| With the development of nanotechnology,nanoparticles have been widely used in biomedical fields for labeling and tracking,drug delivery,cell separation and imaging and some progress has been made.Nanoparticles need to be delivered to a specific site in biological system to play their role,but designing nanoparticles that have the ability of efficient targeting inside cells or animals is still an important challenge.When nanoparticles interact with living cells or animals in biological system,the nanoparticles need to overcome various biological barrier,including cell membranes,crowded cytoplasm,vesicle capture,non-specific binding,organelle membrane and other potential barriers in live cells or animals etc.Therefore,designing novel nanoprobes that can break through various biological barriers and enable specific targeting is an urgent problem to be solved.In this thesis,we designed two kinds of functionalized nanoparticles,CdSe/ZnxC1-xS quantum dots(QDs)and superparamagnetic iron oxide nanoparticles(SPIONs),aiming to solving the problem of nanoparticles delivery in biological systems,including targeting delivery of nanoparticles in live cells and delivery in live animals brain.The main research contents are as follows:(1)This part is mainly about synthesis and basic application display of QDs,mainly preparing for the follow-up research content.CdO was used as the cadmium source.Zn(NO3)2·6H2O was used as the zinc source,ODE was used as the reaction solvent,and selenium powder(Se),sulfur powder,TOP and steric acid are added to synthesize CdSe/ZnxCd1-xS quantum dots.Quantum dots with emission wavelengths from 516 to 650 nm can be obtained by controlling the reaction conditions(reactant amount,reaction time and reaction temperature).We modified quantum dots by thioglycolic acid(TA)through ligand exchange.RGD peptides were coupled with quantum dots modified by TA.The RGD coupled quantum dots(QD-RGD)have good stability,and the cell viability results showed low toxicity.By co-cultivating U87MG and HeLa cells with RGD coupled quantum dots,U87MG cells were specifically targeting by QD-RGD.(2)Intracellular targeted delivery of quantum dots with extraordinary performance enabled by novel nanomaterial design.In this design,unlike the ligand exchange method used in first part,the original surface modification of quantum dots was replaced by a small proportion of ligands,and then coupled with the cell penetrating peptides.An important feature distinguishing new nanoprobe from traditional water-soluble QDs is to maintain a large proportion of hydrophobic surface structures on the nanoscale,adding cosolvent to help the final sample(cS-bQDs-Tat)to disperse.The changes of dynamic particle size,zeta potential and fluorescence performance during the preparation of cS-bQDs-Tat were investigated.XPS and FTIR were used to analysis surface modification of cS-bQDs-Tat.The cS-bQDs-Tat was co-cultured with different kinds of cells(including HeLa,MCF-7,Hep G2 and 3T3 cells).The results revealed that cS-bQDs-Tat could be almost perfectly targeted to the nucleus without causing damage to the cells.(3)Intracellular specific targeted delivery mechanism study of novel nanoprobe(cS-bQDs-Tat).We have studied and analyzed specific targeted delivery mechanism in live cells from the characteristics of the nanoprobe(including particle size,type and amount of cosolvent,and ligand ratio,respectively)and the way that nanoprobe entered the cell.In addition,the integrity of the cell membrane and vesicle membrane during cS-bQDs-Tat delivery to cell nucleus were investigated.The laser confocal microscopy was used to tracking single QD in live cell,the results were used to analyze dynamics of single QD and pair-correlation function(pCF)in the process of targeting delivery.The movement type and vesicle escape of QD in live cell were studied.This gave quantitative information on the intracellular movement of cS-bQDs-Tat.The results showed that the cS-bQDs-Tat could escape from the vesicle and target to the nucleus without destroying the vesicle.(4)Study the ability of novel nanoprobe cS-bQDs-Tat(abbreviated as SDot-Tat)deliver drugs in live cell and in tumor model.We investigated the ability of cS-bQDs-Tat loading of doxorubicin and their ability of delivery doxorubicin.During the delivery of doxorubicin(DOX),SDots-Tat and DOX represented a high colocalization ratio and could be almost perfectly targeted to the interior of the nucleus.Since the mechanism of doxorubicin killing cancer cells was to inhibit the synthesis of DNA or RNA in the nucleus and this new nanoprobe design had the ability to target to the nucleus efficiently,therefore they had a stronger killing effect on cancer cells than free doxorubicin with the same concentration of doxorubicin.At the same time,we also researched the delivery of this new type of probe for large molecular weight biomolecules.The results showed that when loaded with 330,000 molecular weight ova,it still had the ability of tatgeting to the nucleus.At the same time,an in vivo KB tumor model was constructed to study the ability of SDot-Tat-DOX which penetrated the skin and delivered inside the tumor tissue by transdermal administration.The results showed that could penetrate the skin into the tumor tissue.The delivery and targeting ability of SDot-Tat-DOX in tumor tissue far exceeded the water-dispersed QD-Tat-DOX prepared by conventional methods,demonstrating the potential of the novel nanoprobe used for targeted delivery at the animal level.(5)Designing superparamagnetic iron oxide nanoparticles(SPIONs)that can be delivered to the brain of animal to enhance transcranial magnetic stimulation(TMS).TMS is a noninvasive and clinically approved method for treating neurological disorders.But the relatively weak intracranial electric current induced by TMS is an obvious inferiority,which can only produce limited treatment effects in clinical application.Therefore,we designed superparamagnetic iron oxide nanoparticles(SPIONs)coated with carboxylated chitosan and poly(ethylene glycol)and applied to transcranial magnetic stimulation,this is the first time that the effect of clinical brain stimulation has been enhanced with nanomaterials in the brain.To facilitate the nanoparticles' crossing of the blood-brain barrier(BBB)and delivering to the brain regions,an external permanent magnet was attached to the head of rats before intravenous injection of SPIONs.The electrophysiological test showed that the maximum MEP amplitude recorded in an individual rat was significantly higher in SPIONs+magnet group than that in saline group(5.78±2.54 vs 1.80±1.55 mV,P=0.015).The biochemical test detected that in the M1 region,the number density of C-fos positive cells in the SPIONs+magnet group was 3.44 folds of that in the saline group.These results suggest that intravenously injected SPIONs could boost the effects of TMS.In the above research,novel QD probe was successfully designed to overcome the intracellular biological barrier,the novel design enables efficient targeted delivery with the nucleus as the target in cells,and its targeting mechanism and intracellular motion were analysized.The novel nanoprobes had good drug delivery capabilities both in cells and in animal tissues.In addition,we applied magnetic nanomaterials to magnetic stimulation technology for the first time to enhance the effect of transcranial magnetic stimulation. |
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