foreign body response mediated by surrounding tissue 2. Among various synthetic biodegradable polymers, special efforts have been emphasised on modification of Polycaprolactone (PCL) due to its broad spectrum compatibility with a wide range of other polymers6. PCL is thermoplastic semi-crystalline polyester and is characterized by low melting point (55°-60°C), low glass transition temperature (Tg ~ 60°C) and high thermal degradation temperature of 350°C7-8. Although PCL has excellent biocompatibility and processability, but it has limited cell-material interaction. PCL also exhibits slow degradation kinetics due to its hydrophobic and low water absorbing captivity 9. Since PCL degrades at a very slow rate, its use has been restricted only for developing a long-term drug/vaccine delivery vehicle and in other pharmaceutical and biomedical applications8. However, under certain circumstances, enzymes can easily degrade PCL10-11. Several techniques such as copolymerization and physical blending, are being employed and those can improve the final properties (degradation rate and mechanical properties) of native polymer (PCL)

10, 12-13

. For example, Chiari et al.14 and Mondrinos et al. 15 have developed a

composite of PCL with hyaluronic acid and calcium phosphate. Similarly, an electrospun copolymer of Poly(Lactide-co-Glycolide)PLGA/PCL and poly (D,L-lactic acid) (PDLLA)/ PCL blends with good biocompatibility were also developed 16-17. Since polymer modification approach can be adopted to acquire a predefined range of properties, we have made an attempt to design a unique crosslinked polymer based on a 3 ACS Paragon Plus Environment


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specific hypothesis in the present work. This hypothesis is based on the fact that by conjugating salicylic acid (SA) based poly (anhydride-ester) onto a biodegradable amine functionalized polycaprolactone (PCL), using lactic acid as a spacer with such unique design, the problem of hydrophobicity and much slower degradation of native PCL can be controlled. A number of studies have reported the synthesis of salicylic acid based polymers admixtures of SA-based bioactive poly(anhydride esters) and PLGA

18-20

and

21

. The controlled

release of salicylic acid, which is widely classified as non-steroidal anti-inflammatory drugs (NSAIDs), at local site reduces systemic drug absorption and favours the therapeutic efficacy by lowering the probability of drug loss22. Our laboratory have also synthesized a SA-based polymers based on sebacoyl chloride and mannitol and this polymer exhibit good cell compatibility in vitro with C2C12 murine myoblast cells

23

. Further validation of

biocompatibility through in vivo study in a subcutaneous mouse model over 16 weeks showed favourable tissue response with less inflammation, increased angiogenesis and uniform yet well-vascularized capsule around the implanted SA-based polymer Johnson and Uhrich

22

24

. Further,

, while working with salicylate-based poly(anhydride-esters) loaded

with antimicrobials, mentioned that such degradable SA-based polymers as a drug carrier is advantageous because it itself will degrade into active drug molecules, providing localized pain relief and anti-inflammatory effects. Therefore, the rationale behind the present article is to design a novel unique cross-linked polymeric system by cross-linking SA-PCL with lactic acid, which has not been reported yet so far for any biomedical and/or tissue engineering applications, to the best of our knowledge. Furthermore, we expect that the synthesized polymer cross-linked SA-PCL polymer would exhibit an enhanced cytocompatibility, hemocompatibility and antibacterial property, in vitro, enabling their broad spectrum applications for a wide range of pharmaceutical and biomedical fields. 4 ACS Paragon Plus Environment

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2. Materials and Methods 2.1. Materials Salicylic acid (CAS No. 69-72-7) and pyridine (CAS No. 110-86-1) were obtained from Merck Millipore, India, whereas adipoyl chloride (CAS No. 111-50-2) and lactic acid (CAS No.50-21-5)

were procured from Sd-Fine Chem Limited, India. Polycaprolactone

(Mw=80,000 g/mol; CAS No. 24980-41-4), N,N′-dicyclohexylcarbodiimide (DCC; CAS No. 538-75-0), ethylene diamine (CAS No. 107-15-3) and Pseudomonas cepacia lipase (CAS No. 9001-62-1) were obtained from Sigma Aldrich, India. 4-dimethyl amino pyridine (DMAP; CAS No. 1122-55-3), ethylenediamine (EDA; CAS No. 107-15-3), tetrahydrofuran (THF; CAS No. 109-99-9), acetone (CAS No. 67-64-1) and chloroform (CAS No. 67-66-3) were purchased from commercial sources. All the chemicals and reagents were of analytical grade and were used as received. 2.2. Polymer synthesis Melt condensation technique was employed to synthesize salicylic acid-polycaprolactone based polymer (SA-PCL) in three stages. The diacid synthesis is the first step involved the synthesis Scheme I, according to the methods previously reported elsewhere

25

. Typically,

Every 1.2 g salicylic acid was dissolved in 9 mL of pyridine under inert atmosphere. Half the molar ratio as of salicylic acid, adipoyl chloride was added to the above mixture at 0°C. The reaction mixture was allowed to stir for 4 h at room temperature, followed by acidification with hydrochloric acid (HCl; CAS No. 76-47-01-0) over crushed ice untill the pH reached 12. The filtered white powder (diacid) was dried under vacuum and analyzed by fourier transform infrared (FT-IR) and nuclear magnetic resonance (1H-NMR) spectroscopy. By refluxing the product with excess acetic anhydride, the diacid was activated which was followed by distillation of acetic anhydride yielding acetylated diacid.



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In the second step, aminolysis of PCL followed by coupling reaction of aminolyzed PCL and lactic acid were carried out. Essentially, 10% PCL (w/v) was dissolved in a mixture of 40% ethylenediamine (EDA), 30% acetone, and 30% chloroform

26

. In a 60°C pre-heated water

bath, the PCL-mixture was sonicated for two hours. The PCL mixture was incubated at room temperature up to 24 h and then centrifuged to obtain a precipitate. The aminolyzed PCL precipitate was next washed with distilled H2O before being air-dried and weighed. The coupling reaction was then carried out between aminolyzed PCL and lactic acid (Figure 1b). Equimolar lactic acid and aminolyzed PCL were dissolved in THF and stirred for 1h for thorough mixing. Then, 1 mmol each of DCC and DMAP were added and stirred for 3 h. The reaction was stopped by adding cold diethyl-ether (CAS No. 60-29-7) and precipitated. out. The conjugate was then dried for a few hours under vacuum. In the third step, salicylic acid - PCL polymer was achieved after different molar ratios were attempted. Typically, acetylated diacid and PCL conjugate (1: 9 mol ratio) were placed in round bottom flask fitted with an overhead stirrer and the reaction mixture was heated to 160°C using a preheated silicone oil bath under vacuum for 16 h. The solidified melt of the polymer was recovered by cooling down the reaction mixture to room temperature and precipitated into a 20 fold excess of diethyl ether (200 mL). This final SA-PCL polymer thus formed was then cured for 4 days in a vacuum oven at 160°C. 2.3. Polymer characterization Thermo Nicolet 6700 IR spectrometer was utilized for recording the IR spectra of the samples at 4 cm−1 resolution from 4000 to 400 cm−1. The 1H-NMR spectrum for all the samples was recorded on a 400 MHz instrument (Bruker) using CDCl3 as a solvent. Thermal analysis was carried out on an NETZSCH STA 409 PC Luxx at 10 °C min−1 by weighing samples (10 mg) and recorded under dry nitrogen gas.


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The water contact angles of the PCL, intermediate products of PCL and SA-PCL polymer were assessed at 25 °C using the sessile drop method with 3 µl water droplets on a FTÅ 200 contact angle goniometer (Dataphysics, Germany). The contact angles reported in triplicates from three discs and the readings were obtained as soon as the contact angle became static with time. Gel permeation chromatography (GPC, Waters 1515, USA) was used to determine the molecular weights of the PCL before and after aminolysis against polystyrene as a standard using tetrahydrofuran (THF) columns. 2.4. In vitro degradation study In vitro degradation analysis for all the samples (PCL-NH2 and SA-PCL) was carried out in 0.025M phosphate buffer saline (PBS, pH 7.4) at 37°C. The disc shaped polymeric samples of about 4.5 mm diameter and 1 mm thickness were punched out from compression molded sheet. Those test samples were immersed in a test tube containing PBS without (10ml) and with Pseudomonas lipase enzyme (10ml, 1 mg/ml), and placed in an incubator shaker at 100 rpm and 37°C. PBS solution was replaced with fresh solution every 3 days. After incubation for prerequisite time, the samples were removed, washed with distilled water and then vacuum dried at room temperature to attain the constant weight for analysis. The percentage weight loss was calculated as below,

% weight loss =

M − M X 100 M

where Mi is the initial weight of sample and Mf is the constant weight of sample after degradation with PBS hydrolysis at different time intervals. 2.5. Biocompatibility assessment 2.5.1. In vitro Cytotoxicity 2.5.1.1. Cell culture



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For sterilization, all the samples were soaked in 70% ethanol (CAS No. 64-17-5) for 2 h, exposed to UV light for 20-30 min and washed with 1X PBS (pH 7.4) before cell seeding. Sterilized disc shaped samples (4.5 mm diameter and 1 mm thickness) of all the polymers (PCL, PCL-NH2, SA-PCL) were used for conducting all the in vitro cell culture experiments. The mouse myoblast C2C12 cell line, procured from National Center of Biological Sciences (NCBS), Bangalore was used for all the in vitro cytotoxicity experiments. Compared with other cell lines, C2C12 murine myoblast cell line is recommended for assessment of newly synthesized biomaterial in vitro. Importantly, the reasons behind using these cells were, (a) these cells are the only type of cells, which show well-defined phenotypical features in undifferentiated and differentiated forms. (b) Also, because of contractile nature of the myoblast cells, they can easily adhere and proliferate in a reproducible manner on both softer and stiffer surfaces of any bio-mimicking materials. Prior to seeding, the cells were procured from cryo-preserved stock and were expanded in tissue culture graded T25 flask (Eppendorf, Germany) containing complete culture media, supplemented with Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, India), 15% (v/v) Fetal Bovine Serum (FBS; Gibco, India) plus 2 mM L- glutamine (CAS No. 56-85-9; Gibco, India), 1% (v/v) antibiotic antimycotic solution (10 UmL-1 penicillin and 0.1 mg ml-1 streptomycin; Sigma Aldrich, India). Cells were maintained in a 5% CO2 incubator (Sanyo, MCO-18AC, USA) operated at 37°C temperature and 95% humidified atmosphere. The culture media was replaced with fresh one at the interval of every three days. The cells were harvested from the culture flask using 0.05 (w/v) % trypsin-EDTA (Gibco, India) solution, after attaining the 80-90% monolayer cultured confluency. The harvested cells were centrifuged and sub-cultured to passage 2-7 for conducting all the cell culture experiments. The glass coverslip coated with gelatin (0.2%) were used as a control and treated same as sample. 8 ACS Paragon Plus Environment

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2.5.1.2. MTT Assay In order to quantify the viability of the myoblast cells on synthesized polymeric samples, MTT (3(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, CAS No. 298-93-1, Sigma Aldrich, India) assay was performed

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. The cells were cultured on the sterilized

polymeric samples kept in 24 well plates at a approximate density of 3 x 10 3 cells/well with further incubation for 1, 3 and 5 days in 5% CO2 incubator at 37°C temperature and 95% humidified atmosphere. After the desired period of incubation, the culture media was aspirated and replaced by 0.5mg MTT/ml reagent prepared in DMEM without serum. The enzyme mitochondrial succinate dehydrogenase of metabolically active cells reduces the yellowish water soluble MTT to water insoluble blue-violet colored end product, known as formazan crystals. This reaction rate will be directly proportional to the presence of number of mitochondrial active viable cells on the sample. After 3 h of incubation at 37° C, unreacted MTT dye was removed and formazan crystals were solubilized by addition of 200µl dimethyl sulfoxide (CAS No. 67-68-5; DMSO). The optical density (O.D) was quantified spectrophotometrically in an ELISA microplate reader (iMark, Bio-radlaboratories, India) at 595 nm. The results were also expressed as percentage viability and calculated as follows,

% cell viability =

O.D. of the sample X 100 O.D. of the control

2.5.1.3. Immunofluorescence analysis In order to analyze the cell morphology, the sterilized polymer samples and gelatin coated cover slips (control) were placed in 24 well plate and C2C12 mouse myoblast cells were cultured at the approximate density of 3 x 103 cells/ well. The cultured cells were incubated for 1 and 3 days. Then the samples were washed twice with 1 X PBS and adhered cells were fixed for 20 min with 4% paraformaldehyde (PFA; CAS No. 30525-89-4; SD Fine-Chem Limited, India). After fixation, the cells were again washed with 1 X PBS and permeabilized 9 ACS Paragon Plus Environment


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with 0.1% Triton X solution for 5-10 min. To prevent nonspecific binding of dye, the cells were blocked with 1% Bovine Serum Albumin (BSA) for 30 min after a wash with 1 X PBS. The cells were immunostained for 30 min with Hoechst stain 33342/ DAPI (Life technologies, India) and Alexa Fluor 488 (Life technologies, India) to observe nuclei and actin filaments, respectively.

After washing the sample with 1 X PBS, the cells were

observed under fluorescence microscope (Nikon LV 100D, Japan) to study their morphological/phenotypical behaviour. 2.5.1.4. SEM analysis of cell adhesion About 3 x 103 cells/well were cultured on the surface of each sterilized sample kept in 24 well plates for performing the cell adhesion analysis. After 3 days of the culture, the samples were washed with 1XPBS and fixed at room temperature for 30 min with 2.5% glutaraldehyde (CAS No. 111-30-8; LobaChemie, India). For dehydrating the samples, they were washed with a series of ethanol solution (30, 50, 70, 80, 90 and 100% v/v) in distilled water subsequently for 10 min each, which was followed by further drying in a dessicator for overnight

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. The dried samples were sputter coated (Vacuum Tech, Bangalore, India) with

gold and imaged under scanning electron microscope (SEM; Philips, Quanta). 2.5.2. Hemocompatibility assessment 2.5.2.1. Hemolysis test Blood compatibility tests were conducted under the guidelines and policies of Institutional Animal Ethics Committee of laboratory animals, Indian Institute of Science, Bangalore (Approval no. CAF/Ethics/414/2014). Hemolysis assay was carried out as per the procedure reported by elsewhere 29. Healthy whole blood was drawn from New Zealand white rabbits and mixed with a 9:1 volumetric ratio of (3.8% w/v) sodium citrate (CAS No. 64-04-2) followed by dilution with normal saline (CAS No. 8028-77-1) in 4:5 ratio. Disc shaped


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polymer (PCL, PCL-NH2, SA-PCL; 4.5 mm diameter and 1 mm thickness) were kept in a standard falcon tube containing normal saline (10 ml), which were incubated previously for 30 min at 37°C. 0.2 ml of anticoagulated diluted blood was added to these standard tubes containing sample and kept for incubation for 1 h. The negative was normal saline solution, while distilled water was used as a positive control. After stipulated time period of incubation, each tube was then inverted to mix the contents homogenously and centrifuged at 3000 rpm for 5 min (Eppendorf 5810R, USA). The supernatant was carefully collected and transferred to 96 well plates for measuring absorbance by an ELISA microplate reader (iMark, Bio-rad laboratories, India) at 541 nm. The percentage hemolysis was calculated as according to the following relationship,

hemolysis(%) =

O. D. (Test)-O.D. (negative control) X 100 O. D. (positive control)-O.D. (negative control)

2.5.2.2. Platelet adhesion assay Platelet rich plasma (PRP) was obtained after centrifuging the whole blood of rabbits for 10 min at the rate of 1000 rpm. The 3 ml of PRP was added to each of sterilized polymeric sample in 12 well plate. Prior to conducting PRP assay, the sterilization was carried out by soaking them into absolute ethanol for 5 min, followed by equilibration in PBS at room temperature for 3 h. All the samples were incubated for 60 min at 37°C. After washing the samples three times with 3 ml of PBS, the adherent platelet cells were fixed by immersing the samples in 2.5% glutaraldehyde in PBS for 30 min at room temperature. The dehydration of samples were done by gradient washing in ethanol–water solutions series (for 10 min each in 30, 50, 70, 80, 90 and 100% (v/v)) and dried. The samples were coated with sputtering and observed under SEM (Philips, Quanta). The relative density (number) of platelets adhering on the polymer surface was quantified from four SEM images captured on the same polymer at 11 ACS Paragon Plus Environment


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1600 X magnification from different fields. The procedures were repeated using three independent samples of each polymer (n = 15). Average of number was taken to obtain reliable data 30. 2.5.3. Antibacterial study 2.5.3.1. Bacteria Culture For the antibacterial study, the sterilization of polymer samples were done with 70% ethanol and exposed to UV for 2 h before microbial seeding. The two bacterial strains used for present study were Staphylococcus aureus (S. aureus; MRSA USA 300) and Escherichia coli (E. Coli; MG 1655). These strains were cultured at 37°C and revived on Luria-Bertani (LB) broth, supplemented with beef extract plates (Sigma Aldrich, India) and yeast. To obtain pure culture, single colony was picked from the agar plate and used for inoculating 5 ml of freshly prepared LB for overnight suspension culture at 37°C in an incubator shaker at 100 rpm. 100 µL of the overnight cultured cell suspension was sub-cultured further in fresh media for 3 h under same incubating conditions. After incubation, the pure suspension cultures of the bacterial strains were measured for optical density (OD) of 0.5 with an ELISA microplate reader (iMark, Bio-rad laboratories, India) at 670 nm and used for conducting the experiments. . 2.5.3.2. MTT assay For bacterial viability analysis, all the sterilized samples, kept in 24 well plates, were seeded with 200 µl of each bacterial suspension at 0.5 OD670 and incubated at 37°C for 24 h. After incubation, the samples were washed to remove the slackly attached bacteria from their surface and incubated for 1 h with 0.5 mg/ml of MTT at the room temperature. After 1 h, unreacted MTT was aspirated from the samples and the formazan crystals were solubilised using 200 µL of DMSO. The absorbance of colored product was quantified at the wavelength


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of 595 nm using a 96-well ELISA microplate reader (iMark, Bio-rad laboratories, India). The results were expressed as percentage viability, calculated as follows,

% bacterial viability =

O.D. of the sample X 100 O.D. of the control

2.5.3.3. SEM analysis of bacteria adhesion For SEM analysis, E. coli and S. aureus, grown as above, were used for seeding on the samples. After 24 h of incubation at 37°C in standard 24 well plate, all samples were washed three times with PBS solution and fixed by immersing in 2.5% glutaraldehyde at room temperature for 30 min. After washing the samples three times with PBS, the specimens were dehydrated using graded ethanol-water solution series (10 min each of 30, 50, 70, 80, 90 and 100% v/v) and dried. Samples were sputter coated with gold and viewed using a scanning electron microscope (Philips, Quanta). 2.6. Statistical Analysis The quantitative data obtained from all the in vitro experiments, performed in triplicates, were reported as mean ± standard deviation. For comparing significant difference in obtained results between the groups over different time periods on different composition, the analysis of variance (ANOVA) test were carried out followed by Tukey’s post-hoc test (SPSS-16.0; IBM, USA). The p-value at 0.05 and 0.01 was considered as statistically significant. 3. Results The synthesis of biodegradable polymer, in the present case, is based on the hypothesis that with incorporating functional drugs, like salicylic acid into a polymeric backbone may yield a polymeric prodrug with sustained release together with desired cyto and hemocompatibility properties. The alkyl component chosen for the current biopolymeric system, adipoyl 13 ACS Paragon Plus Environment


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chloride of this poly (anhydride esters) is biocompatible and biodegradable, in vivo. Another precursor, lactic acid is a chiral molecule (L,D) that degrades naturally. And LA was used as a spacer between salicylic acid and aminolyzed PCL, which contains abundant ester groups (COO-) (Scheme I). The amine functionalization of PCL enables the establishment of chemical crosslinking among various acid/alcohol functional drugs. It is reported that the reaction between amine and ester groups results in aminolysis31. The possible mechanism is shown in Scheme S1 (see supplementary information). Essentially, our aim was to functionalise PCL with amine groups in order to form chemical conjugates with the carboxyl groups of lactic acid like coupling reaction, followed by the free alcohol group of lactic acid cross-linked with salicylic acid. Thus, lactic acid acts as a spacer in order to connect PCL and salicylic acid. The details of the diacid synthesis were reported elsewhere 23. In the coupling reaction, the carboxylic acid of lactic acid is added to the N, N′-dicyclohexylcarbodiimide (DCC) molecule to form a good living group, which can then be displaced by an amine (PCL-NH2) during nucleophilic substitution 32. The non-cured SA-PCL was thus obtained by melt-condensation reaction. Also, SA-PCL, in non-cured state, was sparingly soluble in chloroform and acetone. But cured cross-linked SA-PCL was insoluble in any solvent (Scheme S2 in supplementary information). While adopting the above synthesis protocol, we were able to prepare synthesized polymers in thin film or disk shaped configuration of various size and shape. The following sub-sections analyse the physio-chemical characteristics of the as-synthesized polymers. 3.1. FT-IR and NMR analysis The FT-IR spectrum of the diacid showed peaks at 1766 cm−1 1685 cm−1 corresponding to the phenyl ester and the carbonyl groups of salicylic acid respectively (Figure 1a). At 3300 cm−1 a broad peak of acid hydroxyl groups was noted. The amine functionalization on PCL was further confirmed in Figure 1b. From the IR spectra, two prominent peaks of amine are well


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evident at 3414 cm−1 and 1670-1570 cm−1 range, respectively (Figure S1 in supplementary information). The alkane, carbonyl and ester linkage of (C-O) were observed at 2945, 1726 and 1170 cm−1, respectively. Further, the coupling of aminolyzed PCL and lactic acid was confirmed from the amide conjugate at 1643 cm−1. In the IR spectrum of the SA-PCL cured system, the existence of both the diacid and PCL functional groups confirmed the formation of the polymer. The results are compared with salicylic acid and pristine PCL (Figure S2 in supplementary information). The proton NMR spectrum of the diacid and synthesized SA-PCL are represented in Figure 2a-d. The 1H NMR spectrum of the diacid (Figure 2a) in CDCl3 showed characteristic peaks of the aromatic and the aliphatic parts of the diacid. A critical aspect of NH2-PCL- lactic acid coupling synthesis was to make sure that only PCL with free amine end conjugate can be obtained. To accomplish this, aminolysis of PCL was carried out (Figure 2b). The formation of PCL-NH2 and the diacid have been compared with the proton NMR of pristine PCL and salicylic acid (Figure S3 in supplementary information). In addition to recorded chemical shifts of ester linkage and inner methylene protons, the free amine group were observed at 2.73 ppm. This confirms that the PCL possesses amine end group. Figure 2c reveals the NMR of amide conjugate of lactic acid and PCL-NH2. The methylene and methyl protons of lactic acid gave a signal at 5.05 ppm and 1.58 ppm, respectively (Figure S4 in supplementary information). The peak at 6.11 ppm can be attributed to amide group. This clearly indicates the formation of amide conjugate between amine-PCL and lactic acid. NMR spectra of the non-cured SA-PCL polymer (CDCl3) are shown in Figure 2d. The aromatic protons resonate between 8.05-7 ppm, which was downshifted relative to the aromatic protons of the acetylated salicylic acid. The aliphatic protons of the salicylic acid backbone at 2.4- 1.3 ppm up shifted as compared to the aliphatic protons of adipoyl chloride at 2.85 ppm and could not be visibly seen as it overlaps with PCL peaks. The acetylation of



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diacid and cross-linking of amide conjugate of PCL showed upshifts, which supported the formation of the crosslinked SA-PCL polymer. 3.2. Thermal analysis and surface wettability studies Thermal degradation profiles of PCL and crosslinked SA-PCL polymers are presented in Figure 3a. The neat PCL showed a one-step degradation at 275 °C, associated with the degradation of 5-hexenoic acid

33

. The aminolyzed PCL degradation profile was unaltered,

except for the fact that it degrades earlier at 248°C. This is presumably due to chain scission of PCL during aminolysis. The PCL-NH2-LA thermogram showed three step degradation at 90°C, 270°C and 407 °C corresponding to loss of amide linkage, PCL ester linkage and lactide (3,6-dimethyl-1,4-dioxan- 2,5-dione), respectively

34

. The SA-PCL polymer also

displayed three step degradation profiles at 275 °C, 394°C and 525 °C. The effect of crosslinking of aminolyzed PCL to salicylic acid was revealed from the thermogram of SAPCL polymer. The cleavage of PCL started at 275 °C, which is followed by degradation of aliphatic chains of adipolyl chloride at 394°C. The aromatic rings decomposed at 525°C, which was slightly lower than reported value (445 °C)

35

. The complete degradation of the

crosslinked polymer took place at 707 °C. The thermograms of salicylic acid and its diacid are shown in Figure S5 (see supplementary information). It can be seen that the thermal degradation of diacid is rapid as compared to salicylic acid. It is attributed to the formation of poly anhydride ester with adipoyl chloride makes the degradation faster than salicylic acid. As compared to the thermal stability of the poly-anhydride ester, the cured SA-PCL polymer is thermally stable over a wide temperature range. In the case of SA-PCL like system, pyrolysis provoked a PCL chain cleavage, which was randomly distributed all along the chain. The evolution of water at the initial phase was not observed in our polymer as neither the free hydroxyl group (from PCL) nor the carboxylic acid (from lactic acid) was available for initial decomposition. We conclude that


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PCL chains depolymerize first, followed by thermal decomposition of aliphatic and aromatic rings of salicylic acid backbone. The water contact angle of the cured SA-PCL and aminolyzed PCL (PCL-NH2) are presented in Figure 3b. With water, the PCL-NH2 exhibited a contact angle of 40.1°, which is half the contact angle of neat PCL (86.1°). This is attributed to the presence of hydrophilic NH2 groups. The wettability of PCL-NH2–LA conjugate was very low (24.2°) due to lactic acid spacer. The polymer SA-PCL showed slightly higher in value (57.3°) than PCL-NH2. This is due to the cross-linking between salicylic acid backbone and PCL. From the results above, it can be concluded that SA-PCL polymer is hydrophilic. The GPC experiment showed that the averaged molecular weight of the PCL is decreased to 42 kDa (Mw) with during the aminolysis, suggesting the degradation of PCL (Table I). Also, the polydispersity index (PDI) increases from 1.7 to 2.2 due to aminolysis. Such observations indicate a decrease in molecular chain conformation due to aminolysis. 3.3. In vitro degradation study The in vitro degradation of PCL-NH2 and SA-PCL over a time period of 40 days was monitored as a function of % weight loss in PBS (pH 7.4) at 37°C and 100 rpm incubating shaker, as shown in Figure S6a and S6b (see supplementary information). The variations of weight loss as a function of degradation time of all the polymers were linearly fitted to obtain rate constant (k) (Figure 4). The apparent degradation profiles of both the polymers were compared and it can be seen that % weight loss for both the polymers enhanced gradually with degradation time. However, until 2 weeks, both the polymers displayed a similar tendency in the rate of degradation with 4.7 ± 1.4 % (PCL-NH2) and 4.4 ± 2.5% (SA-PCL) of weight loss. However, the rate of degradation for SA-PCL polymer enhanced and reached upto 16.2 ± 2.6 %, which was slightly faster than that of PCL-NH2 (6.9 ± 1.4%) at 5 weeks of degradation time (Figure S6a in supplementary information). Both IR and NMR 17 ACS Paragon Plus Environment


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spectroscopic techniques reveal the products are only PCL and not salicylic acid. It is known that salicylic acid degrades rapidly as compared to PCL (Figure S7 in supplementary information). The in vitro degradation of PCL-NH2 and SA-PCL polymers were also conducted in aqueous solutions containing Pseudomonas lipase. The weight of both the polymer in 1mg/ml of lipase solution decreased with time. From Figure S6b (see supplementary information), it is clearly observed that SA-PCL polymer got degraded upto 42.8 ± 1.8%, while PCL-NH2 polymer showed up to 84 ± 2.6 % of weight loss within 5 weeks of degradation. Overall, the rate of lipase-catalyzed degradation for SA-PCL and PCL-NH2 polymers was found to be much higher than the hydrolysis of polymer without lipase. The degradation profile of both the polymers exhibited the following trend: PCL-NH2 in lipase > SA-PCL in lipase> PCLNH2> SA-PCL. At 5 weeks, the weight loss percentage of buffer incubated and lipase incubated samples of SA-PCL polymer were 16.2 ± 2.6 % and 42.8 ± 1.8%, respectively. Whereas, PCL-NH2 polymer underwent respective weight loss of 6.9 ± 1.4% and 84 ± 2.6%, in the absence and presence of lipase, indicating more rapid decrease during enzymatic degradation. 3.4. Compatibility with muscle cell, blood and bacteria 3.4.1. In vitro cytocompatibility analysis The cytocompatibility properties such as cell proliferation and adhesion of synthesized PCLNH2 and SA-PCL polymers with C2C12 myoblast cells were analysed using MTT assay, fluorescence and SEM, respectively. All the in vitro experiments were conducted for different time intervals of up to 5 days. 3.4.1.2. MTT Assay


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In order to quantify the cell survivability and growth on as-synthesized polymer substrate, a quantitative colorimetric assay (MTT assay) was performed with seeded myoblast cells. The mitochondria oxidize the MTT solution into an insoluble product by mitochondrial succinate dehydrogenase enzyme, giving a characteristic blue-violet colored product, known as formazan crystals. This results in absorbance and is presented in Figure 5, which shows the rate of proliferation of murine myoblast cells on different polymeric sample at different time points (1, 3 and 5 days), compared to control (0.2% gelatine coated cover slip). As indicated by ANOVA followed by comparison with Tukey’s post-hoc test, significant differences in cell viability exist amongst PCL, PCL-NH2 and SA-PCL polymers, when compared with each other as well as with control at all the time intervals. The myoblast proliferation rate on SA-PCL polymeric substrate exhibited a marked increase with 72% (p ≤ 0.01; at day 1), 62% (p ≤ 0.01; day 3) and 117% (p ≤ 0.01; at day 5) corresponding to PCL-NH2. In contrast, this growth rate is elevated to 210% (p ≤ 0.01; at day 1), 186% (p ≤ 0.01; day 3) and 270% (p ≤ 0.01; at day 5) when compared to respective rates of PCL. However, a significant increase in cell growth by 338% (p ≤ 0.05; at day 1), 124% (p ≤ 0.05; day 3) and 153% (p ≤ 0.01; at day 5), was also attained by PCL-NH2 polymeric substrate relative to PCL at all the time points (Figure 5). Summarising, SA-PCL was found to support the growth of more number of cultured myoblast cells up to 5 days. Such observation begins to suggest that the assynthesized polymer offers a superior surface for cells to attach and proliferate as compared to PCL and PCL-NH2 substrates. 3.4.1.3. Cell adhesion and proliferation Figure S8 (see supplementary information) presents the SEM micrographs of C2C12 myoblast cell morphology and adhesion on control, PCL, PCL-NH2 and SA-PCL polymer composites after 3 days of culture. The phenotypical features and adhesion of cultured cells were found to be normal on all polymeric samples under investigation, when compared to 19 ACS Paragon Plus Environment


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control. The cultured cells seemed to be better metastasized and had an intense attachment not only on the polymeric surfaces but also with neighbouring proliferating cells (cell-to-cell contact). For conceptualizing cytoskeletal structure (actin filament) and nuclei shape, myoblast cells were cultured for 3 days and immunostained with Alexa Fluor 488 and Hoechst dye after day 1 and day 3 of cell seeding. Alexa Fluor 488 stains actin filament of the cells, whereas nucleus

of

cell

is

stained

by

Hoechst

dye.

Some

representative

images

of

immunofluorescenctly stained cells are shown in Figure 6. Most of th attached cells adopted an elongated threadlike appearance (lamellopodia and filopodia), which reflects the projection of cytoskeletal element (actin) on the leading edge of the cell. However, extensive filopodial projections also known as membrane ruffles were observed only on the surface of PCL-NH2 and SA-PCL polymeric samples, when compared with that of control and PCL polymer. Although, neither of polymeric samples showed a marked shuffle in cell stellate phenotypical features and organization of cytoskeletal at both day 1 and 3, but most promising results were obtained for SA-PCL, as density of cultured cells as well as their spreading increased corresponding to control, PCL, PCL-NH2. Further, marked elevation in cell density on SA-PCL polymer is exhibited at day 3. After 3 days, lamellopodia and filopodia structures with moderate orientation along a specific direction were seen more prominently on the surface of SA-PCL polymer (Figure 6). The cells appear to be evenly distributed with non-aligned actin filaments on PCL and PCL-NH2 polymer substrate, similar to that on control, even after three days of culture. Taken together, these morphological analyses suggest that SA-PCL polymer has the potential to be called as cytocompatible material as the substrate played a favourable role in controlling important cellular functions, such as cell adhesion, proliferation and spreading. 3.4.2. Hemocompatibility analysis 20 ACS Paragon Plus Environment

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3.4.2.1. Hemolysis Assay The hemolysis test was conducted on PCL, PCL-NH2 and SA-PCL to probe the compatibility of polymer surface with RBC of blood from rabbit. Mean percentage of hemolysis, as shown in Figure 7a, seemed to decrease in the case of synthesized PCL-NH2 (0.97 ± 0.03%) and SAPCL (0.39 ± 0.02%) polymers compared with the commercially available PCL (1.7 ± 0.03). This observation indicates lesser damage and interaction between synthesized SA-PCL polymer and RBC (Figure 7a). 3.4.2.2. Platelet adhesion assay Platelet adhesion on PCL, PCL-NH2 and SA-PCL polymeric surfaces from PRP was also evaluated in the present work. Figure 7b shows the respective number of platelets adhering on PCL, PCL-NH2 and SA-PCL polymeric samples. It is distinctly indicated that the amount of platelets that adhered on the surface of PCL-NH2 (23 ± 2.8) and SA-PCL (17 ± 2.9) substrate was significantly (p ≤ 0.01) decreased compared to that on the PCL surface (37 ± 2.9). Furthermore, Figure 8 shows SEM micrographs of the PCL, PCL-NH2 and SA-PCL polymer substrate after 60 min contact with the PRP. While platelet adhesion and spreading with some aggregates were recorded on the surfaces of PCL, platelet adhesion was markedly inhibited on both PCL-NH2 and SA-PCL polymers. Also, the platelets adhered to PCL-NH2 and SA-PCL polymeric samples retained their native round shape compared to those adhered to PCL. To sum up, SA-PCL polymer was found to be better hemo-compatible polymer than PCL polymer. 3.4.3. Antibacterial analysis 3.4.3.1. MTT assay To assess the antimicrobial characteristics of the synthesized polymer, both gram positive (S.aureus) and gram negative bacterial strains (E.coli) were grown on the surface of control, 21 ACS Paragon Plus Environment


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PCL, PCL-NH2 and SA-PCL in stable cultural conditions for 24 h. The growth of E.coli and S.aureus on control and different polymeric substrates was quantified using MTT assay. It is evident from Figure 9, that the measured optical density (540 nm) for both the bacterial strains, corresponding to bacterial viability, showed a marked decrease in case of PCL-NH2 and SA-PCL polymers as compared to control or PCL (highest absorbance). According to Anova test followed with comparison by Tukey’s post-hoc test, both PCL-NH2 and SA-PCL polymers showed a significant decline in E.coli growth with 61 % (p ≤ 0.01) and 58 % (p ≤ 0.01) of viable cells against PCL (102 %). In case of S. aureus strain, both PCL-NH2 and SAPCL polymer substrate were able to reduce the viability of bacteria, with 106 % (p ≥ 0.05) and 102 % (p ≥ 0.05) of live cells compared to PCL, which showed a comparable percentage of viability (119%) on its surface. 3.4.3.2. Bacterial adhesion and proliferation To further confirm the results acquired from the bacterial viability (MTT) analysis, SEM images were taken to observe the adhesion, morphology and number of bacteria on the different polymeric substrates. As shown in Figure 10, both the bacterial strains, E.coli and S. aureus showed their typical rhabditi and coccal forms of phenotypical features, respectively. Although, the growth rate shown by E. coli was lesser compared with that of S.aureus, but the number of adhered bacterial cells of both the strains were observed to be higher on the surface of control and PCL polymer, after 24 h of bacterial culture. Qualitatively, compared to the control and PCL, very less number of adherent bacteria was found on both PCL-NH2 and SA-PCL polymeric surfaces, irrespective of whether it is gram negative or gram positive bacteria. These observations are in accordance with the results from bacterial MTT analysis, giving the signature of antibacterial property of PCL-NH2 and SA-PCL polymers. 4. Discussion 4.1. Physico-chemical characteristics of synthesized SA-PCL polymer 22 ACS Paragon Plus Environment

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A critical analysis of Figures 1-3 led us to summarise the physico-chemical properties in terms of the following key points: a) We have developed a novel system consisting of crosslinked biocompatible and biodegradable salicylic acid–polycaprolactone polymer. Some advantages of the as prepared crosslinked polymer can be expected as salicylic acid backbone would facilitate sustained release of salicylic acid

and allow conjugation with any other polymeric

system. b)

The introduction of amine functionalization on PCL serves not only as non-toxic to cells or tissues but also provides active sites through which other biomolecules can be further immobilized36.

c)

Lactic acid acts as a spacer molecule, which enables to conjugate salicylic acid and PCL.

d)

Formation of salicylic acid-PCL based effective cross linking molecule enhances the stability in the extracellular environment.

e)

Therapeutic efficacy of this cross-linked biocompatible polymer is expected to be higher than those of non-cross linked and free chemotherapeutic drugs vesicles. Overall, the results, as analysed in the preceding section, imply that this cross linked polymer may be a useful drug carrier system for any anticancer drugs incorporation.

4.2. In vitro degradation study In the current study, new formulation of polymer consisting SA and PCL were synthesized and tested to assess its potential to increase the rate of the degradation compared to pristine PCL (a very slow degrading polymer). The hydrolytic degradation of PCL-NH2 and SA-PCL polymers at 37°C in PBS was evaluated for a time period of 5 weeks. The buffer incubated samples of both the polymers showed slow and similar type of gradual weight loss until 2 weeks. However, after 2 weeks, the SA-PCL polymer showed a slightly faster weight loss profile. The weight loss of polymer is the indication of the beginning of “surface erosion”, 23 ACS Paragon Plus Environment


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which can be described as the loss of monomer and oligomers upon degradation. Consistent weight loss upto 2 weeks was due to surface degradation pathway in which polymer was removed from the surface inwards towards the core, throughout degradation periods 7. However, Gopferich 37 had reported that the passive hydrolysis is the most significant way of degradation, whose velocity can be influenced by many factors, such as, kind of chemical bond within the polymer, composition of copolymer and how accessible the polymer is to water. Slightly enhanced rate of hydrolytic degradation after 2 weeks in SA-PCL is attributed to incorporation of the salicylic acid molecules in polymeric backbone which could have accelerated hydrolysis/water ingress into the polymer. This would result in random hydrolytic chain scission to form oligomers and monomers. As the salicylic acid is dislodged from SAPCL polymer, numerous pits would increase the surface area for water intrusion and attack throughout the SA-PCL network7. Enzyme degradation studies have been performed to imitate the in vivo physiological conditions. Although the in vitro degradation results should not be extrapolated to in vivo terms, but the mechanism of degradation can be understood. The data shown in Figure S6b (see supplementary information) indicated that the incubation of the polymer with the enzyme P. lipase has significantly increased the weight loss percentage than the bufferincubated samples. This effect was most pronounced for PCL-NH2 at the end of 40-days time period as compared to SA-PCL polymer, where approximately 84% of weight loss was detected in the enzyme incubated sample. However, SA-PCL polymer was considerably more stable than the PCL-NH2 polymer. These results are interesting and confirm that polymer degradation can be catalyzed in the presence of lipase enzyme as reported for other polymers as well

38

. The weight loss differences reflect the different mechanisms of degradation or

cleavage of the bonds in the polymer molecules and indicates various surface interaction with perhaps the enzyme adsorption on the surface of polymer 39. 24 ACS Paragon Plus Environment

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The rate of enzymatic degradation could also be effected by the differences in the structure of the polymer at the surface compared to the interior39. Enzyme-catalyzed degradation of polymer starts with enzyme adsorption onto the polymer substrate, followed by chain incision of the polymer by transition complex formed between the polymer and enzyme 9. Also, degradation of the PCL-NH2 polymer was faster than the SA-PCL polymer, which can be due to the enhanced enzymatically catalyzed hydrolysis of ester bonds in more hydrophilic PCLNH2 polymer than SA-PCL polymer. It has been reported that an increase in the rate of enzymatic degradation of PCL copolymers containing hydrophilic segments is directly proportional to the presence of total number of the hydrophilic units in that polymer

40

.

Several studies, in line with present study, had reported the degradation of PCL and PCL based polymers

7, 9-11

by lipase, an enzyme responsible for ester bond hydrolysis in various

polyesters. Figure 4 shows the semi-log plot of mass loss with time for both the polymers (PCL-NH2 and SA-PCL). The buffered and enzyme incubated samples of both the polymers showed the linear regions. However, a faster yet linear degradation rate was recorded for PCL-NH2 polymer as compared to SA-PCL, when incubated with enzyme. Whereas, a linear region for buffer incubated PCL-NH2 polymeric sample was much slower than SA-PCL polymer incubated in PBS. Thus, the degradation rate followed zeroth order kinetics represented by the equations as below,

−

[]

=k

[A]= [A] 0−kt A0 is the mass at initial time, A is the mass at any time t, and k is the rate constant of degradation which is obtained from the slope of the plot. The obtained rate constant (k) values (×10-3 h-1) under buffer incubation for PCL-NH2 and SA-PCL polymers are 9×10-2 h-1



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and 1.4 ×10-2 h-1, respectively. Under the influence of enzyme, the rate of degradation is further enhanced and respective rate constant values (×10-3 h-1) for PCL-NH2 and SA-PCL polymers, 9.5×10-2 h-1 and 4.0 ×10-2 h-1 were determined. These rate constant values are in good accordance with the presently obtained degradation rate of the PCL-NH2 and SA-PCL polymers. 4.3. Cell, bacteria and blood compatibility analysis Significant interest in tissue engineering is reflected in the development of novel, biodegradable polymers (polyester, polycarbonates and polyurethanes) with a wide range of physical properties and with better degradation rates 4. All these properties significantly influence the biological response of synthesized polymers 41. However, evaluation of certain critical factors such as in vitro cytocompatibility, haemocompatibility and anti-bacterial properties should be taken into consideration, while introducing any newly synthesized polymeric scaffold and/or implant. Motivated from our previous works, on SA-based polyester exhibiting both in vitro and in vivo biocompatibility, we have developed a novel cross-linked polymer system (SA-PCL), incorporating both PCL and a drug (SA)23-24. In the following, we will critically analyse the compatibility of such polymers with specific components of living systems i.e. cell, bacteria and blood. 4.3.1. In vitro cytocompatibility In the present study, the results of the in vitro cytocompatibility analysis were conducted using MTT assay, fluorescence microscopy and SEM analysis. Overall, the results indicate promising cellular response on SA-PCL polymeric substrates. This aspect has been reflected by enhanced cell proliferation, cell attachment, cell spreading with prominent formation of membrane raffles and cell-to-cell interaction along with specific organisation of cytoskeletal elements. In particular, stellate morphological features were observed for the cells cultured 26 ACS Paragon Plus Environment

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upto 5 days of time period. The synthesized SA-PCL polymer presented a better surface for cellular attachment and proliferation as compared to PCL-NH2 and PCL. To this end, the surface wettability is important as the wettability influences both the attachment and the growth of cells. The hydrophilic polymeric surface (SA-PCL polymer in our case) allows the adsorptions of the serum proteins adsorption with the help of reversible and labile bond42. However, other factors, such as chemistry of polymers can also affect cellular adhesion and proliferation. The presence of carboxyl and hydroxyl groups can also be considered as an important factor in cell adhesion and proliferation, depending on various cell types43. Further, Schmeltzer et al.18 reported enhanced cell proliferation on different salicylic acid-based Poly (anhydride-ester) surfaces and found a correlation between rate of cell proliferation and polymeric structure. They concluded that the presence of more number of aromatic rings in polymeric backbone (two in our case) attributes to increased cell proliferation. In the above backdrop, it is evident that SA-PCL polymer can be the most favorable cell growth substrate. The extensive spreading of murine myoblast C2C12 cells with increased cell density, maintaining their normal stellate morphological features and cytoskeletal organization (filopodia and lamellopodia) and cellular connections with each other as well as with substratum could be observed on SA-PCL polymer substratum. This observation can be explained on the basis of the fact that biomaterial substrate modification plays an important role in controlling important cellular functions, such as cell adhesion, proliferation and spreading through mechano-transduction, by differentially regulating signal transmitting structure-actin 44. By modifying the assembly of focal-adhesion-cytoskeleton, the cells adhere differentially, displaying different phenotypical features on the interfaces of cell and biomaterial44. It has been proposed that the observed cell membrane ruffles (filopodia) on SA-PCL polymer is essential for cell motility and development of substrate adhesions. It displays an active interaction of cell with biomaterial substratum, since it protrudes at the 27 ACS Paragon Plus Environment


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foremost front of the cell 45. Regulated recruitment of accessory chemical molecules, present on any substrate coupled with actin filaments organization into lamella networks are often required for the formation of such lamellopodia and filopodia structures 45. Further, favorable cell adhesion, proliferation and highly organized cytoskeletal

morphological features

substantiate that SA-PCL polymeric sample can provide a good support for increasing the expression levels of NDRG2 (N-myc downstream-regulated gene 2), growth related gene which has been linked to increased myoblast growth and skeletal muscle development 46. 4.3.2. Hemocompatibility The compatibility of the materials with blood is one of the significant problems, which are associated with newly synthesized material. It is therefore essential to analyze the hemocompatibility of newly synthesized materials. Various strategies are continuously being employed to alter the surface so that its compatibility with blood can be improved47. Therefore, hemolysis assay and platelet adhesion studies-two vital characteristics to be considered for long-term biomedical applications were conducted for the present study. The hemolytic potential of any newly synthesized biomaterial is the quantitative measure of the extent of hemolysis that is induced by that material, when it comes in contact with blood 29

. Hemolysis is the problem associated with most of the materials, which occur when

excessive fluid diffusion into the red blood cells causes them to swell and rapture, in presence of that material 48. In general, hemolysis up to

Modulated in Vitro Biocompatibility of a Unique Cross-Linked Salicylic

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