Antithrombotic and antiinflammatory effect of heparin conjugated poly(l-lactide) on inflammation induced cyclooxygenase.
Biomaterial implants are clinically used to alleviate various life-threatening diseases. Implantation of any foreign object into the body causes a strong localized inflammatory response, which leads to insufficient healing of the tissue . To reduce this inflammation, the use of biodegradable polymer has increased significantly during the past few decades. It also helps in controlled release of drug throughout biological degradation of polymer . The biocompatibility of drug carrier, biodegradable polymer, is the major problem seen in blood contacting medical devices. One of the biodegradable polymer used for orthopedic applications in humans is Poly(L-lactide (PLLA), it is found to be biocompatible for at least first few weeks to months after implantation [3-6]. Among various techniques used to enhance the biocompatibility and hemocompatibility of biomaterials, one such technique comprises alteration in polymeric surface by attachment of drug molecules on it [7-9]. Heparin is an established drug molecule that can be used as coating on biomaterial. Experiments have proved anti-inflammatory, anti-coagulant and good biocompatible properties of biomaterials following heparin coating on various polymeric surfaces [10-17]. Investigations on novel heparin-polylactide (PLA) conjugate have confirmed its use as blood/tissue compatible biodegradable material for implantable devices and tissue engineering .
Heparin molecule; being hydrophilic, is critical to deliver at target site and its routine forms of administration is insufficient to observe benefit given by heparin's short half-life and complex pharmacokinetics . The primary data on potential to reduce leukotriene B4, TXB2 and PGE2 synthesis  and antiinflammatory properties of heparin has been observed in many experiments [1,20, 21]. Anti-inflammatory effects were mainly mediated by blocking P- and L-selectin initiated cell adhesion . Heparin has shown to inhibit leukocyte-endothelial adhesion, both in vivo and in vitro [23-25]; lipopolysaccharide (LPS)-induced monocyte activation in vitro  and reduction in TNF-[alpha] elicited inflammatory response in vivo . This characteristic of heparin has increased its use for coating artificial surfaces.
As a result of contact with artificial surfaces important inflammatory mediators, like Prostaglandins (PGs) are produced by enzymatic activity of Cyclooxygenase (COX). COX exists in two isoforms with COX-1 being the constitutive form, found in most normal tissues throughout the body and COX-2 being the inducible form, expressed in normal tissues at low levels and is highly induced by pro-inflammatory mediators in the setting of inflammation, injury and pain. COX-1 & COX-2 catalyze the conversion of arachidonic acid into prostanoid, leading to the formation of thromboxane (TXA2) and prostaglandin E2 (PGE2) mediators, respectively [28, 29, 30, and 31]. The ratio of PGE2/TXA2 depends on the type of COX isoform present. The instable precursor TXA2; one of the most potent platelet aggregating agent and vasoconstrictive substances known [32, 33], is hydrolytically cleaved to stable variant TXB2 in platelets. The measurement of serum TXB2 produced by platelets following blood coagulation is specific and most frequently used test for evaluation of platelet, COX 1 activity in humans and other species [34-36]. COX-2 plays both, a pro-inflammatory and an anti-inflammatory role, depending on the stage of the inflammatory response . COX-2 derived PGs display pro-inflammatory properties .
The present study was conducted as a part of investigation on possibilities for using H-PLLA as coating on medical devices to improve its biocompatibility by means of incorporating antiinflammatory and anti-thrombotic properties of heparin. Highmolecular weight PLLA is found to be well tolerated in the animal as well as human model [39, 40]. Reductions in fibrin formation, fibrinogen binding, including platelet attachment and activation are reported following heparin coated catheter induction [41, 42]. This study involves determination of the inhibitory effects of H-PLLA on products of COX-1 and COX 2 respectively.
Materials and Methods
Poly L-Lactide (PLLA) of 23kDa was purchased from Purac Biochem Bv, The Netherlands. Ethanol, methanol, hexane (HPLC Grade), and ethyl acetate were purchased from J.T. Baker. Sodium acetate was purchased from Sigma Aldrich and DCM was purchased from Merck. PGE2 Express EIA kit, TXB2 EIA kit, SPE C-18 cartridges were purchased from Cayman Chemicals, Ann Arbor, MI, USA. A range of different concentrations (10, 50, 1x[10.sup.2], 1x[10.sup.3], 1x[10.sup.4], 5x[10.sup.4], 1x[10.sup.5] nM) of CP and NCP were dissolved in DCM. CP and NCP were used as test and control material respectively.
The novel concept of H-PLLA is to enhance the biocompatibility and hemocompatibility of the drug carrier biodegradable polymer via integrating thromboresistant action of heparin in conjugation with polymer PLLA. H-PLLA consists of heparin sodium covalently bonded with biodegradable PLLA. Heparin sodium of 26kDa was purchased from Biofer S.p.A., Italy. Heparin sodium is hydrophilic with short half-life in vivo. Heparin-conjugated PLLA was prepared by direct coupling reaction using DCC/DMAP (dicyclo hexyl carbodiimide/dimethyl amino pyridine) chemistry by maintaining its molar ratio . The coupling reaction utilizes terminal hydroxyl groups of PLLA and carboxylic groups of heparin molecules. In brief, accurately weighed heparin sodium was dissolved in N, N-Dimethyl formamide (DMF) and PLLA in dichloromethane (DCM) respectively. Heparin solution was taken in a round bottom flask and heated up to 50[degrees]C. Solution of coupling agent DCC and catalyst DMAP were added to heparin solution. PLLA solution was then added drop by drop in the flask. The reaction mass was maintained at 50[degrees]C for 12 h under inert environment of nitrogen gas. After completion of reaction, the resultant solution was precipitated in excess methanol. The product was then purified by dissolving precipitates in chloroform. Unreacted heparin was removed by extracting it using water. The product was re-precipitated by addition of excess methanol in organic layer. Final precipitates were collected by filtration and drying at 40[degrees]C for 72 h under vacuum to eliminate residual solvents. Covalent bonding of heparin with PLLA improves its pharmacokinetic and pharmacodynamic properties; aid in exerting anticoagulative properties of heparin sodium [8,9,13,18,43].
The amount of PGE2 produced following LPS-treated blood was used as a read-out to measure COX-2 activity in monocytes. COX-2 being constitutively inactive; was activated in monocytes by LPS stimulation. Considerable amount of DCM dissolved CP and NCP was coated by lyophilization on tube to prevent unspecific effects due to solvent induced hemolysis. Non-heparinized human blood was incubated with test item for 24h at 37[degrees]C in the presence of 100 [micro]g/mL LPS to initiate COX2 induction under gentle rocking. Plasma was obtained by centrifugation and purified per instruction given in ELISA kit to determine PGE2 plasma level.
The quantity of TXB2 was used to measure the COX-1 activity in platelets. Considerable amount of DCM dissolved CP and NCP was coated on tube by lyophilization to prevent unspecific effects due to solvent induced hemolysis. Human blood was incubated in tube for 1h at 37[degrees]C in the presence of test item under gentle rocking. The clotted blood was centrifuged to separate serum followed by purification of serum per instruction given in ELISA kit and determine TXB2 serum level.
Results and Discussion
Figures 1 and 2 represent change in COX-2 activity with change in PGE2 concentrations after addition of NCP and CP concentration respectively. Test item-induced changes in PGE2 content were within linear range of the ELISA standard curve. Decrease in PGE2 plasma level was seen at 1x[10.sup.2] nM - 1x[10.sup.3] nM CP concentration compared to NCP. A dose-dependent terminal increase in PGE2 plasma concentration was seen after 1x[10.sup.3] nM concentration of CP. On the other hand figures 3 and 4 show, no specific trends in the TXB2 content after addition of NCP and CP concentration respectively; exposure to samples led to 3 to 9-fold increase of TXB2 production randomly.
Different methods of coating heparin on surfaces of implantable biomaterials have been established [10,19,43]. There are various investigational studies conforming anti-thrombotic properties of modified biomaterials via reduction in platelet adhesion, aggregation and leukocyte recruitment after contact to heparin coated or encapsulated biomaterial as it delivers heparin to a specific site [1, 18, 19,44]. In response to endogenous thrombin formation platelet COX-1 is maximally stimulated to produce TXA2; precursor of TXB2 [32, 33]. In present study, increase
in TXB2 serum level (i.e., increased COX-1 activity) was observed at 1x[10.sup.3] nM concentration of CP compared to NCP demonstrating anti-thrombotic potential of H-PLLA at this dose. Elevated COX-1 concentration helps protecting injured arteries from developing cyclic flow changes and thrombus formation . Anti-inflammatory potential of heparin is established in various animal as well as clinical studies . Mechanism that causes initial prothrombotic vascular response to injury by endothelial cells is very well established through the induction of COX-2 and increased production of PGE2 .
Since, the prostanoid is most generally associated with the inflammatory response the formation of PGE2 is often observed at inflammatory sites of various diseases. Moreover, we noticed that 1x[10.sup.2] nM - 1x[10.sup.3] nM concentration of H-PLLA leads to decrease in PGE2 plasma level compared to NCP. This indicates that 1x[10.sup.2] nM - 1x[10.sup.3] nM of H-PLLA can be a potential therapeutic range in treatment of various inflammatory conditions. However, a dose-dependent terminal increase of PGE2 is seen above 1x[10.sup.3] nM concentration of CP. Thus increase of PGE2 in the concentration range of CP could be owing to a toxic over dose effect of H-PLLA on the COX-2 activity. Over expression of COX-2 or prostacyclin synthase suppresses the development of vascular lesions and growth of vascular smooth muscle cells [46, 47]. The induction of COX-2 is associated with production of deleterious prostanoid because of COX-2 involvement in inflammatory disorders .
The post-exposure response of COX-2 and COX-1 activity to CP indicates that 1x[10.sup.2] nM - 1x[10.sup.3] nM can be a potential therapeutic range in treatment of various inflammatory conditions and 1x[10.sup.3] nM exert anti-thrombotic properties. Dose dependent changes in activity with CP and NCP did not show any conclusive trend. Present study demonstrated improved biocompatibility of H-PLLA in terms of anti-inflammatory and anti-thrombotic properties compared to non-heparinized polymer. This novel formulation can be considered as a potential drug carrier polymer in drug eluting devices and needs further exploration as latent anti-thrombogenic and anti-inflammatory initiative for coating medical devices.
Authors wish to thank Sahajanand Medical Technologies Pvt. Ltd and Harlan Laboratories for conducting this study.
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Ramila D. Mandal (1), Payal J. Patel (1), Nisha R. Patel (1), Himanshu J. Patel (2), Sunil P. Kale (1), Chhaya B. Engineer (1), Ankur J. Raval (1)
(1) Sahajanand Medical Technologies Pvt. Ltd., Sahajanand Estate, W akhariawadi, Nr. Dabholi Char Rasta, V ed Road, Surat 395004, Gujarat, India
(2) Pacific School of Engineering, Kadodara Palsana Road, Surat, Gujarat, India
Received 24 May 2016; Accepted 6 July 2016; Published online 14 September 2016
* Coresponding author: Dr. Ramila Mandal
caption: Figure 1: Effect of different concentrations of Non Conjugated Polymer (NCP) on the enzymatic activity of COX-2 in human monocytes
Caption: Figure 2: Effect of different concentrations of Heparin Conjugated Polymer (CP) on the enzymatic activity of COX-2 in human monocytes
Caption: Figure 3: Effect of different concentrations of Non Conjugated Polymer (NCP) on the enzymatic activity of COX-1 in fresh human blood
Caption: Figure 4: Effect of different concentrations of Heparin Conjugated Polymer (CP) on the enzymatic activity of COX-1 in fresh human blood
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|Title Annotation:||Original Article|
|Author:||Mandal, Ramila D.; Patel, Payal J.; Patel, Nisha R.; Patel, Himanshu J.; Kale, Sunil P.; Engineer, C|
|Publication:||Trends in Biomaterials and Artificial Organs|
|Date:||Apr 1, 2016|
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