Nano-prodrug Systems for Combination Delivery of Anti-cancer Drugs

Author:Xu Yan Zuo

Supervisor:yu jia hui


Degree Year:2019





Due to the molecular complexity of malignant tumors,the multidrug resistance of cancer cells and associated toxicity of clinical uses of single drug,combination chemotherapy has been widely used in the treatment of various malignant tumors,especially there is a significant progress in our understanding of various molecular basis and signal transduction pathways in cancer.The rapid development of nanotechnology promotes the application of nano-preparations in the field of combined chemotherapy.The application of nano-preparations in combination chemotherapy requires sophisticated optimization strategies to identify appropriate drug combinations,the optimal dosages and ratio of different drugs,to achieve accurate controlled drug release,synergistic anticancer effect,and other critical features like safety and stability.Nano-prodrug synchronized the advantages of prodrugs and nanotechnology,and thus able to achieve controlled drug release and enhanced antitumor efficiency.The integration of nano-preparations and combination chemotherapy could effectively load multiple chemotherapeutics,optimize physicochemical property of drugs,improve drug stability and loading capacity as well as deliver hydrophobic drugs and hydrophilic drugs simultaneously.It is convenient to adjust and control molar ratio of different drugs used for combination chemotherapy through co-assemble of different prodrugs or taking nano-prodrug as carrier.Nano-prodrugs with active targeting capacity and responsiveness to various tumor microenvironmental stimuli can achieve more effective treatment of malignant tumors.The following are the main research contents and conclusions of this thesis:Chapter 1:Making an overview of targeting delivery and tumor microenvironment stimuli-responsive controlled release of anti-cancer drugs,traditional combination chemotherapy of malignant tumors,application of nano drug delivery systems and nano-prodrugs for combination delivery of anti-cancer drugs.And finally described the research ideas and contents of this thesis.Chapter 2:Taking CPT and GEM as starting materials,a redox-sensitive amphiphilic small molecule prodrug,CPT-SS-GEM,with disulfide bond as linker was synthesized.Without any other excipients,the CPT-SS-GEM could self-assemble into spherical nano-prodrug micelles with an average particle size of 293±9 nm in water,which could realize combination delivery of CPT and GEM without the help of any other nano-carrier materials.The CPT-SS-GEM nano-prodrug possesses extremely high drug loading capacity with 42.6%of CPT and 32.2%of GEM,respectively.In the intracellular redox microenvironment of tumor cells(pH 6.5,5.0+2 mM DTT),the CPT-SS-GEM nano-prodrug could realize rapidly simultaneous and ratio-metric controlled release of the two pharmacologically active parent drugs,CPT and GEM.The amount of cumulative drug release reached more than 90%within 3 hours.This simultaneously controlled release of CPT and GEM contributes to the significant synergistic anti-proliferation effect of CPT-SS-GEM nano-prodrug against multiple tumor cells.And the CPT-SS-GEM nano-prodrug is helpful to overcome drug resistance of MCF-7/ADR cells at some extent.Chapter 3:Although the CPT-SS-GEM self-assembled nano-prodrug could achieve the combination delivery and simultaneous controlled release of CPT and GEM,the CPT-SS-GEM nano-prodrug has poor colloidal stability and agglomerates or precipitates easily.And,the absolute drug content of CPT-SS-GEM nano-prodrug is very low(<0.7 mg/mL),which made it unsuitable for the subsequent in vivo experiments in animal cancer model.In order to overcome the above deficiencies associated with CPT-SS-GEM nano-prodrug and increase the CPT/GEM molar ratio in nano-prodrug to enhance the synergistic anti-proliferative effect of CPT and GEM,in this chapter,a PEGylated prodrug of CPT with disulfide-bond as linker,CPT-SS-mPEG2000,was synthesized.Based on theπ-πstacking of CPT,the surface PEGylated redox-sensitive CPT-SS-mPEG2000/CPT-SS-GEM co-assembled nano-prodrug was constructed for combination delivery of CPT and GEM.The morphology of this co-assembled nano-prodrug was rod-shaped nano-micelles with average length of 233±29 nm and average aspect ratio of 3.46±1.34.The colloidal stability of CPT-SS-mPEG2000/CPT-SS-GEM co-assembled nano-prodrug was significantly enhanced.Meanwhile,the absolute drug content was greatly increased,the concentrations of CPT-SS-GEM and CPT-SS-mPEG2000 in the aqueous co-assembled formulation were 10mg/mL and 100 mg/mL,respectively.The molar ratio of CPT/GEM was increased to4.2:1.This CPT-SS-mPEG2000/CPT-SS-GEM co-assembled nano-prodrug could rapidly release CPT and GEM in the redox microenvironment of tumor cells.The synergistic anti-proliferative effect of CPT-SS-mPEG2000/CPT-SS-GEM co-assembled nano-prodrug against MCF-7 and MCF-7/ADR cells was further enhanced and superior than CPT-SS-GEM nano-prodrug.Chapter 4:Although the CPT-SS-mPEG2000/CPT-SS-GEM nano-prodrug possesses some merits,for example,its simple and convenient preparation,significantly improved absolute drug content,greatly enhanced colloidal stability,its surface PEGylation is not beneficial for the cellular uptake by tumor cells.In addition,CPT-SS-mPEG2000/CPT-SS-GEM co-assembled nano-prodrug lacks active targeting ability against tumor cells.Therefore,in this chapter,a PEGylated prodrug of CPT with disulfide-bond as linker and 4-carboxyphenylboronic acid as active targeting ligand,CPT-SS-PEG2000-CPBA,was synthesized.The active targeting co-assembled nano-prodrug CPT-SS-PEG2000-CPBA/CPT-SS-GEM was constructed through the co-assembly of CPT-SS-PEG2000-CPBA and CPT-SS-GEM for combination delivery of CPT and GEM.The CPT-SS-PEG2000-CPBA/CPT-SS-GEM co-assembled nano-prodrug showed morphology of rod-shaped nano-micelles with average length of 400-500 nm and average aspect ratio of 2.25±0.42.This co-assembled nano-prodrug still maintained good colloidal stability and high absolute drug content,the concentrations of CPT-SS-GEM and CPT-SS-PEG2000-CPBA were 8 mg/mL and 80 mg/mL,respectively.The molar ratio of CPT/GEM was 4:1.The CPT-SS-PEG2000-CPBA/CPT-SS-GEM co-assembled nano-prodrug could rapidly release CPT and GEM in the redox environment of tumor cells.The CPT and GEM were completely released within 10 h.Due to the specific binding of 4-carboxyphenylboronic acid to sialic acid overexpressed on the surface of tumor cells,the cellular uptake of nano-prodrug was significantly enhanced.Especially against the multidrug resistant cancer cell MCF-7/ADR,the cellular uptake amount of nano-prodrug was 3.26 folds of CPT,4.32 folds of CPT+GEM mixture.The receptor saturation experiment further certified the effectiveness of4-carboxyphenylboronic acid as active targeting ligand.Additionally,this co-assembled nano-prodrug could effectively alleviate the drug reflux against tumor cells,especially multidrug resistant MCF-7/ADR cells.In 4 h,the drug reflux amount of co-assembled nano-prodrug against MCF-7/ADR was only 1/13 of CPT and 1/9 of CPT+GEM mixture.The calculation of combination index verified that the synergistic anti-proliferative effect of this co-assembled nano-prodrug against MCF-7/ADR cells was further enhanced and superior than CPT-SS-mPEG2000/CPT-SS-GEM nano-prodrug.The results of in vivo bio-distribution in 4T1 tumor-bearing mice showed that CPT-SS-PEG2000-CPBA/CPT-SS-GEM co-assembled nano-prodrug could selectively accumulate at tumor site.After intravenous administration for 24 h,the drug concentration at tumor site was about 2.5 folds of free CPT and CPT+GEM mixture.Chapter 5:The metastasis of tumor cells from primary tumor site to distant organs is the main cause that lead to malignant tumors becoming fatal disease.Therefore,anti-metastasis of tumor cells is crucial for the effective treatment of malignant tumors.Hydroxychloroquine has been reported as anticancer drug which could inhibit metastasis of tumor cells through various pathways.However,it usually needs high dose of HCQ to exert its anti-metastatic efficacy,which results in side effects against normal tissues or cells.In this chapter,a reduction-sensitive polymer prodrug with hyaluronic acid as both carrier and active targeting ligand,HA-ss-HCQ,was synthesized.HA-ss-HCQ self-assembled into nano-prodrug capsules with average particle size of 203±30 nm in aqueous solution with physiological pH 7.4.The HA-ss-HCQ nano-prodrug capsules rapidly released hydroxychloroquine in the redox environment of tumor cells,and the cumulative drug release exceeded 90%within 24h.The expression of CD44 receptor on the surface of highly metastatic breast cancer cells 4T1 is greatly increased and the specific binding of HA to CD44 significantly enhanced the cellular uptake of nile red loaded HA-ss-HCQ nano-prodrugs by 4T1 cells.The receptor saturation experiment further demonstrated the effectiveness of hyaluronic acid as active targeting ligand.In vitro cell migration and invasion assays showed that the HA-ss-HCQ nano-prodrug significantly inhibited the migration and invasion of 4T1cells,and the inhibition rates of cell migration and invasion of 4T1 were 82.32±2.92%and 79.85±5.89%,respectively.In the experimental lung metastasis model of 4T1breast cancer,HA-ss-HCQ nano-prodrug greatly reduced the number of tumor nodules at pulmonary of mice,implied it could effectively inhibit the metastasis of 4T1 cells to lungs.Meanwhile,HA-ss-HCQ nano-prodrug alleviated the sharp body weight loss of mice caused by metastasis of tumor cells.aIn addition,HA-ss-HCQ nano-prodrug can be used as nano-carrier for the combination delivery of HCQ and other drugs to achieve the synergistic effect of simultaneous treatment of primary tumors and anti-metastasis of tumor cells.In summary,based on the self-assembly and co-assembly of amphiphilic prodrug molecules,a series of nano-prodrug systems which could response to the stimuli of redox microenvironment of tumor cells were constructed for the combination delivery of anti-cancer drugs in this dissertation.It brings us new ideas for resolving the issues associated with traditional combination chemotherapy,enhancing the synergistic anti-proliferative and anti-metastasis efficacy of anti-cancer drugs,decreasing or avoiding the toxic side effects caused by overuse of nano-carrier materials.