Drug And Gene Co-delivery System For Tumor Microenvironment Remodeling And Therapy

Malignant tumors are widely studied as a major disease that seriously threatens human health.Traditional treatment methods include surgery,chemotherapy,radiation therapy,optical therapy,immunotherapy and gene therapy.Among them,because gene therapy can correct or compensate diseases caused by defects and abnormal genes,it is considered to be the most likely to cure tumors fundamentally.However,successful gene therapy depends on the safe and efficient delivery vectors.Cationic lipid carriers are popular owing to their simple preparation,low immunogenicity,ability to carry large nucleic acids/small molecule drugs,and high biological safety.However,these carrier systems still have disadvantages such as poor stability,poor tumor cell/tissue targeting,poor biodegradability,and low treatment efficiency in in vivo applications.Compared with normal organs and tissues,tumors have complex structures and unique components,forming a special tumor microenvironment(TME).The tumor microenvironment plays a vital role in many progresses of tumors(such as tumor occurrence,development and metastasis),and at the same time affects the delivery efficiency of delivery vehicles and the development of resistance to tumor treatments,resulting in ineffective tumor treatments.In view of the fact that the tumor microenvironment plays a leading role in the fate of the nano-delivery system,various characteristics of the tumor microenvironment are adjusted or utilized,including tumor hypoxia,low p H,high concentration of glutathione,cell matrix and tumor immune microenvironment,etc.It is a hot and difficult point of current research to improve the therapeutic effect of existing tumor therapies.This thesis combined and utilized the advantages of dendritic peptide molecules and environmental responsive delivery systems.Aiming at improving and using the tumor microenvironment to increase the therapeutic effect of conventional tumor treatment methods(chemotherapy and photodynamic therapy),the following three aspects are carried out:(1).Disulfide modified self-assembly lipopeptides with arginine-rich periphery as gene vectorwe demonstrate a prototype of viral envelop & capsid-inspired nanovectors with arginine cluster and disulfide bonds.In brief,amino groups in lysine are conjugated with arginine to form arginine-containing fragments,and both amino groups in lysine were modified with oleic acid in formation of double-tail lipid.These two parts were then conjugated together through disulfide bonds containing cystamine as the linkage to form the final disulfide modified self-assembly lipopeptides with arginine-rich periphery(RLS).For more systematical investigating,a corresponding analog of RLS that without disulfide bond(RL)were synthesized for comparison.Each step of the cationic lipid syntheses was characterized by 1H-NMR spectroscopy and mass spectrum.RLS and RL lipopeptide assemblies had a narrow size distribution with average diameter around 100 nm.However,zeta potentials of RL and RLS were + 46.5 m V and + 27.2 m V.The only difference between RLS and RL in chemical structure lie in the sulfur,which owns lone-pair electrons and might contribute to surface potential decrease.Both of RLS and RL lipopeptide assemblies could form spherical shape.We could confirm the well-defined bilayer structure for RLS lipopeptide from a larger magnification like liposomes,while RL assemblies did not showed apparently outer periphery.From the electrophoresis measurements,both RLS and RL lipopeptide assemblies could condense DNA and completely retard DNA migration as N/P ratio increased to 5,suggesting the difference in zeta potential did not affect DNA binding activity or condensation significantly.It successfully displayed the rapid disassociation of RLS lipopeptide assemblies and further induced liberation of DNA in the stimulated reducing environment of tumor cells.It was surprising that RLS lipoplexes showed higher transfection efficiency at relatively low N/P of 5 and 10 in the presence of serum,compared with the analogs RL lipoplexes that reached the highest transfection efficiency at N/P of 40.The transfection efficiency of RLS cationic assemblies is associated with the intracellular GSH concentration,which induce the cleavage of disulfide linkages,disassembly of the complexes and release of DNA.In the transfection conditions,RLS/DNA complexes(N/P 10)showed even less cellular uptake than RL/DNA complexes(N/P 40).And no difference was detected in buffering effect and endosomal escape between RLS and RL complexes.Examination by gel electrophoresis and fluorescence resonance energy transfer(FRET)image in cell culture conditions confirmed RLS lipoplexes could effectively liberate DNA.Thus,the higher gene transfection efficiency of disulfide modified vectors might be mainly attributed to the cleavage of disulfide bonds and the rapid release of DNA,and the superiority of these design was closely related with the reductive concentration in cells.Encouraged by the excellent in vitro transfection,gene transfer of p CMV-Luc encoding luciferase by RLS and RL lipoplexes was carried out in BALB/c nude mice bearing He La tumor xenografts.As expected,RLS lipoplexes induced the evidently high level of luciferase activity in the tumor with 6.2-fold higher protein expression than that of PEI/DNA complexes.(2).Candesartan conjugate-based si RNA delivery for reconstruct tumor normoxic microenvironmentThe tumor hypoxic microenvironment not only induces genetic and epigenetic changes in tumor cells,immature vessels formation for oxygen demand,but also compromises the efficiency of therapeutic interventions.On the other hand,conventional therapeutic approaches which kill tumor cells or destroy tumor blood vessels to block nutrition and oxygen supply usually facilitates even harsher microenvironment.Thus,simultaneously relieving the strained response of tumor cells and blood vessels represents a promising strategy to reverse the adverse tumor hypoxic microenvironment.In the present study,we designed an integrated amphiphilic system(RSCD)based on Ang-II receptor blocker candesartan(CD)for si RNA delivery against the hypoxia-inducible factor-1 alpha(HIF-1α),aiming at both vascular and cellular “relaxation” to reconstruct a tumor normoxic microenvironment.The RSCD assemblies were dropwise added to si RNA solution followed with gently mixing for gene complexes(RSCD/si RNA)formation.Negatively charged HA-SS-COOH was gradually added into the pre-prepared RSCD/si RNA(N/P 20)complexes for ternary HA-RSCD/si RNA complexes fabrication.The particle sizes of RSCD and HA-RSCD/si RNA were dramatically increased in reductive solution for tumor microenvironment mimicking and such disintegration efficiently led to the si RNA liberation.The interaction between gene complexes and cells were assessed by confocal laser scanning microscopy(CLSM).Hep G2 cells treated with HA-RSCD complexes displayed much higher cellular uptake than those with RSCD complexes incubation,especially at the very beginning(0.5 h),which was probably attributed to HA receptormediated recognition.Data of q-PCR and western blot indicated that the HIF-1α m RNA and protein levels were significantly lower in Hep G2 cells transfected with the HA-RSCD/si HIF-1α and RSCD/si HIF-1α complexes than the positive controls(Lipo/si HIF-1α).Further it has confirmed the co-effect of candesartan and si HIF-1α on the down regulation of VEGF,either in the integrated RSCD/si HIF-1α group or the mixed RLS/si HIF-1α+CD group.The HA-RSCD/si HIF-1α formulation parallelly relieved the stress response of tumor cells and HUVECs to microenvironment,with direct inhibition of cellular proliferation,cascading reduction of abnormal vessel formation,and blocking endothelial cell mitigation.The “relaxation” therapy enabled neovascularization with more complete and organized structures to obviously increase the oxygen level inside tumor,which resulted in a 50% growth inhibition.More convincingly,reconstruction of tumor micro-environment not only significantly improved the chemotherapy but also the oxygen-dependent photodynamic anticancer treatment.Pre-treatment of HARSCD/si HIF-1α could enhance the accumulation of chemotherapeutic agents and photosensitizers in tumor sites and promoted the apoptosis of tumor cells.(3).All-trans retinoic acid conjugate-based si RNA delivery for Anti-stromal treatment together with gene therapyPancreatic ductal adenocarcinoma(PDAC)is characterized by the dense tumor associated stroma and abundant activated pancreatic stellate cells(PSCs)which are the main subset of pancreatic cancer-associated fibroblasts.Activated pancreatic stellate cells play a key role in forming obstacles that restrict the intratumor delivery and attenuate the therapeutic effect.An integrated delivery system(RVA)based on all-trans retinoic acid conjugate for KRAS si RNA delivery has been designed,aiming at simultaneous inhibition of tumor proliferation and reversal of the activated cancerassociated fibroblasts for tumor stromal microenvironment reconstruction.all-trans retinoic acid(ATRA)was modified as the hydrophobic part,while the cationic dendritic arginine was selected as hydrophilic segment.These two parts were linked with disulfide bond to make the product microenvironment responsive to implement co-delivery of ATRA and si KRAS.Poly-glutamic acid(g-PGA)was modified with peptide FH(FH-PGA),which specifically binds to tumor-associated gamma-glutamyl transpeptidase(GGT)and tenascin C overexpressed on tumor cells and CAFs.FHPGA was used to modify the polyplex core via charge interaction(FH-PGARVA/si RNA)to achieve tumor and CAF targeting delivery.This nanosystem simultaneously induces PSC quiescence and inhibits tumor cells proliferation and ECM hyperplasia.RVA assemblies were prepared by injection method.The Z-Average and zeta potential of RVA assemblies were 220.6 nm and + 41.5 m V,respectively.When incubated with 10 m M glutathione,RVA could rapidly release the condensed si RNA due to the cleavage of disulfide bonds.RVA/si RNA(N/P =30)gene complexes were further shielded by FH-PGA corona to construct FH-PGA-RVA/si RNA ternary complexes for co-delivery of ATRA and si RNA targeted KARA.As the w/w ratio of PGA/si RNA increased to 2,the particle size of the formed ternary gene complexes was 201 nm and zeta potential decreased to + 23 m V.The cellular uptake efficiency measured by flow cytometry and CLSM showed that the FH-PGA corona could promote the cellular internalization via the GGT and tenascin C pathway.The intracellular fate of PGA-RVA/si RNA ternary complexes was investigated by FRET technique.The results indicated that the acidic environment in lysosome induce the dissociation of PGA from RVA/si RNA and the exposure of dendritic arginine based RVA could favorable for lysosome escape.Data of q-PCR and western blot suggested FH-PGA-RVA/si KRAS and PGARVA/si KRAS complexes could inhibited KRAS m RNA and protein more effectively than that with treatment of non-targeting RVA/si KRAS delivery system.Subsequently,cell-counting kit-8 was employed to verify the anti-proliferation effect of FH-PGARVA/si KRAS and PGA-RVA/si KRAS complexes.Results from cell viability revealed that si KRAS complexes could damage PSCs and PDAC cells.Followingly,the ability of FH-PGA-RVA/si KRAS to revert activated PSCs back to the quiescent state was assessed.It was found that ARTA conjugate in gene complexes could induce abundant lipid in PSCs and reduce the expression level of α-SMA,collagen and fibronectin,suggesting the quiescent state in PSCs and inhibition of ECM production by PSCs.