Preparation Of Stimuli-responsive Nanocarriers For Anticancer Drug Delivery

To improve the anticancer effects and minimize the side effects of cancer chemotherapy,tumor-specific delivery of toxic anticancer drugs is one of the most effective strategies.Recently,drug delivery systems at the nanoscale,also known as nanocarriers,have shown great potentials in the delivery of drugs to tumor,through either the size-dependent“passive”tumor targeting due to the enhanced permeability and retention（EPR）effect or their surface-modified tumor-specific moieties,e.g.small molecules,aptamers,peptides,and antibodies,-mediated“active”tumor-targeting.Active tumor targeting requires these targeting“ligands”to get close enough to and interact with their corresponding“receptors”on the surface of the tumor cells.An ideal nanocarrier should possess the key characteristics,such as long circulation,high tumor accumulation and cellular uptake.PEGylation is one of the mostly used technologies to engineer the surface of the nanoparticles,to shield the undesired properties.The PEGylated nanoparticles usually h ave long blood circulation time due to the PEG’s hindrance to the plasma proteins and the reticuloendothelial system（RES）,which facilitates the EPR effect-mediated tumor targeting.However,PEGylation impedes the internalization of nanoparticles by cancer cells,and therefore is not beneficial for the cellular uptake.In contrast,the positively charged nanoparticles effectively interact with the negatively charged cell membrane by the electrostatic attraction and trigger the efficient cell internalizatio n.Nevertheless,the positive charge may cause nonspecific interactions of the nanoparticles with serum components and cell membrane of the normal cells,resulting in short circulation time and nonspecific distribution.Similarly,cell-penetrating peptides（CPPs）,such as transactivator of transcription（TAT）peptide,were able to translocate across the cell membrane via various mechanisms.The CPP-conjugated nanoparticles have been extensively investigated as the intracellular drug delivery tools.However,the lack of selectivity of CPPs may cause undesired side effects,which is one of the major obstacles and remains unresolved.With the increasing knowledge of tumor biology,we know that tumor tissues possess a lowered pH in solid tumors with even lower pH value inside tumor cells,overexpressed specific enzymes,high levels of glutathione（GSH）in the cytoplasm,high levels of reactive oxygen species（ROS）derived from by-products of aerobic metabolism,hypoxic tumor microenvironment as well as higher concentration of ATP inside tumor cells.Therefore,these unique features can be utilized as triggers to design stimuli-responsive nanocarriers to overcome the aforementioned crucial challenges of conventional nanocarriers and realize predesigned functions,such as shielding/deshielding of PEG,surface charge-conversion,protection/exposure of the CPPs,and controlled drug release in an on-demand manner,thus enhancing the therapeutic efficacy and reducing the side effects.Accordingly,we developed several stimuli-responsive nano-drug delivery system to realize prolonged circulation,enhanced penetration,increased cellular uptake and controlled drug release,thus increasing anticancer drug（DOX）toxicity against both drug-sensitive and multidrug-resistant（MDR）cancer cells,which can be further categorized into three parts as described below.1.Preparation of PMLA-based charge-conversional nanoconjugates for tumor-specific uptake and cellular deliveryPoly（β-L-malic acid）（PMLA）is a natural aliphatic polyester obtained from the microorganism myxomycete Physarum polycephalum,which degrades into malic acid firstly and then degrades into carbon dioxide and water by tricarboxylic acid cycle in vivo.28 As a novel drug delivery carrier,PMLA was proved to be biodegradable,non-toxic and non-immunogenic.Furthermore,it could provide numerous pendant carboxyl groups,thus,chemotherapeutics,targeting ligands and other functional groups could be easier to decorate on the same polymer backbone.Therefore,in this study,a multi-functional PMLA-based nanoconjugate with a pH-dependent charge conversional characteristic was developed for tumor-specific drug delivery.The short branched polyethyleneimine（PEI）-modified PMLA（PEPM）was first synthesized.Then,the fragment HAb18 F（ab′）₂ and 2,3-dimethylmaleic anhydride（DMMA）were covalently attached to the PEPM to form the nanoconjugate,HDPEPM.In this nanoconjugate,the DMA,the shielding group,could shield the positive charge of the conjugate at pH 7.4,while it was selectively hydrolyzed in the tumor extracellular space（pH 6.8）to expose the previously-shielded positive charge.To study the anticancer activity,the anticancer drug,DOX,was covalently attached to the nanoconjugate.The DOX-loaded HDPEPM nanoconjugate was able to efficiently undergo a quick charge conversion from-11.62 mV to+9.04 mV in response to the tumor extracellular pH.The electrostatic interaction between the positively charged HDPEPM nanoconjugates and the negatively charged cell membrane significantly enhanced their cellular uptake,resulting in the enhanced anticancer activity.Besides,the tumor targetability of the nanoconjugates could be further improved via the fragment HAb18 F（ab′）₂ ligand-receptor mediated tumor cell-specific endocytosis.2.Dual-pH sensitive charge-reversal nanocomplex for tumor-targeted drug delivery with enhanced anticancer activityPMLA has been proven to be a promising carrier for anti-cancer drugs.In spite of excellent bio-compatibility,the application of PMLA as the drug carrier for cancer therapy is limited by its low cellular uptake efficiency.The strong negative charge of PMLA impedes its uptake by cancer cells because of the electrostatic repulsion.In the prious work,DOX was directly conjugated to PMLA via the amide bond and the formed nanoconjugates showed a pH-sensitive drug release pattern.Nevertheless,it’s uncertain whether the released drug formulation was free DOX or not,the pH-sensitive pattern probably caused by the hydrolysis of PMLA at acid environment.Thus,in this study,TAT was introduced to enhance the cellular uptake and DOX was conjugated to PMLA via a pH-sensitive maleic acid amide bond and ensured effective release of free DOX in response to the acidic endosomal/lysosomal pH.We found that the electrostatic interaction might have greater impact on the enhancement of cell uptake than ligand-receptor mediated endocytosis,active-targeting was given up.Besides,pH-sensitive charge-conversional PEG-DMMA was used to wrap the nanoparticles to make it more stable.Therefore,a dual pH-sensitive charge-reversal PMLA-based nanocomplex（PMLA-PEI-DOX-TAT@PEG-DMMA）was developed for effective tumor-targeted drug delivery,enhanced cellular uptake,and intracellular drug release.The prepared nanocomplex showed a negative surface charge at the physiological pH,which could protect the nanocomplex from the attack of plasma proteins and recognition by the reticuloendothelial system,so as to prolong its circulation time.While at the tumor extracellular pH 6.8,the DMMA was hydrolyzed,leading to the charge reversal and exposure of the TAT on the polymeric micelles,thus enhancing the cellular internalization.Then,the polymeric micelles underwent dissociation and drug release in response to the acidic pH in the lyso/endosomal compartments of the tumor cell.Both in vitro and in vivo efficacy studies indicated that the nanocomplex significantly inhibited the tumor growth while the treatment showed negligible systemic toxicity,suggesting that the developed dual pH-sensitive PMLA-based nanocomplex would be a promising drug delivery system for tumor-targeted drug delivery with enhanced anticancer activity.3.pH/enzyme dual-responsive ZnO-based polymer-lipid hybrid nanoparticle system increases cytotoxicity of DOX against multidrug-resistant cancer cellsZinc oxide nanoparticles（ZnO NPs）,a Food and Drug Administration-approved pharmaceutical excipient,are widely used in drug formulations and cosmetics because of their stability,biocompatibility,and safety.ZnO NPs have been investigated as nanocarriers for delivery of a variety of cargoes,including drugs,genes,proteins,and imaging agents.Because ZnO NPs are readily dissolved at low pH,they have been used as an excellent pH-sensitive nanocarrier for tumortargeted drug delivery and intracellular drug release.In fact,ZnO NPs were not inert and showed photocatalytic and photooxidizing abilities against chemical and biological species.The growing evidence indicated that ZnO NPs were able to kill many types of cancer cells via the generation of hydroxyl radicals（OH^·）,superoxide anions（O₂^-）,and perhydroxyl radicals（HO₂^·）from the surface of ZnO NPs,suggesting their potential as an anticancer agent.In addition,ZnO NPs could help to overcome the multidrug-resistanc（MDR）of cancer cells,not only by the inhibition of efflux effect,but also up-regulation of pro-apoptotic protein Bax and down-regulation of antiapoptotic protein Bcl-2,thus increasing intracellular drugs concertration and improving sensitivity of MDR cells to the drugs.Therefore,in this study,we described a pH/enzyme dual-responsive ZnO-based polymer-lipid hybrid nanoparticle system that can efficiently load and release water-soluble anticancer drug doxorubicin hydrochloride（DOX·HCl）and enhance DOX toxicity against MDR cancer cells.DOX-loaded ZnO nanoparticles（ZnO/DOX）,possessing excellent physicochemical and pH-responsive drug release properties,could be effectively internalized by both drug-sensitive and MDR cancer cells and penetrate more efficiently through three-dimensional（3D）cancer cell spheroids compared with free DOX.However,the positive charge of ZnO/DOX may cause nonspecific interactions with serum components and cell membrane of the normal cells,resulting in short circulation time and nonspecific distribution.An anioic phospholipid,1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol（DPPG）,was employed as a coating reagent to modify ZnO/DOX.Then,matrix metalloproteinase 2（MMP2）-sensitive PEG-pp-PE was prepared to load ZnO-DPPG/DOX to form the polymer–lipid hybrid nanoparticle（ZnO-DPPG/DOX@PEG-pp-PE）.The prepared ZnO-DPPG/DOX@PEG-pp-PE showed significant MMP2-dependent cellular uptake,tumor penetration,and anticancer activity in monolayer cancer cells and 3D cancer cell spheroids.