| Gene therapy is a new therapeutic strategy for gene related diseases through gene supplementation or modification.However,whether in gene therapy strategies based on CRISPR/CAS gene editing technology or the introduction of foreign gene fragments(DNA or RNA),the lack of a safe and efficient gene delivery system has been one of the main bottlenecks limiting its clinical application.Compared with viral vectors,non-viral vectors have the advantages of easy preparation,nonimmunogenicity,and large gene loading capacity.Therefore,such as cationic liposomes,cationic polymers,and nanoparticles are used as gene delivery vectors.However,these non viral vectors have shortcomings such as high cytotoxicity and poor biocompatibility.Quantum dots(QDs),as a newly emerging nanomaterial,have been widely studied by scientists due to their advantages and potential in biological imaging,especially in cell imaging.In recent years,low toxicity non-metallic quantum dots,represented by silicon quantum dots(SiQDs),have made significant progress.However,the high surface activity and weak DNA loading ability of quantum dots have greatly limited their use as gene carriers.In order to deeply study the potential of SiQDs as gene carriers,this paper designed a biomimetic gene delivery system based on cell membrane encapsulated quantum dots and designed experiments to evaluate its potential as a gene carrier.The specific work is as follows:(1)In order to improve the DNA loading capacity and transfection efficiency of SiQDs,PEI templated SiQDs samples were designed and synthesized.Subsequently,cell membrane encapsulation strategies were used to improve their biocompatibility.The specific work is as follows: Using 3-aminopropyl-trimethoxysilane(APTMS)as the basic raw material,SiQDs with good water solubility were prepared by hydrothermal reduction.Subsequently,PEI with molecular weights of 10 k and 25k(PEI10k and PEI25k)were selected for template modification of quantum dots to obtain PEI10k-SiQDs(PS10k)and PEI25k-SiQDs(PS25k).Characterization of the samples showed that PS10 k and PS25 k were successfully synthesized.Subsequently,RBMC-SiQDs/DNA(Mc-SiQDs/DNA),RBMC-PS10k/DNA(Mc-PS10k/DNA),and RBMC-PS25k/DNA(Mc-PS25k/DNA)membrane coated composites were prepared by red blood cell membrane(RBCM)encapsulation and co extrusion for three samples(SiQDs,PS10 k,and PS25k).The characterization experiment results showed that the samples were spherical nanoparticles,and the hydrated particle size of the samples after extrusion was maintained at about 200 nm,The cytotoxicity test results showed that the cytotoxicity of SiQDs significantly increased after adding PEI,but it was relieved after being coated with red blood cell membrane.The survival rates of293 T and He La cells were above 80%.In vitro ingestion experiments showed that the samples could be successfully absorbed by 293 T and He La cells;The results of in vitro transfection experiments showed that the optimal transfection efficiency of McPS25k/DNA was 77.32% in 293 T cells at a mass ratio of 4/0.75/1,which was higher than 24.41% in Mc-SiQDs/DNA at a mass ratio of 2/4/1,and 70.34% in McPS10k/DNA at a mass ratio of 4/1/1.The transfection efficiency of the three samples was higher than that of the samples without membrane coating under the same mass ratio,and the results were still expressed in He La cells,The above experimental results indicate that Mc-PS25k/DNA has the potential to be developed as a safe and efficient gene vector.(2)In order to solve the biosafety problems caused by the introduction of PEI,silicon quantum dot nanoclusters(SiNC)were prepared by using disulfide bond graft modification of SiQDs with good biosafety.Subsequently,three different sources of cell membrane encapsulation were used to improve the biosafety of the sample and evaluate its potential as a gene carrier.The specific work is as follows: Using 3,3-dithiodipropionic acid(Dip A)to bridge the amino groups on the surface of SiQDs through an amidation reaction to obtain silicon nanoclusters(SiNC).Through characterization experiments,it was proved that the synthesis of SiNC samples was successful.Subsequently,in order to further improve the stability and in vitro targeting ability of the samples,Three different cell membranes were used to encapsulate samples(SiNC/DNA)with the best mass binding ratio of SiNC to DNA,and liposome extrusion was performed to obtain SiNC/DNA samples(RBCMSiNC/DNA,He La-MSiNC/DNA,and RAW-MSiNC/DNA)encapsulated by red blood cell membrane(RBC-M),He La cell membrane(He La-M),and RAW cell membrane(RAW-M).Characterization experiments showed that the samples were successfully prepared,and the appearance was circular or quasi circular,In vitro cell experiments showed that the cell survival rate of SiNC samples did not significantly decrease compared to SiQDs,and the biological safety was better than that of PEI templated quantum dots.In vitro uptake experiments showed that all three membrane coated samples could be ingested by 293 T and He La cells;In vitro cell transfection experiments showed that the maximum transfection efficiency of the three samples in293 T was similar to the maximum transfection efficiency of the samples in Chapter 2.At the same time,the transfection efficiency of the He La MSINC/DNA complex in He La cells(44.92%)was significantly better than the other two samples(20.08% and32.98%).In comparison,the uptake and transfection efficiency of the RAW group samples in cancer cells were also better than those of the RBC group,This indicates that the addition of tumor cell membrane and immune cell membrane can make the sample exhibit better biological targeting.In addition,comparing the results of transfection experiments on red blood cell membrane coated PS25 k and SiNC samples,it can be found that the transfection efficiency of RBC-MSiNC/DNA has been significantly reduced after the removal of PEI to improve the biological safety of the sample,but the transfection efficiency of cells coated with He La and RAW cell membranes has been significantly improved,especially in He La MSiNC/DNA samples,compared with the former(Mc-PS25k/DNA)in 293 T cells,there is no significant difference in transfection efficiency,However,the transfection efficiency in He La cells is superior to that of Mc-PS25k/DNA.In summary,The He La cell membrane coated SiNC/DNA membrane coated composite(He La MSINC)is expected to be developed as a safe,efficient,and targeted gene delivery system.tumor cell membrane coated SiNC/DNA complexes are expected to be developed as a safe,efficient,and targeted gene delivery system. |
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