Study On Construction,characterization And Their Regulation On Cancer Progression Of Polysaccharide Biomaterials And The Related Mechanism

The development and evolution of tumors are closely associated with by synchronous changes in extracellular microenvironment,and the relationship between the two is particularly close.It is of great significance to study how ECM components regulate tumor development for effective control of tumor recurrence and metastasis Therefore,the research system constructed in vitro must simulate ECM of tumor in material composition,morphology,biological stimulation and other aspects.Inspired by this,this paper,based on lentinan(LNT),chitosan(CS)and hyaluronan acid,simulated the extracellular matrix(ECM)to regulate the tumor cell behaviors and studied its underlying regulation mechanism,which could provide new ideas for the treatment of solid tumor.1)In the treatment of breast cancer(BC)currently,local administration system is the research focus of the most difficulf problem--postoperative in-situ recurrence.Due to its excellent biosafety and plasticity,chitosan has been widely used in the local delivery of chemotherapeutics for BC.However,there are still many problems to be solved for chitosan-based systems,such as multidrug resistance of tumor cells and off-target toxicities of chemotherapy.Therefore,a system with the ability of long-term inhibiting tumor recurrence and without cytotoxicity is expected to solve these problems.Lentinan has good biocompatibility and significant anti-tumor activity,however,LNT bioavailability is low after intravenous or oral administration.The composites composed of chitosan and polysaccharide could better simulate the cell microenvironment,which showed better biosafety,prolonged local release enhanced bioavailability of LNT in the local treatment for long-term inhibition of postoperative breast cancer recurrence(BCR).In this part,a sponge-like lentinan/chitosan composite(LNT/CS)was successfully prepared by physical mixing of macromolecular solution and freeze drying.The results showed that after lyophilization,LNT and CS were thoroughly interacted through intermolecular hydrogen bonds and van der Waals forces.LNT/CS had uniform texture and the water absorption ratio was over 30 folds of initial weight.The porosity was higher than90% and the curve of compression deformation was smooth.LNT/CS was completely degraded within 2 weeks in vitro enzyme environment,and the release of LNT showed sustained and controlled characteristics.In the in vivo experiment,the recurrence model of breast cancer after xenotransplantation was established using nude mice,and LNT/CS was given locally.The in vivo compatibility of LNT/CS was characterized by observing the fluctuation of body weight,local inflammatory reaction,wound healing process and serum indexes in nude mice.The release characteristics of LNT/CS complex were characterized using Cy7 labeling.The results showed that local administration of LNT/CS showed good biocompatibility in vivo,and did not cause tissue inflammation or obvious changes in serum indexes.During 4 weeks,LNT showed long-acting sustained release.Meanwhile,the recurrence rate was notably inhibited,with 100% in the control group and 66.7% in the LNT/CS group.In addition,the change of tumor volume growth curve was more significant,and finally the tumor volume of the LNT/CS group was reduced by more than 60%compared with other control groups.In the mechanism study,we focused on the expression of breast cancer stemness biomarker such as CD24/CD44,ALDH1 and Ki67.The molecular mechanism of LNT inhibiting breast cancer recurrence was characterized at multiple levels by immunohistochemistry of the final recurrent tumor tissues,immunofluorescence and western blot experiments of MDA-MB-231 cells.The results showed that LNT controlled the recurrence and proliferation of residual tumor cells by inhibiting the expression of biomarkers of cancer stem cells.Therefore,sponge-like LNT/CS complex has great potential as an alternative treatment for postoperative BCR,and also provides reference for other local treatment strategies in this field.2)In this part,a multi-layer film for mimicking ECM in vitro was constructed through the layer-by-layer self-assembly of poly-lysine and hyaluronic acid.The matrix stiffness was adjusted by controlling the crosslinker concentration,and the surface was modified with polydopamine to immobilize transforming growth factor β1(TGF-β1)as a biological signal.Finally,the development of ephithelial-mesenchymal transition(EMT)of hepatoma cells under the dual regulation of growth factors and environmental mechanical stiffness and the underlying mechanism was explored.The loading efficiency of TGF-β1 was higher than 90%.The atomic force microscopy results showed that the modification of polydopamine and the loading of TGF-β1 did not affect the surface morphology of the substrate.The behaviors of Hep G2 cells were regulated by different stiffness(Soft/Stiff)and different presentation states of TGF-β1 and related molecular mechanisms were explored.The results showed that the matrix stiffness had a significant effect on cell morphology.And the cell area and aspect ratio on the stiff matrix are significantly higher than those on the soft matrix.Through analyzing the cellular EMT marker proteins such as E-cadheirn,N-cadherin and vimentin,it was found that either the soft matrix with immobilized TGF-β1(i-TGF)or the stiff matrix alone could only promote the formation of epithelial phenotype in Hep G2 cells,while the stiff matrix with i-TGF was the only condition for inducing a mesenchymal phenotype.Further researches on cellassociated receptors indicated that i-TGF could increase the expression of TGF-β1-specific receptor TβRI and activate the PI3 K pathway.The i-TGF-TβRI interaction also promoted Hep G2 cell adhesion,expanded the cell’s contact area for stiffness sensing,and leads to increased mechanosensor(β1 integrin)expression.Furthermore,mechanotransduction is enhanced by the β1 integrin-vinculin-p-FAK pathway,leading to the activation of PI3 K.Taken together,this part of the study found that there is a clear crosstalk between the cell’s mechanotransduction pathway(stiffness-integrin-vinculin-p FAK)and the biochemical signaling pathway(TGF-TβRI-PI3K).The coordination of the two pathways together stimulates the adhesion,spreading,migration and phenotypic transformation of Hep G2 cells.Using this model,this study proposes a novel mechanism to explain the process by which Hep G2 cells regulate cell fate by coupling mechanotransduction and biochemical signaling.In summary,based on macromolecular polysaccharides(chitosan,lentinan or hyaluronic acid),this paper mainly describes the construction of biomimetic multifunctional composite materials that simulates tumor microenvironment.The regulation of tumorigenesis and development of polysaccharide materials,the interaction between tumor cells and materials and their underlying mechanisms have been studied in vitro and in vivo.And they may provide a new strategy and basis for the preparation of tumor biomimetic materials and the treatment of tumor recurrence and metastasis.