Construction And Characterization Of Smart Self-Assembly Drug Delivery Systems For Cancer Therapy

Chemotherapy remains the crucial approach for the treatment of cancers,a major threat to human health and life.In state-of-the-art chemotherapy,the conventional free antitumor drugs often suffer from several drawbacks,including lack of aqueous solubility,highly non-specificity and undesirable adverse effects,which significantly limited their wide applications in clinic.Hence,it has been a hot research topic to construct highly efficient drug delivery systems with low toxicity for cancer therapy.Up to now,numerous strategies have been put forward to realize this goal.Among them,environment-responsive drug delivery systems adaptable to microenvironmental stimuli associated with tumor tissues and cells have gained significant attention.They have not only demonstrated good water solubility and enhanced stability,but also targeted the tumor site and released the loaded drug in a controlled manner.In this dissertation,we chose chemotherapeutic agents used in clinic and constructed a serious of stimuli-responsive drug delivery systems,which can be divided into two categories:?1?smart small molecular prodrugs by introduction of stimuli-responsive functional groups;?2?smart polymeric nano-drug delivery systems using stimuli-responsive polymers as carriers.The main contents are described as below:In the second chapter,a novel nanoparticle formulation of paclitaxel?PTX?based on dual paclitaxel succinate glycerophosphorylcholine?di-PTX-GPC?prodrug was developed.The di-PTX-GPC prodrug was synthesised by conjugating PTX with GPC through esterification under N,N?-carbonyldiimidazole?CDI?and 1,8-diazabicyclo[5.4.0]undec-7-ene?DBU?catalytic system.Di-PTX-GPC liposomes were prepared by thin film method and characterized by dynamic light scattering?DLS?and transmission electron microscope?TEM?.The results indicated that the liposomes have an average diameter of 157.9 nm with well-defined spherical morphology.In vitro drug release studies confirmed that the di-PTX-GPC liposomes have controlled release profile of PTX at a weakly acidic environment,which formulates them suitable for sustained drug delivery.Additionally,in vitro cellular uptake analysis and cytotoxicity evaluation showed that di-PTX-GPC liposomes were internalized successfully into tumor cells to induce the apoptosis against MCF-7,HeLa and HepG-2 cells.In vivo pharmacokinetics study revealed that such nanoparticle formulation of di-PTX-GPC has longer retention half-life in bloodstream,which subsequently leads to accumulate in tumor sites due to enhanced permeability and retention?EPR?effect.More importantly,di-PTX-GPC liposomes demonstrated excellent in vivo anticancer activity compared to Taxol with reduced adverse effect.Conclusively,these results suggest that di-PTX-GPC liposomes could be an effective PTX delivery vehicle in clinical cancer chemotherapy.In the third chapter,the co-assembled combination strategy involved thiol-sensitive paclitaxel-lipid prodrug?PTX-ss-PC?with typical lipids was designed for PTX to improve loading and respond to the reducing environment of the cell to trigger the release intraliposomal content upon cellular uptake by cancer cells.The sterically stabilized PEGylated liposomes?PTX-ss-PC/Liposomes?comprised of EPC:Chol:mPEG₂₀₀₀-DSPE components were prepared by conventional dehydration-rehydration method.Physicochemical characterization of the PTX-ss-PC/Liposomes was carried out.It was demonstrated that these liposomal formulation possessed 234.9 nm size,zeta potential of-29.1 mV and 2-fold increase in drug PTX loading due to co-assembled loading manner.In vitro GSH-mediated release revealed that an efficacious system with improved loading and pharmacokinetics was formulated as potential alternative for currently marketed PTX formulation.In the fourth chapter,the reduction-responsive disulfide core-cross-linked micelles derived from the dimeric lipoic acid-glycerophosphorylcholine?di-LA-PC?conjugate were developed.Di-LA-PC conjugate was first synthesized by a facial esterification of lipoic acid?LA?and glycerophosphorylcholine?PC?and its structure was established by MS,1H NMR and 13C NMR spectra.The amphiphilic conjugate can self-assemble into nanoscale micelles that were easily core-cross-linked under the catalytic amount of dithiothreitol?DTT?.Due to the PC shell and core crosslinking,the assembled reduction-responsive micelles maintained highly stability against extensive dilution under physiological environment but rapidly disassembled within 10 mM DTT,highlighting their potentials as a drug delivery system.Paclitaxel,a clinically first-line antitumor drug,could be stably encapsulated into the core of disulfide cross-linked di-LA-PC micelles with impressive drug loading content?8.13%?and loading efficiency?88.6%?.The in vitro release displayed that less than 20%of PTX released from the core-cross-linked micelles after incubation at 37°C for 68 h,whereas over 80%PTX released within 10 mM DTT under otherwise the identical condition.Moreover,PTX-loaded micelles exhibited capably uptake behavior into tumor cells and inhibited the growth of MCF-7,HepG-2 and A549 cells,which is demonstrated by in vitro MTT assay,apoptosis and cell cycle results.Pharmacokinetics in vivo studies demonstrated that disulfide core-cross-linked di-LA-PC micelles prolong the circulation of incorporated PTX in bloodstream.More importantly,the developed micellar formulation loaded PTX displayed the enhanced antitumor activity while reducing side effect in xenograft models of 4T1 breast tumor.Therefore,such reduction-responsive degradable core-cross-linked di-LA-PC micelles with excellent extracellular colloidal stability and triggered intracellular drug release are potential candidates for the tumor-targeted delivery of therapeutic agents in clinic.In the fifith chapter,a novel disulfide cross-linked liposomes?CLs?assembled from dimeric lipoic acid-derived glycerophosphorylcholine?di-LA-PC?conjugate was developed.The conjugate was synthesized by a facial esterification of lipoic acid?LA?and glycerophosphorylcholine?GPC?and characterized by MS,¹H NMR and ¹³C NMR.Featuring the enhanced serum-stability and intracellular drug release determined by in vitro stability and GSH-responsive behavior,CLs prepared with dried thin film technique following 10 mol.%DTT cross-linking can attain effective delivery of anticancer candidates.Notably,CLs stably encapsulated doxorubicin?Dox?in their vesicular structures and showed a remarkable thiol-sensitive release of payload upon cellular uptake by cancer cells,compared to that of uncross-linked liposomes?uCLs?or Doxil-like liposome?DLLs?.The cell viability and apoptosis of Dox-loaded CLs worked the pronounced cytotoxic effects to MCF-7 cells with an IC50 value of 10.8?g Dox equiv./mL comparable to free Dox and 2.8-fold higher than DLLs.More importantly,it is demonstrated that the nanoscale characteristics of Dox-loaded CLs could prevent the proliferation of adriamycin-resistant MCF-7/ADR cell line,due to their high cellular uptake efficiency and reduction-triggered release under intracellular GSH environment.Furthermore,in vivo tests showed that disulfide cross-linked liposomal formulation of Dox?Dox-CLs?significantly improved the therapeutic efficacy compared to free Dox and DLLs in a human breast carcinoma xenograft mouse model.Therefore,the current thiol-responsive cross-linked liposome may provide a robust drug delivery platform for cancer therapy.In this thesis,we developed the self-assembled nano-delivery system based on drug-phospholipid conjugate and the redox-responsive cross-linked nanocarrier for PTX or Dox delivery,exhibiting high drug loading and high stability.Aditionally,the amphiphilic phospholipid conjugates designed herein have good expandability,and provide a novel strategy for the construction of a variety of drug-phospholipid prodrug and different stimuli-responsive nanomedicines.