Construction And Application Of Polycations-based Gene/Protein Delivery Vectors For The Treatment Of Tumor And Osteoarthritis

Therapeutic biomacromolecules,such as proteins,nucleic acids,etc.,have shown great potential in disease treatment because of their wide therapeutic range,low side effects,and high specificity.However,exogenous biomacromolecules often suffer from fast clearance from the blood,easy degradation in the body,and difficulty in passing through the biological barriers,greatly limiting their therapeutic efficacy.Therefore,the development of safe and efficient delivery systems is essential for the potentiation of therapeutic biomacromolecules.The ideal delivery systems should possess good biocompatibility,and can efficiently and selectively target the drugs to lesion sites,cells,or subcellular organelles,improving drug efficacy while reducing toxicity and side effects.Cationic polymers are a class of important and widely research materials for the delivery of biomacromolecules with the advantages of simple preparation,easy modification and variable structures.However,multiple biological barriers and different pharmacokinetic processes in the body put forward contradictory requirements on the performance of cationic polymer-based nanocarriers,so that cationic polymer-based nanocarriers still generally suffer from defects such as high toxicity,unsatisfactory delivery efficiency,and low stability.This thesis addresses the physiological barriers encountered by cationic polymerbased nanocarriers in the delivery of therapeutic biomacromolecules and regulates the interaction between cationic polymer-based nanocarriers,cargoes(proteins and nucleic acids),and biological microenvironment(tissues,cells,body fluids,etc.)through structural design and modification,self-assembly and stimuli-triggered drug release.A series of efficient and safe cationic nanocarriers are designed to achieve effective delivery of nucleic acids and proteins both in vitro and in vivo(systemic administration or local drug delivery in the joint cavity),and excellent efficacy is achieved in animal models of tumor and osteoarthritis(OA).Firstly,to improve the intracellular gene delivery efficiency of cationic polymers,fluoride-modified,ROS-degradable cationic polymers were designed to optimize various aspects of intracellular delivery.Therefore,the contradictions between nucleic acid condensation and intracellular nucleic acid release,gene transfection efficiency,and material toxicity were resolved.Then,protein and nucleic acid delivery systems with high blood circulation stability,tumor targeting,and selective drug release were designed to overcome the physiological barriers facing in the systemic(intravenous)administration for cancer treatment in vivo.On the one hand,hypoxia-degradable cationic polymers,combined with pH-responsive block polymers and photosensitizers,were designed to construct micellar nanocomplexes with high in vivo stability for light-controlled,hypoxia-responsive tumor gene therapy.On the other hand,a hypoxia-activated protein prodrug and hypoxia-degradable polymers were designed to construct the hypoxiaactivated,programmed self-accelerating nanocomplexes was by co-packaging glucose oxidase and protein prodrugs.The resulting nanocomplexes achieved programmable hypoxia-triggered cargo release and protein prodrug activation,and tumor-targeting,selective intracellular delivery of proteins.Finally,considering the advantages and clinical relevance of local drug delivery in the treatment of specific diseases,fluorocationic nanocapsules and nanocomplexes were explored and developed for joint cavity delivery of therapeutic proteins and nucleic acids,respectively,to promote cartilage penetration and synergistic OA treatment.The research content of this thesis is listed as follows:In Chapter 1,the cationic polymer-based biomacromolecules delivery systems were described,and the current status and challenges of therapeutic proteins/nucleic acids in tumor therapy and OA therapy were introduced.Besides,the types of cationic materials used in protein/nucleic acid delivery,as well as the challenges,advantages,and disadvantages of cationic materials,were reviewed.Moreover,the research progress of cationic nucleic acid/protein delivery systems in the treatment of cancer and OA was also introduced.In Chapter 2,a reactive oxygen species(ROS)-responsive diselenide-cross-linked fluorinated cationic polymer(DSe-PEI-F)was designed and synthesized to improve the properties of anti-serum,cell uptake,endosomal escape of cationic polyplexes,and ultimately enhanced the gene delivery efficiency.ROS-responsive cationic polymers containing diselenide bonds were prepared by chemical cross-linking of low molecular weight polyethyleneimine(PEI)with associated diselenide cross-linkers,followed by the modification of fluoroalkane chains at varying contents.Chemical cross-linking greatly improved the DNA condensation capacity of the polymer,and the fluorinated modified DSe-PEI-F completed the efficient condensation of DNA at a low ratio(N/P=1),while greatly improving the cellar uptake capacity(4-5 times).After internalization,the high levels of intracellular ROS promoted the degradation of DSe-PEI-F into low molecular weight fragments,accelerated the release of DNA and improved the transfection efficiency,and reduced the long-term toxicity of the polymer.The transfection efficiency of DSe-PEI-F was improved with the increase of fluorination degree,and at the optimal N/P ratio,the transfection efficiency of DSe-PEI-F was improved by-4 orders of magnitude compared with PEI 25k,a commercial transfection reagent.In addition,the resulting nanocomplexes effectively resisted the adsorption of serum proteins,due to the hydrophobic and oleophobic properties of the fluoroalkane chains.As a result,DSe-PEIF still maintained a good gene transfection efficiency in the presence of serum,effectively solving the poor serum stability and the defect of cationic polymers that cannot be effectively transfected in serum.In Chapter 3,a hypoxia-responsive cationic polymer-based siRNA delivery system was developed for tumor-targeted siRNA delivery in vivo.Low molecular weight PEI was chemically cross-linked with azobenzene-containing domains,yielding the azobenzene-containing,hypoxia-responsive cationic polymers(AOEI).Then an amphiphilic block copolymer(PPDHP)was also synthesized,which coupled with a photosensitizer(pheophorbide a,Pha),AOEI,and siRNA against XIAP(siXIAP)to form the micellar nanocomposites(PAX MPs)with a colloidal size of about 110 nm.PAX MPs showed excellent salt and serum stability,due to the hydrophobic effect of micelles and the polyethylene glycol(PEG)decoration on the surface of the micelles that resists the adsorption of serum proteins,facilitating their long circulation in the blood after intravenous injection and passive accumulation at the tumor sites.After internalization,the hydrophobic block of the PPDHP copolymer was protonated and turned into hydrophilic,which can promote the shedding of PEG,expose the positively charged groups of AOEI,and promote the escape of endosomes through the "proton sponge"effect.Upon light irradiation(660 nm,2 mW/cm2,30 min),the photosensitizer Pha produced a lethal amount of ROS and consumed oxygen,which aggravated the degree of intracellular hypoxia,which in turn caused the rapid rupture of azobenzene in AOEI,and the dissociation of nanocomposites and release of siXIAP,ultimately improving gene silencing efficiency and tumor cell apoptosis rate and reducing the long-term toxicity of cationic polymer materials.After intravenous injection of PAX MPs in Skov-3 tumorbearing mice and exposure to light,the XIAP gene silencing efficiency increased from～50%to～70%,and photosensitizer-mediated photodynamic therapy and siXIAPmediated gene therapy played a synergistic effect and significantly inhibited the growth of Skov-3 tumor,yet with negligible side effects.In Chapter 4,a hypoxia-degradable polymer-based cascade protein delivery system was designed for the precise regulation of protein activity and tumor-targeted intracellular delivery.The hypoxia-activated protein prodrug(RPAB)was produced by reversible chemical modification of protein ribonuclease(ribonuclease A,RNase A)with an azobenzene-containing moiety.The resulting RPAB was subsequently assembled with the above-mentioned hypoxia-responsive cationic polymer(AOEI),hyaluronic acid(HA),and glucose oxidase(GOx)through electrostatic interaction to construct the nanocomposites(HARPG NCs)with a particle size of about 180 nm and a negatively charged surface.After intravenous injection,HARPG NCs passively accumulated at the tumor sites,where HA-mediated the active targeting and cellular uptake by recognizing the over-expressed CD44 on the surface of tumor cells.The excessive reductases in tumor cells promoted the degradation of AOEI and released GOx and RPAB under the hypoxic condition of a tumor.GOx catalyzed the decomposition of glucose and consumed the intracellular oxygen,which further aggravated the hypoxia level in the tumor cells and caused the rapid recovery of RPAB activity.At the same time,hypoxia also accelerated the degradation of AOEI and released more proteins,thereby accelerating the selfcirculation process,and finally GOx-mediated cell starvation and RNase A-mediated cell apoptosis played a synergistic anti-cancer effect.As a result,HARPG NCs significantly increased the apoptosis level of HeLa cells(51%),which was better than HAG NCs(GOx alone,32%)and HARP NCs(RPAB alone,8%),proving the synergistic effect of RPAB and GOx.In the HeLa tumor-bearing mouse model,HARPG NCs significantly reduced the oxygen level at the tumor site to～10%,and the tumor suppression effect was significantly better than that of HAG NCs and HARP NCs.The survival rate of mice within the 45-d observation period after the treatment of HARPG NCs was 80%.In Chapter 5,an enzyme-responsive fluorine-containing cationic nanocapsule was designed to encapsulate a therapeutic protein IGF-1,improve cartilage penetration efficiency,and locally administered through the joint cavity to enhance OA treatment efficacy.The monomers of acrylamide and acrylate were designed,and nanocapsules with with different numbers of fluorine chains were formed by in-situ polymerization and cross-linking on the surface of IGF-1.The particle size of the resulting nanocapsule was 13-16 nm with the surface potential of 20-31 mV.Matrix metalloproteinase(MMP)responsive moieties slowly released IGF-1 in the articular cartilage,prolonging the action time of the drug.Cationic monomers provided electrostatic interaction with the articular cartilage and promoted its retention in the joints.The hydrophobic and oleophobic properties of the fluorine chain in the fluorine-containing monomer can significantly enhance the penetration efficiency of nanocapsules in synovial fluid and cartilage.The nanocapsules with the strongest synovial fluid and cartilage permeability(NPs-F3)were screened out through multiparticle tracking study,photobleaching study,and other experiments,and its cartilage adsorption capacity was increased by-50%compared with that of nanocapsules without fluorine.In in vitro experiments,IGF-1 was continuously released within 15 days when MMP2 was present,and the cumulative release reached 80%.In the rat OA model,after a single administration of the joint cavity,the nanocapsules stayed in the rat joints for up to 35 days,the production of cartilage matrix was increased,matrix degradation was inhibited,and the condition of articular cartilage was significantly improved.In Chapter 6,a fluorine-containing poly(β-amino ester)(F-PBAE)was designed for the condensation and local delivery of anti-miR-204(anti-204)to the joint cavity to realize the local gene therapy for OA.The hyperbranched structure of F-PBAE donated itself with a higher charge density and a stronger anti-204 encapsulation capacity than linear polymers.Due to the presence of the fluorinated alkyl chain,the F-PBAE/anti-204 complexes exhibited stronger stability in serum,ions,and synovial fluid,and therefore possessed a stronger cartilage penetration ability,which was 5-fold stronger than that of their non-fluorinated counterparts.In the chondrocyte endocytosis experiment,the FPB AE/anti-204 complexes were quickly distributed into the cytoplasm within 1 hour,and the miR-204 inhibition rate reached 25%.Inhibition of anti-204 expression led to an increase in the synthesis of sulfated proteoglycan(PG)in chondrocytes,and a significant reduction in inflammatory senescence-associated secretory phenotype(SASP),thereby preventing cartilage suffers further damage.In the rat OA model,after continuous injection of F-PBAE/miR-204 complexes,the miR-204 inhibition rate reached 18%,and The equilibrium between degradation and synthesis of the articular cartilage matrix was sustained,which provided a better joint microenvironment for chondrocytes,and remarkably changed the damage of articular cartilage.In Chapter 7,we summarized the doctoral thesis and provided perspectives for future work.In this doctoral thesis,a series of structural design and optimization strategies for cationic polymers are proposed to overcome multiple physiological barriers in vivo and in vitro and to improve the delivery efficiency of therapeutic proteins and nucleic acids.This thesis expands the application of cationic carriers in systemic drug delivery and local drug delivery in the joint cavity,explores the application of protein and nucleic acid drugs in diseases such as cancer and osteoarthritis,and obtains a series of innovative findings for the design and application of cationic materials.