Covalently Conjugation of Genistein with Biodegradable ... - medIND

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Dec 27, 2009 - A novel drug (Genistein) conjugated biodegradable polymer (PLLA) was synthesized by the direct coupling of Genistein with Poly L-Lactide ...
P. Sojitra, A. Raval, D. Kothwala, A. Rawal, H. Kotadia, S. Adeshara 144 Trends Biomater. Artif. Organs, Vol 23(3), pp 144-148 (2010)

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Covalently Conjugation of Genistein with Biodegradable Poly L-Lactide Prakash Sojitra*, Ankur Raval, Devesh Kothwala, Haresh Kotadia, Subodhchandra Adeshara Research & Development Division, Sahajanand Medical Technologies, Surat GJ 395003, India *corresponding author: [email protected] (Prakash Sojitra) Received 20 February 2009, Accepted 13 June 2009, Published online 27 December 2009 A novel drug (Genistein) conjugated biodegradable polymer (PLLA) was synthesized by the direct coupling of Genistein with Poly L-Lactide (GEN-PLLA) and was characterized by FTIR, HPLC and GPC study. These results indicate that the unique property of bound Genistein has an inhibiting influence on the coagulation, plasma protein adsorption, and subsequent platelet adhesion systems. This novel GEN-PLLA conjugate could be applied as blood/tissue compatible biodegradable materials for implantable medical devices and tissue engineering, especially to medical devices like coronary stents. © Society for Biomaterials and Artificial Organs (India), 20090220-38.

Introduction Drug delivery plays an important role in the development of pharmaceutical dosage forms for the animal health care industry because often the duration of drug release needs to be extended over days up to several months. The application of polymeric materials for medical purpose is growing very fast. The biodegradable polymers have found applications in such diverse biomedical fields as tissue engineering, implantation of medical devices and artificial organs, prostheses, ophthalmology, dentistry, bone repair, and many other medical fields. All these applications benefit from the fact that the polymer “disappears” after providing a desired function. Biodegradable polymers are widely used as drug careers, wound dressing [1], medical devices [2] and scaffolds in tissue engineering [3]. Aliphatic polyester contains flexible ester bonds and easily degrades in to nontoxic matter in the body. Using PLLA directly as drug career, burst release or biphasic release occurred [4]. Covalently attachment of the drug to lactic acid oligomers with the aim of achieving temporary hydrophobization and slower release controlled by the separation of the drug from the

degradable link within the polymer matrix [5] can be a promising option to the present drug delivery stent base systems. By reviewing the field of oncology, conjugation method of Drug-Polymer is an important aspect to control the cell growth by target site drug delivery. Conjugated polymer gives pro-long release of drug at the local site. Polymer–drug conjugation is one of the major strategies for drug modifications, which manipulates therapeutic agents at molecular level to increase their solubility, permeability and stability, and thus biological activity. The most attractive and commonly used biodegradable polymers are polyesters such as poly-l-lactide, poly (lactide-co-glycolide) (PLGA) and poly caprolactone (PCL). These materials are commercially available in different compositions and molecular weights which allow control degradation of the polymer [6,7]. Genistein (5,7,4'-trihydroxy isoflavone) are synthesized by a wide variety of dietary plant species such as grapes, mulberries and

Covalently Conjugation of Genistein with Biodegradable Poly L-Lactide

peanuts [8] and in dietary isoflavones such as beans and legumes [9]. Phenolic rings in Genistein is are responsible for estrogen receptor binding [10]. The hydroxyl group at the C-4’ position enhanced the antioxidant properties of Genistein drug. The structure of Genistein consisting of two benzyl rings by a three- carbon bridge, which is simplified as C6C3-C6. It is also classified as a phytoestrogens due to its weak estrogenic activity in mammalian systems [11]. Genistein is a potential flavonoid which possesses anti-thrombotic and antiproliferative properties. Data suggest that Genistein may exert a strong anti-carcinogenic effect, and that this effect possibly involves an induction of p21, which inhibits the threshold kinase activities of Cdks and associated cyclins, leading to a G2/M arrest in the cell cycle progression [12, 13]. It inhibited collage induced human platelet aggregation in a dosedependent manner [14,15]. It has been shown to enhance NO production from the endothelium. NO is a potent inhibitor of platelet adhesion, aggregation and thrombosis. Impaired platelet production of NO has been associated with acute coronary syndromes. Thus Genistein may affect platelet aggregation via an NO-dependant signal transduction pathway, a potential mechanisam. The antitumor effect of Genistein has been reported through the inhibition of protein kinase pathways leading to gene expression modification of many proteins, including VEGF. Novel drug-biodegradable polymer conjugates can serve the purpose of sustained drug delivery simultaneously with the polymer degradation period. In the present study, Genistein was conjugated with biodegradable PLLA and was performed as coating on coronary stent. Materials and methods The Stainless steel 316L electropolished stents (16mm length) were used in the present study. L-Lactide (Boehringer-Ingelheim, Germany) was purified by recrystallization from dry ethyl acetate and dried in vacuo at room temperature. Genistein was obtained from Ren Young Pharmaceutical Co Ltd, China and used without

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further purification process. Stannous octoate (Sn-oct, Sigma), Dicyclohexylcarbodiimide (DCC, FLUCA), 4-(dimethyl amino) pyridine (DMAP, FALUCA), dichloromethane (DCM), Dimethyl Sulphoxide (DMSO) and other chemicals used in the current investigation were of HPLC grade procured from Ranbaxy Fine Chemicals Ltd, India. Methods PLLA was synthesized by the reported method [16]. Briefly, L-lactide (10 g, 0.07 mol) was melted at 180 °C for 30min in a nitrogen atmosphere. Stannous octoate (0.1 wt % of Llactide) was added to the melted L-lactide solution. The mixture was degassed and stirred at 150 °C for 30 min. The product was dissolved in chloroform and then precipitated in excess methanol. The precipitate was filtered and dried overnight under vacuum. A DCC, DMAP chemistry was used to synthesize the PLLA-Genistein. Genistein C 15 H 10 O 5 (0.02702 gm 1x10 -4 mol) and PLLA (0.1g, 2.0x10-4 mol) was dissolved in the DMSO (50 ml), N, N-dimethyl formamide (DMF, 50 ml) respectively. DCC (2x10-4mol) and DMAP (2x104 mol) were added to drop by drop PLLA solution in DCM was added to Genistein solution with stirring for 10 min at constant 50°C. The reaction was further carried out for 18 hours under nitrogen atmosphere. After the coupling reaction, the reaction solution was precipitated in excess methanol. The precipitate was dissolved in chloroform and the solution was precipitated in excess methanol. After filtering, the precipitate was dried at 35°C for 24 h in vacuum to eliminate the residual solvent. HPLC analysis of Genistein were performed on HPLC-LC-2010 AHT [Shimadzu (Asia Pacific) Pvt. Ltd.] consisting of X-Terra RP-18 (250*4.6 mm) analytical column having particle size 5µm. The drug content was analyzed using mobile phase consisting of acetonitrile: methanol: water (45:35:20 v/v) at flow rate of 1.2 ml/min and oven temperature 40°C. The retention time for Genistein was kept at 2.9 minute and was detected at 254 nm by UV absorption. This analysis was performed on mother liquor to set molar ratio of genistein and PLLA. Liquor was taken from the reaction mixture at each 18

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hours after reaction time and was quantified by HPLC analysis for free genistein. To set Molar ratio of Genistein and PLLA various experiments were carried out in 1:1, 1:2 and 1:3. FTIR spectra of the product was recorded on ‘‘Perkin Elmer FTIR Paragon 1000 SPIR S. No. 42825’’ using KBr pellet technique. The thermograms were obtained on a thermobalance ‘‘Metler TA-4000 system’’ at a heating rate of 10 deg. C/min.

Figure 1: Chromatogram of Genistein Standard

Coating Technique The Genistein-PLLA conjugation was dissolved in 100 ml HPLC grade DCM and coated on stent using spray coating technique to check the feasibility of conjugation coating on stent. The coating process is carried out using aseptic conditions under controlled environment and class 1000 clean room conditions. The temperature and humidity were maintained below 23°C and 60% RH respectively in clean room.

Figure 1: Chromatogram of GEN PLLA

The OLYMPUS SZX12 microscope was used for photography and surface cherecterization.

observed in vibrational frequencies of their functional groups are explored in detail.

Results and Discussion

There is a broader absorption in the 3300-3500 cm-1 which can be identified by characteristic of O-H bond stretching adsorption in PLLA. The bands at approximately 2997 and 2945 cm -1 arise from the -CH3 asymmetric and symmetric stretch. These signals are related to both CH3 groups on the PLLA. However, the bands at approximately 2858 and 2928 cm-1, related to CH2 symmetric and asymmetric stretch, 2881 cm-1 show -CH absorption.

HPLC analysis for molar ratio determination Free amount of genistein was observed in 1:1 and 1: 2 molar batches while there was no quantification was observed in 1:3 molar ratio so the reaction was further proceed with molar ratio experiments. From the figures 1 and 2, it can be concluded that in molar ration 1:3 there is no free genistein at the end of the reaction was detected. There for one mole of genistein has consumed three mole of PLLA during conjugation reaction. GPC analysis The GPC data suggest weight average molecular weight has decreased from 247 KDa to 22 KDa. This is due to degradation of PLLA during reaction.

FTIR Spectra of GEN-PLLA Conjugate shows 1651 cm -1 & 1760 cm -1 shows presence of lactones ring and carbonyl of PLLA. The C-O stretching vibration frequency of free drug at 1260-1000 cm-1 (17). The infrared spectrum of GEN-PLLA implies that the vibration in GEN at 3412-3104 cm -1 has disappeared in the conjugated polymer. This also reveals the conjugation of OH group of genistein. On the other way absorption of –OH of free –COOH has also disappeared.

FTIR analysis Coating Surface Characterization FTIR spectroscopic method is employed to study the Genistein(Fig. 3), PLLA (Fig. 4) and PLLAGEN (Fig. 5) complexes wherein the changes

The stents were subjected to microscopic analysis after coating. It can be easily seen from

Covalently Conjugation of Genistein with Biodegradable Poly L-Lactide

Figure 3: FTIR spectra of Genistein

Figure 4: FTIR spectra of PLLA

Figure 5: FTIR spectra of GEN-PLLA

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P. Sojitra, A. Raval, D. Kothwala, A. Rawal, H. Kotadia, S. Adeshara

the figures that the coating surface is free from lumps, crack, and webbing or bridging. It is proved the feasibility of Genistein-PLLA conjugation on stent surface. Conclusion From FTIR analysis Genistein was successfully conjugated with PLLA. The GEN-PLLA can be applied to stent coating, which may release genistein from the stent surface after stent implantation, which may reduce the risk of restenisois and other implications which are chalanges of post implantation of stent after angioplasty.

Acknowledgement We express our sincere gratitude to Mr. Dhirajlal Kotadia, Chairman, Sahajanand Group of Companies for providing financial assistance to carry out the research work. Also thankful to Mr.Jaynish Tailor for the HPLC analysis and Mr.Arpit Jariwala for the literature survey and other technical help. Contributions from research team of Sahajanand Medical Technologies (Kamal Revdiwala, Ami Raval, Jaynish Tailor, Laxman Upale, Nilesh Kadu and Rahul Patel) are also gratefully acknowledged.

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