Design And Synthesis Of Novel Paclitaxel-loaded NO-donating Polymeric Micelle And Study On Its Antitumor Effect And Mechanisms

Objective:We designed and synthesized of NO donor(m PEG-PLA-NO)biodegradable polymer micelles encapsulating paclitaxel as a nano-drug delivery system(NO/PTX),aims to enhance the solubility,reduce toxicity and improve anti-tumor activity of paclitaxel.Acute toxicity studies,pharmacokinetics studies,drug nature evaluation and anti-tumor mechanism studies were conduct to provide experimental basis for the development of new drugs.Methods:Part 1 Preparation and characterization of paclitaxel-loaded NO-donating polymeric micelleThe m PEG-PLA-NO amphiphilic polymer were prepared by using furazan nitric oxide donors by the active anhydride method,and the nitric oxide donor paclitaxel containing paclitaxel in the core of the polymer micelles(NO/PTX)was prepared by the self-emulsification method.The structure of the NO/PTX was investigated in detail by using ¹H NMR.The morphology of NO/PTX was investigated by transmission electron microscope(TEM)and Malvern laser scattering particle size analyzer.The drug loading amount and the entrapment efficiency of NO/PTX was detected by the high performance liquid method(HPLC).The level of NO release abilities from PTX and NO/PTX were evaluated by griess method.Part 2 Acute toxicity study of paclitaxel-loaded NO-donating polymeric micelleThe acute toxicity for m PEG-PLA and m PEG-PLA-NO were investigated in 60healthy male SPF KM mice.Mice were injected the solutions via caudal vein at the dose starting with 800 mg/kg.Each metering group with a minimum of 6 mice.The acute toxicity for PTX and NO/PTX were investigated in 120 healthy male SPF KM mice.Mice were received a daily injection with Saline or 20,30,36.3,38.84,40.55,42.3 and 45 mg/kg of PTX or 60,80,120,141.72 and 160 mg/kg of NO/PTX respectively.The death wre Observed and recorded.The body weight was observed14 days,as well as the coat color,limb activity,eating,drinking,excretion,poisoning,death,etc.The median lethal dose(LD₅₀)and the maximum non-lethal dose(MNLD)were determined by the Graph Pad 6.Part 3 Study on the Targeting pharmacokinetics of paclitaxel-loaded NO-donating polymeric micelleThe pharmacokinetics and tissue distribution were studied in male KM mouse xenograft model established with H22 cells to delineate the disposition of the NO/PTX in vivo.The mice were injected with PTX(20 mg/kg calculated as paclitaxel)or NO/PTX(50 mg/kg calculated as paclitaxel)solution in saline via tail vein.Plasma and tumor were collected at 0,0.05,0.5,1,2,3,4,8,12 and 24 h.At every sampling time point,3 to 4 mice were anesthetized with ether and blood was collected via cardiac puncture from every mouse.Then,these mice were sacrificed,and all tumors were also collected.After centrifugation,about 100?L plasma from every mouse was collected and frozen.The concentrations of paclitaxel in plasma and in tumor were determined by HPLC and the concentrations of paclitaxel in heart,liver,spleen,lungs and kidneys were determined by HPLC-MS/MS with docetaxel as internal standard.The pharmacokinetic parameters were processed by non-compartmental analysis using the DAS 2.0 software package.Part 4 paclitaxel-loaded NO-donating polymeric micelle anti-tumor cells in vitro and its effect on P-glycoproteinCulture 12 kinds of tumor cell lines,SMMC-7721,SCG-7901,SW480,Sk-ov-3,A549,Bel-7402,HCT-116,MCF-7,Hela,HEPG-2,HT29 and NCI-H460.Using MTT assay to detect the inhibition rate of PTX and NO/PTX on tumor cells,and the IC₅₀was calculated.The experiment was repeated at least 3 times.Culture 12 kinds of tumor cell lines,SMMC-7721,SCG-7901,SW480,Sk-ov-3,A549,Bel-7402,HCT-116,MCF-7,Hela,HEPG-2,HT29 and NCI-H460.Western blotting was used to detect the expression of P-glycoprotein after PTX and NO/PTX intervention.Part 5 Study on the mechanism of action of paclitaxel-loaded NO-donating polymeric micelle against colon cancer and paclitaxel-resistant colon cancerPTX,NO/PTX or PTX Nano intervene human colon cancer HCT116 cells for 24hours in vitro,using Hoechest/PI staining,the number and morphology of cells was observed under fluorescence microscope;The influence of cycle was observed by flow cytometry;The expression of proteins related to cell apoptosis and proliferation and migration detected by Western blotting.Culturing human HCT116 cells and Taxol/HCT116 cells in vitro,and the differences in cell morphology between the two cells was observed under the inverted fluorescence microscope.The suppresion rate(%)of PTX and NO/PTX in HCT116and Taxol/HCT116 cells were detected by CCK8 assay and the half suppression rate IC₅₀(?g/m L)were calculated.Western blotting was used to detect the difference in the expression of P-glycoprotein between HCT116 and Taxol/HCT116 cells.Western blotting was also used to detect the expression of P-glycoprotein and??-Tublin protein in Taxol/HCT116 cells.Part 6 Study on the anti-hepatocellular Mechanism of action of paclitaxel-loaded NO-donating polymeric micelleHuman Bel-7402 cells were Cultured in vitro.The half suppression rate of PTX and NO/PTX on Bel-7402 cells were detected by CCK8 assay.Hoechst 33258staining,morphology and number were observed under the inverted fluorescence microscope.Establish H22 hepatocellular carcinoma model in vivo to evaluate the antitumor effect of PEG-PLA and m PEG-PLA-NO.Give corresponding drugs 3 times with an interval of 72 hours each time.The tumor volume and tumor inhibition rate(%)were calculated.Establish H22 hepatocellular carcinoma model in vivo to evaluate the antitumor effect of PTX,Genexol?-PM and NO/PTX.Give corresponding drugs 3 times with an interval of 72 hours each time.The tumor volume and tumor inhibition rate(%)were calculated.Culturing human Bel-7402 cells in vitro,and giving PTX and NO/PTX for 48hours,extracting Bel-7402 cell proteins,using kits to detect the levels of SOD,MDA and GSH-PX,Using Annexin V/PI double staining to observe the effect of PTX and NO/PTX on Bel-7402 cells apoptosis by flow cytometry.Western blotting was used to detect the expression of NO/PTX on iron death,pyrolysis,and endoplasmic reticulum stress related proteins.Part 7 Discriminantion of paclitaxel-loaded NO-donating polymeric micelle nature by metabonomicsForty SD rats were divided into blank group,PTX group,NO/PTX group,and PTX Nano group with 10 rats per group.It is administered once every three days for a total of seven administrations.The changes in body temperature and weight of rats were detected,Agilent 1290 tandem 6460 triple quadrupole mass spectrometer was used to detect rat urine metabolome.Orthogonal Partial Least Squares(OSC-PLS-DA)was used to analysis data for drug nature discrimination.Results:Part 1 Preparation and Characterization of paclitaxel-loaded NO-donating polymeric micelleThe m PEG-PLA-NO was prepared and synthesized.¹H NMR spectra of the polymer m PEG-PLA-NO was recorded on a 600 MHz spectrometer using deuterated chloroform(CDCl₃)as solvent and tetramethylsilane(TMS)as the internal standard.The PDI of m PEG-PLA-NO were determined to be 1.13 by gel permeation chromatography(GPC).The average diameter of NO/PTX micelles,determined by dynamic light scattering(DLS),was less than 50 nm.Transmission electron microscopy(TEM)investigation showed that the micelles were spherical and had sizes consistent with that of DLS measurements.The encapsulation efficiency of NO/PTX was 98.6%,and the paclitaxel drug loading of NO/PTX is 4.69%.The level of NO release abilities from NO/PTX were evaluated by Griess method,it's results show that compared with nitroglycerin and PTX,NO/PTX has a better release performance within 10 hours,and the release rate is the highest within 6 hours,reaching 66.8%.Part 2 Acute toxicity study of paclitaxel-loaded NO-donating polymeric micelleThe median lethal dose(LD₅₀)and the maximum non-lethal dose(MNLD)were determined by the CALC 2.0 software and the BLISS method.The MNLD of m PEG-PLA and m PEG-PLA-NO were 2000 mg/kg and 1500 mg/kg respectively,which indicated that both m PEG-PLA and m PEG-PLA-NO were nontoxic amphiphilic copolymer.The maximum tolerated dose LD₀of PTX was 36.3 mg/kg,the absolute lethal dose LD₁₀₀was 45 mg/kg.The maximum tolerated dose LD0 of NO/PTX is 80 mg/kg,the absolute lethal dose LD₁₀₀is 160 mg/kg.The LD₅₀of PTX and NO/PTX in KM mice were determined to be 39.9 mg/kg and 137.1 mg/kg respectively.The acute toxicity of NO/PTX reduced 3.43 folds than that of PTX.Part 3 Study on the Targeting pharmacokinetics of paclitaxel-loaded NO-donating polymeric micelleAfter pretreatment of serum samples,the endogenous substances in them do not interfere with the determination of paclitaxel and internal standard.Under these chromatographic conditions,the retention times of docetaxel and paclitaxel in plasma were 19.43 min and 22.08 min,respectively.The regression curve equation:Y=0.0618X-0.0648(R²=0.9986)(n=3),indicating that the mass concentration of plasma paclitaxel has a good linear relationship within 0.5?150?g/m L.The regression curve equation:Y=0.0783X-0.2581(R²=0.9961)(n=3),indicating that the mass concentration of paclitaxel in tumor tissue has a good linear relationship within1?150?g/m L.The concentration of paclitaxel was gradually diluted from high to low,and the lowest detection limit was 0.25?g/m L,the lowest quantification limit was 0.5?g/m L for plasma and 1?g/m L for tumor tissue.The accuracy was between 85%and115%.The precision RSD is less than 15%.The recovery rates of plasma extraction were 103.2%,99.4%,95.7%and the recovery rates of tumor extraction were 112.0%,95.2%,and 95.5%,respectively.The recovery rate of internal standard extraction was92.55%.The DAS 2.0 pharmacokinetic program was used to fit the plasma concentration-time to a compartment model,and the results showed that the metabolism of mice after the tail vein injection of paclitaxel in vivo conformed to the two-compartment model.After the administration of 50 mg/kg of NO/PTX,the peak plasma concentration(C_max)obtained was 105.2?g/m L,and after the administration of 20 mg/kg of PTX,the C_maxwas 71.7?g/m L.The elimination half-life of PTX(t_1/2?)is 1.5 h,and the t1/2?of NO/PTX is 1.7 h.The areas under the plasma concentration-time curves of NO/PTX and PTX(AUC_(0-?))were 128.1?g.h/m L and86.9?g.h/m L/kg,respectively.The AUC_(0-?)of NO/PTX is 1.35 times that of PTX.After administration,paclitaxel is widely distributed in most tissues.Among them,the highest concentration of paclitaxel was clearly found in tumors.At all time points,the concentration of paclitaxel in the NO/PTX group was higher than that in the PTX group,and reached the maximum paclitaxel concentration at 3 h.In all tissues and organs except tumors,the concentration of paclitaxel is low.The highest paclitaxel concentration in all tissues and organs is less than 10?g/m L,most of which are eliminated after 24 hours.After administration(after 3 minutes),the distribution order of PTX in tissues and organs is:tumor>kidney>lung>heart>spleen>liver,and the distribution order of NO/PTX in each tissue and organ is:tumor>lung>kidney>heart>spleen>Liver.Part 4 paclitaxel-loaded NO-donating polymeric micelle anti-tumor cells in vitro and its effect on P-glycoproteinAnti-tumor in vitro showed that the IC₅₀values of NO/PTX in human breast cancer cell MCF-7,human gastric adenocarcinoma SCG-7901,human colon cancer cell SW480,human colorectal adenocarcinoma HCT-116,and human liver cancer cell Bel-7402 are lower than PTX,indicating that NO/PTX have a better anti-tumor effect than PTX.The results of WB showed thatl at concentration of 0.01?g/m L in 7402 cells,SW480 cells,Hela cells,SMMC-7721 cells,HCT-116 cells,Hep G2 cells,MCF-7cells,HT29 cells,SCG-7901 cells,the expression of P-glycoprotein in NO/PTX group was lower than that in PTX group.It indicates that NO/PTX enhances the anti-tumor effect of paclitaxel may be related to the inhibition of P-glycoprotein.Part 5 Study on the mechanism of paclitaxel-loaded NO-donating polymeric micelle against colon cancer and paclitaxel-resistant colon cancerFirst of all,through Hoechst/PI staining,it can be observed that NO/PTX has a better anti-colon cancer HCT116 effect than PTX.Through cell cycle experiments,we detected that NO/PTX at 0.001?g/m L and PTX 0.01?g/m L has a greater inhibitory effect on the G₂M phase of cells than PTX(NO/PTX inhibition rate is 39.6 and 50,PTX is 30.2 and 36.3).Western Blot detection results showed that compared with the control group,the NO/PTX group was more able to reduce the expression of P-gp protein in HCT116 cells.Western Blot detection also showed that compared with the control group and PTX group,NO/PTX can increase the expression of pro-apoptotic protein BAX,reduce the expression of anti-apoptotic protein BCL-2,increase the ratio of BAX/BCL-2,and increase the Cleaved Caspase 3 Expression,showing better anti-human colon cancer HCT116 results than the commercially available paclitaxel injection PTX.Compared with the blank group and the PTX group,the paclitaxel polymer nanomicelle PTX Nano wich without nitric oxide donor can also reduce the P-gp protein and increase the expression of Cleaved Caspase 3 in HCT116 cells,but reduce the P-gp protein and increase the Cleaved Caspase The effect of 3 expression is not as good as NO/PTX.PTX Nano proves that m PEG-PLA polymer micelles have certain advantages in anti-tumor preparations.It also shows that the anti-tumor effect of NO/PTX is not only due to the advantages of preparations,but the result of double blockade of paclitaxel and nitric oxide donors.Secondly,the cell scratch test showed that NO/PTX has better anti-HCT116 cell proliferation and migration ability than the blank group and PTX group.Western Blot detection results showed that NO/PTX decreased the expression of Vimentin protein,increased the expression of ZO-1 protein,and decreased the expression of?3-Tublin and phosphorylated GSK-3?.PTX Nano showed better reduction of Vimentin protein expression and increased ZO-1 protein expression ability than NO/PTX,but it did not show the effect of reducing?3-Tublin and phosphorylated GSK-3?expression.Furthermore,the advantage of NO/PTX against colon cancer tumors is also reflected in its better antitumor effect on colon cancer drug-resistant cells.We first studied the cell morphology,drug resistance coefficient and P-gp protein expression of Taxol/HCT116 cells resistant to paclitaxel.It is confirmed that Taxol/HCT116 cells are more resistant to paclitaxel than HCT116 cells.The CCK8 experiment analyzed the effects of PTX and NO/PTX on the proliferation of taxol/HCT116 cells,and the results showed that the IC₅₀value of NO/PTX(IC₅₀:1.2±0.4?g/m L)was significantly lower than that of PTX(IC₅₀:5.6±1.9?g/m L),NO/PTX has an IC₅₀value that is 4.66times lower than that of PTX,Indicating that NO/PTX has a more significant anti-colon cancer drug resistance effect than PTX.Western Blot detection results showed that compared with the blank group and PTX group,NO/PTX significantly reduced the expression of P-gp protein and?3-tublin protein,the key proteins of paclitaxel resistance.Part 6 Study on the anti-hepatocellular mechanism of paclitaxel-loaded NO-donating polymeric micelleFirst,CCK8 assay showed the anti-Bel-7402 liver cancer cells effects of NO/PTX and PTX.The IC₅₀of PTX is 7.8±0.4?g/m L and the IC₅₀of NO/PTX is3.7±1.1?g/m L.These results indicate that NO/PTX is more toxic to Bel-7402 cells than PTX.Using Hoechst 33258 staining method to detect cell apoptosis have the same conclusion.Secondly,in H22-bearing liver cancer mouse model,the experimental results showed that the polymer m PEG-PLA did not show any anti-tumor response,and a slight anti-tumor effect was observed in the m PEG-PLA-NO group.Significant anti-tumor activity was observed in the PTX,Genexol^?-PM and NO/PTX groups.The PTX,Genexol^?-PM and NO/PTX groups showed comparable anti-tumor activity at low doses(10 mg/kg)(inhibition rates were 39%,36%and 41%,respectively).In addition,when the dose reached 15 mg/kg,the anti-tumor effect of NO/PTX was significantly stronger than that of the PTX and Genexol^?-PM groups(PTX,Genexol^?-PM and NO/PTX groups were 53%,41%,and 67%,respectively),proving that the synergistic effect of NO and paclitaxel enhances anti-tumor activity.Furthermore,we illustrated that NO/PTX inducing liver cancer cell death was mediated by ferroptosis,pyroptosis,endoplasmic reticulum stress(ERS)and apoptosis-associated networks.The mechanism of ferroptosis was confirmed by increasing the level of reactive oxygen species(ROS)and malondialdehyde,and by decreasing the level of glutathione peroxidase,superoxide dismutase and glutathione peroxidase-4.The use of ferroptosis inhibitor ferrostatin-1 or ROS inhibitor N-acetyl cysteine could reduce the anticancer activity of NO/PTX.Moreover,NO/PTX up-regulated the expression of caspase-1 and down-regulated inflammatory cytokines IL-1?,which are the key proteins of pyroptosis.In addition,NO/PTX up-regulated the expression of a series of modulators such as calnexin,inositol-requiring enzyme 1a(IRE1a),glucose-regulated protein 78(GRP78),activated caspase-7,cleaved caspase-3 and decreased B-cell lymphoma 2(BCL-2),Nuclear NF-?B,which induced endoplasmic reticulum-mediated stress and apoptosis against Bel-7402.More importantly,we demonstrated the enhanced anti-tumor effect of NO/PTX might be related to down-regulation of multidrug resistance transporter P-glycoprotein,?3-tubulin,sensitizing paclitaxel chemotherapy.Finally,we used flow cytometry to detect the apoptosis rate of Bel-7402 cells after 48 hours of paclitaxel intervention.The apoptosis rate of NO/PTX at each concentration is higher than that of PTX,indicating that NO/PTX has a better anti-Bel-7402 cell effect than PTX.Part 7 Discriminantion of paclitaxel-loaded NO-donating polymeric micelle nature by metabonomicsThe PTX group,NO/PTX group and PTX Nano group were all located in the cold medicine area,and there were no significant difference.It shows that PTX,NO/PTX and PTX Nano belong to cold drugs.Preparation of paclitaxel polymer micelles or paclitaxel polymer micelles connected with nitric oxide donors through preparation methods did not change the drug nature.Conclusion:1.NO/PTX is a paclitaxel-loaded NO-donating polymeric micelle with a particle size of 30±0.58 nm,spherical shape and uniform distribution.The encapsulation rate of NO/PTX is 98.6%,and the paclitaxel drug load of NO/PTX is 4.69%.NO/PTX has good monoxide Nitrogen release performance.Both the drug-carrying materials m PEG-PLA and m PEG-PLA-NO have good safety.NO/PTX is well tolerated,and the maximum tolerated dose is 2.2 times that of PTX.NO/PTX is mainly distributed in tumor tissues,and the drug concentration in tissues and organs is low.2.The anti-tumor effect of NO/PTX on MCF-7,SCG-7901,SW480,HCT-116,Bel-7402 cells is better than domestic paclitaxel injection PTX,and its enhancement of paclitaxel anti-tumor effect is related to the inhibition of P-glycoprotein expression.3.Compared with PTX,NO/PTX has better anti-colon cancer HCT116 effect and anti-HCT116 cell proliferation and migration ability.This anti-tumor effect not only has the advantages of polymer micelles,but also has the advantages of paclitaxel and nitric oxide donors.Double blocking is related.Compared with PTX,NO/PTX has better anti-paclitaxel resistance than PTX.The anti-paclitaxel resistance effect of NO/PTX is related to the inhibition of P-gp and?3-tublin protein expression by NO/PTX.4.The death of liver cancer cells induced by NO/PTX is mediated by iron death,pyrolysis,endoplasmic reticulum stress and apoptosis-related networks.Inhibition of iron death can significantly reduce the toxicity of NO/PTX to Bel-7402 cells.High concentration of NO/PTX causes the main death mode of Bel-7402 cells to be apoptosis,while pyrolysis occurs in low concentration of Bel-7402.5.PTX,NO/PTX and PTX Nano are all cold medicines.Preparation of PTX Nano or NO/PTX connected to a donor by means of preparations did not change the properties of the medicine.