Research On Drug Delivery, Breast Cancer Imaging And Bioseparation Based On Biofunctionalized Nanoparticles

Bionanotechnology is a new interdiscipline,which has provided new opportunities and scientific methods for the deep extensive development of biomedicine research.As the novel products of bionanotechnology,bionanomaterials, especially biofunctionalized nanoparticles with specific physical,chemical or bioactive properties,have shown great potential in the researches of cancer diagnosis and treatment,cells and proteins separation,gene therapy and controlled drug delivery.However,many of these researches are still in their infants.Further studies need to be extended.Recently,our research group has developed systemic researches based on the biofunctionalized silica nanoparticles and chitosan nanoparticles with the combination of nanotechnology,analytical chemistry,biotechnology and material science.On the basis of our correlative researches,this dissertation has focused on the target imaging of breast cancer and bioseparation with biofunctionalized silica nanoparticles,and the further studies of the drug delivery with biofunctionalized chitosan nanoparticles.1.Imaging breast cancer cells and tissues using biofunctionalized fluorescent silica nanoparticles.A method using RuBpy doped silica nanoparticles（FSiNPs） as novel fluorescent labels of peptide has been developed for in vitro and ex vivo imaging of breast cancer cells and tissues.The biofunctionalized FSiNPs（RGD-FSiNPs） were prepared by covalent immobilization of arginine-glycine-aspartic acid（RGD） peptide onto FSiNPs according to the cyanogens bromide（CNBr） method.In vitro cell imaging showed that RGD-FSiNPs exhibited high target binding to fixed and living cells ofα_vβ₃ integrin receptor（ABIR）-positive MDA-MB-231 breast cancers.Further study regarding the ex vivo imaging of tumor tissue samples was also carried out by intravenously injecting PEGylated-RGD-FSiNPs into athymic nude mice bearing the MDA-MB-231 tumors.Tissue images demonstrated that it could reduce the phagocytosis of nanoparticles by liver and spleen and the high integrinα_vβ₃ expression level of the MDA-MB-231 tumors was clearly visible due to the special targeting effects of the RGD-FSiNPs.This RGD peptide-oriented fluorescent labeling technique based on the biofunctionalized FSiNPs will be promising for tumor cell imaging and cancer metastasis,etc.2.Targeted separation of protein based on biofunctionalized magnetic silica nanoparticles. A method of silica coated magnetic nanoparticles（MSiNPs）-based targeted separation of proteins has been developed based on the technology platform of our research group.The biofunctionalized MSiNPs（IgG-MSiNPs） were synthesized by modification of human IgG onto the surface of MSiNPs with CNBr method.Target protein（goat-anti-human IgG-FITC） was specifically recognized with IgG-MSiNPs and isolated under magnetic field.Then the target protein could be dissociated from the nanoparticles with suitable dissociation agent.The effects of separation and dissociation conditions on the separation efficiency,selectivity and dissociation effiency were studied.At the optimal conditions,the relatively high separation and dissociation efficiency and good selectivity were obtained.This study will further explore the application of functionalized magnetic silica nanoparticles in direct bio-separation of proteins from complex samples.3.Synthesis of biofunctionalized fluorescent magnetic nanoparticles and their potential applications in separation of breast cancer cells.We have developed a simple method to synthesize FITC dye doped silica fluorescent magnetic nanoparticles（FMSiNPs） with chemical precipitation method and reversed microemulsion technique under mild condition.FMSiNPs were formed by employing human IgG modified FITC dye and magnetic fluid as core and the silica as shell.The biofunctionalized FMSiNPs（RGD-FMSiNPs） were prepared by immobilizing of RGD peptide onto the surface of FMSiNPs.The specific enrichment ofα_vβ₃ integrin receptor-positive MDA-MB-231 breast cancer cells using RGD-FMSiNPs were carried out due to the specific recognition of RGD peptide andα_vβ₃ integrin.Then the target cells were isolated with RGD-FMSiNPs under magnetic field and detected by FCM based on the fluorescence properties of RGD-FMSiNPs. Results indicated that RGD-FMSiNPs exhibited high target binding to MDA-MB-231 breast cancer cells and high separation efficiency.This new kind of RGD based biofunctionalized FMSiNPs could be widely used for the magnetic separation and fluorescent detection of tumor cells.4.Research on protein drug carriers based on biofunctionalized chitosan nanoparticles.A new type of biocompatible chitosan（CS） nanoparticles encapsulated with bovine serum albumin（BSA）（CS-BSA-TPP NPs） was prepared through ionic cross-linking technique based on CS and sodium tripolyphosphate（TPP） at room temperature.The influence of formulation conditions on the properties of CS-BSA-TPP NPs was investigated.Results showed that CS-BSA-TPP NPs with diameter of below 100 nm and good dispersion were prepared at the optimal conditions,and the BSA encapsulation efficiency was more than 50%.This system was also applied in the preparation of CS nanoparticles encapsulated with Gamma Seroglobulin（CS-IG-TPP NPs）,the CS-IG-TPP NPs presented the encapsulation efficiency of IG with 55.0%and displayed sustained-release property.Thus,this protein delivery system based on biofunctionalized CS nanoparticles will be promising for protein drug delivery.5.A novel anticancer drug delivery system based on biofunctionalized chitosan nanoparticles.A chitosan-methotrexate covalently conjugated nanoparticles（CS-MTX-TPP NPs） has been developed as a potential delivery system for methotrexate（MTX）. MTX was first conjugated to CS by using glutaraldehyde as cross-linked agent,and followed by the process of ionic gelation between MTX-conjugated CS and sodium tripolyphosphate（TPP） to form CS-MTX-TPP NPs at mild reaction conditions.At the optimal conditions,CS-MTX-TPP NPs with diameter of sub-200-nm and encapsulation efficiency of 53.0%were obtained.Additionally,in vitro release test revealed that the stable covalent bonding of CS and MTX was beneficial for providing slow release for MTX.Especially,cellular toxicity study in MCF-7 cancer cells further demonstrated the effective anticancer efficacy of this new type of delivery for MTX.In the future,this work could be expected to broaden the applications of biofunctionalized CS NPs in anticancer drug delivery.