Targeting cancer cells using PLGA nanoparticles surface modified with monoclonal antibody

https://doi.org/10.1016/j.jconrel.2007.03.012Get rights and content

Abstract

Targeting drugs to their sites of action is still a major challenge in pharmaceutical research. In this study, polylactic-co-glycolic acid (PLGA) immuno-nanoparticles were prepared for targeting invasive epithelial breast tumour cells. Monoclonal antibody (mAb) was used as a homing ligand and was attached to the nanoparticle surface either covalently or non-covalently. The presence of mAb on the nanoparticle surface, its stability and recognition properties were tested. Protein assay, surface plasmon resonance, flow cytometry and fluorescence-immunostaining confirmed the presence of mAb on nanoparticles in both cases. However, a binding assay using cell lysate revealed that the recognition properties were preserved only for nanoparticles with adsorbed mAb. These nanoparticles were more likely to be bound to the targeted cells than non-coated nanoparticles. Both types of nanoparticles entered the target MCF-10A neoT cells in mono-culture. In co-culture of MCF-10A neoT and Caco-2 cells immuno-nanoparticles were localized solely to MCF-10A neoT cells, whereas non-coated nanoparticles were distributed randomly. Immuno-nanoparticles entered only MCF-10A neoT cells, while non-coated nanoparticles were taken up by both cell types, indicating specific targeting of the immuno-nanoparticles. In conclusion, we demonstrate a method by which mAbs can be bound to nanoparticles without detriment to their targeting ability. Furthermore, the results show the effectiveness of the new carrier system for targeted delivery of small or large active substances into cells or tissues of interest.

Introduction

The field of therapeutics has seen an exponential growth in new molecular entities, ranging from low molecular weight drugs to macromolecules like proteins and genes. However, the ideal drug substance which interacts site-selectively with its molecular target at a therapeutically-relevant level has not yet been established, at least not in clinical practice. Some degree of site-selective delivery has been achieved only with “targeting homing drugs” that specifically recognize their pharmacological target [1]. An important impetus since 1975 has come from the development of monoclonal antibodies (mAb) and the exploitation of their targeting properties and hence therapeutic potential [2]. The specificity of delivery using nanoparticles was initially a coincidental property, active targeting has now become a central concept in therapeutic research. This concept has been developed into practical application with the construction of a variety of immuno-conjugates, also known as drug-attached antibodies (Abs) [3], [4], [5], [6], [7]. Certain mAbs have been shown to initiate specific signalling cascades, which can potentiate the therapeutic effect of the attached drug [8], [9]. The latter has been confirmed for chemotherapeutic drugs and tumour-targeting antibodies [8]. However, the number of drug molecules that can be attached to an antibody molecule is usually the limiting factor for such a strategy, especially for low potency drug molecules [9], [10]. For high potency drug molecules such as proteins, the coupling reaction could affect the pharmacological and immunological activities of the drug molecule, as well as the in vivo fate [11].

Nanoparticles with specific recognition ligands bound to the surface have a good potential for site-selective delivery, and offer higher drug carrier capacity than bioconjugates, as well as improved specificity for drug targeting [12]. The carrier material used can additionally protect the drug from premature release and degradation [13], [14]. Ligands attached to the surface can include any molecule that selectively recognizes and binds molecules on target cells [9], [15]. Of the different targeting ligands, such as peptides, glycoproteins, carbohydrates and polymers, mAbs have been the most widely studied [8].

In our previous study we developed poly(lactic-co-glycolic acid) (PLGA) nanoparticles as a delivery system, and showed that they could be used to deliver a protein drug intracellularly more efficiently and faster than could be achieved with the free protein [16]. However, the formulated delivery system was not able to distinguish between different cells. We report here the preparation of PLGA nanoparticles designed specifically to target breast tumour cells. For this purpose we used a mAb recognizing the specific profile of the cytokeratins expressed by these cells. Covalent and non-covalent binding were both employed to attach mAb to the surface of pre-formed PLGA nanoparticles. The amount of mAb attached to the nanoparticle surface, the stability and recognition properties of the formulated systems were all characterized, as well as their ability to target and enter antigen-rich MCF-7 and MCF-10A neoT cells in mono-culture and, specifically, in co-culture with Caco-2 cells.

Section snippets

Materials

Poly(lactic-co-glycolic acid) (PLGA), 50:50, Resomer RG™ 503H) was obtained from Boehringer (Ingelheim, Germany), polyvinyl alcohol (PVA, Mowiol™ 4–98) from Hoechst (Frankfurt, Germany), ethyl acetate from Merck (Darmstadt, Germany), EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) from Fluka Chemie AG (Switzerland), Alexa Fluor® 546-labelled goat anti-mouse immunoglobulin and Blue Cell Tracker from Molecular Probes (Carlsbad, CA, USA), bovine serum albumin (BSA) and fluorescein from Sigma

Results

In order to achieve an immuno-nanoparticle system able to target desired cells, the binding procedure must preserve the biological activity of targeting ligand. In the present study a special focus was oriented to the selection of a suitable method for modification of the nanoparticle surface with mAb under aspect of an effective binding to particle surface and still preserving its biological activity. For this purpose mAb was bound to the surface of PLGA nanoparticles either by adsorption or

Discussion

Drug targeting can improve the efficacy of therapy and reduce side effects associated with drugs [8], [14]. Various carriers can be used to deliver a drug in a stable and protective form, however, it is nanotechnology which offers the most unique and intriguing approach in the area of nanomedicine [21], [22].

In an attempt to formulate a carrier system for site-selective delivery, fluorescein-loaded PLGA nanoparticles capable of targeting invasive breast epithelial cell lines, such as MCF-7 and

Conclusion

New PLGA nanoparticles, having the ability to recognize and target specific antigens on breast epithelial cancer cell lines, were prepared by attaching mAb on the nanoparticle surface via the adsorption process. Attempts to attach mAb to nanoparticles by covalent bonding were less successful, since the biological activity of the bound mAb was inactivated. The specificity of the immuno-nanoparticles was seen from their selective distribution in a co-culture of MCF-10A neoT and Caco-2 cells,

Acknowledgements

The authors thank Prof. Dr. Roger Pain for critical reading of the manuscript, Prof. Dr. Slavko Pečar for helpful discussion on covalent coupling, Urška Repnik from Institute Jožef Stefan, Department of Biochemistry, Molecular and Structural Biology, for assistance in flow cytometry, Mojca Lunder for assistance in surface plasmon resonance experiments, and Alenka Kužnik for isolation and characterization of mAb. This research work was supported by Ministry of Higher Education, Science and

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