Preparation and in vitro characterization of HSA-mPEG nanoparticles

https://doi.org/10.1016/S0378-5173(99)00253-7Get rights and content

Abstract

Surface modified human serum albumin (HSA) nanoparticles with a size of approximately 150 nm in diameter were prepared from a PEG-HSA conjugate, methoxy-polyethylene glycol modified human serum albumin (HSA-mPEG) using a coacervation method and crosslinked with glutaraldehyde. The ζ-potential of the surface modified nanoparticles was significantly lower than that of unmodified HSA nanoparticles. The existence of a hydrated steric barrier surrounding the nanoparticles was confirmed by electrolyte and pH induced flocculation tests. The surface modified nanoparticles showed a reduced plasma protein adsorption on the particle surface compared with unmodified particles.

Introduction

The biodistribution of colloidal drug delivery systems after intravenous administration is mainly determined by their physicochemical properties such as size and surface characteristics. This is achieved through recognition or non-recognition of the colloidal system by the body’s defence system (Davis and Illum, 1986, Moghimi et al., 1991). Particles that are small enough to escape the capillary beds of the lungs are normally sequestered rapidly by the cells of the reticuloendothelial system (RES), particularly the Kupffer cells of the liver (Illum et al., 1982). This sequestration has been identified as a major obstacle to targeting cells or tissues elsewhere in the body such as the bone marrow and solid tumours (Artursson, 1987). On intravenous administration, particles are normally rapidly coated by the adsorption of specific blood components known as opsonins and then recognized and taken up by the RES. The adsorption of amphipathic copolymers, such as polyoxyethylene–polyoxypropylene (PEO-PPO) block copolymers (commercially available as poloxamer and poloxamine surfactants), onto polystyrene latex can form a hydrophilic steric barrier. This hydrophilic layer can dramatically affect the opsonization of particles by plasma components (Moghimi et al., 1993a, Moghimi, 1995). The uptake of the particles by the RES is consequently reduced and provides particles with a significantly longer circulation half-life (Illum and Davis, 1984). In some cases such particles can deposit preferentially in a specific organ site such as the bone marrow (Porter et al., 1992) and spleen (Moghimi et al., 1993b).

Human serum albumin (HSA) is widely used as a material for microsphere preparation since it is considered to be non-antigenic and biodegradable, and is readily available (Arshady, 1990, Bogdansky, 1990). HSA nanoparticles with a size less than 150 nm in diameter can be prepared using a pH-coacervation method (Lin et al., 1993). Particles of this size have a good chance to escape from the vascular system to target to sites outside the circulation, provided they have not been sequestered previously by the RES (Artursson, 1987). However, since poloxamer or poloxamine surfactants are poorly adsorbed on the hydrophilic surface of albumin particles, the required steric PEO barrier on albumin nanoparticles cannot be created simply by adsorption of block copolymer surfactants. We previously reported the preparation and characterization of dextran-PEG, poly(amidoamine)-PEG and poly(thioeramido acid)-PEG modified HSA nanoparticles (Lin et al., 1994, Lin et al., 1997). This paper describes the surface modification of HSA nanoparticles by PEG using a PEG-HSA conjugate: methoxy-polyethylene glycol modified human serum albumin (HSA-mPEG). This material offers the possibility of producing HSA nanoparticles with a sole PEO surface layer which will be similar to those created on polystyrene or PLGA nanoparticles by coating with poloxamer and poloxamine surfactants and, therefore, nanoparticles prepared from this conjugate may show a similar in vivo behaviour to that of PEO-PPO surfactant coated polystyrene or PLGA nanoparticles (Illum and Davis, 1984, Porter et al., 1992, Stolnik et al., 1994). The PEG-HSA conjugates used in the present study were synthesised using mPEG with a molecular weight of 2000 and 5000 Da and with different HAS:mPEG ratios. HSA-mPEG nanoparticles were prepared using modified coacervation methods. The surface characteristics of the nanoparticles produced using these new conjugates have been investigated and the adsorption of plasma proteins on the HSA-mPEG nanoparticles compared with unmodified HSA nanoparticles was also studied.

Section snippets

Materials

HSA (Albutein, 20% albumin solution, BP) was supplied by Alpha (Thetford, UK). Methoxy-polyethylene glycol 2000 modified human serum albumin (mol. wt. 115 000 Da, 40% lysine modified, HSA content 60% w/w, HSA60-mPEG2000), methoxy-polyethylene glycol 5000 modified human serum albumin (mol. wt. 81 500 Da, 5% lysine modified, HSA content 80% w/w, HSA80-mPEG5000) and methoxy-polyethylene glycol 5000 modified human serum albumin (mol. wt. 173 700 Da, 35% lysine modified, HSA content 50% w/w, HSA50

Results and discussion

HSA is characterized as having a high content of charged amino acids and is insoluble in organic solvents. HSA nanoparticles may be prepared by adding water miscible organic solvents such as alcohol or acetone to HSA aqueous solution (Lin et al., 1993, Chen et al., 1994). However, when HSA is modified with mPEG, the amphipathic nature of the PEG molecule confers a higher solubility of HSA-mPEG in hydrophobic solvents. This makes it difficult for HSA-mPEG to coacervate and thereby form

Conclusion

Surface modified albumin nanoparticles with a size approximately 150 nm in diameter were prepared from HSA-mPEG conjugates using modified coacervation methods. The ζ-potential of the surface modified nanoparticles was significantly lower than that of the unmodified HSA nanoparticles. The molecular weight of the PEG component of the conjugate seems to be a more important factor for the ζ-potential than the degree of PEG modification. The existence of a hydrated steric barrier surrounding the

Acknowledgements

This research was supported by a European Community Brite/Euram Programme (BE-3348-89). We would like to thank T. Gray for assistance with the photographic work.

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