Water-based nanoparticulate polymeric system for protein delivery

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COMMUNICATION TO THE EDITOR Water-Based Nanoparticulate Polymeric System for Protein Delivery: Permeability Control and Vaccine Application Ales Prokop,1 Evgenii Kozlov,1 Gale W. Newman,2 Mark J. Newman3 1 Chemical Engineering Department, Vanderbilt University, Nashville, Tennessee 37235; telephone: 615-343-3515; fax: 615-343-7951; e-mail: [email protected] 2 Morehouse School of Medicine, Atlanta, Georgia 30310 3 Epimmune Inc., 5820 Nancy Ridge Drive, San Diego, California 92121

Received 15 June 2001; accepted 15 November 2001 DOI: 10.1002/bit.1200

Abstract: The idea of using polymeric nanoparticles as drug carriers is receiving an increasing amount of attention both in academia and industry, Nanoparticles have a number of potential applications in protein, drug and vaccine delivery, as well as gene therapy applications. In this article, we focus on this unique drug delivery technology as a method to control the release rate of substances, not only for protein delivery but also for delivering an experimental vaccine immunogen. Nanoparticles were assembled on the basis of ionic interaction between water-soluble polymers so that the resulting particles were stable in physiologic media. Among the typical polymers used to assemble nanoparticles, different polysaccharides, natural amines, and poly-amines were investigated. The entrapped substances tested included a protein and antigens. Polydextran aldehyde was incorporated into the particle core, to enable physiologic cross-linking as a method to control permeability. This resulted in long-term retention of substances that would otherwise rapidly leak out of the nanoparticles. Results of cross-linking experiments clearly demonstrated that the release rate could be substantially reduced, depending on the degree of crosslinking. For vaccine antigen delivery tests, we measured an antibody production after subcutaneous and oral administration. The data indicated that only the crosslinked antigen was immunogenic when the oral route of administration was used. The data presented in this article address primarily the utility of nanoparticulates for oral delivery of vaccine antigen. ã 2002 Wiley Periodicals,

Inc. Biotechnol Bioeng 78: 459±466, 2002.

Keywords: nanoparticle; water-based; polymeric; antigen delivery; protein delivery; crosslinking; permeability control

Correspondence to: A. Prokop Contract grant sponsor: Vaxcel (Norcross, 6A; to A.P., E.K.); National Institutes of Health (to A.P., E.K.) Contract grant number: HL65982-02

ã 2002 Wiley Periodicals, Inc.

INTRODUCTION Vaccines that are composed of protein, protein±carbohydrate conjugates, or inactivated pathogens are almost universally delivered by needle injection (Kassianos, 1998; Plotkin and Mortimer, 1998). Although vaccines delivered using this route are generally capable of inducing systemic immune responses, generally antibody responses, they are not useful for inducing mucosal immune responses. The mucosal tissues are the most common port of entry for infectious agents and as such, the induction of mucosal immune responses could be highly advantageous and increase vaccine ecacy. The delivery of vaccines orally is the most logical approach, but this route presents numerous challenges for vaccine delivery (Woodley, 1994). Firstly, vaccine immunogens do not routinely survive the acidic and proteolytic environment of the stomach. Secondly, e€ective mucosal delivery of vaccines requires that the products be delivered to the gut-associated lymphoid tissues (GALT); for vaccines delivered orally the product must be taken up by the M cells located in the Peyer's patches of the intestine (Haneberg et al., 1995; Neutra et al., 1996). Delivery of vaccine immunogens in an encapsulated or particulate form is an approach that has been used successfully in animal models because the product can be protected from degradation and particles are most e€ectively taken up through the Peyer's patches (Santiago et al., 1995). One of the best-characterized methods is based on the use of poly-lactide-co-glycolide (PLG) polymer encapsulation (Duncan et al., 1996; Eldridge et al., 1991, 1992). The polymers used are biodegradable and the encapsulation processes can be controlled so that the particles formed are of the size for ecient uptake by M cells, 80% of the mice (data not presented). However, the cross-linked nanoparticulate formulation clearly augmented immunogenicity of the vaccine compared to soluble product and the CRL-1005, and approximately 70±100% of the immunized animals responded to levels comparable to those given CTO-adjuvanted formulations. With respect to biocompatibility and in¯ammatory responses, qualitative observations con®rmed absence of such the following reactions: absence of sustained

Figure 4. Anti-OVA titers in mice at day 21, 28, 49, and 56 for di€erent experimental groups immunized orally. Groups are the same as in Figure 4. Data are mean ‹SD. Number of responders as a fraction. Nanoparticulate chemistry was as follows: Core: 0.025% HV, 0.025% CS, 0.67% OVA, 0.014% PDA; Shell: 0.065% PMCG, 0.05% CaCl2, 1% F-68. Antigen was crosslinked by PDA.

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mononuclear cell, eosinophils, and neutrophil in®ltration into the injection sites. DISCUSSION A new core-shell type of nanoparticles (assembled in a top-down process) could be considered as a matrix type device, because the vaccine antigen is part of the particle structure and the nanoparticulate shell. The vaccine immunogen is contained in the reservoir (core) from which it can exit. The manner in which the protein is released from the nanoparticulates is dependent on the chemical nature of the product. If the release of the protein is faster than the matrix degradation process, then the mechanism of release is most likely di€usion. However, the release of materials from nanoparticles involves the process of partitioning because partitioning is based on a high af®nity of drug, or protein, to the matrix. In case of drug release from hydrogels, such release occurs mainly due to the particle swelling, which can be controlled by formulation chemistry of the matrix (ionizable groups, degree of crosslinking) and by the environmental conditions (pH, ionic strength, etc., Peppas et al., 2000). Hydrogels possessing ionizable and often pendant groups exhibit increased swelling due to localization of charges within the hydrogel. The release of a drug from such a formulation involves the dissociation of the drug-polymer(s) complex through the exchange of the drug with incoming counterions from the dissolution medium. Based on our data, we believe that the release mechanism of proteins from nanoparticles that were not cross-linked is due to a combined e€ect of swelling, di€usion from the matrix-associated complex, and hydrophobic interactions. Ionic strength and pH in¯uenced both swelling and ion exchange phenomena, e€ects observed in our case. In addition, there was a sizable contribution of hydrophobic e€ects. As the opposite charges of interacting molecules are neutralized, and hydrophobicity rises, the particle is instantaneously formed. This is supported by our observations on the e€ect of ionic strength (Fig. 3), leading to an enhancement of release when the salt concentration is lowered. Hydrophobic interactions, in addition to ionic forces, were identi®ed as important mechanism for drug release from ion exchange resin (Jaskari et al., 2001). We used two di€erent methods to adjust the release kinetics. The polymeric matrix itself served as a slowly sequestering chemical container. As the protein was incorporated into the core polyanionic polymer mixture, it formed a complex. An equilibrium-binding constant between the protein and the polycationic matrix polymer(s) then may control the release rate. In addition, we used a reactive step using PDA (Fagnani et al., 1990), applied into the core polymeric formulation (Prokop, 1999). A short incubation of recovered nanoparticles at an elevated pH 8.3 provided physiologic cross-linking 464

conditions. The ®nal product is assumed to feature Schi€-base linkage between the carboxyl group of polysaccharides of the polymeric mixture and the amino groups of proteins. The development of linkages, which can be easily hydrolyzed, was our goal. Instead to developing a permanent covalent bond under the reductive conditions such as in presence of cyanoborohydrate, non-reductive conditions were used in our studies and this allowed us to take advantage of relatively labile Schi€-base. The choice of acidic and alkalic conditions for the in vitro OVA release experiments (Fig. 1) was in¯uenced by a desire to simulate environment relevant 4 to gastrointestinal passage (GI). It should be noted that the release rates were higher at acidic pH, however (a comparison data not reported here). On the other hand, the residence time in stomach is relatively short, compared to intestines. Drug loading capacity for water-soluble drugs is encouraged by creating an environment within the nanoparticles that is highly compatible with the solubilizate. We achieved reasonable levels of loading eciency, Table I, in some cases close to 50%. This high loading eciency is due to the fact that the charged entrapped proteins, OVA or TT, contributed to the structure of particle and become an integral part of the nanoparticles (Prokop, 1997). Calvo et al. (1997) described CT±TPP nanoparticulate delivery vehicles for protein delivery with high entrapment eciencies. Their particles, however, dissolve at low pH and lack an adjustment in controlled-release kinetics. Several key surface properties of importance to nanoparticulate delivery vehicles are surface charge and degree of hydrophilicity (hydrophobicity). The presence of polyethylene oxide (PEO, e.g., F-68), co-entrapped onto the surface of nanoparticles, provided a means of increasing surface hydrophilicity and produced a steric barrier preventing interaction with various biological components and, particularly, enhancing the particles stability in suspensions. The presence of long ¯exible PEO chains at the surface may stabilize the vehicle by creating entropic and osmotic forces that outweigh van der Waals or hydrophobic interactions (Lee et al., 1995). The surface density of PEO and its e€ect on biodistribution for our nanoparticulate systems is at present under study. Likewise, the e€ect of positive charge is not known (we cannot formulate a neutral control vehicle). Calvo et al. (1997) reported on PEO entrapped nanoparticles. The presence of PEO chains, although not detected directly, was inferred from a stoichiometry of the assembly process (Prokop et al., 2001). The presented technology provides a compatible environment that is suitable for protein entrapment. TT is known to be sensitive to denaturation during the processing (Tobio et al., 2000). We suspect that our vaccine data indicate that TT antigen is, at least partially, available for presentation in the GI system (intact) and that our water-based technology provides a

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gentle method for entrapping this antigen, opposite to 5 solvent-based technologies (Singh et al., 2000), and its subsequent bioavailability in vivo. The major obstacles for the delivery of protein vaccines orally are the acidic and proteolytic environment of the stomach and limited uptake of proteins by the GALT. The majority of available evidence suggests that the Mcells within the Peyer's patches are the predominant site of uptake of microparticulates (1±5 lm). However, nanoparticles may also access GALT via a paracellular mechanism (Damgge et al., 1996; Jani et al., 1990) and by transcytosis (Florence, 1997; Puchel et al., 1997). In both instances, particle uptake observed can be improved using particles with mucoadhesive properties. Many polymers we used to fabricate nanoparticles are mucoadhesive (Capron et al., 1996; Hunt et al., 1987). Among them are alginate, carrageenans, and pectin. Although these materials were often used as the core polymers in our nanoparticulates, and thus hidden inside the nanoparticulate structure, the interior can be exposed when the shell is lost or degraded. However, there is no doubt that the shell polymer Pluronic F-68, typically used to prevent particle aggregation, exhibits very high degree of mucoadhesion, as other members of the PEO family of polymers (Capron et al., 1996). The current encapsulation-based vaccine delivery have several limitations, which include relatively low bioavailability, less than 10% of the total protein dose in most cases (Gomez±Orellana and Paton, 1998), and encapsulation methodologies that destroy protein through the use of organic solvents or extreme temperatures. Such apparent low level of bioavailability appears to be sucient to initiate immune responses using our nanoparticles. Also, the production method used is based on water-soluble chemistry which should eliminate, or reduce greatly, destruction of the protein. This bene®t of this later property will need to be proven on a vaccine by vaccine basis as we did incorporate a crosslinking step that might alter protein structure and e€ect immunogenicity. However, the cross-linking is a gentle process and should provide a reliable means for limiting protein denaturation.

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