Preparation And Characterization Of Multifunctional Ph-responsive Nanocarriers

One of the most critical challenges in cancer treatment is the design anddevelopment of a delivery system with multiple functionalities. This is becausecurrent chemotherapeutical agents have notorious undesirable side effects likelimited accessibility to tumor tissues, systemic toxicity, development ofmultidrug resistance and non-specifc targeting. Ideal drug delivery systemsshould be stable with a long circulation time, and should keep the loaded drugsunreleased during circulation in the bloodstream or in normal tissues. Uponreaching and accumulating in tumor tissues by passive and active targeting, andafter being taken up by cancer cells, the systems should release the drugs rapidlyin response to the local environment. To achieve this goal, variousmultifunctional drug delivery systems including liposomes, polymericnanoparticles, block copolymer micelles, and polymer-drug conjugates havebeen developed for cancer chemotherapy. Among them, pH-sensitive polymericnanoparticle is the most attractive candidate due to intrinsic differences betweenvarious solid tumors and the surrounding normal tissues in terms of their relativeacidity. The extracellular pH (pH_e) in most solid tumors is more acidic （pH6.5-7.2） than in normal tissues. When the polymeric nanoparticles are taken upby cells there is also pH variation at different states. The endosomal pH is about 5.0-6.5, whereas lysosomes have an even lower pH （4.5-5.0）. In this respect, thedevelopment of drug-carriers that respond to the pH in tumors may be favorablestrategy to trigger the extensive release of anticancer drugs, increase anticancerefficacy and to decrease the side effect in normal tissues.This paper was based on the study of pH sensitive multifunctionalnanocarriers. In the present study, three different pH sensitive nanocarriers weredeveloped, of which pH sensitivity arised from acid-sensitive materials or anacid labile linkage between the drug and the polymer. The first part of theresearch work is based on the acid-sensitive materials. Comparedphysicochemical properties of nanoparticles composed of dextran and threesulfadimethoxine derivatives, the most appropriate nanocarrier was chosen forfurther study. Meanwhile, polymeric nanoparticle was developed frompolymeric conjugate, which pH sensitivity arised from acid labile linkage in thesecond part of research work. To be effective, some cytotoxic drug must haveanticancer activity within the cells. The desired intracellular drug concentrationwas not easily achieved if the nanocarrier relied solely on a pH-responsivemanner as mentioned above. Therefore, we used the new strategy to develop pHsensitive nanocarrier in the third part of research work, of which combined theacid-sensitive materials and an acid labile linkage in one system. This paper alsointroduced a simple method for using TAT, which made the drug could enrich inthe intracellular area and play the efficacy. Details are as follows:（1） Preparation and characterization of maleilated dextran/sulfadimethoxine （Dex-MA/SD） nanocarrier.A pH-sensitive drug targeting system for solid tumors was establishedbased on dextran and sulfadimethoxine conjugates. Three graft polymers werefirstly synthesized: Dex-MA/SD polymer, Dex-MA/PSD polymer and Dex/COOH-OSDM polymer. The suitable ratio of three polymer systems （i.e.Dex-MA:SD=1:3, Dex-MA:PSD=2:3, Dex:COOH-OSDM=4:3） was obtainedby adjusted the balance between the hydrophilic segments and hydrophobicsegments. The structure of nanoparticle was different from that reported in theprevious reference because of the different dialysis environment pH. Here pH7.4aqueous solution was used as dialysis medium.The critical aggregationconcentration of three systems was3.0μg/mL,4.9μg/mL and5.2μg/mL,respectively. And the zeta potential was-47.1mV,-28.3mV and-8.18mV.Dex-MA/SD nanoparticle was chosen for the further study because of itsfavourable stability. Its particle size was92nm at pH7.4, and reached222nm atpH6.8, which showd pH sensitivity. The Dox-release profiles from nanoparticlewere pH-dependent. The cumulative released Dox from pH7.4, pH7.0, pH6.8or pH6.0medium was6.0%,13.2%,20.0%,24.0%. The study of antitumoractivity in vitro was also confirmed the Dex-MA/SD nanoparticle waspH-sensitive. The cellular uptake efficacy of drug-loaded nanoparticle at pH6.8was higher than that at pH7.4. These results suggested Dex-MA/SD was apromising pH-sensitive tumor-targeted drug delivery system.（2） Self-assembled nanoparticle from Folate-decorated maleilatedpullulan–doxorubicin conjugates for co-delivery doxorubicin and PDTC.Multidrug resistance is one of the bottleneck problems in cancerchemotherapy. How to overcome the protein efflux function and how to improvethe drug concentrations in the cell are the key issues. Nanoparticles modified bythe specific ligand can be used for overcoming MDR to some extent. In addition,when the NF-κ B inhibitor in combination with chemotherapeutic drugsapplication, NF-κ B inhibitor can enhance the radio chemotherapy treatmenteffect, also can alleviate the effects of drug resistance. But there is a problem to solve urgently for present drug delivery system, which is the lower drug loadingcapacity （e.g. the drug loading capacity was less than10%in some nanoparticlesor liposomes carrier）. The goal of this study was to develop doxorubicinconjugate nanoparticles with increased anti-tumor effect, reduced side effectsand overcoming multidrug resistance （MDR）. In this regard, folate-decoratedmaleilated pullulan-doxorubicin conjugate nanoparticles were developed ascarriers for co-delivery pyrrolidinedithiocarbamate and doxorubicin（abbreviated as FA-MP-Dox/PDTC+Dox NPs）. The Dox or FA conjugateefficiency was7.5wt%,2.0wt%, respectively. The resultant nanoparticlesshowed spherical geometry, with an average size of152nm in diameter. Twodrugs-releases from nanoparticles were slow, pH-dependent sustained release.After30h, the cumulative released Dox from pH5.0and pH7.4medium were53%and26%. To test the efficacy of these nanoparticles, in vitro tests includingcell viability, folate receptor-mediated endocytosis were conducted against bothA2780cells and A2780/Dox^Rcells. For A2780/Dox^Rcells,FA-MP-Dox/PDTC+Dox NPs （IC50=9.8μM） exhibited superior cytotoxicactivities to MP-Dox/PDTC+Dox NPs （IC50=20μM）. This result revealed thatFA moieties in FA-MP-Dox/PDTC+Dox NPs played an important role inenhancing cytotoxic effect by binding of FA-MP-Dox/PDTC+Dox NPs with FAreceptors on A2780/Dox^Rcells, and subsequently increasing their intracellularuptake as a result of the receptor-mediated endocyctosis. The results suggestedthat folate moiety was indeed on the surface of nanoparticles. On the other hand,for A2780cells, the cytotoxicity of FA-MP-Dox/PDTC+Dox NPs （IC50=2.0μM）was significantly higher than that of MP-Dox/PDTC+Dox NPs (IC₅₀=7.5μM).In spite of this result, FA-MP-Dox/PDTC+Dox NPs did not surpass thecytotoxicity of free doxorubicin (IC₅₀=0.17μM). These results suggested that co-delivery of PDTC and Dox may further overcome the MDR by transportingan increased amount of Dox within cells besides the folate receptor mediatedendocytosis process.（3） Preparation and characterization of multifunctional pH-responsivemicelles for tumor-specific uptake and enhanced cellular deliveryIn our study, we found some drug delivery system would release drugsinevitably in the extracellular area and reduce the specificity of drug targeting,even though resistance reversal agents and chemotherapy drugs wereco-deliveried by nanocarrier. Therefore, it is a key issue for overcomingmultidrug resistance to transfer biologically active molecules or drugs to theintracellular area. Cell penetrating peptide （TAT）, which has a high membraneaffinity, fast-penetrating, rapid degradation and other advantages, could directlytransfer macromolecules or drugs to pass through the cell membrane, especiallyeffective for resistant cells. Therefore, combining the more suitable acidsensitive materials and acid-labile bond to build more sensitive to pH responsivenano-drug carriers is our new target. A novel multifunctional delivery systemwas developed for tumor-specific uptake and enhanced cellular delivery. In thisregard, the luteinizing hormone-releasing hormone modified poly （ethyleneglycol）-poly （L-histidine）-doxorubicin micelles （LHRH-PEG-PHIS-Dox） wereprepared and used for doxorubicin-TAT conjugate （Dox-TAT） delivery. Themorphology and properties of the micelles such as pH sensitivity, zeta potentialand mean diameters were investigated. Dox-TAT loaded micelles were alsoevaluated with regard to their anticancer efficacy against A2780/Dox^Rcells tostudy the in vitro anti-tumor effect. The results showed LHRH-PEG-PHIS-Doxhad lower critical aggregation concentration of4.9μg/mL. The size and zetapotential increased with the decrease in pH. Its particle size grew down from 158nm at pH5.0to78nm at pH8.0. At an acidic pH （<7.0）, the micelle waspositively charged, which would enhance Dox-TAT release. So the in vitroDox-TAT release was significantly accelerated by decreasing pH from7.4to5.0.After20h, the cumulative released drug from pH5.0and pH7.4medium were84%and19%, which showd pH dependent release profile. This resulted inincreased cytotoxicity against A2780/Dox^Rcells at pH6.8and low cytotoxicityat pH7.4. In summary, Dox chemically conjugated to the polymer backbone（LHRH-PEG-PHIS） not only improved the stability of the micelles, but alsoincreased the drug concentration. A novel and simple method was firstly usedfor the introduction and protection of TAT by utilizing the different solubility ofPHIS at different pH. The results show that the development ofLHRH-PEG-PHIS-Dox/Dox-TAT, combined the advantage of pH sensitivematerial and TAT, may be beneficial for tumor-specific uptake and enhancedcellular delivery.