Established methods of protein chemistry can be used for the effective attachment of drug targeting ligands to the surface of protein-based nanoparticles. In the present work gelatin nanoparticles were used for the attachment of biotinylated anti-CD3 antibodies by avidin-biotin-complex formation. These antibody modified nanoparticles represent a promising carrier system for the specific drug targeting to T-lymphocytes. The objective of this work was the comprehensive quantification of every chemical reaction step during the preparation procedure of these cell specific nanoparticles. Gelatin nanoparticles were formed by a two-step desolvation process. After the first desolvation step the remaining sediment and the supernatant were analysed for molecular weight distribution by size exclusion chromatography (SEC). Nanoparticles then were formed using the high molecular gelatin fraction and subsequently were stabilised by glutaraldehyde crosslinking. A part of the detectable amino groups on the particle surface was reacted with 2-iminothiolane in order to introduce reactive sulfhydryl groups. The thiolated nanoparticles were coupled to NeutrAvidin (NAv) which previously was activated with the heterobifunctional crosslinker sulfo-MBS. All these reaction steps were quantified by photometry or gravimetry. The functionality of NAv after covalent conjugation was confirmed by a biotin-4-fluorescein assay. The NAv-modified nanoparticles then were used for the binding of biotinylated anti-CD3 antibodies by avidin-biotin-complex formation. A highly effective attachment of the ligand was ascertained by different, indirect methods: immunoblotting and fluorimetry. Therefore, a well-defined nanoparticle system with drug targeting ligand modification was established that holds promise for further effective preclinical testing.

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.

Membrane transport of antisense oligonucleotides (ODN) is an inefficient process which requires special carriers for their intracellular delivery. We have developed a delivery system for AS-ODN and their phosphorothioate analogues (AS-PTO) directed against human immunodeficiency virus type 1 (HIV-1) tat mRNA for efficient transfection of HIV-1 target cells. Protamine was used to complex AS-ODN and AS-PTO to form nanoparticles with diameters of about 180 nm and surface charges in the range of −18 to +30 mV. Cellular uptake of these nanoparticles was significantly enhanced compared to naked oligonucleotides. A double labeling technique with fluorescently tagged protamine and AS-ODN was used to follow the intracellular fate of the nanoparticles. Protamine/AS-ODN nanoparticles showed release of the antisense compound leading to specific inhibition of tat mediated HIV-1 transactivation. In contrast, protamine/AS-PTO complexes were stable over 72 h, and failed to release AS-PTO. These results demonstrate that protamine/AS-ODN nanoparticles are useful for future therapeutical application to inhibit viral gene expression.