Selective targeting of antibody-conjugated nanoparticles to leukemic cells and primary T-lymphocytes

Abstract

In the present study, surface-modified nanoparticles based on biodegradable material were used for antibody coupling in order to get a selective drug carrier systems. Gelatin nanoparticles were prepared by a desolvation process. Sulfhydryl groups were introduced which enabled the linkage of NeutrAvidin (NAv). Antibodies specific for the CD3 antigen on lymphocytic cells were conjugated to the nanoparticles surface. The binding of biotinylated anti-CD3 antibody was achieved by NAv–biotin-complex formation. Cellular binding and uptake were determined by flow cytometry and confocal laser scanning microscopy (CLSM). Cell-type-specific targeting of anti-CD3-conjugated nanoparticles into CD3-positive human T-cell leukemia cells and primary T-lymphocytes could be shown. Celluar uptake and effective internalization of antibody-conjugated nanoparticles into CD3 expressing cells were demonstrated. Uptake rates of about 84% into T-cell leukemia cells were observed. To confirm selectivity of T-cell targeting, competition experiments were carried out adding excessive free anti-CD3 prior to nanoparticle incubation leading to significantly reduced cellular uptake of antibody-conjugated nanoparticles. Further analysis on the mechanism of uptake confirmed a receptor-mediated endocytotic process. Protein-based nanoparticles conjugated with an antibody against a specific cellular antigen hold promise as selective drug delivery systems for specific cell types.

Introduction

The concept of drug targeting and controlled drug delivery is used in attempts to improve the therapeutic index of drugs by increasing their localization to specific organs, tissues or cells and by decreasing their potential toxic side effects at normal sensitive sites. As in the field of cancer therapy, chemotherapeutic agents have toxic side effects for tumor cells as well as for normal cells, the controlled delivery of these agents to diseased sites would enable the use of higher doses for increasing therapeutic efficacy. Controlled drug delivery involves the association of a drug with a carrier system, thereby allowing modulation of the pharmacokinetic properties and biodistribution of the drug. Among several drug carriers and drug delivery systems, nanoparticles are very attractive particulate carrier systems under investigation. In principle, the nanoparticle technology has been used in recent years with great promise in promoting the efficacy of drugs [1], [2]. Nanoparticles are solid colloidal particles in size of 10–1000 nm. They consist of macromolecular materials in which the active principle is dissolved, entrapped, or encapsulated, or to which the active principle is adsorbed or attached [3]. The body distribution of these carriers can be controlled by size and surface properties [4]. The particulate drug carrier systems are characterized by considerable payload and enable a controlled release of the drug as well as protection from degradation [5]. Following intravenous application, nanoparticles are known to accumulate in the tissues of the mononuclear phagocyte system (MPS). Also in tumor tissue, which is often characterized by badly formed and leaky vasculature, nanoparticles can leave the blood stream and accumulate in tumor sites. This process which is due to an enhanced permeability and retention effect, is called “passive targeting” [6]. To enhance the targeting of nanoparticles to specific cells or tissues, target-specific ligands must be linked to the nanoparticle surface (“active targeting”). Antibody-coupled liposomes (so called “immunoliposomes”) were first described in early 1980s [7]. However, fundamental problems regarding immunoliposomes preparation and application such as stability and pharmacokinetics are still remaining obstacles which could not be overcome completely until now. Among several coupled homing devices, antibody-coupled nanoparticles can be regarded as a very attractive drug-targeting system due to their advantageous properties (e.g. stability). Nanoparticles can be made from biopolymers, such as gelatin or human serum albumin [8]. These materials offer the opportunity for easy surface modifications by the introduction of sulfhydryl groups which can be used for the covalent attachment of functional proteins. Binding of these proteins can be achieved employing a bifunctional crosslinker characterized by two specific binding sites, one for amino and another for sulfhydryl groups. In previous work, we used the avidin derivative NeutrAvidin (NAv) as functional protein for the covalent coupling to gelatin nanoparticles. For covalent coupling to gelatin nanoparticles, amino groups of NAv were activated via the succinimidyl structure of the cross linker followed by a conjugation reaction to the sulfhydryl groups on the particle surface [9]. Due to its high binding affinity for biotin ($K_{\mathrm{D}}=10^{-15}\text{<mi mathsize="small" is="true">M</mi>}$), biotinylated compounds such as antibodies can be attached by strong avidin–biotin-complex formation. Therefore, protein-based nanoparticles such as gelatin nanoparticles which are surface-modified using NAv offer the opportunity to attach a multitude of biotinylated compounds to a fixed carrier system.

The objective of the present work was the development of a cell-specific drug carrier system on the basis of gelatin nanoparticles. Biotinylated antibodies were coupled to the nanoparticle surface via NAv in order to achieve specific receptor-mediated cellular uptake into lymphocytic cells.