Smart Nanovectors For Gene Delivery And Cancer Therapy

Tumor is one of the first leading causes of human death and can survive after several treatment methods. Gene therapy primarily emerges as a treatment for tumor, while naked gene can’t be used for tumor therapy because of the enzymolysis in vivo, so it is necessary of some gene carriers to protect delivered gene. Normal human tissues are so tensely arranged that only small molecules can permeate, while, the structural" integrity of tumor tissues disrupts and large molecules can also pass through, making it possible that gene delivery by nanoparticles.It is of unique advantages of nanoparticles for gene delivery, while like other gene delivery materials, nanoparticles also face some obstructions for gene delivery, such as cell membrane inhibits the nanoparticles to enter cell, endosomal membrane prevents their endosomal escape to plasma and nuclear membrane blocks the nuclear translocation of nanoparticles.To avoid the major troubles in gene delivery, we designed nanoparticle delivery system with HA-HP, charge reversible CS-Aco, Dexa and PEI-SS. For details, our works are introduced in following four parts:(1) Nanoparticles need to overcome the blockade by cell membrane for gene delivery, so we established DNA/PEIS/HA-HP nanocomplex, this nanocomplex was stable in an extracellular environment and degraded by HAase after targeted HA receptor CD44-mediated cell endocytosis, causing the outer shielding of the nanocomplex to loosen. The resulting partially exposed disulfide-linked DNA/PEIS nanocomplexes efficiently ruptured the endosome, facilitating the cleavage of disulfide bonds and the release of DNA/PEI polyplexes into the cytoplasm, where DNA release from the polyplexes was remarkably enhanced due to strong electrostatic competition of HP with PEI. We took use shBmi-1 to test our gene delivery efficiency. We found PEIS/HA-HP nanoparticles can deliver shBmi-1 into cells efficiently and knowdown the expression of Bmi-1 which is of critical role for cancer stem cells, triggering reduced sphere formation ability in vitro and attenuate tumor initiating capacity in vivo and good prognosis approved by mouse model.(2) Cationic polymer can be used for shRNA delivery. However, it is not easy for nanoparticles to escape from endosome into plasma, resulting low delivery efficiency. Herein, a three-layered polyethyleneimine (PEI)-coated gold nanocomplex interlaid with a pH-responsive charge-reversible chitosan-aconitic anhydride (CS-Aco) is constructed:a Au-PEI/CS-Aco/PEI/shRNA nanoparticle. The negatively charged CS-Aco hydrolyzes into positively charged CS in lysosomes, causing the nanocomposite to disassemble. The released Au-PEI nanoparticles and CS efficiently rupture the lysosomes and thus release the PEI/shRNA polyplexes into cytoplasm, where they quickly disassociate because the PEI chains are short (1.2 kDa). For application, we constituted shABCG2 and designed nanocarriers for tumor therapy. We found the nanocomplexes efficiently deliver shABCG2 to tumors and markedly silence ABCG2 expression, which sensitizes HepG2 cells to the drugs. The experiment data in vivo proved that three-layered structural nanoparticles can signifiently enhance the cancer therapy efficiency.(3) All gene delivery system should carry gene into nucleus where the gene can be transcripted. Dexamethasone (Dexa), which has nucleu targeting property, was added into nanocomplex for gene delivery. Truely, we found Au-PEI/DNA/PEI-Dexa could enhance nuclear translocation of shRNA by confocal microscope and cellular fractionation. To examine the gene delivery of pTRAIL, we cloned and delivered TRAIL, an important regulator of cell death and apoptosis, especially in tumor cells. We found Au-PEI/DNA/PEI-Dexa can carry more pTRAIL into nucleus, promote the expression of TRAIL and thus trigger apoptosis and death of tumor cells. Mouse model showed small volume of Au-PEI/DNA/PEI-Dexa treated xenograft tumors, indicating that Au-PEI/DNA/PEI-Dexa can reduce the tumor volume through TRAIL overexpression. What is more, comparable bodyweight between treatment and control groups indicates little side effect of our gene delivery system.(4) In blood system and tumor tissues, the protection by gene carriers is of great concern for gene. Nonetheless, the protective effect becomes a reversed element when the gene is delivered into cytoplasma, where exogenetic genes should be released from nanoparticles and then transcription occurs. To found the balance of protection and releasing, we designed gene carrier PA-PEI-SS, which can response to reductive environment and pH changes. Agarose gel electrophoresis was used to analyze the protection and releasing function by gene carriers, respectively. Experimental results showed that PA-PEI-SS gene carrier, which protects gene in normal environment, can releases gene into cytoplasma to promote transcription when the external environment change. This gene carrier system demonstrates no side effect to cells proved by cytotoxicity examination, and enhanced transfection efficiency than PEI 25KDa.Above all, on the basis of common cationic polymer PEI, we developed smart nanoparticles to overcome the obstructions of gene delivery. Taking use of some important biomolecules, we examined the influence to corresponding function with our designed nanocomplex.