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Cationically Modified Solid Lipid Nanoparticles for Intestinal Delivery of an Antiplatelet Agent, Clopidogrel: Preparation and Characterization.

Introduction

The major concerns in the design and development of novel drug delivery systems are the controlled and targeted delivery of the pharmacological agent at a therapeutically optimal rate and dose regimen and its bioavailability. This site specific or targeted delivery combined with delivery at an optimal rate would not only improve the efficacy of a drug but would also reduce the possibility of unwanted toxic side effects of the drug, thus improving the therapeutic index. The most promising system to achieve this goal is the colloidal drug delivery systems [1, 2]. Colloidal drug delivery systems include the drug and the carrier systems like liposomes, niosomes, nanoparticles and micro emulsions. Solid lipid nanoparticles (SLN) are effective carrier systems that can replace the traditional colloidal drug carriers. SLN combine the advantage of traditional systems, like production with organic solvents, long term physical stability, and the possibility of protection of chemically labile actives inside the particles [3]. SLN's are also used as carrier moieties for therapeutic peptides, proteins and antigens [4].

Lauric acid is widely used in cosmetic, food and pharmaceutical applications. It has been used as a penetration enhancer for topical and transdermal absorption, rectal absorption, buccal delivery and intestinal absorption [5, 6]. Lauric acid enhances the intestinal absorption and lymphatic transport [7] and among thirteen fatty acids tested, lauric acid showed the highest percentage of mucosal absorption [8]. Lauric acid based solid lipid nanoparticles with poloxamer coating can be effectively absorbed through the intestinal tract, prolong the circulation of drug in blood by acting as a reservoir and can effectively deliver the drug by the combined effect of lipophilicity and surface charge. Addition of stearylamine makes the matrix more cationic. Catatonically charged SLN nanoparticles and negatively charged mucin can provide a prolonged contact time, thereby increasing the bioavailability of drug. It has also been reported that positively charged nanoparticle enhances the intestinal absorption [9, 10].

Clopidogrel besylate is an anti-platelet drug that inhibits the ability of platelets to clump together as part of a blood clot [11]. It prevents blood clots by irreversibly binding to the P2Y12 receptor on platelets, preventing adenosine diphosphate (ADP) from activating platelets [12]. It belongs to a class of drugs called P2Y12 inhibitors. The risk of heart attacks and strokes (which usually are caused by blood clots) is increased in patients with a recent history of stroke or heart attack, and patients with peripheral vascular disease. Clopidogrel is used to reduce the risk of heart attacks and strokes in these patients. However, its bioavailability is low. Clopidogrel and other antiplatelet drugs are normally administered orally with about 50% bioavailability [13]. Since highly lipophilic drugs like clopidogrel are easy to entrap within the solid lipid matrix, we have studied the application of cationically modified solid lipid nanoparticles prepared from lauric acid for intestinal delivery of clopidogrel, in an attempt to increase its bioavailability. Cationic solid lipid nanoparticles with clopidogrel were formulated by hot homogenization followed by ultrasonication with a combination of lauric acid and stearylamine. Physicochemical characterization, cytotoxicity and limited blood compatibility of the SLN formulation were performed along with the in vitro release kinetics of clopidogrel. Caco-2 cell experiments on tight junction integrity in the presence of these preparations were evaluated for their ability to open the tight junctions. Whole blood clotting time and bioavailability studies were performed on normal Wistar rats.

Materials and Methods

Clopidogrel was kindly supplied by Zynus Cadula Inc.Mumbai, India. Lauric acid, stearylamine, and Pluronic F 127 were purchased from Sigma-Aldrich (Bangalore, India). Purified water used for the experiments was obtained from MilliQ Plus (Millipore). All other chemicals and reagents were of analytical grade and used without further purification.

Preparation of clopidogrel loaded SLN

SLNs containing clopidogrel were prepared by the high shear hot homogenization and ultrasound method following a standard procedure [14]. Briefly, 75 mg clopidogrel was added to lauric acid (250mg) or a mixture of lauric acid and stearylamine (50 to 200 mg) and melted at 55[degrees]C. This was added to a Pluronic F127 solution at 55[degrees]C in a beaker and homogenization was carried out at 10,000 rpm for 5 min. After 5 minutes the suspension was cooled under the refrigerated temperature to obtain the solid lipid nano suspension. The obtained pre emulsion was ultrasonicated using a bath sonicator for 3 min. The nanoparticles suspension was stored at 4[degrees]C for further studies.

Particle size analysis and Zeta potential measurements

The mean particle size distribution and zeta potentials were determined using laser diffraction particle size analyzer (Nano ZS, Malvern Instruments, UK). Zeta potential measurements were carried out at a pH range between 6.8 and 7.4 in phosphate buffered saline.

Freeze-drying and drug content estimation

SLN dispersions were filtered and freeze-dried using Labconco tabletop freeze drier (4.5 L capacity) at -40[degrees]C for 24 h. The obtained freeze-dried SLN were stored at 4[degrees]C. Accurately weighted 250mg of freeze-dried product was dissolved in 30 ml of chloroform:methanol mixture(1: 1 ratio) at 45[degrees]C. The solution was then cooled down to 25[degrees]C so that the lipid was precipitated. The mixture was then vortexed and filtered using a 100nm syringe filter. The filtrate was then appropriately diluted and analyzed using HPLC at 220nm (United States Pharmacopoeia, 2005) for drug content. The encapsulation efficiency (EE) was calculated by determining the amount of non-encapsulated clopidogrel in the aqueous surfactant solution, against the total amount of drug added to the formulation using a standard calibration curve by HPLC at 220nm.

Physico-chemical characterization

SLN nanoparticles were characterized using DSC (Q20, TA instruments) and X-ray diffractometer (Bruker D8 Advance).

In vitro release studies

In vitro release of clopidogrel from SLN formulation was evaluated in phosphate buffer pH 7.4 (SIF) and in 0.1N HCl pH 1.2 (SGF) for 8h each at room temperature using dialysis bag method. The SLN formulation (2.5mg) as well as clopidogrel suspension was taken in dialysis bags (MW cutoff 12,000 Daltons, Sigma, USA) that were previously soaked in distilled water for 12hours. The dialysis bags with samples were placed in beaker containing 100 ml of dissolution medium and magnetically stirred at 100 rpm. After each sampling the dissolution media were completely replaced with 100 ml of fresh media, to maintain sink conditions. Samples were mixed with acetonitrile in the volume ratio 70:30, and analyzed using HPLC.

Red blood cell aggregation, hemolysis, complement activation and platelet factor 4 release

SLN nanoparticles were incubated with washed RBCs for 30 minutes at 37[degrees]C. Polyethylene imine (PEI) and saline were taken as positive and negative controls respectively. Aggregations if any were observed through a phase contrast microscope (Leica DM IRB, Germany) at a magnification of 20X. Hemolysis assay was done on the particles as reported elsewhere [15]. Normal saline was used as negative control (0% lysis) and distilled water as positive control (100% lysis). Complement activation by SLN nanoparticles was determined by the turbidimetric method, assessing the depletion of complement protein C3 on incubation with the nanoparticles [16]. Platelet factor 4 on incubation with SLN nanoparticles was assayed by enzymelinked immunosorbent assay (ELISA) kit (Diagnostica Stago, France) according to manu-facturer's instructions [16].

Mucoadhesion experiments

Mucoadhesion testing of the nanoparticles was carried out using a texture analyzer (TA XT plus, Stable Micro Systems, UK) with 50 N load cell equipped with mucoadhesive holder.

Visualization of tight junction

Caco-2 cells were seeded (at 20,000 cells/well) onto four well cell culture plates (Nunc) and maintained in an incubator at 37[degrees]C under 5% C[O.sub.2] [17]. Cells were treated with 500 ql of SLN suspension at a concentration of 10 mg/ml for 1 h. The particles were removed by washing the cells three times with phosphate buffered saline (PBS). The cells were fixed with 250 [micro]l of 4% paraformaldehyde for 20 min at room temperature. Then the cells were permeabilised using 0.2% Triton X-100 in blocking solution, made of 1% (w/v) bovine serum albumin (BSA) in PBS, for 20 min, so as to make the cell wall permeable to the stain. The permeabilised cells were then washed twice with PBS and incubated with 250 [micro]l of 1% BSA for 30 min. For actin filament visualization, the blocking solution was removed and cells were incubated with 200 [micro]l rhodamine phalloidin solution (0.2 [micro]g/ml) for 20 min at room temperature. After removal of rhodamine phalloidin, the cells were treated with 1% BSA as before. The cells were washed with PBS, and dried overnight at 4[degrees]C. Images were obtained using Carl Zeiss LSM Meta 510 inverted confocal laser scanning microscope (Carl Zeiss, Germany), equipped with He/Ne laser 543. The visualization of rhodamine phalloidin was done using excitation and emission wavelengths of 543 and 605 nm respectively.

In Vitro Cytotoxicity Studies

The L929 fibroblast cells were seeded in 24 well plates at a density of 5 x [10.sup.5] cells/well, cultured for 24 h in incubator at 37[degrees]C under 5% C[O.sub.2]. The medium was replaced with SLN nanoparticles suspension in the medium at a concentration of 5 mg/ml/well and incubated for 20h. Medium alone was used as control. The particles were removed and 3-(4,5-dimethythiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was done.

In vivo studies on Wistar rats

Bioavailability and whole blood clotting time has been evaluated on male Wistar rats weighing 200 [+ or -] 20g. All surgical and experimental procedures were reviewed and approved by the Animal Ethics Committee, Department of Pharmaceutics, Nehru College of Pharmacy. The rats were maintained on pellet diet and water ad libitum. Animals were housed in propylene cages under controlled environment (temperature 30 [+ or -] 2[degrees]C and 60 [+ or -] 5% RH).

Determination of in vivo effectiveness in whole blood clotting time

Whole blood clotting time of rat blood after administration of SLN formulation and clopidogrel suspension was evaluated in male Wistar rats that were fasted overnight. Two groups containing 6 animals each were used for the study. The first group was given clopidogrel suspension and the second group with SLN formulation. Normal clotting times of each animal were noted first. The pure drug and the formulation were administered orally with the aid of a syringe and infant feeding tube. Blood samples (0.5ml) were withdrawn by retro-orbital plexus puncture with the aid of capillary tube at one hour intervals post oral dose and the clotting time for each time interval was determined by capillary tube method.

Oral bioavailability studies

Single oral dose bioavailability study was carried out as per the reported procedure [18]. Three groups of male Wistar rats with each group containing 6 animals were fasted overnight. The first group was administered with blank SLN (placebo). The second group was administered with clopidogrel suspension and the third group with SLNs formulation. The formulations were administered orally with the aid of a syringe and infant feeding tube. Blood samples were drawn by retro-orbital venous plexus puncture with the aid of capillary tube at one hour interval post oral dose. Clopidogrel was extracted into phosphate buffer (pH 7.4) and analyzed using HPLC at 220 nm.

Accelerated stability studies

The accelerated stability study of the SLN formulation was carried out according to International Conference on Harmonization (ICH) Q1A (R2) guidelines (2003) for a three month period. The shelf life of the samples, based on change in drug content, was also determined.

Results and Discussion

Lauric acid (C12) has been reported to cause significant increase in the absorption of various hydrophilic drugs compared to C8, C10 and C14 [19, 20]. Pronounced effect on transepithelial transport of drugs have also been reported for lauric acid [21]. However, lauric acid is negatively charged. Zeta potential has been suggested to play an important role in particle uptake because the surface of the intestinal mucosa is negatively charged owing to the presence of glycocalix [22]. Particles with a high positive surface charge like chitosan are usually attracted by the intestinal mucosa which helps in increasing the intestinal absorption of the encapsulated drug. However, the strong electrostatic interaction between the positively charged particles and the negatively charged glycocalix may slow down the progression and penetration of these particles towards the epithelial cell surface reducing their uptake. Also it has been shown that non-ionized particles have a greater affinity for M cells than for ionized particles and positively charged particles [23]. Therefore, cationically modified SLNs were prepared from lauric acid by adding suitable quantity of stearylamine. SLNs prepared with lauric acid alone had a zeta potential of 27.3 mV at neutral pH. However, addition of stearylamine produced SLNs with a positive zeta potential. Increasing the stearylamine content also increased the zeta potential of the SLN nanoparticles as shown in table 1. A low positive zeta potential could effectively help in bioadhesion and uptake by the intestinal Peyer's patches or by paracellular route, thereby increasing the bioavailability of the drug.

SLN nanoparticles prepared with varying ratio of lauric acid and stearylamine were attached to the cylindrical probe using double sided adhesive tapes. This probe was immersed in the test medium for 10 min prior to attachment to a duodenal part of small intestinal mucosa. This was pulled out at a speed of 0.5mm/s after attachment to the mucosa at a contact force of 0.05 N and contact time of 1s. Table 1 shows the [F.sub.max] of pre-max hydrated nanoparticle discs of SLN nanoparticles, lauric acid and chitosan nanoparticles (as control) after attachment to duodenal mucosa. The results showed that the [F.sub.max] of the SLN formulation (250:200) was significantly higher than lauric acid SLN (200:0) and chitosan. Work of adhesion ([W.sub.ad]) for the SLN formulation (250:200) was also significantly higher at 428.8 mN.mm than 132.1 and 144.4 mN.mm for lauric acid SLN (200:0) and chitosan respectively. The [F.sub.max] and [W.sub.ad]. was increasing with respect to the content of stearylamine and was proportional to the zeta potential of the particles. The significant increase in bioadhesion for optimized SLN formulation could be because of its cationic nature that enhances its electrostatic interaction with negatively charged mucosal surface. Mucoadhesion involves various interactive forces between mucoadhesive materials and the mucus surface, including electrostatic attraction, hydrogen bonding, Van der Waals forces, mechanical interpenetration and entanglement [24]. Based on the zeta potential and the bioadhesion results, SLN formulation prepared with the specific ratio (250:200) was selected for further experiments.

The SLNs formulated in this study were found to be in the size range of 90 to 500nm. The mean particle size of the optimized formulation was found to be 244 [+ or -] 3 nm with a PDI of 0.159 as shown in figure 1. The morphology of the particles was evaluated using AFM (not shown). It shows irregular particles of around 200nm. It has been reported that nanoparticles mainly distribute into liver (60-90%), spleen (2-10%), lungs (3-20% and more), and bone marrow (>1%) [25]. However, solid lipid nanoparticles are composed of triglycerides that orient to form a polar core with polar heads oriented toward the aqueous phase, resembling chylomicrons. This composition decides the course of drug absorption to and through lymphatic route, following a transcellular path of lipid absorption, especially by enterocytes and polar epithelial cells of the intestine [26]. Mechanism of transcellular absorption of SLN is similar to that of dietary lipids. The lipid vehicle enhances the stimulation of chylomicron formation by enterocytes, which dissolve and assimilate lipophilic molecules into their nonpolar core and thus promote the absorption of clopidogrel into the intestinal lymphatics and organs [27].

The clopidogrel content in the optimized SLN formulation determined by HPLC was found to be 7.4172 mg/250mg. The entrapment efficiency was found to be 48%. The melting and re-crystallization behavior of crystalline materials like SLNs were investigated with DSC. Figure 2 shows an overview of the melting process of drug-free SLN and SLNs containing clopidogrel along with clopidogrel. The melting process took place with maximum peak at 52[degrees]C for drug-free SLN as well as for clopidogrel loaded SLN. The melting point of the drug was 149.5[degrees]C. However, the melting point of pure lauric acid and stearylamine was 44[degrees]C and 52.5[degrees]C (not shown) respectively.

The diffraction pattern of bulk lauric acid showed significant difference from those of SLN nanoparticles. Both drug-free and drug loaded SLN's showed an amorphous state compared to bulk lauric acid as shown in figure 3. It has been reported that the SLNs with amorphous state would contribute to the higher drug loading capacity [28]. It has also been observed that the highly crystalline clopidogrel did not show in the SLNs and the sharp peaks of the drug disappeared. The decrease in crystallinity indicates an enhanced solubility of clopidogrel in lauric acid. Polymorphic crystalline changes occur in the lipid structure which upon heating is converted into more stable form [29].

A comparison of FTIR spectrum of clopidogrel sample with that of the reference standard showed characteristic peaks at relevant regions as shown in figure 4. The band due to aromatic C-H stretching vibrations was present at 3121[cm.sup.-1]. A broad absorbance band at about 2500-2550[cm.sup.-1] associated with stretching vibrations of bonded [N.sup.+]-H occurring due to salt formation between the quaternary nitrogen of clopidogrel and OH of hydrogen sulphate. A strong absorbance band due to C=O stretching vibrations at 1752 [cm.sup.-1]. The band associated with C-O stretching appeared at 1175 [cm.sup.-1], 1187 [cm.sup.-1] and 1155 [cm.sup.-1]. SLNs (figure 5b) shows C-O,C-C stretching, CH rockimg at 842 [cm.sup.-1], C=O stretching in dimmers at 1694 [cm.sup.-1], C-O stretching in systems with hydrogen bond at 1276 [cm.sup.-1] corresponding to lauric acid and C[H.sub.2] deformation at 1473, C[H.sub.3] at 1382, C[H.sub.2] asymmetric stretching at 2915, C[H.sub.3] assymetric stretching at 2955, and C[H.sub.2] symmetric stretching at 2848 [cm.sup.-1] corresponding to stearylamine.

In order to obtain some preliminary information about the potential use of the SLN formulation as a delivery system for oral administration of clopidogrel, in vitro hydrolysis studies were performed, subjecting the hydrolysis in simulated gastric (SGF, pH 1.2), and intestinal fluids (SIF, pH 7.4). A moderate sustained release of clopidogrel was observed in the intestinal as well as gastric fluid as shown in figure 5 indicating no pH sensitivity. Clopidogrel may be embedded within the outer shell of the lipid rather than in the inner core.

The aggregations of the red blood cells on interaction with the nanoparticles are shown in figure 6. It revealed no gross aggregation of blood cells on incubation with SLN nanoparticles. Polyethyeleneimine (PEI) which was used as positive control showed aggregation whereas saline used as negative control did not show any aggregation. The same pattern was visible with the hemolytic property of the nanoparticles as given in table 2.

The hemolysis induced by SLN formulation was significantly low at 0.34% which was well within the acceptable limits of 1%. The interactions of solid lipid nanoparticles with blood cells, particularly with platelets are of significant importance as blood cell aggregation is crucial for vascular hemostasis and thrombosis. It has been reported that translocated ultrafine particles in the systemic circulation can directly play an important role in the clotting system.

The C3 adsorption data is given in table 2. The amount of C3 in blood (pre-incubation) was 116 mg%. After incubation with SLN formulation and lauric acid SLNs the amount of C3 were 115 and 116 mg% respectively indicating no significant depletion of C3 protein, which indicates that SLN nanoparticles are not complement activating. Measuring C3a or C5a in blood or serum after contact with a material has been the most usual way of assessing complement activation. It has been claimed that a surface is biocompatible if these markers are not increased in the fluid phase [30]. Since C3 is cleaved to C3a and C3b by the contact of the surface with blood, irrespective of whether the activation occurs via classical or alternative pathways, and also C3a could be adsorbed on to the material surface just like any other proteins, C3 depletion in the medium can be taken as an indirect measure of complement activation. Adsorption of C3 triggers complement activation [31]. It has been demonstrated in this study that the adsorptions of C3 on SLN were insignificant, indicating that these nanoparticles do not induce any complement activation when it comes in contact with blood. The platelet factor (PF4) level in control plasma was 15.4 IU/ ml and after incubation with SLN formulation for 15 min it was 16.1 IU/ml and 15.8 IU/ml for lauric acid SLN nanoparticles as shown in table 2. Activation of platelets initiates the deformation of the cells with pseudopod formation and ends with blood coagulation or thrombus formation [31]. In the present study it has been observed that the SLN nanoparticles are compatible with the blood cells which do not initiates any aggregation of cells and also the nanoparticles do not seem to activate the platelets. This is an indication of the very high blood cell compatibility of the SLN nanoparticles.

Tight junction visualization studies demonstrated that SLN nanoparticles are capable of opening tight junctions. The control Caco-2 cells stained with rhodamine phalloidin, to visualize actin protein, showed uniform staining pattern as shown in figure 7(a). This was similar to the cells treated with clopidogrel drug suspension. However, cells treated with SLN nanoparticles showed disrupted staining pattern (figure 7(b)). Actin filaments were observed to be discontinuous and disrupted as evidenced from the staining pattern and the clumping. To further investigate the effect on the tight junction proteins, immunofluorescence studies using anti ZO-1 were done. ZO-1 is a tight junction associated protein which plays an important role in tight junction functional regulation. The effect of nanoparticles on ZO-1 tight junction proteins was evaluated on Caco 2 cell monolayers and is also shown in figure 7. The untreated cells as well as cells treated with clopidogrel drug suspension were observed as smooth lines at the cell-cell junction (figure 7(c)); whereas, for SLN nanoparticles-treated cells the staining intensity was weaker at cell-cell contact sites (figure 7(d)). Tight junctions are composed of transmembrane proteins occludin, claudins and junctional adhesion molecules which intercalate with corresponding proteins from adjacent cells to form the intercellular barrier. These proteins associate with peripheral membrane proteins including the membrane proteins zonula occludens (ZO-1 to 3), which joins the transmembrane proteins, to the actin cytoskeleton. ZO-1 and occluding phosphorylation are associated with stimulus-induced tight junction disassembly and paracellular permeability changes. In the untreated cells, ZO-1 was observed as smooth lines at cell-cell junctions. The immunofluorescent staining intensity of SLN nanoparticles treated cells were observed to be weaker, when compared to control which indicated the loss of ZO-1 from sites of cell-cell contact.

L929 cell is an established cell line which has been extensively used for cytotoxicity studies. The percentage of viable cells compared with negative control represented the level of cytotoxicity of the particles. Percentage of viable cells on incubation of SLN formulation was more than 98%, which confirms that, they are non-toxic to the L-929 cells. Red-blood cell lysis experiment has been done and reported in table 2. Hemolysis caused by SLN formulation was practically nil. This results support the cytotoxicity results. Thus, the cationically modified SLN formulation seems to be a better candidate for intestinal delivery, since it could be highly compatible with intestinal cell walls.

Animal studies

The reported per oral dose of clopidogrel alone administered to human rats was 75 mg/kg [32], and the oral plasma bioavailability has been reported as <50% [13]. Based on this an oral dose of 10mg/kg was selected for our study. The plasma concentration of clopidogrel at different time intervals is shown in figure 8. Free clopidogrel administered rats showed almost a steady concentration of clopidogrel whereas rats administered with SLN nanoparticles showed an initial concentration of 9.45 [+ or -] 0.3 ng/ml at 120 minutes. The [C.sub.max] was 9.58 [+ or -] 0.4 and 9.39 [+ or -] 0.5 ng/ml with a [T.sub.max] of 180 and 60 min respectively for SLN formulation and clopidogrel suspension. The [AUC.sub.0-480] was 3533 [+ or -] 160 and 1258 [+ or -] 65 ng.min/ml respectively for the SLN formulation and clopidogrel suspension. The difference was significant (p<0.05) and the calculated relative bioavailability was 280%. A significant enhancement in the bioavailability of clopidogrel was observed on administration of SLN formulation compared to drug suspension. The result indicated that cationic lauric acid SLNs enhances the intestinal absorption of the drug. The relative percentage bioavailability of clopidogrel was about 280% which assures the potential use of cationic lauric acid SLN as a novel carrier for the poorly bioavailable antithrombotic drug, clopidogrel. The absorption of lipids via intestinal lymphatics might be the reason for improved oral bioavailability.

Determination of in vivo clotting time revealed that a controlled release and prolonged action could be achieved using SLN formulation compared to clopidogrel suspension as shown in the figure 9. Prolonged antiplatelet therapy with high plasma concentration has important implications for the subsequent performance of surgical procedures that requires stoppage of the therapy which may lead to thrombosis. Excess bleeding in cases of intracranial hemorrhage, peptic ulcer etc. is also a significant problem. Significant liver impairment has also been reported. Therefore maintaining the clotting time at the minimum required level using SLN formulation is an advantage.

Freeze dried SLN formulation was dispersed in PBS and the EE was determined for each evaluation period. The amount of clopidogrel was assayed by HPLC as shown in figure 10. Estimation of shelf life was assessed on the basis of drug loading (ie; the ratio between the mass of entrapped drug and the total mass of the lipid) and also particle size variations during the three months of storage at 4[degrees]C. Corresponding data are given in the table 3. The obtained results indicated no significant change in the mean particle size and PDI after storage at refrigerated conditions. The decrease in the drug content may be due to the expulsion of the drug from the lipid matrix during storage, which projected a shelf life of 13.25 months.

Conclusion

Solid lipid nanoparticles have been widely accepted for the delivery of several bioactives. In the case of clopidogrel, bioavailability of the marketed formulation is significantly low at <50%. Hence the role of colloidal drug delivery systems of suitable particle size and lipophilicity becomes significant as effective carriers for delivery of antiplatelet drugs. Since highly lipophilic drugs like clopidogrel are easy to entrap within the solid lipid matrix it can be used for the intestinal delivery of this group. For effective targeting of these drugs to the intestine, development of a SLN formulation is necessary. Rapid gastrointestinal emptying and rapid metabolism by liver etc. are the obstacles in developing effective nanoparticle formulations of this group of drugs. Lauric acid based cationic solid lipid nanoparticles with a combination of stearylamine can prolong the residence time of the nanoparticles in the intestine and may also help in effective uptake of these particles by the Peyer's patches or via paracellular route. Thus the drug can be effectively absorbed through the lymphatic system, prolonging the circulation of drug in blood by acting as a reservoir, and can effectively increase the bioavailability of the drug approximately 3 fold due to the combined effect of lipophilicity and surface charge. The high biocompatibility, biodegradability and nontoxicity again make the cationically modified lauric acid SLNs an excellent carrier for intestinal delivery of clopidogrel.

Acknowledgment

We are grateful to the Director and the Head BMT Wing of SCTIMST for providing facilities for the completion of this work. This work was supported by the Department of Science & Technology, Govt. of India through the project 'Facility for nano/microparticle based biomaterials--advanced drug delivery systems' #8013, under the Drugs & Pharmaceuticals Research Programme.

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Joshah Varghese (1), Willi Paul (2), Deepa Karthikeyan (1) and Chandra P. Sharma (2) *

(1) Department of Pharmaceuitics, Nehru College of Pharmacy, Pampady, Thrissur 680588, India

(2) Division of Biosurface Technology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Thiruvananthapuram 695012, India

Received 27 November 2017; Accepted 30 December 2017; Published online 31 December 2017

* Coresponding author: Dr. Chandra P. Sharma; E-mail: drsharmacp@yahoo.com

Caption: Figure 1: Particle size distribution of the optimized clopidogrel-SLN formulation

Caption: Figure 2: DSC thermograms of clopidogrel-SLN formulation and placebo SLN along with clopidogrel

Caption: Figure 3: X-ray Diffraction spectra of (a) the drug, clopidogrel (b) the optimized SLN formulation and placebo SLN

Caption: Figure 4: FTIR spectra of (a) the drug, clopidogrel (b) the optimized SLN formulation and placebo SLN

Caption: Figure 5: In vitro release kinetics of clopidogrel from optimized SLN formulation and the drug suspension

Caption: Figure 6: RBC aggregation on incubation with (a) SLN formulation, (b) placebo SLN, (c) saline (negative control) and (d) PEI (positive control) with RBC suspension

Caption: Figure 7: Confocal images (20 x) of Caco 2 cells--Actin. a) Caco cells without any treatment (control). b) Caco cells exposed to 5 mg of SLN formulation for 1h.--Tight-junction protein ZO-1. c) Caco cells without any treatment (control). d) Caco cells exposed to 5 mg of SLN formulation for 1h

Caption: Figure 8: Plasma concentration versus time curves of clopidogrel obtained after administration of the optimized clopidogrel-SLN formulation and of a suspension of clopidogrel

Caption: Figure 9: Whole blood clotting time of rat blood versus time curves of clopidogrel monitored after administration of the optimized clopidogrel-SLN formulation and of a suspension of clopidogrel

Caption: Figure 10: Estimation of the shelf life of clopidogrelSLN formulation on the basis of drug loading
Table 1: Zeta potential, mean particle size and p.d.i of SLN
nanoparticles prepared with different ratios of lauric acid
and stearylamine

Lipid:Lipid Ratio          Zeta                 Mean
(Laurie acid:           potential          Particle Size
Stearylamine)        (mV [+ or -] SD)     (nm [+ or -] SD)

0:250               +35.4 [+ or -] 0.3           --
250:0               -27.3 [+ or -] 1.1   136.4 [+ or -] 5.1
250:50              +4.2 [+ or -] 0.9    93 5 [+ or -] 4 1
250: 100            +6.3 [+ or -] 4.2    357.2 [+ or -] 6.4
250: 150            +8.7 [+ or -] 4.9    166.2 [+ or -] 7.6
250: 200            +12.2 [+ or -] 4.1   244.5 [+ or -] 3.1
Chitosan                    --                   --

Lipid:Lipid Ratio   Polydispersity          [F.sub.max]
(Laurie acid:          (p.d.i.)                (mN)
Stearylamine)

0:250                     --                  --
250:0                   0 174        528.0 [+ or -] 23.7
250:50                  0 125        723.2 [+ or -] 22.8
250: 100                0 195        795.6 [+ or -] 28.5
250: 150                0 124        831.4 [+ or -] 35.1
250: 200                0 159        1402.6 [+ or -] 56.1
Chitosan                  --           432 [+ or -] 34

                      [W.sub.ad]
Lipid:Lipid Ratio     (mN.mm)
(Laurie acid:
Stearylamine)

0:250                     --
250:0               132 [+ or -] 13
250:50              165 [+ or -] 10
250: 100            181 [+ or -] 15
250: 150            199 [+ or -] 14
250: 200            429 [+ or -] 16
Chitosan            144 [+ or -] 11

Table 2: Hemolysis, cytotoxicity and bioadhesiveness of SLN
nanoparticles

Samples                Hemolysis           Cell Viability
                          (%)                   (%)

SLN formulation   0.3414 [+ or -] 0.10   102.1 [+ or -] 1.2
SLN (blank)       0.2674 [+ or -] 0.14   101.5 [+ or -] 2.1
Laurie acid SLN   0.3123 [+ or -] 0.08    98 [+ or -] 2.6
Control                   100                   100
                        (water)               (medium)

Samples           Platelet Factor 4         C3
                       (TU/ml)             (mg%)

SLN formulation   16.1 [+ or -] 1.2   115 [+ or -] 15
SLN (blank)              --                 --
Laurie acid SLN   15.8 [+ or -] 1.5   116 [+ or -] 12
Control           15.4 [+ or -] 1.5   116 [+ or -] 9
                      (plasma)           (plasma)

Table 3: Stability characteristics of freeze dried
SLN formulation (n=3)

Drug content                          Test period

                            0 month               1st month

Particle size(nm)     231.2 [+ or -] 1.836   231.1 [+ or -] 1.004
PDI                          0.232                  0 212
Active drug content           100                   99.65

Drug content                          Test period

                           2nd month              3rd month

Particle size(nm)     234.0 [+ or -] 2.542   236.07 [+ or -] 2.07
PDI                          0.241                  0.284
Active drug content          99.23                  98 41
(%)
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Title Annotation:Original Article
Author:Varghese, Joshah; Paul, Willi; Karthikeyan, Deepa; Sharma, Chandra P.
Publication:Trends in Biomaterials and Artificial Organs
Date:Oct 1, 2017
Words:6320
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