Crystallised chitosan + Vitamin-E Coated Drug-Eluting Stents to Prevent Restenosis and Stent Associated Infections

Drug-Eluting Stents (DES) was developed to reduce re-endothelialisation and thrombosis. Chitosan due to its biocompatible and biodegradable property, it is used in drug delivery and wound healing applications. Chitosan-Vitamin E (alpha-tocopherol) coated stents were prepared by seeding and crystallisation process. Drug release pro ile, anti-bacterial activity and biocompatibility were analysed. FESEM analysis of the coated stent evidenced the homogenous coating of the drug-polymer mixture does not provide any surface space on the stent for the bacterial adhesion. Maximumbacterial reduction percentagewas observed for bio ilm-producing Staphylococcus aureus (96.6±1.04%). Escherichia coli andPseudomonas aeruginosa showedbacterial reduction percentage of 93.3±2.51% and 93.6±2.08% respectively. Release pro iles of crystalline chitosan from the coated stents indicated that the rate of chitosan release was sustained and constant with the release time. Chitosan coated stent samples showed signi icant biocompatibility indicated by the cell viability percentage of 96.6±1.04% when compared with the control samples (99.1±0.76%). To conclude, the drug-releasing phenomenon aided by the vitamin was correlated with its ability to prevent the formation of restenosis in atherosclerosis cases.

A Drug-Eluting Stents (DES) was developed to reduce re-endothelialisation and thrombosis. Chitosan due to its biocompatible and biodegradable property, it is used in drug delivery and wound healing applications. Chitosan-Vitamin E (alpha-tocopherol) coated stents were prepared by seeding and crystallisation process. Drug release pro ile, anti-bacterial activity and biocompatibility were analysed. FESEM analysis of the coated stent evidenced the homogenous coating of the drug-polymer mixture does not provide any surface space on the stent for the bacterial adhesion. Maximum bacterial reduction percentage was observed for bio ilm-producing Staphylococcus aureus (96.6±1.04%). Escherichia coli and Pseudomonas aeruginosa showed bacterial reduction percentage of 93.3±2.51% and 93.6±2.08% respectively. Release pro iles of crystalline chitosan from the coated stents indicated that the rate of chitosan release was sustained and constant with the release time. Chitosan coated stent samples showed signi icant biocompatibility indicated by the cell viability percentage of 96.6±1.04% when compared with the control samples (99.1±0.76%). To conclude, the drug-releasing phenomenon aided by the vitamin was correlated with its ability to prevent the formation of restenosis in atherosclerosis cases.

INTRODUCTION
Cardiovascular diseases are the primary cause of death, accounting for 20% of deaths globally (Roth et al., 1990). Atherosclerosis is the signi icant cardiovascular disease; deposition of fat in the coro-nary vessels results in a build-up of plaque, reducing the blood low in the arteries causing symptomatic coronary artery disease. Repeated plaque formation causes total blockage of the artery leading to atherosclerosis (Rosenthal, 2014).
A stent implantation is considered as one of the successful methods for atherosclerotic cases Stefanini and Holmes (2013) by improving constant blood low through arteries (Kastrati et al., 2007). This immediate treatment after a heart attack can be done by stent implantation, though it has some disadvantages. Hyperplasia occurs at the site of injured arterial blood vessels. These cells migrate and increase with the extracellular-matrix formation, which results in the development of neointimal tissue-this impact of the development of hyperplasia results in restenosis. About 35% of patients treated for atherosclerosis had re-occlusion within six months (Durgin and Straub, 2018).
Other treatments like bypass surgeries, atherectomy and revascularisation process were considered to be the cost increasing factors (Jones et al., 2014). To reduce the restenosis, the stents containing drugs are developed to reduce the formation of neointimal tissues known as drug-eluting stents (DES), and they were applied clinically (Mongrain and Rodés-Cabau, 2006). Drug-Eluting Stents (DES) was developed to reduce re-endothelialisation and thrombosis. Sirolimus and Paclitaxel drugs are used commonly for coating on the stent surface. These drugs reduce the complications like the growth of neointimal tissue, and restenosis (Honda, 2009).
Heparins are anti-coagulant agents preferentially used for angioplasty cases for the prevention of restenosis. He et al. (2019) synthesised different heparin-like polymers; among them, chitin derived chitosan having good biocompatibility and biodegradability was analysed for its anti-coagulant properties. In support, Wang et al. (2018) earlier reported that chitosan has an excellent performance in anticoagulation with a similar backbone structure to that of heparin. Zheng et al. (2011) stated that chitosan could also promote the growth of endothelial cells.
Based on this biological and medical signi icance, the heparin-like chitin derived chitosan was investigated for the ef icacy of preventing restenosis in the present research. This approach was carried out for the irst time by coating chitosan onto metal stents, and the drug-releasing behaviour was studied using vitamin E as a drug carrier. Tocopherol acetate (Vitamin E) has been approved by FDA as a safe adjuvant and widely used for different applications in drug delivery like high biocompatibility, antimicrobial activity, and improvement of drug permeation, antitumor activity and enhancement of drug solubility.
In the account of its anti-bacterial nature against several Gram-Positive and Gram-Negative bacterial species, Apart from the anti-coagulant properties of chitosan, it has high bio-degradability (Shi et al., 2006) and antimicrobial property (Acharya et al., 2005). Another signi icant risk associated with the coronary stents is their high infection rate due to bio ilm-forming organisms like Staphylococcus aureus and Staphylococcus epidermidis. The patients' proteins play a signi icant factor in bio ilm formation leading to associated stent infections (Mack et al., 2004).
Based on these therapeutic applications of chitosan, prevention of restenosis and bio ilm formation was considered as the main objectives in the present research. Chitin derived chitosan was earlier described in our previous studies. Thus extracted chitosan was mixed with vitamin-E and coated onto stainless steel fabricated metal stents. The coated stents were analysed for drug release, anti-bacterial activity and biocompatibility.

MATERIALS AND METHODS
The present research work was carried out in the Department of Microbiology, Annamalai University, Chidambaram, Tamil Nadu, India. The work was done from November 2019 to February 2020. Highquality medical grade type 304VSS stainless steel material (Sigma SS straight length wire #0.018) was commercially procured and used as a coronary stent. The stents were coated with chitosan and Vitamin-E (alpha-tocopherol) using a modi ied dipcoating method as described below.

Coating the stents with Chitosan and Vitamin-E
The stents are coated with chitosan, and a drugcarrier called alpha-tocopherol (Vitamin-E). Two different phases of the coating were carried out for the effective and constant release of drugs from the stent surface. Seeding is the initial coating process followed by crystallisation of drugs and carriers on the surface of the stent (Su et al., 2019).

Seeding of the stent surface with chitosan
For seeding, 1g of chitosan was prepared as described in our previous studies (Amrutha et al., 2018) was mixed with 5ml of n-Hexane (Sigma-Aldrich) and sonicated (amplitude 60 for 15min and then for 2-5min at amplitude 100) until homogeneous dispersion of the chitosan in hexane was formed. After sonication, stents were mounted on shrinkable tube placed on a needle. The needle loaded with a stent was placed at the centre of the vial containing the dispersed chitosan in hexane (one stent per trial). All the vials were kept in an ultrasonic bath (Shimadzu) for 10min at 30 • C to form a seeding layer. Stents were gently taken out of the vial and allowed to dry at room temperature. The dried and seeded stents were used for the crystallisation process in the next step.

Crystallisation of chitosan and vitamin E on stent surface
Over the seeded layer, a secondary layer using chitosan with a drug carrier (alpha-tocopherol) was coated as crystals on the surface. For crystallisation, 1g of Chitosan and 1g of Vitamin-E (alphatocopherol) was weighed and dissolved in 3ml of ethyl acetate. This solution was transferred to 20ml screw-capped glass tube illed dropwise with 5ml nhexane to form a homogenous solution. Stents were placed in this solution at 25 • C/5 min for crystallisation of chitosan-vitamin E (CV E ) on the seeding layer to form crystal-carpet formation. The stents were dried at room temperature under strict sterile conditions and stored at refrigeration temperature before testing.

FESEM analysis of chitosan-Vitamin E coated stents
The crystallised chitosan-Vitamin E mixtures coated on the stent surface was observed using Field-Emission Scanning Electron Microscopy (FESEM). The topographic analysis was also used to identify the uniform coating of the mixtures on the stent surface. Chitosan-Vitamin E crystals on the stent were observed under the magni ication of 6000X.

Quantitative anti-bacterial activity of chitosan-Vitamin E coated stents.
Anti-bacterial activity of chitosan-Vitamin E coated, and bare (uncoated) stents were quantitatively measured using standard bacterial adherence test against test organisms. The coated materials were placed separately in a tube with 5ml of each of the test bacteria and incubated at 37 • C for 18h. During the incubation period, the bacterial cells adhere to the surface. The stent materials were separated and washed to remove the non-adherent cells.
The washed pieces were sonicated for 30 seconds to dislodge the sessile adherent. After sonication, a serial dilution of the sonicated saline was made, and the number of sessile bacteria was detected to determine the degree of adherence by viable count technique. The similar experimental set up was run in parallel for bare stent materials. Chi-square method is used to estimate the number of adhered cells on both coated and bare stents (Elayarajah et al., 2011).
The percentage reduction of adhered organisms on the coated materials was determined using a standard percentage reduction formula.

Bacterial reduction (%) = A − B/A X 100
Where, A = number of adhered organisms (in CFU) obtained from the bare stents, B = number of adhered organisms (in CFU) obtained from the coated materials.

Statistical evaluation of total viable bacteria
The chi-square test can determine the effect of an anti-bacterial drug on bacterial adherence. The difference in the bacterial reduction percentage between the chitosan-vitamin E coated and bare stents was statistically calculated with P< 0.05 considered signi icant.

Drug releasing ef icacy of coated stents for the prevention of restenosis using Highperformance liquid chromatography
The ef icacy of chitosan concentration released from the coated drug-eluting stents was analysed using High-performance liquid chromatography (HPLC) with a known standard. The release pro ile of chitosan from the biodegradable polymer matrix for a period of 120hours was studied under simulated biological conditions. Standard solutions were prepared by dissolving 10mg of crystallised CV E drug in 10ml of the mobile phase. This mobile phase was then diluted up to 100ml. Diluted 20µl standards were injected in the HPLC column, and standard chromatogram for this standardised solution was obtained. For in-vitro kinetics study, stents were incubated in 50 ml of phosphate buffer saline (PBS -pH 7.0) solution at 37 • C with constant agitation at 300 rpm in a thermal mixer. Each CV E coated stents were removed at 30min, 1,2,4,8,12,24,48,72,96 120hours from their release vials and analysed for the amount of drug release in PBS. The experiment was carried out at room temperature. Chitosan was extracted using dichloromethane, which was later evaporated using dry nitrogen gas. The mobile phase was added to this, and the resultant supernatant was analysed for drug content by Highperformance liquid chromatography (HPLC). The chitosan released at regular interval from each stent was calculated (Ankur et al., 2007).
MTT assay is used to evaluate the in-vitro biocompatibility of polymeric components as it is a quick, effective method for testing mitochondrial impairment and correlates quite well with cell proliferation. It is based on the use of tetrazolium salt 3-[4,5dimethylthiazolyl-2]-2,5-diphenyl tetrazolium bromide (MTT), which can be converted to an insoluble blue formazan product by mitochondrial enzymes in viable cells.
L 929 ibroblast cell line is often used to evaluate cytotoxicity/biocompatibility of any drugs and drugcoated medical devices. The ibroblast cell lines were cultivated in 12-well-microtitre plates to reach con luence growth. The samples were applied directly to the developed ibroblast monolayer. The specimens immersed in 1ml Dulbecco's modi ied eagle medium (DMEM) ibroblast medium in 24well plates for 2hours in an incubator at 37 • C. The specimens were then seeded with L 929 ibroblast cell line at 10,000 cells per well according to routine cell-culture methods. The plates were incubated at 37 • C and 5% CO 2 for ifteen days. Well, without drugs were included in this study. The effect of drugs in the samples on the biocompatibility of ibroblast was evaluated using the photometric MTT assay. At each time point, samples were taken from the 24-well plates and transferred into new plates for the MTT study. The MTT solution was prepared by dissolving the powder in phosphatebuffered saline at a concentration of 1 mg/ml. After 1hr of incubation, the purple crystals were dissolved by adding sodium dodecyl sulphate (SDS) in a 1:1 mixture of water and dimethylformamide (DMF) at a concentration of 20% w/v. After adding 1ml of MTT medium (0.0005mg/ml) to each well, the plates were incubated for 3hrs, rinsed and desorbed in 100ul of 70% isopropanol. After being agitated rapidly at 400rpm/min for 40min, the dyed medium was transferred to 96-well plate and read at 550nm. The biocompatibility or cell viability is expressed as a percentage of the control sample (100%) (Budman et al., 2012).
The cell viability was determined and the percentage cell viability was calculated using the below formula,

FESEM analysis of chitosan-Vitamin E coated stents
The stents were coated with Chitosan-Vitamin E particles, and the topographical analysis was carried out using FESEM analysis. In our previous studies, only the surface coating of the stents with chitosan-Vitamin E particles was presented; despite the crystallised forms of chitosan on the coated stents were signi icantly evident during the present research work. The present analysis revealed the presence of crystallised drug particles on the stent surface; exhibiting adherence to the greatest possible extent. Chitosan coatings as large uniform and continuous layer of parallelogram shapes on the stents were observed (Figure 1). The coatings in crystal forms also increased the thickness of the stent. FESEM analysis of the coated stent also evidenced that the homogenous coating of the drugpolymer mixture does not provide any surface space on the stent for the bacterial adhesion or bio ilm deposition. This was mainly by reducing the depressions on the material surfaces by the crystallised deposition of chitosan on the stent. The obtained results were found supportive when compared to the results of Basalus et al. (2009). The surface charge of anti-bacterial coatings was found to be effective in reducing bacterial adhesion and bio ilm formation.

Quantitative anti-bacterial activity of Chitosan-Vitamin E coated stents
Anti-adherent activity for each coated stent materials was analysed using bacterial adherence test. The anti-adherent activity was calculated by bacterial reduction percentage. The anti-adherent activity of surface-modi ied stent materials against the test organisms was concentration-dependent as the reductive effect of drugs and carriers was in the range of 93.0% to 96.6% (Table 1).

Figure 1: FESEM analysis of Chitosan-Vitamin E coated stents
Bacterial reduction percentage calculated from the CFU (colony forming units) of the Chitosan-Vitamin E coated stents against the test organisms was measured. Maximum bacterial reduction percentage was observed for the high bio ilm producer, Staphylococcus aureus (96.6±1.04%). Other cultures also showed signi icant reduction percentage. Escherichia coli and Pseudomonas aeruginosa were reduced up to 93.3±2.51% and 93.6±2.08% respectively. Using chi-square statistical analysis, the effect of an anti-bacterial drug on bacterial adherence was determined. The difference in bacterial reduction percentage of Chitosan-Vitamin E (CV E ) coated stents and bare stents were taken as the experimental design. The hypothesis selected was "There is a signi icant effect of chitosan on the test organisms". The difference in the bacterial reduction percentage between the coated stents and bare stents were calculated with P<0.05 considering signi icant. For all the test organisms, the calculated Chitosan is a natural polysaccharide widely known for its inhibitory activity against a wide range of microorganisms. The presence of charged groups in the polymer and their ionic interactions with the bacteria cell wall constituents causes a drastic effect to microorganisms. This interaction leads to hydrolysis of the peptidoglycans in the microorganism wall, provoking the leakage of intracellular electrolytes which results in the death of the microorganism. Chitosan was found to inhibit the growth of both Gram-positive and Gram-negative microorganisms. Similarly, the effect of chitosan-coated stents on microorganisms was determined by Prasanth and Saravanakumari (2017). Anti-bacterial activity of the drug-eluting stents coated with 1X and 2X rapamycin showed signi icant inhibitory zones against the bio ilm-producing test organisms. The inhibitory zones ranged from 18.9mm to 31.3mm during the analysis. The obtained inhibitory zones for 2X coated drug-eluting stents were found to be high when compared to that of stents coated with 1X concentration. Govindarajan et al. analysed the antibacterial activity of kanamycin-chitosan nanoparticles (KMCSNPs) on catheters (Kumar et al., 2016). The surface-modi ied stents showed signi icantly increased anti-bacterial activity against Escherichia coli MTCC-729 and Proteus mirabilis MTCC 425 relative to the surface of an unmodi ied stent.

Drug releasing ef icacy of drug-eluting coated stents
In vitro release study was conducted on developed Chitosan+Vitamin-E crystallised stents. Release concentration of Chitosan in PBS at a speci ic temperature was determined. The release study was conducted for 120h in PBS at 37 • C. Release proiles of crystalline chitosan from the coated stents indicated that the rate of chitosan release was sustained and constant with the release time ( Figure 2). The lag phase exhibited an initial burst effect from 0.5h to 4h (45µg, 55µg, 55µg and 55µg). Fol-lowed this lag phase, an increase in drug concentration was observed from 8 hours to 48h (85µg, 100µg, 105µg and 110µg). In PBS at pH 7.0, the hydrophilic polymer, Vitamin-E undergoes degradation during the log phase. Due to the rate of polymer degradation, the release of drugs was facilitated at a higher rate than the initial burst level concentration. During this phase, the release concentration of chitosan remained almost constant (115µg, 120µg and 120µg) from 72h to 120h, indicating the sustained rate of drugs from the coated stents (Table 2). The rate of degradation of Vitamin E in the PBS was considered to be directly proportional to the rate of release of drugs. When the rate of degrada-  tion was high, then the release concentration was also found out to be high (Szymańska and Winnicka, 2015). This drug-releasing phenomenon aided by the vitamin was correlated with its ability to prevent the formation of restenosis in atherosclerosis cases. In another study, the release of rapamycin and heparin from the stent surface for the prevention of restenosis and thrombosis was investigated by Bae et al. (2017). They reported that about 67.3% of rapamycin was released at seven days interval.
This release of rapamycin signi icantly inhibited the growth of smooth muscles and platelet adhesion in the heparin-coated group than the uncoated heparin group; thus proving the prevention of restenosis and stent thrombosis. In another study, Elayarajah et al. (2011) analysed that cyclodextrin as a carrier was used for effective drug delivery mechanisms. Due to their ability to form a complex with drugs, it can act as functional carrier material and also in formulations of new drug mixtures.
These properties proved that cyclodextrin could serve as a potential carrier for effective and constant drug delivery at the targeted site. The use of tocopherol acetate (vitamin E) as a polymer in the present research offers the possibility to place hydrophilicdrugs on the surfaces of hydrophobic stents, building up a slow-release drug deliv-ery system independent of the drugcharge. This slow-release drug delivery system containing crystallised chitosan-Vitamin E mixtures from stent surface could have a cytostatic effect on the neointimal growth, which is desirable in the later stages of vascular healing. Protective polymer (vitamin) in the drug-polymer mixture being hydrophilic would degrade within a short time in a vascular environment. Crystallised chitosan-Vitamin E from the coated stents slowly gets diffused into the surrounding tissues, thus preventing restenosis (Puranik et al., 2013).

Biocompatibility of Chitosan-Vitamin E coated stents
The cell viability percentage was found to be highly similar and signi icant (99.6±0.57%) when compared to that of control (99.1±0.25); indicating the biocompatible properties of CV E coated stents (Table 3). Also, no signi icant difference in the morphology of the L 929 ibroblast cells after 24hours of cell culturing in the cell culture media (DMEM) was evident when compared to the control samples (Figure 3).
The following literature cited observations were found to be signi icantly supportive of the results of our present research. To overcome the reendothelialisation and to promote the healing, coat-ing of chitosan-heparin in coronary stents was investigated by Joner et al. (2006). During the second week of observation, the growth of neointimal tissues was observed on bare-metal stents, whereas the growth of intimal tissues was integrated on the coated stent surfaces. No in lammation response was recorded in coated stents, indicating the significant biocompatibility of the coating. Fiqrianti et al. investigated the effect of Poly l-lactic acid with chitosan-coated on coronary stents., Fiqrianti et al. (2018). Cytotoxicity test was carried out by MTT Assay method. Results revealed the cell viability of the test samples in this experiment reached 90%, which is considered to be non-toxic and safe to use in the human body. And hence, as chitosan shows high cell viability, signi icant biocompatibility and gradual degradation it can be used for various applications like tissue engineering, wound healing, drug delivery and gene delivery systems (Mi et al., 2001).

CONCLUSION
As chitin derived chitosan has good biocompatibility and biodegradability, the anti-coagulant properties of chitosan were studied in the present research. To prevent restenosis due to atherosclerosis and bio ilm-associated infection, chitosanvitamin E coated drug-eluting stents were designed. The coating of the stent was carried in a two-step process: seeding and crystallisation for the effective uniform coating on the stent surface. FESEM analysis of the coated stent revealed the presence of crystallised drug particles in large uniform and continuous layer of parallelogram-shaped. Maximum bacterial reduction percentage during quantitative anti-bacterial assay showed that the homogenous crystal coating of the drug-polymer mixture does not provide any surface space on the stent for the bacterial adhesion. Release pro iles of crystalline chitosan from the coated stents indicated that the rate of chitosan release was sustained and constant with the release time. Chitosan coated stent samples showed signi icant biocompatibility indicated by the cell viability percentage of 96.6±1.04% when compared with the control samples (99.1±0.76%). The researchers emphasise that the drug-releasing characterisations of the stents could able to prevent the bio ilm formation and restenosis. However, this should be studied in detail with more optimised conditions in future.