Research Paper
A new preparation strategy for surface modified PLA nanoparticles to enhance uptake by endothelial cells

https://doi.org/10.1016/j.ijpharm.2017.11.047Get rights and content

Abstract

Nanoparticles are promising drug delivery systems to overcome physiological barriers such as the blood-brain barrier. In this respect nanoparticle uptake into endothelial or epithelial cells is the first necessary step to overcome these obstacles. Therefore, a new strategy for the covalent attachment of drug targeting ligands on poly(lactic acid) (PLA) nanoparticles was developed and the influence of the resulting surface properties on the uptake behaviour in cerebral endothelial cells was investigated. PLA nanoparticles were modified on their surface by apolipoprotein E, penetratin, or ovalbumin using a newly developed vinyl sulfone-modified poly(vinyl alcohol)-derivative (VS-PVA) as steric stabilizer. With this approach an easy option for ligand coupling reactions to PVA-stabilized nanoparticles was achieved. All obtained formulations showed a favourable behaviour concerning cytotoxic effects on endothelial cells, not compromising their viability. Furthermore, a clear relation between cellular uptake and surface coupled functional ligand could be determined: Penetratin- and apolipoprotein E-modified nanoparticles showed a distinct higher cellular uptake than ovalbumin-modified or unmodified nanoparticles, which both can be explained by mechanistic reasons. Overall the use of the reactive VS-PVA as stabilizer for nanoparticle preparation is an universal and effective approach to couple several functional ligands to the particles’ surface for targeting applications.

Introduction

The blood-brain barrier (BBB) is a major obstacle in the treatment of several central nervous disorders, because it restricts the capability of many molecules to enter the brain (Pardridge, 2012). In order to deliver therapeutically active substances to the brain several quite invasive methods, like an intracerebral injection (Begley, 2004) or a reversible osmotic opening of the BBB (Rapoport, 2000), can be used. In this context nanoparticles are a promising – because more gentle – approach to enable drug transport into the brain, because of the feasibility to simply modify their surface characteristics by the attachment of drug targeting ligands. Usually most cells take up nanoparticles unselectively, especially in liver and spleen (Alexis et al., 2008). In order to achieve an active targeting to the brain a covalent linkage of drug targeting ligands such as functional proteins or peptides to the particles´ surface is a common way, thus enabling the nanoparticles to enter the central nervous system by receptor-mediated transcytosis (Kreuter, 2013).

Nanoparticles based on poly(lactic acid) (PLA) are an appropriate vehicle for in vivo use, because of the polymers´ characteristics as a biodegradable starting material, which is metabolized to lactic acid in physiological systems exhibiting a good biocompatibility (Kumari et al., 2010). Besides the pure polymer additives are needed to stabilize nanoparticles during preparation and storage. For example poly(vinyl alcohol) (PVA) is a commonly used steric stabilizer for the preparation of PLA nanoparticles (Lourenco et al., 1996). Unfortunately, after particle preparation PVA is mainly found on the particles´ surface, which leads to an insufficient accessibility of the polymers´ carboxylic groups, which restricts the possibility to use such particles for covalent surface modifications (Sahoo et al., 2002). Therefore in the past several groups proposed different strategies to make functional groups on the surface of PLA or PLGA nanoparticles accessible. For instance the number of carboxyl groups at the surface of PLGA based microspheres can be increased by replacing the traditional stabilizer PVA with poly(ethylene-alt-maleic acid) (PEMA), a stabilizer bearing carboxylic acid side chains which can be used for a subsequent carbodiimide reaction (Keegan et al., 2004). Furthermore, PEMA modified PLGA nanoparticles can be used for electrostatically stabilized surface coatings with polycationic polymers (Lo et al., 2010). Another strategy for ligand attachment can be seen in the application of modified surfactants for the preparation of nanoparticles: A thiol-reactive Pluronic® F-108 modified with pyridyl disulfide groups was described by Gullberg et al. (2006) which can be covalently linked to thiol group containing ligands whereas Chittasupho et al. (2009) converted terminal hydroxyl groups on Pluronic® F127 to carboxyl groups by reaction with succinic anhydride. Furthermore in some studies the hydroxyl groups of PVA were used for ligand conjugation using the epoxy based compound Denacol® EX-521 (Sahoo and Labhasetwar, 2005, Rao et al., 2008). In a first step the hydroxyl groups of PVA were activated by Denacol® in the presence of zinc tetrafluoroborate while in a second step amino group-containing ligands were attached to the epoxy activated nanoparticles.

In order to cross the BBB, the uptake into endothelial cells is a prerequisite for a nanoparticulate carrier to reach the brain (Abbott, 2002). In this regard the extent of nanoparticle uptake into cells can be influenced by their size and surface properties (Ahn et al., 2013). Furthermore the presence of certain functional ligands on the surface may allow the nanoparticles to enter the cells through a receptor-dependent endocytotic pathway (Xu et al., 2013). An approach described in literature is the modification with apolipoprotein E (ApoE), which results in an accumulation of nanoparticles in the central nervous system in in vivo studies (Kreuter, 2001). Another but less selective approach to increase the intracellular accumulation of nanoparticles is the linkage of cell-penetrating peptides (CPP) resulting in an easier cell entry due to interaction with the cell membrane (Farkhani et al., 2014).

The aim of this study was to perform three different surface modifications on PLA-based nanoparticles using ovalbumin, ApoE, or penetratin as surface ligands. To enable the surface modification a new strategy was developed by using vinyl sulfone-poly(vinyl alcohol) (VS-PVA) as a reactive steric stabilizer, thus enabling an effective surface modification with amine- or thiol-containing ligands like proteins and peptides. To evaluate the different cellular uptake properties, the surface modified nanoparticles were compared with unmodified ones using a cerebral endothelial cell culture model (HBMEC).

Section snippets

Materials

Poly(vinyl alcohol) (PVA, average molecular weight 31 kDa, 85–89% hydrolysed) and trehalose were purchased from Merck KGaA (Darmstadt, Germany). Divinyl sulfone (DVS) was obtained from Alfa Aesar GmbH & Co. KG (Karlsruhe, Germany). The fluorescent dye Lumogen® Red was kindly provided by BASF SE (Ludwigshafen, Germany). Acid terminated poly(D,L-lactide) (PLA, Resomer® R 203H), which was used as particle matrix, was purchased from Evonik Industries AG (Essen, Germany). The ɑ-amino-ω-carboxy-PEG265

Functionalization of PVA with DVS

In the present study a reactive stabilizer for nanoparticle preparation was developed in order to enable an effective surface modification of PLA nanoparticles. Therefore the commonly used stabilizer poly(vinyl alcohol) (PVA) was modified with DVS, introducing reactive vinyl sulfone-groups (Fig. 1A). In preliminary experiments different molar ratios of DVS to PVA were used representing 1.25fold, 2.5fold, and 2.75fold molar excess of DVS over the hydroxyl groups of PVA. All of these conditions

Preparation and characterization of VS-PVA

The commonly used stabilizer PVA is often utilised in nanoparticle preparation (Lourenco et al., 1996). Because of its localization on the particle surface after preparation (Spek et al., 2015), on the one hand PVA prohibits direct covalent drug targeting ligand conjugation to the carboxylic groups of the PLA matrix but on the other hand represents potential as point of action for surface modification reactions. In the present study, we investigated an innovative way to create a novel PVA-based

Conclusion

In this study the stabilizer PVA, commonly used for the preparation of PLA nanoparticles by emulsion-diffusion techniques, was successfully modified with divinyl sulfone to the more reactive VS-PVA derivative. In a new approach the surface of the obtained nanoparticles could be easily functionalized with different targeting ligands such as ApoE or penetratin using the reactive VS-PVA properties. All designed formulations exhibited a good biocompatibility, not compromising the viability of brain

Acknowledgements

The authors like to thank Dr. Jens Köhler and Claudia Thier from the Institute of Pharmaceutical and Medicinal Chemistry (University of Münster) for performing the NMR measurements.

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