Trastuzumab-modified nanoparticles: Optimisation of preparation and uptake in cancer cells

Abstract

Nanoparticles consisting of human serum albumin (HSA) represent a promising strategy for targeted drug delivery to tumour cells. The coupling of the antibody trastuzumab to HSA nanoparticles takes advantage of the capability of HER2-positive cells to incorporate substances binding to HER2. In our present study, we developed nanoparticles based on HSA which were covalently modified on their surface with thiolated trastuzumab. A special focus was on the optimisation of the thiolation procedure of the antibody under the aspect of an effective binding to particle surfaces. Different thiolation conditions were evaluated and the degree of antibody dimerisation was determined. We analysed the thiolated antibody by size exclusion chromatography (SEC) and identified the best thiolation procedure for the preparation of trastuzumab-conjugated nanoparticles. Over a storage period of 6 weeks the resulting particles were stable and physico-chemical properties such as size and zetapotential did not show any changes. Biological activity was confirmed under cell culture conditions: antibody-conjugated nanoparticles showed a specific targeting to HER2-overexpressing cells with cellular uptake by receptor-mediated endocytosis. These data provide the basis for the development of stable and biological active carrier systems for directed targeting of tumour cells using trastuzumab-conjugated HSA nanoparticles.

Introduction

Antibodies are well-established systems to target drugs or colloidal carriers to specific cell types. This principle is based on a defined receptor ligand interaction which enables the surface binding and even cellular internalisation of drugs or drug carriers conjugated to the antibody. On the one hand chemical modification of the antibody is necessary to enable conjugation with a drug carrier. On the other hand it is a prerequisite to perform conjugation reactions maintaining biological activity and full receptor binding of the antibody.

Trastuzumab (Herceptin^®) is a humanised IgG1 monoclonal antibody directed against the extracellular domain of the human epidermal growth factor receptor 2 (HER2). HER2 or ErbB2 (Neu) belongs to a receptor tyrosine-specific protein kinase family, the EGFR (epidermal growth factor receptor) family, which consists of four EGF receptors, EGFR (ErbB1), ErbB2 (Neu), ErbB3, and ErbB4. Members of the EGFR family contain a cytoplasmic tyrosine kinase domain, a single transmembrane domain, and an extracellular domain that is involved in ligand binding and receptor dimerisation [1]. High-affinity ligands are not yet known for HER2 but it acts as co-receptor of other members of the EGF receptor family through heterodimerisation. Overexpression of HER2 at the cell surface may lead to a spontaneous formation of homodimers, but more likely the availability of the receptor for ligand-driven heterodimerisation increases [2]. Moreover, HER2 signaling is less accessible to normal inactivation processes ensuring rapid termination of signals. Therefore, HER2 overexpression translates into signals that potentiate dysregulated growth, oncogenesis, metastasis and possibly resistance against apoptosis-inducing therapeutic agents [2].

HER2 is amplified at 20–30% incidence in human breast cancer. Amplification also occurs in ovarian, lung and gastric cancers, the normal expression in adult tissue is weak [3]. HER2 amplification leads to a shortened time to relapse and overall survival.

Trastuzumab represents a humanised anti-HER2 antibody for the treatment of patients with metastatic breast cancer. The mechanism of trastuzumab is incompletely defined. It acts in a cytostatic manner by blocking and downmodulation of the HER2-receptor and by antibody-dependent cellular cytotoxicity as major mechanism of antibody action [4], [5]. It induces HER2 internalisation and degradation in BT-474 and SK-BR-3 cells. Trastuzumab offers an excellent strategy for drug targeting to cancer cells because HER2 is an easily accessible cell surface receptor overexpressed on the primary tumour as well as on metastatic sites [6]. The internalisation ability of HER2 allows an efficient uptake of the antibody alone as well as conjugated to drugs or drug carrier systems. Direct linking of several drug molecules to an immunoglobulin is possible but may lead to a decreased biological activity and receptor specificity.

Colloidal systems such as nanoparticles or liposomes provide higher drug carrier capacities than antibodies and, therefore, are especially suited for conjugation to antibodies. Human serum albumin (HSA)-based nanoparticles represent a colloidal drug carrier system with high drug loading capacity. These particles are biodegradable, non-antigenic, non-irritative for tissues and non-toxic [7]. Drugs can be incorporated within the particle matrix, adsorbed on the particle surface or bound by covalent linkage. Especially incorporation in the particle matrix protects the drug against degradation and enables controlled release.

The aim of the present study was the direct covalent coupling of the antibody trastuzumab to the surface of HSA nanoparticles in order to achieve a cell type specific drug carrier system.