Synthesis And Functional Research Of Monoclonal Antibody Against Human CD40Molecule5C11Immunized Chemical Drug Encapsulated Nano Polymeric Micelle

As a special tumor biotherapeutic choice of lymphoma, solid tumor and multiplemyeloma, monoclonal antibodies against human CD40molecule have been drawing theresearchers’ attention since its therapeutic effects had been identified. The previousstudy results had shown that5C11, a kind of agonistic monoclonal antibody againstCD40molecule produced by our laboratory could definitely induce the apoptosis ofmultiple myeloma cell line and enhance the chemical drugs sensitivity against tumorcells. For these sakes,5C11had the potential value for tumor treatment and couldexhibit a better anti-tumor bioactivity via using it with adjuvant and anti-tumor drugs.Nanoparticle drug delivery system had a great many of useful advantages, such assmall size, stability in aqueous environment, wide range of drug encapsulability,long-time circulation in vivo to accumulate these nano-scaled drug delivery system attumor site via the enhanced permeability and retention effect and so on. These benefitscould overcome the toxic effects of free chemical drugs treatment and this drug deliverysystem could be considered as a kind of prosprctive adjuvant.In order to achieve the advantages of nanoparticle drug delivery system andmonoclonal antibody targeting drug to enhance the cellular cytotoxicity to tumor cells atthe same time, we focus on synthesizing a kind of nanoparticle to encapsulate chemical drug then coupling with antibody against human CD40molecule without changing anybiologic activities of it.Objective: Synthesize the polymeric micelle as the drug delivery system toprepare the chemical drug encapsuled and5C11coupled polymeric micelle complex.Evaluate the cytotoxicity and cellular uptake of it in vitro.Method: Heterobifunctional PEG with a tosyl group at one terminus andtosyl-terminated poly （lactic acid）-poly （ethylene glycol）（PLA-PEG） block co-polymer（PLA-PEG-OTs） was Synthesized. Doxorubicine （DOX） were encapsulatedinto polymeric micelles（DOX-PMs） by solvent evaporation method then coupled with5C11to obtain immunized DOX-PMs （DOX-PMs-5C11）.¹H NMR analyzed and gelpermeation chromatography （GPC） measured the chemical groups or molecule weightof the copolymers. Fluorescent spectroscopy using pyrene as a probe was adopted todetermine the critical micelle concentration （CMC） of PMs. The mean diameter of PMswas determined by dynamic light scattering （DLS）. MTT assay was used to test thecytotoxicity of DOX,DOX-PMs and DOX-PMs-5C11to breast cancer cell line MCF-7and MCF-7/ADR in vitro. Fluorescence microplate reader, flow cytometry and laserconfocal microscope were adopted to evaluate the CD40expression ratio on humanbreast cancer cell line, the coupling efficiency of polymeric micelles with protein, theactivity of5C11conjugaed polymeric micelles and the tumor cellular uptake efficiencyof DOX-PMs, DOX-PMs-5C11in vitro.Results:1H NMR analyze and gel permeation chromatography （GPC） analysisdisplayed that the the molecule weight of the copolymers range spread from（4000¹0000）g/mol. CMC of the PMs were （3.2⁵.5）×10^-3g/L, diameter of polymericmicelles were （21.2³1.8）nm and up to range from （21.4³3.1）nm after beingconjugated with protein. The best pH value for the reaction of polymeric micellescoupling with protein was8.5and under this condition the coupling efficiency were13.4%¹7.1%. The PMs could well encapsulate doxorubicine and then couple with5C11, their loading capacity and encapsulation efficiency were （3.75±0.27）%,（75.12±2.38）%respectively. CD40expression ratio was54.5%on MCF-7and73.6% on MCF-7/ADR. DOX-PMs-5C11exhibited the superior cytotoxicity compared toDOX-PMs especially onto MCF-7/ADR （P<0.05）. After being coupled with5C11,cellular uptake efficiency of PMs could be enhanced compared to unconjugated ones.Conclusion: The chemical drug encapsuled5C11polymeric micelle complexpossessing a superior cytotoxicity compared to unconjugated ones attributed to itsenhanced cellular uptake efficiency. This effect exhibited more significantly in thebreast cancer cell line with doxorubicine resistance and higher CD40expression.