Hybrid Nanomaterials forAntibody Drug Delivery Systems

Abstract

Antibody therapeutics have been widely investigated for biomedical applications due to specific antigen biding ability. The specific antigen targeting of antibody makes drug delivery systems (DDSs) more effective and reduces side effect of cytotoxic drugs to normal cells. The versatility of antibody for drug targeting and the verified clinical success open up the door to targeting strategies. Furthermore, antibody can be easily modified for various functional DDSs. Antibody-drug conjugate (ADC) is one of the most promising DDSs with high targeting effect on intractable diseases. In addition, antibody has been conjugated to various nanoparticles such as gold nanoparticles and magnetic nanoparticles for targeted delivery. During the Ph.D. course, various DDSs using antibody therapeutics were developed for the effective therapeutic applications. In part I, hyaluronate - gold nanoparticle / Tocilizumab (HA-AuNP/TCZ) complex was prepared for the treatment of Rheumatoid arthritis (RA). RA is a chronic inflammatory immune disease causing the inflammation of synovial membrane and the articular cartilage destruction. AuNP was used as a drug carrier with anti-angiogenic effect. TCZ is a humanized monoclonal antibody against the interleukin-6 (IL-6) receptor and used as an immunosuppressive drug by interfering IL-6 in the pathogenesis of RA. HA is known to have cartilage-protective and lubricant effects. HA was modified with cystamine via reductive amination, which was reduced with dithiothreitol (DTT) to prepare end-group thiolated HA (HA-SH). AuNP was chemically modified with HA-SH and physically modified with TCZ. The formation of HA-AuNP/TCZ complex was corroborated by UV-Vis spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). The therapeutic effect of HA-AuNP/TCZ complex on RA was confirmed in collagen induced arthritis (CIA) model mice by ELISA, histological, and Western blot analyses. In part II, Tocilizumab (TCZ) – Alendronate (ALD) conjugate is synthesized for the early intervention of RA. A humanized monoclonal antibody of TCZ shows an immunosuppressive effect, targeting interleukin-6 (IL-6) receptor in the RA pathogenesis. ALD is an anti-inflammatory bisphosphonate drug which can bind to the exposed bone surface. ALD is conjugated selectively to N-glycan on Fc region of TCZ using a chemical linker of 3-(2-pyridyldithio) propionyl hydrazide (PDPH) – poly(ethylene glycol) – N-hydroxysuccinimide (PDPH-PEG-NHS). The successful synthesis of TCZ-ALD conjugate is corroborated by 1H NMR, the purpald assay, mass spectrometry (MS), and high performance liquid chromatography (HPLC). In vitro binding affinity and cell viability tests confirmed the biological activity of TCZ-ALD conjugate. Furthermore, in vivo efficacy of TCZ-ALD conjugate is confirmed by micro computed tomography (CT), histological, and western blot analyses for the treatment of RA. In part III, hyaluronate (HA) - death receptor 5 antibody (DR5 Ab) conjugate was synthesized as a dual targeting therapeutic agent to treat the liver metastasis. Liver is the most frequent site of metastasis with a 5-year survival rate of only 20~40 %. Dual targeting is achieved by DR5 Ab, a humanized agonistic monoclonal antibody binding to DR5 frequently overexpressed in many kinds of cancer cells, and by HA, a natural polysaccharide binding to HA receptors highly expressed in both the liver and cancer cells. Thiol end-modified HA was site-specifically conjugated to N-glycan on Fc region of oxidized DR5 Ab using a hetero-bifunctional linker of 3-(2-pyridyldithio)propionyl hydrazide (PDPH). The successful synthesis of HA-DR5 Ab conjugate was confirmed by 1H NMR, purpald assay, dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC). In vitro analysis of HA-DR5 Ab conjugate revealed that the conjugation of HA to DR5 Ab did not affect the binding affinity and anti-cancer efficacy of DR5 Ab. Remarkably, according to in vivo bioimaging study, HA-DR5 Ab conjugate appeared to be highly accumulated in the liver and dramatically effective in inhibiting the tumor growth in liver metastasis model mice. In the last part, hyaluronate - gold nanorod / death receptor 5 antibody (HA-AuNR/DR5 Ab) complex was developed for transdermal theranosis of skin cancer. Non-invasive transdermal delivery is a promising method with distinct advantages including patient compliance over other delivery routes. The successful formation of the complex was corroborated by 1H nuclear magnetic resonance (NMR), UV-Vis spectroscopy, dynamic light scattering (DLS), zeta potential, and transmission electron microscopy (TEM). In vitro biological activity of the complex was verified by ELISA and MTT assay using HCT116 cancer cells. In addition, in vivo photoacoustic imaging and two-photon microscopy clearly visualized the transdermal delivery of HA-AuNR/DR5 Ab complex through the inevitable barrier of stratum corneum in the skin. Furthermore, in vivo anti-tumor effect on skin cancer model mice was confirmed from statistically significant decrease of tumor-reflecting luciferase expression levels and apoptotic signals in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Taken together, we could confirm the feasibility of HA-AuNR/DR5 Ab complex as a novel theranostic platform for non-invasive transdermal treatment of skin cancers.