| Objective:Breast cancer is a malignant tumor in which breast epithelial cells proliferate out of control under the action of a variety of carcinogenic factors.It has become one of the most common cancers in the world,and it is also one of the hotspots and difficulties in current basic and clinical research.The current treatment methods for breast cancer mainly include surgery,radiotherapy,chemotherapy and immunotherapy.Although these treatments have obtained good treatment effects in some degree,there are still problems such as tumor metastasis,easy recurrence and immune damage.At present,photodynamic therapy(PDT),as an emerging treatment method,has attracted more and more attention from researchers owing to the advantages of high selectivity,low systemic side effects,reproducibility and synergistic effect.After irradiated by a specific laser,a photosensitizer can produce a large amount of reactive oxygen species(ROS)this is the mechanism by which PDT uses to cause cell apoptosis and DNA damage.Currently,PDT is mainly used in the treatment of skin tumor or other skin diseases.We believe that it is feasible and promising to wide the application of PDT to other types of cancer.However,most of currently used photosensitizer still has some problems that may limited its application,such as poor water solubility,low aggregation at the tumor site,low ROS yield and no specificify,consequently insignificant tumor cell killing effect.Therefore,how to improve the aforementioned shortcomings needs to be solved urgently.Macromolecular drug delivery systems are able to load a variety of drugs,and can accumulate at the tumor site by means of the enhanced permeability and retention(EPR)effect of tumor tissue,which is a feasible strategy to solve the above-mentioned problems.Among them,the glycosyl polymer drug delivery system has the advantages of good biocompatibility,degradability and non-immunogenicity,and can be used to construct the drug delivery system herein.In addition,glycopolymers with different structures have differences in assembly behavior,drug loading efficiency and biological behavior in vivo.Therefore,to explore the effects of different glycopolymers structures on the performance of glyco-based polymer delivery systems in PDT,and to screen the optimal PDT carrier that can be used to deliver dual drugs is one of the objectives of this study.The unstablity of the genomic DNA chain of tumor cells makes tumor cells more sensitive and vulnerable to a variety of treatment strategies related to DNA synthesis or repair inhibition,especially under the action of targeted DNA-based combination therapy.In this study,we design and synthesis a glycosyl-based polymers drug delivery system loading with a photosensitizer and poly-ADP-ribose polymerase(PARP)inhibitor to combine the effects of DNA damage and repair inhibition,which may improve the therapeutic effect of PDT.On one hand,it can increase the accumulation of therapeutic drugs at the tumor site;on the other hand,it can enhance the killing effect of PDT by inhibiting PARP.In addition,due to the properties of reserving cell membrane antigens and structure,ligand recognition,long blood circulation,and immune escape functions,bio-membrane based nano-delivery systems have developed rapidly.The adhesion molecules expressed on the surface of tumor cells,such as Ncadherin,galectin-3,epithelial cell aggregation molecules etc.contribute to the formation of multiple tumor cell aggregates because of their homologous adhesion domains.Tumor cells can use cell membrane proteins to achieve homologous binding between tumor cells and change the intracellular pathway of drugs.The bio-membrane coasted nanoparticles can avoid the phagocytosis of macrophages,target interesting tumor cells and can be used as an alternative and effective anti-tumor therapy.However,it’s worth nothing that the existing reports of biomimetic nanoparticles also have the shortcoming of low drug loading capacity and the inability to intelligently release drugs.How to further improve the performances of biomimetic nanoparticles is of great significance.To sum up,in view of the inefficiency of photodynamic therapy caused by the drawbacks of photosensitizer,such as low water solubility,low aggregation at tumor sites,low ROS yield,lack of targeting and easy quenching,we have designed and synthesised biodegradable glycosyl polymers with different structures and control materials to explore the advantages of branched/cross-linked sugar-based polymers combined with PDT and small molecule inhibitors as a means of anti-tumor.The combined mechanism of PDT’s damage and olaparib’s inhibition of DNA damage repairing can enhance the overall therapeutic effect while achieving high accumulation of therapeutic drugs in tumor sites through the glyco-based polymer dilivery system.Furthermore,tumor cell membranes are used as the biomimetic carrier to encapsulate the glycosyl polymers and achieve "homologous aggregation" through the specific proteins on the surface of tumor cell membranes to further increasing the therapeutic effect of the glycosylpolymer-based dual drug system.Materials and Methods:The design and preparetion of glycosyl polymer drug delivery systems with different structures and the exploration their structurefunction relationships.In this paper,a novel enzyme-sensitive chain transfer agent and crosslinking agent were prepared.On this basis,we constructed a degradable branch/cross-linked structure by reversible addition-fragmentation chain transfer polymerization(RAFT)polymerization.A relatively superior structure composition was obtined by adjusting the ratios of chain transfer agent,crosslinking agent and other monomers to ensure the polymers with appropriate molecular weight so that they can be effectively metabolized after degradation while avoiding the low solubility caused by over-crosslinking.Similarly,a linear structure glycosyl polymer and a branched/crosslinked structure poly N-(2-hydroxypropyl)methacrylamide(p HPMA)were prepared separately and used as control materials to explore the differences between these materials in the respects of physicochemical properties,biological characteristics and functions.A series of reseacher instruments and methods,such as dynamic light scattering,transmission electron microscopy,fluorescence spectroscopy ultraviolet spectroscopy were used to characterize the physical and chemical properties and structures of related compounds and polymers,and to compare the ROS generation rate,cytotoxicity,and tumor site aggregation Based on the differences in the structure and properties of the polymer photosensitizer,including physical and chemical properties and biological behavior in vivo and in vitro.The glycosyl polymer photosensitizer BSP which can effectively balance the π-π stowage between ROS production and olaparib loading was screened and glycosylpolymer-based dual drug system BSPO was synthesised.Finally,tumor cell membranes were encapsulated on the surface of BSPO to form CM-BSPO.With the help of the "homogeneous aggregation" characteristics of tumor cell membranes.The successful encapsulation of tumor cell membrane on the polymer surface was confirmed by modifying magnetic resonance contrast agent on the surface of the tumor cell membrane and and detecting with imaging experiments in vivo and in vitro.The in vitro and in vivo anti-tumor properties of tumor cell membrane coated glycosyl polymers drug delivery systemWe use the glycosyl branched/cross-linked PDT treatment system BSP selected in Chapter 1,the BSPO dual drug system after encapsulating olaparib,and the CM-BSPO coated with cell membranes to conduct a series of cell experiments,their differences in repect of cellular behaviors were studied through comparing of cellular uptake,cytotoxicity,apoptosis detection,cell cycle detection,ROS production rate detection,cell entry pathway detection and other methods.Cell cycle detection,single cell gel electrophoresis,DNA fragment immunofluorescence staining dnd DNA damage marker immunofluorescence staining were performed to investigate the differences of DNA damage among these compounds.In vivo anti-tumor therapeutic effect was studied using a mouse breast cancer model.A mouse subcutaneous 4T1 breast cancer model was established,and different treatment compounds were injected through the tail vein.The aggregation of each compound at the tumor site was monitored by a small animal in vivo imager.In addition,the real-time changes of tumor volume during treatment were monitored by manual measurement and 3.0 T magnetic resonance imaging instrument,and small animal computed tomography was used to observe the status of tumor metastasis in the lungs of mice after treatment.,CD31 immunohistochemical staining and Ki-67 immunohistochemical staining were used to observe the angiogenesis of tumor sits and the proliferation of tumor cells respectively.The study of the combined anti-tumor mechanism of PDT with olaparib using transcriptome technologyUsing transcriptome sequencing technology to study the combined antitumor mechanism of PDT and olaparib through bioinformatics analysis methods,and use Western Blot technology to study the expression of related proteins in cells of different treatment groups.Results:In the first part of the study,we successfully designed and synthesized three different structures of sugar-based polymer carriers BSP,BSh P and LSP,and p HPMA-based control material BSHP.The particle sizes of the three polymer carriers measured by dynamic light scattering(DLS)were 238 nm,164 nm and 374 nm,and the zeta potentials were-16.81 m V,-19.23 m V and-21.97 m V,respectively.Next,the differences between the fluorescence spectrum properties of the three polymer carriers,the efficiency of ROS generation,the killing effect of tumor cells,the concentration of tumor sites and the amount of olaparib encapsulation were studied.The experimental results show that under the same photosensitizer pyropheophorbide-a(Ppa)concentration,the fluorescence spectrum intensity of BSP is the highest,and the efficiency of ROS generation by BSP is also the highest by detecting ROS generation after light.The half-inhibitory concentrations(IC50)of the three polymer carriers of BSP,BSh P and LSP were 0.25 μg/m L,4.8 μg/m L and 0.91μg/m L,respectively.Finally,we compared the olaparib loading capacities of BSP,BSh P,LSP and the control material BSHP.The results show that the olaparib loading capacity of the four materials were 4.03%,2.17%,0.33% and0.13%,respectively.From this result,we screened out BSP as a carrier.By loading with olaparib,a dual-drug delivery system BSPO was prepared.In order to further enhance the anti-tumor effect,we wrap the surface of BSPO with the tumor cell membrane to form CM-BSPO macromolecules.The results of transmission electron microscopy showed that the outer layer of CM-BSPO was covered with a double-layer membrane structure,which indicate that BSPO was successfully wrapped by the cell membrane.In addition,we modified the MRI contrast agent on the surface of the membrane by bioorthogonal reaction,and observed that the signal of the polymer carrier wrapping the cell membrane was significantly enhanced by MRI,which once again demonstrated the successful wrapping of the tumor cell membrane on the polymer surface.In vitro experiments demonstrated that under the same Ppa concentration and light conditions,comparing with BSP,BSPO has higher cytotoxicity(IC50 is 0.43 μg/m L and 0.93 μg/m L,respectively),and more cells that produce apoptosis(5.39%±4.82% vs.14.49%±1.47%,p<0.01,n=3),the fluorescence intensity of dead cells in the staining of dead cells was also higher(45.6 vs.38.3,p<0.01,n=3),the proportion of S phase in the cell cycle increased(47.8% vs.26.7%),comet experiments showed that the proportion of DNA damage was higher(71.88%±6.72% vs.43.96%±11.95,p<0.01,n=30),and the expression of DNA damage related proteins marker was higher(33.98±5.70 vs.16.65±3.06,p<0.01,n=3),which is obviously related to the synergy of olaparib.These results indicated that olaparib can inhibit the activation of PARP,thereby inhibiting DNA repair and aggravating cell death.At the same time,CM-BSPO can be taken up by tumor cells more than BSPO and BSP,and the production of ROS in tumor cells has been significantly increased.Tumor cytotoxicity results found that the IC50 value was about 0.20μg/m L,and the number of apoptotic cells caused by the same light conditions also increased significantly(up to 40.11%).Through the study of its entry pathway,we found that,compared with BSP and BSPO,CM-BSPO can enter cells through clathrin-mediated endocytic pathways in addition to macropinocytosis and caveolin-mediated pathways.Experimental results showed that encapsulating olaparib can enhance the killing effect of photodynamic therapy on tumor cells,and the surface tumor cell membrane wrapping strategy can further enhance the therapeutic effect by increasing the uptake of the drug-loading system by tumor cells.The anti-tumor effects of photodynamic therapy combined with olaparib in different treatment groups of BSP,BSPO and CM-BSPO were studied used mouse breast cancer models.The results showed that the BSP + L,BSPO + L and CM-BSPO + L groups all showed good therapeutic effects after illumination.Among them,the CM-BSPO + L group had the highest tumor inhibition rate of 87.66%,the tumor inhibition rate of BSPO + L and BSP+L groups were about 81.34% and 69.55% respectively.In the BSPO + L group containing olaparib,the tumor volume was significantly reduced compared with the BSP + L group(p<0.01,n=5),probably it is because that olaparib can enhance the DNA damage of tumor cells.The tumor volume in the CM-BSPO+ L did not increase significantly during the whole treatmen period and was smaller than that of the BSPO + L after treatment.With significant difference between the two groups(p<0.01,n=5).The therapeutic effect of CM-BSPO +L was significantly improved,which may be related to the increase of the aggregation intensity in the tumor site.The changes of tumor volume were also detected by magnetic resonance imaging and the results indicated that the tumor volume in the CM-BSPO group was the smallest after the treatment,which was consistent with the manual measurement results.Lung metastasis was detected with computed tomography imaging and it did not observed in the CM-BSPO + L group mice,and the specimen tissues were also observed with naked eyes and no abnormal tissue was observed.In Chapter 4,we use transcriptome sequencing technology to study the combined anti-tumor mechanism of PDT and olaparib through bioinformatics analysis methods.The results of bioinformatics analysis indicated that the differential genes of the BSPO + L group vs.CON + L group were more obvious in the pathways of DNA damage,cell cycle,and apoptosis than the differential genes of the BSP + L group vs.CON + L group.In addition,Western Blot experiments showed that the expression of DNA damage-related proteins in the BSPO + L group was higher than that of the BSP + L group,in which H2 A.X/β-actin(0.99±0.14 vs.0.23±0.19,p<0.01,n =3),Chk1/β-actin(0.31±0.15 vs.0.27±0.09,p<0.01,n=3),Chk2/β-actin(0.47±0.08 vs.0.45±0.04,p<0.01,n=3)And Brca1/β-actin(0.54±0.07 vs.0.51±0.03,p<0.01,n=3).The overall data show that compared with pure photodynamic therapy,photodynamic therapy combined with olaparib can inhibit the repair of damaged DNA by inhibiting the activity of PARP enzyme,thereby enhancing DNA damage,and finally achieving synergistic enhancement of PDT antitumor effect.Conclusion:Taking advantages of tumor genome instability and the DNA damage characteristics of PDT,a glycosyl polymer-based drug delivery system loaded with photosensitizer and olaparib is constructed,and an anti-tumor strategy that combined DNA damage and repair inhibition is proposed to enhance the anti-tumor effect of drug delivery system.We designed and prepared new glycosyl polymers with different structures,and studied their structure-function relationship through physical and chemical characterization.On this basis,we screened out branched/cross-linked polymers that can take into account both ROS generation and PARP inhibitor encapsulation performance.The cross-linked structure make the glycosyl polymer carrier degradable,and the appropriate hydrophilicity and hydrophobicity,and the assembly structure can make the coupled photosensitizer have excellent optical properties,and is in favour of effective encapsulationof the small molecule inhibitor.The dual-drug system achieves the combined treatment effect of PDT and PARP inhibitors against DNA damage.In order to further increase the degree of polymer aggregation at the tumor site,the tumor cell membrane is wrapped on the surface to achieve "homologous aggregation" of the tumor site,and further enhance the anti-tumor effect.A series of research results such as tumor cell killing,cell entry pathway,animal anti-tumor and molecular mechanism show that the dual-drug system encapsulated by cell membrane has obvious therapeutic effect in the treatment of breast cancer.The tumor cell membrane-glycosyl polymer drug delivery strategy proposed in this study provides a new idea for the treatment of breast cancer. |
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