Tertiary Amine-oxide-based Nanocarriers For Drug Delivery And Microfluidics-based Stepwise Preparation For Polyplexes

This thesis includes two parts:the application of polymer nanocarriers to deliver chemotherapeutic drugs in cancer therapy and the construction of polymer nanocarriers in gene deliveryThe first part of this thesis focuses on tertiary amine-oxide-based nanocarriers for drug delivery.Anticancer nanomedicines based on water-soluble polymers have urgent and widespread demands in clinics,because they can effectively improve the solubility of hydrophobic drugs,prolong the circulation time,increase the concentration of drugs in tumor and greatly mitigate the adverse effects of chemotherapeutic drugs.However,current anticancer nanomedicines cannot significantly improve curative effect and patients' median survival time.It is necessary to further engineer the properties of nanomedicines to improve the antitumous effect.One key approach to break the delimma is to achieve nanomedicines delivery efficiently in a 'CAPIR' cascade(Circulation,Accumulation,Penetration,Internalization and Release).Therefore,the nanomedicine surfaces are generally designed to achieve a stealthy-to-sticky transition:in blood circulation,nanomedicines should be made stealthy by modification with antifouling materials;once accumulated in tumors,naomedicines should be restored with their stickiness to cell membrane for internalization.Several strategies,such as PEGylation/dePEGylation,charge-reversal and shielding/exposing targeting ligands are devoted to meet this requirement.However,such integrated nanomedicines with complicated structures are difficult in chemical characterizations,pharmacokinetic evalutions and reproducible fabrication process establishments for clinical translation.Therefore,design of distinct carriers with simple structures,but possessing all the required functions,i.e.one-for-all strategy,is demanded for the further development of nanomedicines.Meanwhile,the subcellular organelle-targeted release of nanomedicines after endocytosis is also a key factor to the therapeutic effect.Mitochondria,the energy factory of cells,participates in the genesis,progression and apoptosis of tumors.The mitochondrial targeted delivery has been proved effectively to improve the therapeutic efficacy.Nonetheless,most of traditional mitochondrial targeting groups are positively charged and thus cannot be used intravenously.To realize direct intravenous applications,the targeting ligands must be first modified to shield the cationic groups in blood circulation but deshielded after endocytosis.However,such complex design and limited deshileding ability make it less useful.Therefore,it is significant to develop mitochondrial targeting nanomedicines with a neutral or negatively charged surface.In the first part of this thesis,we designed tertiary amine-oxide-based(water-soluble and zwitterionic)amphiphilic block polymers,poly[2-(N-oxide-N,N-dimethylamino)ethyl methacrylate]-block-poly(?-caprolactone)and poly[2-(N-oxide-N,N-diethylamino)ethyl methacrylate]-block-poly(?-caprolactone),and prepared the doxorubicin(DOX)-loaded micelles(OPDMA-PCL/DOX and OPDEA-PCL/DOX).OPDMA-PCL/DOX and OPDEA-PCL/DOX micelles formed with a diameter about 30 nm and slightly negatively charged in aqueous solutions.They not only had a long blood circulation like PEG,but also effectively adhered to the surface of red blood cells to avoid the immune recognition and elimination.In the in vitro and in vivo tumor penetration models,they showed an outstanding permeation in tumor tissues to the areas far away from the tumor blood vessels.They were fast internalized by tumor cells mainly through macropinocytosis,which did not lead to lysosomes'degrading.Meanwhile,they showed a strong mitochondrial targeting ability,effectvely inducing mitochondrial dysfunction and cell apoptosis.In the tumor models of adriamycin-resistant human breast cancer cell line MCF-7/ADR,OPDMA-PCL/DOX and OPDEA-PCL/DOX showed stronger anti-tumor effects and lower side effects than traditional DOX-loaded micelle PEG-PCL/DOX and DOX.The second part of this thesis focuses on microfluidics-based stepwise preparation for polyplexes.Cationic polymers such as polyethyleneimine(PEI)are excellent carriers for gene delivery,because they can effectively compress DNA to form complexes and prevent DNA from being degraded by nucleases.However,the in vivo applications of cationic polymer/DNA polyplexes are limited by their positively charged surface,and thus they are generally coated with neutral or negatively polymers such as hyaluronic acid(HA)to shield the positive charges and overcome the blood barriers.The HA/PEI/DNA complexes prepared by current traditional preparation methods(vortex mixing and piping by pipette,etc.)generally have shortcomings such as large sizes and poor uniformity.In the second part of this thesis,we explored a microfluidics-based stepwise layer-by-layer assembly method for preparation of HA/PEI/DNA polyplexes with uniform particle size and negatively charged surface.We first optimized the preparation of the microfluidic devices,and selected a microfluidic chip with suitable channel dimmensions.Then PEI and DNA solutions were mixed in the microfluidic channel to form the polyplexes,which then assembled with HA in the second microfluidic chip to obtain the HA/PEI/DNA polyplexes.The parameters including microfluidic channel dimmensions,flow rates,the flow rate ratio R,PEI:DNA ratio(nitrogen to phosphorus ratio,N:P)and HA:DNA mass ratio(HA:DNA),were investigated to study their correlations with the size,uniformity and surface potential of the formed polyplexes.The size and uniformity of HA/PEI/DNA polyplexes were adjusted effectively by tuning the flow rate ratio N:P and HA:DNA.Compared with traditional vortex mixing method,HA/PEI/DNA polyplexes prepared by microfluidic chips were much smaller and more uniform with better stability and higher repeatability.