Preparation Of Multi-functional Nano-drug Delivery System Based On Natural Polymer And Their Antitumor Efficacy Study

In recent years,with the development of materials science and nanotechnology,the research of nano-drug delivery system has been deepening and has become a research hotspot of tumor therapy.Compared with traditional small-molecule chemotherapeutic drugs,nano-drug carriers can significantly improve the tumor transmission efficiency of loaded drugs and reduce drug's side effects.It is based on the enhanced permeation and retention(EPR)effect on tumor tissues that the therapeutic drugs can be delivered to the tumor site using the nano-drug carriers.Due to abnormal vasculature and impaired lymphatic drainage around the tumors,nano-drug carriers can be penetrated in the tumor tissue and release the drug at specific locations.As a result,reduce the exposure to normal tissues,and decrease the side effects.However,three major transport steps govern how effectively an agent will navigate this path:vascular transport,transvascular transport,and interstitial transport.Unfortunately,the abnormal physiology of tumors gives rise to a set of transport barriers,such as compact extracellular matrix(ECM),and high interstitial fluid pressure(IFP)caused by impaired lymphatic system,that limit the rate and extent of drug delivery to tumor mass.Although nano-carriers intend to accumulate in the tumour tissue due to the EPR effect,and can be leaked out from vessels.Unfortunately,the nano-carriers can't be effectively penetrated into the extracellular matrix(ECM)and absorbed by cancer cells,which could be stacked around the uneven blood vessels,it greatly reduce the anti-tumor effect.In addition,since EPR effect is affected by many factors such as tumor location,amount of infiltration by macrophages,as well as pore size of tumor vessels,the passive targeting is limited to some types of tumors.Furthermore,EPR effect is only restricted to some solid tumors which are larger than approximately 4.6 mm in diameter.In order to overcome these drawbacks,in this dissertation,tumor-homing ligands(phenylboronic acid,lactobionic acid)and bromelain were decorated the surface of nanoparticles to give multi-functional nanoparticles,that is expected to overcome the single function of nano-drug carriers in obstacles of tumor infiltration,and can enhance the cellular uptake,accumulation and permeation.Therefore,multi-functional nanoparticles can improve the anti-tumor effect.In addition,due to the natural polymer has many advantages,such as good biocompatibility and biodegradability,ample abundance,inexpensive as well as can be easily modified,can be separated and purified in large quantities,nontoxic.Therefore,a series of nano-drug delivery systems were prepared by natural polymer in this dissertation.In sum,the multifunctional nanoparticles designed in this dissertation with some advantages,such as can be easily designed,biodegradability,precisely controlled structure and function,which may have great significance for the application of nano-carriers in the field of anti-tumor therapy.The main content is described in three parts as below:(1)In this part,we chose gelatin(Type B)as an initial material to prepare gelatin nanoparticles(NP1)by a desolvation method.Thereafter,a tumor-homing ligand,3-Carboxyphenylboronic acid(3-CPBA)was modified on the surface of NP1 to give tumor-targeting nanoparticles(NP2).The morphology and stability of NP1 and NP2 were investigated by transmission electron microscope(TEM),scanning electron microscope(SEM),and dynamic light scattering(DLS).The results show that both NP1 and NP2 are spherical-like,and kinetically stable in various conditions.DOX was used as a model anticancer drug,and loaded into NP1 and NP2 to give NP1-DOX and NP2-DOX.The drug release behavior of these nanoparticles were investigated under different pH values,and found the DOX release rate from NPs increases significantly with the change of pH value from 7.4 to 5.0.In vitro cellular uptake and cytotoxicity of doxorubicin hydrochloride(DOX)-loaded NP1 and NP2 were evaluated in conventional monolayer cell models(SH-SY5Y cell?H22 cell and HepG2 cell)and three-dimensional(3D)cultured multicellular spheroids(SH-SY5Y MCS)via Laser Scanning Confocal Microscope(CLSM),Flow Cytometry(FCM)and MTT method,respectively.At last,tumor penetration,accumulation,and antitumor activity were investigated using H22 tumor-bearing mice via Near Infrared Living Imager,Tumor Histopathological Analysis,and anti-tumor assay.All results demonstrated that the conjugation of 3-CPBA can efficiently enhance non-targeted NPs' tumor-homing activity,thus improve their tumor accumulation and antitumor effect.(2)In this part,we chose carboxymethyl chitosan(CMCS)as an initial material to prepare conventional nanoparticle drug delivery system(CM NP)by a desolvation method.Thereafter,a tumor-homing ligand,3-carboxyphenylboronic acid(3-CPBA),was decorated on the surface of prepared CM NP to give tumor-targeting NPs(CB NP).Particle sizes were measured by dynamic light scattering(DLS),while the morphology was observed via transmission electron microscopy(TEM)and scanning electronic microscope(SEM).The results show that CM and CB NPs are spherical-like,and kinetically stable in various conditions.Doxorubicin(DOX)as a model drug was successfully encapsulated to give CM-DOX and CB-DOX NPs.The biological effect of these DOX-loaded NPs was then investigated by monolayer cell model and three-dimensional multicellular spheroids(MCS).The results demonstrate that 3-CPBA modification can improve NPs' accumulation and penetration ability.In vivo antitumor effect was evaluated by H22 lung metastasis tumor-bearing mice.The results show that CB-DOX NPs can deliver more drug than CM-DOX NPs,and retain for a long time in lung tissue,thus remarkably reducing the size of tumor mass of H22 metastasis lung tumor.All results demonstrate that the obtained CB NPs would be potentially useful as nano-scaled drug carriers in chemotherapy.(3)Tumor extracellular matrix(ECM)still remains a major barrier that hinders the diffusion of drug in tumor parenchyma.In order to solve this problem,we chose chitosan(CS)as an initial material.Firstly,because of the specific recognition effect of lactose acid and tumor cell surface overexpression receptor(asialoglycoprotein),lactobionic acid(LA)as a tumor-homing ligand was decorated on the surface of chitosan to give tumor-targeted polymer(CLA).CLA-based nanoparticles(CLA NPs)were simply prepared by a desolvation method.Thereafter,bromelain-decorated hybrid nanoparticles(CLAB NPs)were obtained by the modification of the surface of CLA NPs to improve their penetration ability.The size distribution,morphology and stability under various conditions of these two nanoparticles were investigated by transmission electron microscopy(TEM),scanning electronic microscope,and dynamic light scattering(DLS).Doxorubicin(DOX)as a model drug was successfully encapsulated into these two nanoparticles and drug release profiles were investigated under three different pH values(7.4,6.5 and 5.5).Enzymatic activity experiment demonstrated that free bromelain still remained relative appropriate activity after immobilization.Cellular uptake and MTT results against HepG2 cells and SH-SY5Y cells demonstrated that the LA-conjugated tumor-targeting nanoparticles can be efficiently internalized into hepatoma carcinoma cells and leading to higher cytotoxicity than free DOX.Three dimensional cell models(SH-SY5Y and HepG2 MCS)results demonstrated that CLAB-DOX NPs significantly improved the penetration of DOX in MCS.In vivo drug distribution and antitumor activity results demonstrated that bromelain increased nanoparticles penetration and diffusion ability in tumor area upon the digestion of ECM,leading to higher drug concentration in tumor area and superior antitumor effect.