Esterase-responsive Tumor Cell Selective Gene Delivery System For Cancer Gene Therapy

Fibroblasts are important supporting cells in tumor. Recent studies show that cancer treatment damaging fibroblasts induces them to secret WNT16B, a signal strongly promoting tumor cell survival and metastasis. Futhermore, preserving fibroblasts correlates well to good prognosis. However, most therapies cannot avoid damaging fibroblasts due to indiscrminated killing, causing quick tumor relapse and poor prognosis. Therefore, a therapy benign to fibroblasts would avoid their damages to have better prognosis.Cationic polymers have been extensively used as nonviral vectors for gene delivery. The intracellular dissociation of cationic polymer/DNA complexes (polyplexes) to release DNA is essential for the DNA to be efficiently expressed. Generally, the dissociation is difficult due to their inherent strong electrostatic interactions and thus has been the bottleneck to effective transcription. Thus, we proposed that polyplexes carrying a cancer suicide gene that can efficiently dissociate in cancer cells but hardly dissociate in tumor fibroblasts would selectively kill tumor cells but avoid damaging fibroblast. We found that fibroblasts had low esterase levels than many cancer cells including HeLa cells. Thus, a cationic polymer that can respond to the esterase (i.e. as a trigger) in the cytosol to remove its cationic charges would be able to efficiently release DNA in cancer cells but not in fibroblasts.Based on these three hypotheses, we synthesized two esterase-responsive cationic polymer (ERP) consisting of quaternary amines carrying N-propionic 4-acetoxybenzyl ester moiety. Esterases can quickly hydrolyze the phenolic acetate moieties in ERP, subsequently trigger elimination of p-quinone methide and thus producing carboxylates, transforming the cationic polymer to a zwitterionic one. Therefore, ERP/luciferase DNA polyplexes expressed protein 103 higher in HeLa cells than in fibroblasts. For in vivo uses, the lipid coated polyplexes (LERPs) were further developed and they still maintained such a discriminated transfection in the two types of cells in vitro and in vivo. In an intraperitoneal (i.p.) tumor model, LERPs carrying cancer suicide gene TRAIL plasmids effectively induced apoptosis of cancer cells but did not harm tumor fibroblasts, which were hypoactivated in expression of WNT16B, resulting in much lower tumor recurrence, longer survival and less adverse effects than the three first-line chemotherapy drugs, irinotecan, paclitaxel and cisplatin, which cause fibroblast hyperactivation to excrete WNT16B. Thus, these results have confirmed these hypotheses.Using the same concept, in the third part of this thesis we synthesized a series of poly{(4-acetyloxy)benzyl-N,N-dialkylammonium] alkyl acrylate} (alkyl= methyl (PQDMA), ethyl (PQDEA) and butyl (PQDBA)) with increased hydrophobilities. For in vitro HeLa cells transfection, PQDEA polyplexes have the best transfection efficiency, following by PQDBA and PQDMA polyplexes. PQDEA made from PDEA with a molecular weight of 14KDa can obtain 108 RLU/mg protein expression level. Meanwhile the cellular uptake and lysosomes escape abilities of PQDEA polyplexes are superior to PEI25K/pLUCI. The lipid coating used in LERPs can also be coated to PQDEA/pDNA polyplexes to form stable lipidic polyplexes (LPQDEAs/pDNA). LPQDEAs/pTRAIL can also effectively transfect and induce HeLa cells apoptosis and have a better therapeutic efficacy and much longer survival time than chemotherapy drugs in HeLa intraperitoneal tumor model. Pegylating lipids DSPE-PEG and TPGS are further introduced to LPQDEAs/pDNA to form pegylated lipid polyplexes. The in vitro transfection results confirm the pegylation layers have no obvious influence on the LPQDEAs/pDNA transfection efficiency and can be applicated in intravenous systematic administration.