Cystatin incorporated in poly(lactide-co-glycolide) nanoparticles: development and fundamental studies on preservation of its activity

https://doi.org/10.1016/j.ejps.2004.04.003Get rights and content

Abstract

Preservation of biological activity is still a major challenge for successful formulation and delivery of protein drugs. Cystatin, a potential protein drug in cancer therapy, was incorporated in poly(lactide-co-glycolide) nanoparticles by the water-in-oil-in-water emulsion solvent diffusion technique. In order to preserve the biological activity of cystatin, a specific modification of the method of producing nanoparticles was introduced. The activity of cystatin was strongly influenced by the stirring rate during preparation and, to a lesser extent, by selected organic solvents. A synergistic effect of mechanical stirring and sonication, both at low energy levels, enabled nanoparticles to be formed without denaturing the cystatin. Nanoparticles produced by the optimised method ranged from 300 to 350 nm in diameter with 85% of the starting cystatin activity. The loading efficiency of cystatin depends on polymer type and ranged from 12 to 57%, representing an actual loading of 0.6–2.6% (w/w). Among various cryo-/lyoprotectants bovine serum albumin was identified as the most successful. The use of a protein protectant prior to nanoparticle formation was essential to maintaining the biologically active three-dimensional structure of cystatin. In addition, a specific type of poly(lactide-co-glycolide) polymer, particularly in terms of its functional groups, was identified to be important in retaining cystatin activity. Cystatin incorporated into nanoparticles in this way maintains its structural integrity, making it suitable for effective drug delivery.

Introduction

Rapid advances in the fields of biotechnology and genetic engineering have generated an interest in peptide and protein drugs. They are an important class of therapeutic agents and are likely to replace many existing drugs due to their specific actions in disease treatment. Cystatins, which are extracellular inhibitors of cysteine proteases, have been suggested to be involved in various diseases, including cancer (Kos et al., 2000). In tumour cells cysteine proteases are overexpressed and translocated to the plasma membrane or secreted from the cells, and take part in the degradation of components of the extracellular matrix and basement membrane, which is deemed to be a crucial step in the metastatic process. Thus, inhibitors of cysteine proteases could possibly be new drugs for use in cancer therapy. However, cystatins, like other proteins, require individual, innovative approaches to their formulation in delivery systems.

Molecules of any size, including globular proteins such as insulin and calcitonin (Dorkoosh et al., 2002), tetanus toxoid (Vila et al., 2000, Tobio and Alonso, 1998), somastatin acetate (Herrmann and Bodmeier, 1998), cyclosporin A (Lee et al., 2002), HIV-1 proteinase inhibitors (Jaeghere et al., 2000), interferon-α (Sanchez et al., 2003) and others can now be formulated into delivery systems. Protein encapsulation and delivery with poly(lactide-co-glycolide) nanoparticles appears to be a very promising approach (Alonso, 1996). Important advantages associated with the use of nanoparticles (NPs) include their high stability during storage, protection of the protein when incorporated inside the carrier matrix, variable release kinetics by using different polymers, and small particle size (Kristl et al., 1996). The latter shows the potential not only for parenteral but also for non-parenteral administration. It has been shown that the size of nanoparticles is a crucial parameter for oral administration, since it determines the uptake of nanoparticles across the mucosal and intestinal epithelia (Norris et al., 1998, Florence, 1997, Jung et al., 2000).

A major challenge in protein formulation is to preserve protein activity during the preparation of small size nanoparticles. Unlike low molecular weight drugs, proteins have relatively large globular structures, possessing complex internal architecture that defines their unique biological functions. Techniques generally used for producing nanoparticles involve processing conditions that are frequently inappropriate to maintaining protein stability (Quintanar-Guerrero et al., 1998, Murakami et al., 2000). If the protein is denatured during encapsulation in NP, it will be therapeutically inactive, and may cause unpredictable side effects, such as immunogenicity or toxicity. Although some successful guidelines are available, development of a particular protein formulation still must be determined experimentally because proteins differ in many aspects, such as stability, solubility, affinity to polymer matrices, etc. (Alonso, 1996). Among various techniques available to entrap water-soluble bioactive agents into biodegradable NP the w/o/w double emulsion solvent diffusion technique is the most popular. It consist of four steps: (1) primary emulsification: water solution of the active agent is emulsified into an organic solution containing PLGA polymer, (2) re-emulsification: primary emulsion w/o is further emulsified into a second aqueous phase containing a stabilizer PVA to form w/o/w double emulsion, (3) solidification: the organic solvent is extracted in the continuous aqueous phase resulting in precipitation of polymeric particles, and finally (4) separation and purification: nanoparticles are collected by centrifugation or filtration and subsequently lyophilised (Quintanar-Guerrero et al., 1998, Alonso, 1996).

Chicken cystatin is a reversible, tight-binding, protein inhibitor of papain-like cysteine proteases. A model, based on the X-ray crystal structure, was proposed for its interaction with papain (Bode et al., 1988). Predicted model of interaction of cystatin with cysteine protease clearly indicates that cystatin must be preserved in the native three-dimensional state to be biologically active, since several distinct parts in its structure should participate in the interactions with the enzyme (Fig. 1).

Our primary goals were to incorporate cystatin in poly (lactide-co-glycolide) (PLGA) nanoparticles, to adjust the processing conditions in order to produce small size nanoparticles, and to preserve the activity of the protein. To this end, the process variables were investigated systematically, to modulate the size of the nanoparticles and the protein loading capacity, and to study the effects of PLGA type and lyoprotectants, and the structural stability of cystatin in NP during release in vitro.

Section snippets

Materials

Chicken cystatin, MW 13 kDa, was isolated from chicken egg white (Kos et al., 1992). PLGA (lactic acid/glycolic acid, 50/50) copolymers (Resomer RG® 503 H with MW 48 kDa, Resomer RG® 502 H with MW 12 kDa, and Resomer RG® 502 with MW 12 kDa) were obtained from Boehringer (Ingelheim, Germany), polyvinyl alcohol (PVA) (Mowiol® 4–98) from Hoechst (Frankfurt, Germany), ethyl acetate, dichloromethane and acetone from Merck (Darmstadt, Germany), bovine serum albumin, trehalose, mannose, fructose and

Preparation of nanoparticles

Chicken cystatin was selected as a testing protein for enhanced delivery with NPs due to its high potential for inactivation of cysteine proteases, enzymes that are involved in progression of malignant disease (Premzl and Kos, 2003). In an attempt to developed new dosage form, PLGA polymers were chosen for nanoparticles due to their biocompatibility and biodegradability as approved from FDA, and since they have also been the most extensively investigated for drug delivery. Optimisation of

Conclusion

We have identified several process variables that are crucial for preserving the biological activity of cystatin, a potential protein drug, during the production of nanoparticle-formulations. An optimised method was introduced to adjust the processing conditions to give small size nanoparticles and to preserve the activity of cystatin. The results also show that appropriate selection of cryo-/lyoprotectants protects nanoparticle-entrapped cystatin during lyophilization. Additionally, we have

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