The Design And Synthesis Of Nano Drug Carriers With PH-responsivity

The pH-repsonsive polymer, a kind of intelligent polymer, is able to undergo large and abrupt physical or chemical changes in response to pH change in the environmental conditions. In combination of nano technology, the pH-responsive polymers have been widely applied for drug delivery. These "smart" drug vectors can site-specifically release anticancer drugs or therapeutic DNA by taking advantage of the difference among the slightly acidic environments of tumor tissues (pH 6.8), intracellular environment (pH 4.5-6.5) and that of bloodstream and normal tissues (pH 7.4). As a result, the pH-responsive carriers have been an effective strategy to improve therapeutic efficacy and reduce adverse toxicity toward normal healthy cells.In this thesis, four novel pH-responsive nanocarriers for the triggered delivery of anticancer drugs or DNA are designed and evaluated in vitro. The pH-responsive components are poly(β-amino ester) (PBAE), poly(disulfide amine) (PSSA) and polyethyleneimine (PEI). Due to containing biodegradable linkages such as disulfide bond, ester and carbamate, these nanocarriers can degrade in response to specific environment and have good biocapatibility. A large number of primary amines, secondary amines or tertary amines protonate in response to slightly acidic environment of tumor tissues, endosomes and lysosomes, which change the physical or chemical properties of these nanocarriers and thus trigger the release of anticancer drugs or DNA.(1) Noncovalent construction of hepatoma-targeted polymer micelles for drug deliveryAiming at premature release of drug and inefficient uptake by tumoral cells, novel hepatoma-targeted micells for drug delivery were prepared via noncovalent interaction. We synthesized a series of poly(ethylene glycol)-poly(β-amino ester) (MPEG-PBAE) containing high hydrophobic alkyl carbon chain. These polymers can load DOX and form micelles in water. At very low concentration, the pH-reponsive micelles still remained stability and tiggerred the release of DOX without burst release at slightly acidic environment (pH 4.5). Then amphiphilicN-(1-deoxylactitol-1-yl) dodecylamine (Gal-C12) as targeting ligand was successfully bound to the micellar surface by hydrophobic interaction. The results of cellular uptake, in vitro cytotoxicity and cell cycle analysis indicated that the Gal functionalized micelles effectively transferred DOX into hepatoma cells (e.g. HepG2 cells) via asialoglycoprotein receptor (ASGPR) mediated endocytosis, released DOX from the micelles and resulted in enhanced proliferation inhibition efficacy.The ease of surface functionalization and enhanced drug efficacy make the present platform promising for hepatoma-targeted drug delivery in cancer therapy.(2) Preparation of PEI-g-PSSA-g-PEI vesicles for gene deliveryBoth of polymer vesicles and liposomes have hollow vesicular structure. Cationic liposomes have been one of the most studied nonviral vectors and liposomal transfection reagent Lipofectamine 2000 is commercially available. However, to our knowledge seldom reports focus on polymer vesicles as gene carrier. The novel polymer vesicles were prepared by the self-assembly of amphiphilic polyethyleneimine-g-poly(disulfide amine)s-g-polyethy leneimine (PEI-g-PSS As-g-PEI) for gene delivery. To investigate the effect of the hydrophobicity on transfection efficiency, a small series of PEI-g-PSSAs-g-PEI were prepared under uniform conditions and contain PEI fragments with the same molecular weight. The hydrophobicity of PEI-g-PSSAs-g-PEI was adjusted by varying the hydrophobic content in poly(disulfide amine)s backbone (100%-60%) and by choosing hydrophobic monomers ranging in length from C12 to C16. The hydrophobicity of polymers is also related to DNA binding affinity. Polymer vesicles attained from the water insoluble polymers condensed DNA into polyplexes with the size below 200 nm and the surface charge ranging from+10 to+35 mV that were suitable for cell endocytosis. DNA-polyplexes exhibited inverted hexagonal structure, observed by TEM. The results of in vitro transfection demonstrate that the hydrophobic/hydrophilic balance of copolymers greatly affects their transfection property. Top-performing polymer Ⅱ-70% showed improved transfection efficiency and significantly lower cytotoxicity on COS-7 cells, when compared to commercial reagents polyethyleneimine (PEI 25k) and Lipofectamine 2000.These results indicate that cationic polymer vesicles with tunable hydrophobicity are promising materials for gene delivery.(3) Synthesis of thio-specific degradable PEI derivatives for gene deliveryDisulfide crosslinked PEI is an important kind of cationic polymeric gene vectors. Disulfide bonds can stay stable in blood circulation and degrade rapidly (minutes to hours) by reduced glutathione (GSH) in the intracellular environment. These reducible polymers exhibit potentials as safe and efficient gene vectors. However, disulfide bonds in these crosslinked PEIs cleave into the free thiol groups in the reductive intracellular environment. The external thiol groups can readily destroy the disulfide bonds in protein by thio/disulfide exchange or form new disulfide with cysteine residues of protein via oxidative reaction, which may disturb protein function and normal cellular process.To resolve this problem, we apply thio-specific linker (-NH-CO-O-CH2CH2SS-) to connect low molecular weight PEI into high molecular weight PEI derivatives. ESI-MS analysis showed that after incubated with 1,4-dithio-DL-threitol (DTT), the degradable linkages in the polyethyleneimine (PEI) derivatives underwent disulfide bond cleavage followed by intramolecular cyclization and cleavage of the neighboring carbamate bond. Thus, the free thiol groupswere converted into 1,3-oxathiolan-2-one. In vitro transfection results indicateed that PEI-SS-CL-1.5 (at crosslinker/PEI feed molar ratio of 1.5) can transfect different cell lines (COS-7, HepG2 and Raw246.7 cells)and exhibited higher transfection efficacy than the commercially available reagents such as the high molecular weight PEI with molecular weight of 25 kDa (PEI 25k) and the versatile liposomes Lipofectamine 2000. PEI-SS-CL-1.5 also demonstrateed significantly lower cytotoxicity, compared with PEI 25k and Lipofectamine 2000.Our study indicates that incorporating the thio-specific cleavable disulfide bond into crosslinked PEIs is an effective strategy for designing safe and effective gene vectors.(4) Heterobifunctional crosslinked PEI for gene deliveryTo develop safe and effective gene vectors, most researchers crosslink low molecular weight PEI with homobifunctional compound, which make it difficult to obtain the crosslinked polymers with narrow molecular weight distribution. We synthesized heterobifunctional crosslinker with acrylate and chloroformate. Due to higher reactivity than acrylate, chloroformate preferentially reacted with the amine groups of PEI at -10℃ and then acrlate was conjugated to PEI when the reaction was transferred to room temperature. The controlled crosslinking strategy makes it promise to obtain crosslinked PEI with moderate molecular weight andgood water solubility.The molecular weight of our synthesized PEI derivatives were 30-90 fold that of the starting material and these crosslinked PEI derivatives exhibited good water solubility (> 1 mg/mL).In vitro transfection results indicateed that PEI-HCL-1.5 (at crosslinker/PEI feed molar ratio of 1.5)exhibited higher transfection efficacy than the commercially available reagents such as the high molecular weight PEI with molecular weight of 25 kDa (PEI 25k) and the versatile liposomes Lipofectamine 2000. PEI-HCL-1.5 also demonstrateed significantly lower cytotoxicity, compared with PEI 25k and Lipofectamine 2000. These results indicate that heterobifunctional crosslinked PEIs are high molecular weight and exhibit good water solubility, which have good transfection efficiency.