Detection and determination of surface levels of poloxamer and PVA surfactant on biodegradable nanospheres using SSIMS and XPS

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Abstract

The surface chemical characterisation of sub-200 nm poly(DL-lactide co-glycolide) nanospheres has been carried out using the complementary analytical techniques of static secondary ion mass spectrometry (SSIMS) and X-ray photoelectron spectroscopy (XPS). The nanospheres, which are of interest for site-specific drug delivery, were prepared using an emulsification-solvent evaporation technique with poly(vinyl alcohol), Poloxamer 407 and Poloxamine 908 respectively as stabilisers. The presence of surfactant molecules on the surface of cleaned biodegradable colloids was confirmed and identified on a qualitative molecular level (SSIMS) and from a quantitative elemental and functional group analysis (XPS) perspective. SSIMS and XPS data were also used in combination with electron microscopy to monitor the effectiveness of cleaning procedures in removing poorly bound surfactant molecules from the surface of nanospheres. The findings are discussed with respect to the development of nanoparticle delivery systems, particularly the composition of the surface for extending blood circulation times and achieving site-specific deposition.

Introduction

The applications of injectable, biodegradable microparticles have until recently been limited to depot-type preparations for controlled drug and protein release 1, 2, 3, chemoembolisation, tumour targeting 4, 5and vaccine delivery 6, 7. Over the last decade however microspheres have been proposed and investigated as vehicles for site-specific delivery of therapeutic agents following intravenous (i.v.) injection [8]. The concept of targeted drug delivery using particulate carrier systems has proved problematical in practice due to rapid particle sequestration, following i.v. administration, by the cells of the reticuloendothelial system (RES), primarily the Kupffer cells of the liver [9]. Extensive investigations with liposomes and model, non-degradable polystyrene particles have indicated however, that the surface properties of an injected colloid can mediate interactions with serum components (opsonisation) and, as a consequence, influence the biodistribution of the system. Recently, successful avoidance of Kupffer cell uptake has been achieved using surface modified liposomes 10, 11, 12and model polystyrene nanospheres where the surface has been modified by adsorption of poly(ethylene oxide)–poly(propylene oxide) [PEO–PPO] block copolymers 9, 13. More specifically, Poloxamer 407 has been reported to redirect polystyrene latex to the endothelial cells of the bone marrow [14]and Poloxamine 908 coated systems can be retained by the spleen [15]. The mechanisms by which such surface modifications prove effective in promoting targeted delivery are still unknown, but a hypothesis based on processes of differential opsonisation promoted by the introduction of hydrophilic and steric surface groups has been proposed 8, 9, 14, 15.

Critical to the development of clinically relevant dosage forms is the production of biodegradable nanospheres which exhibit the desirable surface properties of the model, non-degradable systems. This requirement has stimulated development of resorbable sub-200 nm particles of poly(DL-lactide co-glycolide) [PLGA] [16]and poly(β-malic acid) nanospheres surface modified by PEO–PPO copolymers and polylactic acid–poly(ethylene glycol) (PLA–PEG) block copolymers respectively 17, 18. Smaller, sub-100 nm colloids have been prepared directly from PLA–PEG copolymers using the PEG component as an integral stabiliser 19, 20. In all cases, one of the major criteria to be satisfied in the development of these delivery systems is the correct surface expression of PEO chains for achieving site-specific deposition. It would clearly be of great interest, therefore, to be able to characterise the distribution and presentation of functional groups at the nanosphere surface for correlation with in vivo behaviour. It would also be advantageous to gain an insight into the strength of association between colloidal PLGA particles and the surface-bound stabiliser used in their preparation. At the preparation stage, for example, nanospheres are often cleaned by gel filtration or centrifugation and resuspension techniques 21, 22but little is known about the effect of such harvesting procedures on the concentration, conformation and distribution of residual surfactant.

The complementary surface analytical techniques of static secondary ion mass spectrometry (SSIMS) and X-ray photoelectron spectroscopy (XPS) have been applied extensively for surface analysis of biomedical materials 23, 24. SSIMS analysis provides a mass spectrum of the top 10 Å of a surface and provides detailed molecular information on polymer structure 25, 26. In XPS experiments, the binding energy of electrons associated with atoms in the top 20–100 Å is measured allowing quantitative elemental and chemical state information to be gathered regarding the surface composition. We have previously shown that SSIMS and XPS techniques can be employed specifically and successfully for analysis of colloids. Examples include the assessment of preferential surface migration of one or more polymer components in copolymer colloids and confirmation of the presence of immobilised sugar residues at colloid surfaces 27, 28. Of particular relevance to the work presented here is the application of SSIMS and XPS for characterising the interfacial chemistry of non-degradable nanospheres, surface modified by grafted PEO chains; the aim being to provide structure–activity relationships which enable prediction of biological fate in-vitro and in-vivo 29, 30.

We have previously reported the preparation of biodegradable sub-200 nm nanospheres from PLGA by an emulsification / solvent evaporation technique using poly(vinyl alcohol) [PVA] as a stabiliser [16]. By utilising the surfactant properties of Poloxamer and Poloxamine PEO–PPO block copolymers, it may be possible to prepare biodegradable nanospheres which present an appropriate distribution pattern of surface groups (as elucidated by studies with model systems) for achieving targeted delivery. Other activities in our group and elsewhere are examining PLGA–PEG block copolymers for forming and/or coating such nanoparticles 19, 20. Here we report on the surface chemical characterisation of sub-200 nm PLGA nanospheres prepared using PVA, Poloxamer 407 and Poloxamine 908 respectively as particle stabilisers. SSIMS and XPS were employed to define the surface chemistry of the colloids which are currently being developed for site-specific drug delivery.

Section snippets

Materials

75:25 poly(DL-lactide-co-glycolide) (MW 10–15 000 D and polydispersity 1.45) was prepared as described previously [31]. PVA (75% hydrolysed from the acetate, MW 3,000 D) was obtained from Polysciences (Warrington, PA, U.S.A.). PVA (87–89% hydrolysed, MW 13–23 000 D) was obtained from Aldrich (Gillingham, U.K.). Poloxamer 407 (weight average molecular weight (MW) 12 600, PEO block length (moles): 98, PPO: 67) and Poloxamine 908 (MW 25 000, PEO:122, PPO: 67) were obtained from B.A.S.F

Results and discussion

Particle size was determined to be 142.1±2.1, 161.1±3.0 and 137.2±2.5 nm for nanospheres prepared using PVA, Poloxamer 407 and Poloxamine 908 respectively.

The surface characterisation of the PLGA matrix copolymer, PVA and PEO–PPO surfactants was first performed in isolation. The surface of the PVA and PEO–PPO stabilised colloids was subsequently analysed to determine the relative contribution of each component to the surface chemical composition.

Concluding remarks

This paper has illustrated the potential of SSIMS and XPS for defining the surface chemical structure of biodegradable nanospheres which may be of interest for targeted drug delivery, diagnostic applications and biomedical implants. XPS provided quantitative elemental and functional group analysis which elucidated the changes in surface chemistry with formulation procedure for PLGA nanospheres stabilised with PVA and PEO–PPO copolymer surfactants. This information was complemented by

Acknowledgements

The authors acknowledge the financial support of the SERC and the BRITE–EURAM Programme of the European Community. Thanks are due to Dr. T. Grey, Dept. of Anatomy, University of Nottingham for providing the TEMs of Poloxamer-stabilised nanospheres.

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    1

    Present address: Pharmaceutical Development Dept., 3M Healthcare, Loughborough, LE11 OSF, U.K.

    2

    Present address: Dept. of Pharmaceutical and Biological Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK.

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