OHA Modified Polymeric Micelle With ATP-sensitivity To Enhance Tumor Therapy Efficiency

Objective:Adenosine triphosphate(ATP)is a complex organic chemical that provides energy to drive many processes in living cells,e.g.muscle contraction,nerve impulse propagation,and chemical synthesis.ATP is the most direct functional substance in life,it exists extensively in human body,and here is a significant difference in intracellular and extracellular concentrations.We have discovered an aptmer that responds to the difference in intracellular and extracellular ATP concentrations,and combines it with the corresponding cDNA to form a duplex(DNA)rich in GC fragments,whichcan deliver doxorubicin into cells while responding to ATP concentration differences.In order to better deliver this DNA duplex with ATP aptemer,we synthesized a polyethylene diamine(PEI)lauric acid(LA)polymer micelle as a carrier to deliver doxorubicin-loaded composed ofDNA duplex with ATP aptemers.To provide active targeting of the drug-loaded micelles while reducing toxicity,an oligomeric hyaluronic acid(OHA)layer is coated on the drug-loaded micelles.This structure is both passive and active targeting,and has environmental sensitivity,and responds to the concentration difference inside and outside the cell to achieve targeted drug delivery.Methods:(1)The ATP aptamer is mixed with its corresponding cDNA to form an ATP-sensitive double helix structure.It is mixed with a certain ratio of doxorubicin hydrochloride solution to form an ATP-sensitive double helix structure loaded with doxorubicin.Mixing it with different concentrations of ATP solution and observe the response.(2)Synthetic polyethylene diamine lauric acid polymer micelles(PEI-LA)were characterized by 1H-NMR,and the critical micelle concentration and particle size potential were measured to observe the morphology.(3)Evaluation of micelle safety using MTT.(4)The adsorption of polymer micelles on drug-loaded ATP-sensitive double helix was investigated by agarose gel electrophoresis.(5)Examine the release of drug-loaded micelles in different intracellular ATP concentrations.(6)Investigate the uptake of the drug-loaded micelles by the cells and the distribution of the cells after entering the cells.The cytotoxicity of drug-loaded micelles and the apoptosis of cells after administration were investigated.(7)The permeability of drug-loaded micelles was examined by a 3D tumor sphere model.(8)Establish subcutaneous and orthotopic lung cancer models,and use Dir-labeled polymer micelles to investigate the in vivo distribution of polymer micelles using small animal in vivo imaging techniques.(9)Construction of nude mice in situ lung cancer and subcutaneous tumor models,respectively,given saline,DOX,oligo-hyaluronic acid-coated drug-loaded micelles,no oligo-hyaluronic acid-coated drug-loaded micelles for treatment,through small Animal in vivo imaging was used to observe the fluorescence changes of the orthotopic lung cancer model and the subcutaneous tumor size changes to investigate the drug efficacy,monitor the body weight changes of mice,and evaluate the quality of life of mice.Finally,the experimental animals were dissected,and the tissues were taken out for section staining to observe the tumor necrosis.Results:(1)ATP-sensitive loadable DOX double helix structure was formed.The optimum loading ratio was determined when the ratio of DOX to ATP adapter was 3:1 by detecting the fluorescence intensity of DOX.With the increase of ATP concentration,DOX increased gradually,and the structure was ATP sensitive.(2)PEI-LA was synthesized and its structure was verified by 1H-NMR.The critical micelle concentration was determined and the polymer was determined as micelle.The average particle size was(188.6+3.86)nm and the PDI was 0.280.After OHA coating,the average particle size was(152.1+0.5)nm and PDI was 0.110.The morphology of the micelles was observed by transmission electron microscopy(TEM).The appearance of the micelles was round and uniform.After coating the OHA layer,an obvious outer layer could be seen at the edge.(3)Through MTT experiments,the polymer micelles with ATP-sensitive double helix adapters but without DOX were less toxic and safe to cells.(4)When the ratio of ATP aptamer double helix to PEI-LA micelle is 60:1,the optimal adsorption ratio is achieved.(5)When the cells were placed at 4? and 37 ?,the micelles in the ATP-rich environment were more released than the ATP-inhibited environment,indicating that the ATP-sensitive double-helical aptamer was adsorbed.The drug micelles do have ATP concentration responsiveness.(6)Flow cytometry and laser confocal microscopy were used to investigate the uptake and subcellular localization of the drug-loaded micelles.The results showed that the coating of OHA could improve the targeting of the carrier,increase the uptake,and have lysosomes.Escape phenomenon.The drug-loaded micelles coated with the OHA layer are more cytotoxic than the uncoated and DOX drug substances.At the same time,the uptake of DOX by cells was investigated.The drug-loaded micelles coated with OHA had the best intake and could enter the nucleus faster.Apoptosis assays have shown that administration of oligo-hyaluronic acid-coated drug-loaded micelles can accelerate cell entry into the apoptosis program.(7)3D tumor sphere sections showed that the drug-loaded micelle coated with OHA was more penetrating and DOX was the weakest.(8)Small animal in vivo imaging experiments show that micelles have good targeting to both subcutaneous and orthotopic lung cancer.(9)The nude mice inoculated with orthotopic lung cancer showed bioluminescence change of the tumor site after administration,and the bioluminescence of the drug-loaded micelle coated with OHA was weakened and the area was weakened,which was the most effective compared with other groups.it is good.At the same time,the body weight of the mice was not significantly reduced,and there was no obvious organ damage in the HE-stained tissue sections.Conclusions:ATP-sensitive drug-loaded micelles can quickly respond to the difference of ATP concentration inside and outside the cell to achieve targeted drug release.At the same time,PEI-LA micelles can adsorb ATP adapter double helix and provide carrier for its entry into the cell.OHA coating can reduce the toxicity of carrier and make it have active targeting.The results show that ATP-sensitive polymer micelles coated with OHA and loaded with DOX have good targeting ability and can be used as potential new sensitive nano-preparations.