Preparation of biodegradable cyclosporine nanoparticles by high-pressure emulsification-solvent evaporation process

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Abstract

The cyclic endecapeptide cyclosporine (CsA), a potent immunosuppressive drug, was incorporated into biodegradable poly (dl-lactide-co-gylcolide) (dl-PLG) 50/50, 65/35 and PEG 5000-70/30 dl-PLG to improve the oral bioavailability and pharmacokinetics. Nanoparticles were prepared by a high-pressure emulsification-solvent evaporation (HPESE) process. The CsA-loaded nanoparticles were evaluated for particle size, zeta potential, surface morphology by scanning electron microscopy (SEM), thermal characterizations by differential scanning calorimetry (DSC), encapsulation efficiency (E.E.%) and in vitro release. The amount of CsA loaded into the nanoparticles was determined using high-performance liquid chromatography (HPLC) at a detection wavelength of 210 nm. The mobile phase was acetonitrile–water (70:30% v/v) and flow rate was set at 1.5 ml min−1. The photon correlation spectroscopy showed that the particles size were <250 nm and polydispersity index (PI) <0.14. The zeta potential was positive for 200 mg and negative for 400 mg of polymer composition, respectively. The SEM micrographs revealed that the nanoparticles were spherical and smooth. The drug loading was between 82% and 92%. Differential scanning calorimetry (DSC) studies did not show the melting endotherm for CsA in the drug-loaded nanoparticles. In-vitro release in intestinal fluid pH 6.8 (USP XXIV) showed a cumulative percent release of 30–45% CsA in 8 h. The physicochemical properties showed that the dl-PLG and PEG-DLPLG nanoparticles could be an effective carrier for oral CsA delivery. The reported method is easy, reproducible and can be automated for batch scale production.

Introduction

Biodegradable, oral polymeric nanoparticle delivery systems provide an attractive alternative for long-term delivery of therapeutic agents for chronic administration. These nanoparticles offer many advantages over conventional oral dosage forms. Drugs can be delivered in a sustained and continuous manner, encapsulated drugs are protected in the polymer network from gastric and enzymatic degradation and daily administration may not be required.

Cyclosporine (CsA) is a neutral, lipophilic, cyclic endecapeptide used as a first line immunosuppressive drug to prevent allograft rejection in organ transplantation. To be clinically effective, CsA required the development of a proper delivery system for both the oral and intravenous routes. Oral dose forms should produce both, a reasonably high bioavailability as well as a low inter and intrapatient variability in absorption. With conventional dose forms CsA bioavailability after oral dosing is low with a high inter patient variability (20–50%) although CsA is a critical dose drug with narrow therapeutic window [1], [2], [3]. Blood levels above the therapeutic range have been related to adverse effects, such as nephrotoxicity, neurotoxicity and hypertension [4], [5], [6], [7], [8]. Low levels of CsA are associated with danger of organ rejection [9], [10]. Due of these problems, transplant patients on CsA require careful monitoring.

Recently, a new microemulsion based oral CsA formulation (Neoral™, Novartis, Switzerland) was marketed. This preconcentrate spontaneously forms a microemulsion upon contact with water. Neoral is less dependent on bile for its absorption as compared to the conventional oil based Sandimmune™ (Novartis). This microemulsion formulation displays significantly less inter and intra individual variation [11], [12]. However, this new formulation also shows toxic and sub-therapeutic levels, and clinicians still cannot be sure of a uniform CsA exposure. The reasons for this has been attributed to high molecular weight, high lipophilicity, low intestinal permeability, extensive metabolism by CYP 450 3A, and efflux by a transporter, p-glycoprotein (P-gp) present in the gut wall [13], [14], [15]. Theoretically, protection against metabolism and bypassing drug efflux transporters should improve oral bioavailability of CsA.

Microparticles and nanoparticles were shown to enhance the oral absorption of a number of macromolecules and vaccines that are either poorly absorbed or are susceptible to gastrointestinal degradation [16], [17], [18], [19], [20], [21], [22].

Several studies done at the tissue and cellular levels have demonstrated that, latex, polystyrene and dl-PLG copolymer particles in a size range of 50 nm to 20 μm are absorbed mainly through the Peyer's patches found in small intestine, with little translocation occurring through non-lymphoid gut tissues [23], [24], [25], [26]. A recent study with differently sized colloidal gold has shown the particle uptake in small intestine by persorption through holes created by single, degrading enterocytes in the process of being extruded from a villus [27]. These studies have also explained that translocation is largely dependent on particle size: smaller particles are more readily absorbed. Particle composition and charge have been found to be important factors. Hydrophilic particles show a higher uptake, whereas negatively charged particles experience reduced absorption. Following absorption these particles localize mainly within the Peyer's patches and mesenteric lymph nodes, while small amounts are found in the liver and spleen [25], [26]. The immunosuppressive activity of CsA is related to a selective inhibitive effect on T lymphocytes, which circulate mainly in the lymphatic system [28]. Increasing the lymphatic level of CsA as well as reducing its blood concentration could enhance the therapeutic value of the drug. Therefore, targeting the lymphatic system through gut associated Peyer's patches could improve the therapeutic potential of CsA. The formulation of CsA as nanoparticles has received much attention in recent years as a suitable alternative system for cyclosporine delivery [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39]. Biodegradable nanoparticles have shown their ability to increase the oral bioavailability of CsA in rats. An in vivo study with CsA loaded polycaprolactone (PCL) nanoparticles showed a significant increase of the oral bioavailability after therapeutic dosing [32], [37]. Besides PCL, several CsA delivery systems using PLGA or PLA microparticles and nanoparticles have been reported. Recently, chitosan nanoparticles have been shown to increase the oral bioavailability by 1.8-fold when compared with reference Neoral microemulsion in beagle dogs [38].

The present study provides an improved method for producing CsA-loaded biodegradable nanoparticles and their physical characterization.

Section snippets

Material

Cyclosporine, CsA, (purity >99.32%) was kindly donated by RPG Life Sciences (Bombay, India). Polyvinyl alcohol, PVA (Av. MW 30 000–70 000 Da, Lot 042K0086) was purchased from Sigma, St. Louis, MO, USA). Poly (dl-lactide-co-glycolide) copolymer (dl-PLG) 50/50 M/M% (Purasorb, Purac, Holland, Lot no. 82/112). Poly (dl-lactide-co-glycolide) (dl-PLG) 65/35 M/M% (Birmingham Polymers, USA, Lot no. D95086) and PEG 5000/70:30 M/M% dl-lactide-co-glycolide (PEG-DLPLG) (Birmingham Polymers, USA, Lot no.

Results and discussion

Several studies have demonstrated an improved bioavailability of drugs encapsulated in nanoparticles and subsequent oral administration [41], [42], [43], [44]. dl-PLG nanoparticles coated either with albumin, an easily digestible protein, or PVA, a non-digestible synthetic polymer, have already been evaluated as a potential carrier system for oral administration [45]. These nanoparticles can be easily prepared by high-pressure emulsification-solvent evaporation (HPESE) process. The obtained

Conclusion

The HPESE process presented here produces small, monodisperse nanoparticles combined with a high encapsulation efficiency, easy control, and reproducibility. The process can be automated and scaled up for producing large amount of nanoparticles required for animal and human studies. Stability studies and in-vivo study may be reported in future publications.

Acknowledgements

Jagdish Jaiswal gratefully acknowledges DAAD, Germany for providing research fellowship (Kennziffer A/02/29754) to carry out this work in the laboratories of Prof. Dr. Kreuter. We thank Dr. Manfred Ruppel, Botanical Institute, Department of Biology and Computer Science, J.W.G. University for SEM pictures.

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