Investigation On Delivery System For Antitumor Drug Based On Hybrid Particles And Biological Vesicles With The Ability Of Active Tumor Deep Penetration

Recently,nano-drug delivery systems have been widely used in tumor therapy and achieved good therapeutic effect,because they can significantly improve drug distribution and behavior in vivo and improve drug retention rate in tumor tissues.With the deep study of tumor tissue pathology,it is found that the tumor microenvironment is complex,highly viscous,and contains multiple barriers such as tumor extracellular matrix?ECM?,which limits the penetration of traditional nano-drug delivery systems in tumor tissues.To solve the penetration issues of nano-drug delivery systems,many nanocarriers have been designed to achieve deep tumor penetration by destroying the ECM barrier or precisely regulating the size of nanomaterials.However,the destroying of tumor ECM may bring the risk of accelerated metastasis of tumor cells,while the change of particle size is limited by the response sensitivity to tumor microenvironment.In this thesis,hybrid nanoparticles?NPs?and yeast cell vesicles?YCV?were used as nanocarriers to develop nano-drug delivery systems with the ability of active tumor tissues deep penetration,and the efficiency and mechanism of deep tumor penetration in vivo and in vitro,in vivo distribution behavior,the loading and release rate of chemotherapy drugs,and the anti-tumor efficiency were systematically investigated.The details are as follows:1.Anti-tumor research of self-driven hybrid nanoparticles with the ability of enhanced tumor penetration.In this study,F68@TA@Ag hybrid nanoparticles with asymmetric structure was first constructed by reduction in situ,in which TA was tannic acid and Ag was argentum.TEM and TEM mapping were used to confirm the successful preparation.Further research demonstrated that the asymmetric Ag NPs on F68@TA@Ag NPs movement of F68@TA@Ag.Cisplatin?CDDP?was loaded into the hybrid nanoparticles to obtain F68@TA@Ag/CDDP nanosystem,in which the loading efficiency of CDDP was about 8.5%,and the diameter and zeta potential of the nanosystem were about 80 nm and-30 m V,respectively.The results of in vitro studies on ion regulation properties and motion characteristics of F68@TA@Ag/CDDP showed that:?1?The nanosystem was stable at p H7.4 and cumulative release rate of CDDP was only 16.5%at 12 h,while at p H5.0,the nanosystem was swollen and broken,leading to 82.0%of CDDP release.p H sensitive CDDP release lay the foundation of intracellular drug release.?2?F68@TA@Ag/CDDP was movable with the dependent manner of H₂O₂ concentration,and thus Ag⁺was released.It can be seen from the typical trajectory of F68@TA@Ag and relative analysis of MSD and diffusion coefficiency that directional movement distance of the self-driven F68@TA@Ag/CDDP increased significantly with the increase of H₂O₂ concentration and reaction time.The driving motion of H₂O₂ lays the foundation of the deep tumor penetration of F68@TA@Ag/CDDP.Moreover,Ag⁺generated by the reaction of Ag and H₂O₂ can significantly consume Cl^-in tumor cells.?3?F68@TA@Ag/CDDP showed significant Fe²⁺complexation.ICP-MS results displayed that F68@TA@Ag/CDDP?1?mol?could chelate about 50?g of Fe²⁺within 12 h,which was mainly attributed to polyphenol hydroxyl structure of TA.In this study,4T1 cells were selected as tumor cell model,and Hs578Bst cell as normal cell model for investigation on in vitro deep penetration capacity,ion modulation-mediated CDDP self-amplifying antitumor effect,and tumor-selective killing capacity of F68@TA@Ag/CDDP.The results of distribution of the nanosystem in 3D cell spheres showed that compared with F68@TA/CDDP,F68@TA@Ag/CDDP with self-driven motion showed stronger permeability.After12 h incubation,the penetration depth of F68@TA@Ag/CDDP was 50?m,significantly higher than that of F68@TA/CDDP?30?m?.Next,the internalization ability of F68@TA@Ag/CDDP by 4T1 tumor cells was investigated.Both CLSM and flow cytometry results showed that F68@TA@Ag/CDDP could be rapidly internalized by 4T1 cells and entered lysosomes,with the highest colocalization efficiency at 4 h.However,after 7 h,a large number of lysosomes were broken and the colocation rate decreased significantly,confirming the drug release of F68@TA@Ag/CDDP in lysosomes and its lysosome escape effect.The study of antitumor mechanism of F68@TA@Ag/CDDP confirmed that CDDP significantly improved the level of intracellular H₂O₂,and further accelerated the decomposition of Ag nanoparticles and release of Ag⁺.The abundant release of Ag⁺significantly decreased the level of intracellular Cl^-,improved the dechlorination efficiency of CDDP,and increased the formation of Pt-DNA adducts.On the other hand,F68@TA@Ag/CDDP significantly decreased intracellular free Fe²⁺,inhibiting the activity of DNA repair enzyme,which was beneficial for the maintenance of Pt-DNA adducts.The content of Pt-DNA adducts was further investigated by ICP-MS,and the results showed that F68@TA@Ag/CDDP induced highest Pt-DNA adducts.In vitro antitumor assays showed that,when the inhibition rate of CDDP on4T1 cells was about 50%,that of F68@TA/CDDP was 58.3%.However,in the F68@TA@Ag/CDDP group,the inhibition rate reached to 67.2%,significantly higher than that of CDDP and F68@TA/CDDP.In addition,the inhibition effect on4T1 cells was stronger with the increase of CDDP concentration.All above proved that ion modulation led to strong CDDP self-amplifying antitumor effect.4T1 cells bearing BALB/C mice were used as the animal model to evaluate in vivo deep tumor penetration capacity and dual ion modulation anti-tumor effect of F68@TA@Ag/CDDP.In vivo distribution experiments showed that F68@TA@Ag/CDDP had the property of tumor accumulation and the deep tumor permeability.The dosage of CDDP was set as 2 mg/kg.After 2 weeks of treatment,the tumor inhibition rate of F68@TA@Ag/CDDP was about 77.9%,significantly were significantly down-regulated by F68@TA@Ag/CDDP treatment.The preliminary results of in vivo biosafety evaluation test showed that F68@TA@Ag nanocarriers had good biosafety and significantly reduced the toxic side effects of CDDP on normal tissues.2.Anti-tumor research of yeast cells derived vesicles with the ability of enhanced tumor penetration.In this study,S.cerevisiae cells were selected as the source of nanovesicles,because they are easy to be cultured on a large scale and have high biological safety.YCV was extracted by the alkaline lysis method,and its morphology and particle size distribution were investigated.The results of TEM and NTA analysis showed that YCV was spherical with uniform size of about 100 nm.Then the chemotherapeutic drug doxorubicin?DOX?was loaded into YCV to obtain YCV/DOX with the loading rate of 82.5%.The drug release profile of YCV/DOX implied that DOX release was significantly p H-dependent.Compared with that p H 7.4,the cumulative DOX release rate increased from 5.4%to 65.7%under p H 5.0.4T1 cells were used as tumor cell model to construct 3D cell spheres.Firstly,the deep penetration capacity of YCV was investigated in vitro.The results showed that compared with liposomes?Lipo?,YCV from natural sources showed stronger permeability.After incubation for 12 h,the penetration depth of YCV was 50?m,significantly higher than that of Lipo?30?m?.It is speculated that the mechanism may be good deformation capacity of YCV and repelling behavior of natural membrane to the extracellular matrix of tumor tissues.Cellular uptake experiments showed that YCV/DOX could be efficiently internalized by tumor cells with a time dependent manner.Under lysosomal acidic conditions,DOX was released to kill tumor cells,with the inhibition rate of 64.2%for 24 h and 89.5%for 48 h on 4T1cells.4T1 cells bearing BALB/C mice were used as the animal model.In vivo distribution experiments showed that YCV had stronger tumor accumulation than Lipo.Further fluorescence colocalization results of tumor vessels and YCV showed that YCV had stronger tumor deep penetration ability,which played a good role of efficient DOX delivery to superficial and deep tumor tissues.The dosage of DOX was set as 5 mg/kg.After 2 weeks of treatment,the tumor inhibition rate of YCV/DOX was about as 67.4%,significantly higher than that of the other groups.In addition,pathological analysis of tumor tissues and TUNEL results showed that compared with Lipo/DOX,YCV/DOX could more effectively induce apoptosis of tumor cells and significantly reduce the number of tumor cells,which also confirmed the high efficiency of DOX delivery by YCV.In this thesis,two kinds of nano-drug delivery systems with active deep penetration ability were constructed and studied based on hybrid nanoparticles and yeast cell vesicles respectively,which provided a new idea and experimental basis for the enhanced treatment of chemotherapy drugs.