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Preparation of stable insulin-loaded nanospheres of poly(ethylene glycol) macromers and N-isopropyl acrylamide

https://doi.org/10.1016/S0168-3659(02)00028-7Get rights and content

Abstract

A series of nanospheres composed of temperature-sensitive poly(N-isopropylacrylamide), poly(ethylene glycol) 400 dimethacrylate, and poly(ethylene glycol) 1000 methacrylate was prepared by a thermally-initiated free radical dispersion polymerization method. Insulin was loaded into the nanoparticles by equilibrium partitioning. The loading capacity of insulin into the nanoparticles was 2.1% (2.1 mg insulin/100 mg nanoparticles). The stability of the loaded insulin at elevated temperatures was investigated by reverse phase high pressure liquid chromatography. The nanoparticles were able to protect the loaded insulin, as more than 80% of the loaded insulin could still be detected compared to 0% for the control (0.1% insulin solution in PBS) when heated to 80 °C for 5 h. The stability of the loaded insulin at high shear stress (289 1/s) was also investigated. No significant loss of insulin was detected both from nanoparticles loaded with insulin sample and the control (0.1% insulin solution in PBS). The results showed that shear stress alone did not have a major effect on insulin denaturation. The ability of the nanoparticles to protect the insulin from high temperature and high shear stress made the system a good candidate as a carrier for insulin for fluidized bed coating technology.

Introduction

The stability characteristics of proteins and peptides are important in the formulation of protein delivery products. Chemical stability refers to reforming and breaking of covalent bonds due to deamidation, oxidation, and disulfide exchange. The sensitivity of particular amino acid residues towards deamidation and oxidation is affected by the accessibility of a protein domain to exogenous oxidants, presence of metal binding sites and potential neighboring group effects [1]. The ability to create a system that will be able to provide an extra barrier in order to limit the accessibility of denaturing agents to sensitive sequence of amino acids of a protein or peptide is very important to increase the chemical stability.

Numerous researchers have reported studies on the damaging effects of shear stresses in fluid flow on globular proteins [2], [3], [4], [5], [6], [7], [8], [9], [10]. For example, Alder and Lee [4] studied the stability of lactate dehydrogenase (LDH) during the spray drying process and also on subsequent dry storage. They found that the process temperature has a measurable effect on LDH inactivation. At a high operating temperature (Tout=95 °C), 25% of the LDH was lost due to denaturation. At a lower operating temperature (Tout=70 °C), 11% of the LDH was lost. However, the storage stability was a problem for the system prepared at a lower operating temperature. At a lower operating temperature, the system could not dry effectively, and hence, resulted in high moisture content in the system. This remaining moisture content caused the protein to denature during the storage period. They also found out that the air–liquid interface had an adverse effect on LDH stability. LDH is surface active which causes the LDH to assemble on the interface. This caused the LDH to be shear-denatured during the spray-drying process.

Clarkson et al. [5] tried to elucidate the mechanism by which protein molecules became denatured during foaming. They found that the damage to the protein was due to surface denaturation at the air–liquid interface. They hypothesized that a fraction of the proteins absorbed to the interface did not refold to their native state when they desorbed. They found a direct correlation between the degree of denaturation and the interfacial exposure. Oxidation was eliminated as the major cause of denaturation because the same degree of denaturation was observed when the air was replaced with nitrogen.

Maa and co-workers [6], [7], [8], [9], [10] investigated the effect of shear and air–liquid interface on protein denaturation in detail. They found no major loss of anti Ig-E antibody, rhGH and rhDNase due to shear stress alone. However a significant aggregation was observed for rhGH when the protein was exposed to shear stress combined with high air–liquid interface. They found that the degree of aggregation was proportional to the total air–liquid interface and was independent of the concentration of the rhGH.

The work of Alder [4], Clarkson et al. [5], and Maa and colleagues [6], [7], [8], [9], [10] clearly substantiated the significance of the protein stability problem posed by elevated temperature, air–liquid interface and shear stress. In our work, these three problems were addressed by the synthesis of poly(ethylene glycol)-containing nanoparticles.

With respect to the stability of proteins and peptides, the major challenges of a microencapsulation process with a fluidized bed coating are the stability against elevated temperatures and high shear stresses encountered during the coating process. In this study, we designed two sets of experimental set-ups that allowed these harsh conditions of the real coating process to be studied independently of each other. The protective effect of the synthesized nanoparticles on the thermal and mechanical stability of insulin was then assessed.

Section snippets

Nanoparticle preparation

A free radical dispersion polymerization was used to prepare nanoparticles of poly(N-isopropyl acrylamide-co-poly(ethylene glycol) 1000 methacrylate) (P(NIPAAm-co-PEGMA)). The crosslinker used was poly(ethylene glycol) 400 dimethacrylate (PEGDMA). Prior to the reaction, NIPAAm (Fischer Scientific, Pittsburgh, PA) was recrystallized in benzene/hexane. PEGDMA and PEGMA (Polysciences, Warrington, PA) were used as received. In a typical experiment, NIPAAm, PEGDMA, and PEGMA (70:20:10 w/w%) were

Insulin loading into the nanoparticles

The temperature sensitivity of the nanoparticles was used for the loading of the insulin. The loading process was conducted at 4 °C for 24 h and then the nanoparticles were collapsed at 37 °C. The temperatures of 4 and 37 °C were selected because they were the lowest temperatures providing maximum volume swelling. Indeed we wanted to avoid higher collapsing temperature and avoid induction of a possible thermal denaturation of insulin in the loading process (Fig. 1). The loading efficiency

Conclusions

The PEG-nanoparticles showed good insulin protecting properties from high temperature and high shear stress and could be used as a carrier for sensitive proteins and peptides during fluidized bed coating process. The PEG-nanoparticles could be loaded with insulin with a high loading efficiency at 65%. The loading capacity was 2.1% (w insulin/w nanoparticles). The PEG-nanoparticles also showed remarkable results in protecting the insulin from elevated temperature. After 8 h of heating at 60 °C,

Acknowledgments

This work was supported in part by a grant from the National Science Foundation of the USA (BES-97-06538) and a Grant-in-Aid for Scientific Research (C-12672098) from the Japan Society for the Promotion of Science.

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