Nano Drug Delivery Systems Based On Chitosan For Targeted Therapy Of Breast Cancer

Cancer is the leading challenge threatening human health,and it is estimated that millions of people die of cancer every year.Especially for the breast cancer,which is not only the malignant tumor with the highest morbidity among women,but also one of the diseases with the highest mortality among women,seriously endangers women's health.On account of the high cost,long cycle and high risk of developing new therapeutic drugs,the development of new delivery systems for existing drugs has become the preferred strategy.At present,most clinical anticancer drugs,such as paclitaxel?PTX?,methotrexate?MTX?,5-fluorouracil?5-FU?,doxorubicin?DOX?,etc.,exhibit extremely low solubility in water.In addition,due to the distribution of chemical drugs throughout the body during the treatment process,there are many side effects,such as the toxicity to normal tissues and cells as well as other adverse reactions in the body,which have been the limiting factors for the clinical application of chemotherapy drugs.In order to improve the water-solubility and targeting of drugs as well as reduce the side effects of drugs,the design and application of nanoscale intelligent drug carrier have attracted more and more attention.As a tactic for targeted delivery and controlled release of drugs,nano carriers can solve many limitations of chemotherapy drugs,such as enhancing water-solubility,reducing side effects,increasing passive accumulation of drugs in tumor tissues,and prolonging blood circulation time.This study develops a series of intelligent drug delivery systems for hierarchical target therapy,based on the tumor specific physiological environments of pH,temperature and hypoxia.We employ the biocompatible and pH sensitive chitosan as the base material,combined with temperature-sensitive polymers and target peptides,so as to obtain novel breast cancer target therapy systems with passive targeting?level 1?,active targeting?level 2?and cellular microenvironment responsive drug release behavior?level 3?.As anti-tumor drug delivery systems,these platforms own a good application prospect,and can also provide a feasible technical strategy and a steady theoretical basis for the treatment of breast cancer.This paper is divided into six chapters.Four breast cancer targeted intelligent nano drug delivery systems are introduced in total,and their efficacy is studied.According to the characteristics of the tumor tissue?hyperpyrexia and weak acid?,a thermally responsive PNIPAM has been grafted onto the chitosan via a facile RAFT process.The CS-g-PNIPAM copolymer conjugate self-assembles into nano-spheres with a favorable size distribution and smart stimuli-responsive drug release profiles that are highly desired for intravenous administration.Taking advantage of the hydrophobic core it formed,the hydrophobic drug PTX can be loaded.Moreover,L-peptide,which can bind to a verified specific marker GRP78 on the surface of cancer cells,is coated with PTX loaded CS-g-PNIPAM NPs can induce potent antitumor effects via both intra-tumor and systemic administration.The results show that,the as-prepared L-CS-g-PNIPAM-PTX NPs have a relative high drug loading?13.5%?and excellent encapsulation efficiency?74.3%?and an average diameter of275 nm.The release of PTX is slow at pH 7.4 and 25°C but greatly accelerated at pH5.0 and 37°C.MTT assays and confocal experiments showed that the L-CS-g-PNIPAM-PTX NPs possessed high targetability and antitumor activity toward GRP78 overexpressing MDA-MB-231 human breast cancer cells.As expected,L-CS-g-PNIPAM-PTX NPs could effectively treat mice bearing MDA-MB-231human breast tumor xeografts with little side effects,resulting in complete inhibition of tumor growth and a high survival rate over an experimental period of 60 days.These results indicate that L-peptide-functionalized acid-and thermally activated-PTX prodrug NPs have a great potential for targeted chemotherapy in breast cancer,and can be applied in future clinical trials.In breast cancer patients,targeted therapy for certain tumor cell types are difficult because they lack the expression of many key biomarkers,and these receptors are often used in targeted therapy for tumors.In this paper,we chose a cascade response,transmembrane targeting approach for the targeted therapy of these cancer cells.Similarly,PNVCL was grafted to a chitosan backbone according to the characteristic microenvironment of tumor.The ratio of CS to NVCL was optimized to give a formulation which underwent a phase transformation at the elevated temperature of the tumor microenvironment,providing a route to targeted drug delivery.Considering that,carcinomas are surrounded by a dense extracellular matrix full of matrix metalloproteinases?MMPs?and with a high interstitial fluid pressure?IFP?.This makes it challenging for a drug delivery system to penetrate the tumor.A cell penetrating peptide?CPP?was conjugated to CS-co-PNVCL through an amide bond.Thus,as the NPs penetrate into the tumor tissue,the amide bond between CPP and chitosan will be cleaved by the MMPs in the ECM.The hydrophobic state of the polymer carrier at the elevated tumor temperature should enhance the interactions between the cell surface and NPs,extending the residence time in the tumor.The mildly acidic conditions therein will cause the dissolution of CS,thereby triggering the release of the drug inside the target cells.The results indicate that,we obtained high drug loading and encapsulation efficiency values?14.8±1.8%and 85.3±9.7%respectively?,and observed drug release to be accelerated in acidic and hyperpyrexic conditions.The uptake of the NPs is elevated in breast tumor cells,which leads to the selective death of cancer cells in vitro.In vivo experiments in a MCF-7 xenograft mouse model showed that the NPs accumulate at the tumor site after intravenous administration,and are highly effective in halting and reversing the growth of cancerous cells.The NPs have good hemocompatibility and biocompatibility in vivo,with no adverse off-target effects noted.Thus,the nano-drug delivery system developed in this study is suitable for targeted therapy of breast cancer that lacks the expression of biomarkers?such as triple-negative breast cancer?,and has potential therapeutic value for cancers that are not easy to cure.Multidrug resistance?MDR?is mainly related to the overexpression of membrane transporter proteins?P-glycoprotein?P-gp?and the multidrug resistance protein?MRP??,which act to expel anticancer drugs from cancer cells.This profoundly decreases the sensitivity of the tumor to chemotherapy.In this study,we applied oleanolic acid?OA?,a natural chemotherapeutic sensitizer that can reduce MDR of cancer cells,to enhance the chemotherapy effect of nanomedicine.Chitosan was frst functionalized with folic acid to allow selective uptake by cancer cells,and then subsequently with OA.These FA-CS-g-OA@DOX NPs had appropriate size?180 nm?and size distribution?PDI<0.45?for tumor therapy,as well as a high drug-loading efciency?15.6%w/w DOX;5.1%w/w OA?and pH-responsive release properties.In breast cancer MDA-MB-231 cells,more efcient uptake of FA-CS-g-OA@DOX NPs than of free DOX was observed by confocal laser scanning microscopy and flow cytometry.The in vitro cytotoxicity of FA-CS-g-OA@DOX NPs against MDA-MB-231 cells was higher than with free DOX and free OA,while the NPs were less harmful to healthy HUVEC cells.In vivo pharmacokinetic studies showed that FA-CS-g-OA@DOX NPs had a much longer circulation time than free DOX,while biodistribution results revealed that FA-CS-g-OA@DOX could actively target a MDA-MB-231 xenograft tumor in mice.The NPs are found to have apoptosis-enhancing and anti-proliferative capacities in vivo.The presence of OA in the formulation both sensitizes cancer cells to DOX and mitigates DOX-induced damage to healthy tissues.The FA-CS-gOA@DOX NPs generated in this work hence have great potential for the treatment of MDR breast cancers,and further offer a platform to target other cancers.Among a cluster of deaths from breast cancer,metastasis is the main cause of the most tumor-related deaths and the least effective treatment.In this case,cancer cells detach from the primary tumor and spread to the nearby organs and tissues aimlessly,which makes them hard to track and accurate therapy.A potential tactic to solve this puzzle is the application of theranostics,which platforms contain both an anti-tumor ingredient and an imaging agent.In this study,chemotherapy drug PTX was grafted onto chitosan to form the prodrug CS-PTX,thereafter modified with the small tumor targeting peptide K237.Meanwhile,photoheat and imaging reagent MoS₂ was loaded into the hydrophobic core formed by the self-assembly of polymer K237-CS-PTX in aqueous solution.The as prepared K237-CS-PTX@MoS₂ not only enhances the solubility of hydrophobic PTX,but also provides a“hide”effect for cytotoxic PTX in physiological condition.The results indicate that,K237-CS-PTX@MoS₂ switched from the“silent state”before reaching the tumor to the“activated state”within the tumor respond to these stimulus including low pH,redox gradient,hypoxia,overexpression of enzymes,etc.In vitro experiments show that our smart nanomaterials show high affinity and specificity for breast cancer cells,and effectively killed tumor cells.In addition,since the unique photothermal properties of MoS₂,we also conducted photothermal therapy,photoacoustic imaging,CT imaging and other experiments on tumor-bearing mice,and the results showed favorable effect.All these indicate that the multi-functional NPs designed by us can significantly improve the synergistic therapeutic effect of breast cancer,and achieve the theranostic effect.In conclusion,we have successfully prepared a series of smart nanomaterials taking advantage of the excellent properties of chitosan.These nanoplatforms combined active and passive targeting,exhibited the controlled drug release behavior response to the stimuli of tumor microenvironment,and addressed the efficient treatment of different types of breast cancer.According to the characteristics of acidic environment in tumor tissues,chitosan was used as the base material of drug delivery system to study the pH responsive drug release behavior of chitosan nanomaterials.The inclusion of disulfide bond enables the synthesized nanomaterials exhibit the responsive drug release behavior,respond to the redox microenvironment of tumor.For the characteristic of hyperpyrexia in tumor cells,PNIPAM and PNVCL were grafted with chitosan,respectively,making the drug delivery system capable to accelerate drug release when the temperature increases.Folate,L-peptide,CPP and K237 peptide were employed to modify chitosan materials to improve the active targeting ability of drug delivery systems respectively,and the results showed favorable results.While OA can be used as a chemosensitizing agent to enhance the efficiency of therapy,suppress the MDR of cancer cells.In addition,the inclusion of MoS₂ can endow our materials with photothermal properties,so as to conduct a series of theranostic applications such as photothermal treatment,photoacoustic imaging,and CT imaging on tumor tissues.We hope that this study can provide certain reference for the preparation and application of more and more valuable nano-chitosan theranostic materials in the future,as well as theoretical support for the therapy of breast cancer.