| At present,cancer is still a major threat to human health and life safety with high lethality,and its occurrence and development can cause both huge physical and psychological trauma to patients.Chemotherapy is still the main strategy in clinical cancer treatments.Though the application of various anti-tumor drugs could inhibit the growth of tumor cells,they could also cause certain damage to the normal tissues inside the human body.As a drug delivery method with high-security,nano drug delivery systems have gained extensive attention in the field of cancer treatment,which could achieve long circulation of the drug.The system could also enhance the drug stability,realize the sustained drug release and improve the pharmacokinetic and pharmacodynamic performances of original drugs in vivo.However,it is still a huge challenge to design a nanoplatform with the properties of active tumor targeting,tumor microenvironment-responsive drug release,strong safety,as well as combined therapy.The self-assembled nanoparticles with polysaccharide as skeleton manifested high biocompatibility,low toxicity and biodegradability and have been extensively studied in the delivery of anti-tumor drugs.The hydrophilic organic material of chondroitin sulfate(CS)always worked as the backbones in polymeric nanoplatforms,and showed favorable active targeting ability to tumor cells.Rhein(Rh)not only showed good anti-tumor and anti-metastasis ability,but was firstly discovered as an effective sonosensitizer in sonodynamic therapy(SDT)against tumors.Therefore,in this subject,several environmentally responsive drug delivery systems were designed and constructed with CS as the hydrophilic skeleton and various small molecules including Rh,which worked as the hydrophilic and hydrophobic groups,respectively.The nanoplatform could not only release the encapsulated drugs in the presence of high concentration of reduced glutathione(GSH)in tumor tissues quickly and completely,realizing good tumor targeting and specific tumor suppression effect,but also achieve SDT via ultrasonic intervention.Therefore,the combined therapy would be used to strengthen the tumor suppressor effect.This project mainly carries on the research from two parts.In the first part,the amides of CS were constructed by linking with the adipic acid dihydrazide(ADH).And the hydrophobic small-molecules of Rh and β-lipoic acid(LA)were separately grafted to the amino terminals on CS backbones.Therefore,the CS-ADH-Rh-LA conjugates were synthesized.As a crosslinking agent,LA could be crosslinked under specific conditions and formed the intermolecular disulfide bonds within the nanoparticles,achieving the redox-sensitive drug release and enhancing the stability of the nanosystem.Combining the original anti-tumor and SDT properties of Rh,as well as the function of loaded antineoplastic drug,the nano drug delivery system was expected to achieve efficient tumor inhibition ability through the chemo-sonodynamic combined therapy,and the system could inhibit the tumor metastasis and invasion to a certain extent.Studies found that the nanoplatform could achieve good chemo-sonodynamic combined therapy,but the hypoxia character of tumor microenvironment still limited the therapeutic effect of the nano drug delivery system.Therefore,a multifunctional nano drug delivery system with oxygen-carrying ability was further constructed on this basis.Based on the first part,the second part was intended to graft Rh and perfluorooctanoic acid(PFC)to the CS backbone successively with the help of cystamine(cys),and the new redox-sensitive and oxygen-carrying amphipathic CS-cys-Rh-PFC polymer was synthesized.After replacing the connecting arm,the degree of substitution of Rh increased,and the anti-tumor effect was improved.Besides,the grafting of cys could increase the introduction rate of small hydrophobic molecules,therefore acquiring the self-assembled nanoparticles with smaller size and higher stability.The addition of PFC could improve the hypoxic conditions at the tumor sites,and the sufficient oxygen molecules are also regarded as a necessary prerequisite for increasing the yield of reactive oxygen species(ROS)molecules produced by SDT.Based on this hypothesis,the stability,drug-carrying ability and oxygen-carrying capacity of the nanoparticles were evaluated,and the cell and animal experiments were conducted to evaluate the efficacy of the nanoparticles in improving hypoxia in vivo and the combined chemo-sonodynamic therapy against tumor.Significant differences between the data during the experiment were obtained by SPSS 26.0 software after analysis,and the corresponding results were presented in the form of mean ± standard deviation(SD).The main research methods and experimental results are exhibited as follows:1.The construction of docetaxel(DTX)-loaded redox-responsive crosslinked CS-ADH-Rh-LA nanoparticles and study of their chemo-sonodynamic combined therapy against tumorThrough the amidation reaction,Rh was connected to the hydrophilic backbone containing carboxylic terminals on CS via ADH as the linking arm,and the CS-ADH-Rh polymer was formulated.Then by controlling the feeding ratio of Rh and CS,the CS-ADH-Rh polymer with the highest degree of Rh substitution(3.15)was obtained for subsequent experiments.Then,through another amide reaction,LA was reacted with CS-ADH-Rh at different feed ratios and connected to the amino groups in ADH which were not occupied by Rh in different feed ratios.The degree of substitution of LA ranged between 5.14%and 7.38%,while the corresponding critical aggregation concentration(CAC)values varied between 33.74 and 66.54 μg/mL.The CS-ADH-Rh-LA polymer could form self-assembled nanoparticles under the stimulation of probe ultrasound,which were regarded as the non-crosslinked nanoparticles(NC-NPs).While LA could form intermolecular disulfide bonds after damaging the intermolecular disulfide bonds under the catalysis with appropriate amount of reducing substances,and the redox-sensitive crosslinked nanoparticles(C-NPs)were synthesized with improved the stability.In solution,NC-NPs usually distributed in an irregular elliptical shape,while C-NPs were uniformly distributed in a spherical shape.As determined,the particle size of NC-NPs with different LA feed ratios varied from 165 nm to 185 nm,and the zeta potential was between-15 mV and-21 mV.After crosslinking,the particle size of C-NPs reduced to 138-179 nm,and the zeta potential varied from-25 mV to-29 mV.The hemolysis rate results were all below 5%,indicating the good biocompatibility of C-NPs.Based on the synthesis of C-NPs,the drug loading method,good drug solvent,ultrasound time and drug/carrier feed ratio were all screened and optimized,and finally the DTX/C-NPs(drug-loaded crosslinked nanoparticles)were prepared by the ultrasonic-dialysis method.When the feeding ratio was 10:3 and the polymer was dissolved in methanol followed by ultrasound for 3 times,the optimized DTX/C-NPs showed the highest drug loading of 10.07%with the encapsulation efficiency of 35.37%.C-NPs could maintain good stability under the conditions of phosphate buffer,high ionic strength solvent and 10%fetal bovine serum,and also showed good anti-dilution stability.While in the presence of a high concentration of reducing agent,C-NPs were cleaved and their morphology changed significantly,indicating their good redox-sensitive characteristics.Subsequently,the dynamic membrane dialysis method was used to study the release behavior of DTX/C-NPs under different conditions.The results showed that the release behavior of DTX was almost the same in the solutions containing no dithiothreitol(DTT)and low concentration of DTT(20μM).After 72 h of incubation,only less than 40%of DTX could be released.However,in a solution containing high concentration of DTT(20 mM),the cumulative release rate of DTX could reach to nearly 80%at 12 h,and could even reached to 95%at 72 h.Therefore,C-NPs might realize specific-drug-release at the tumor sites,improving the stability of nanoparticles and preventing drug leakage.Through the measurement and comparison of singlet oxygen contents in the deionized water(DW)solutions of different substances,it was found that C-NPs produced more singlet oxygen compared with the previously reported sonosensitizers of emodin(EMO)and chlorin e6(Ce6).The effective singlet oxygen generating capacity proved the ultrasound sensitivity of Rh,which lays the foundation for subsequent chemo-sonodynamic therapy.As observed from the cellular level,the tumor cells showed good uptake capacity for C-NPs,and C-NPs could target the Golgi apparatus after entering into the cytoplasm.Under the effect of SDT,C-NPs could destroy the organelle structure and interfere with the key protein synthesis process of tumor cells,promoting the cellular apoptosis process.However,C-NPs exhibited poor targeting effects on lysosomes and mitochondria.At the same time,C-NPs could be transmitted into the deeper tumor cells and play anti-tumor roles via transmembrane transport.Later,the DTX/C-NPs could play an ideal tumor killing effect under the cooperation of SDT,and the cytotoxicity strengthened with the increase of nanoparticle concentration and ultrasound time,while C-NPs behaved in a similar way.Compared with free DTX and Rh,DTX/C-NPs could produce good cytotoxicity and improve tumor inhibition rate at low concentrations,which could be attributed to the targeting effect,high cell uptake ability and sustained release effect of nanoparticles.After the tumor cells were incubated with DTX/C-NPs and treated with ultrasound,their apoptosis rate increased by 4.3 times.In a short period of time,ultrasound could induce the production of ROS molecules by C-NPs and free Rh to a similar extent,and the Rh effect limited by the chemical bonds formed between ADH and Rh in C-NPs were gradually enhanced after the breakage of the bonds along with incubating time.Therefore,the ROS production ability was positively correlated with the concentration of C-NPs and incubation time.After incubating with free Rh or DTX/C-NPs,the mitochondrial membrane potential(MMP)in tumor cells significantly decreased,and the MMP values reduced with the prolong of SDT time,while the C-NPs and SDT had no significant effect on the cell cycles.The results of microtubule experiments showed that DTX/C-NPs could act on the specific part of microtubulin,which promoted the microtubule polymerization and inhibited tumor cell mitosis,and SDT treatment played a certain role in the process as observed.Both C-NPs and DTX/C-NPs could significantly inhibited the metastasis and invasion of tumor cells with the help of SDT treatment,and the decrease in the expression level of MMP9 protein in western blot experiments also confirmed the above results.In addition,DTX/C-NPs were proved to promote cell apoptosis through the caspase-3 pathway.After the tumor-bearing mice were injected with free iodide[1,1‘-dioctadecyl-3,3,3’,3‘-tetramethylindotri-carbocyanine iodide(DiR)and DiR/C-NPs,they were imaged and observed at different time points,and the DiR fluorescent intensity and distribution in each mouse were recorded.In addition,the mice models were dissected after 24 h of the injection,and the fluorescent intensity of the isolated organs was observed.The results showed that DiR/C-NPs improved the tumor targeting ability and exhibited longer residence time compared with the free DiR group.The in vivo anti-tumor experiment showed that the chemo-sonodynamic therapy significantly improved the anti-tumor ability to model mice compared with traditional single chemotherapy.At the end of the experiment,the relative tumor volume of DTX/C-NPs and DTX/C-NPs+SDT groups decreased to 1.62 and 0.74,respectively,compared with 4.40 of the normal saline(NS)group.And the tumor tissues were detected using q-PCR technique.The results showed that compared with NS group,the expression of COX-2 and uPA was significantly reduced after treating by DTX/C-NPs+SDT,indicating that this combined therapy could achieve the optimal anti-tumor effect.Combining the H&E staining results of the major organ sections and the serum contents of glutamic oxaloacetic transaminase(AST),glutamic pyruvic transaminase(ALT),creatinine(CREA)and urea nitrogen(BUN)in the mice models with different treatment,all preparations showed good in vivo safety.While,the TUNEL staining was applied to further verify the apoptosis promoting effect of the nanoparticles.In addition,according to the results of distal tumor volume change,the related components of macrophages in serum of IL-12 and IL-10,and observation of various tumor sections,DTX/C-NPs+SDT could significantly inhibit the distal tumor growth,which might be related to the inhibition of M2 type macrophages in mice by SDT treatment,therefore the nanoparticles could play a role in systemic immunity.DTX/C-NPs+SDT not only killed the tumor cells violently,but performed extremely safety in vivo.However,this part of the subject was only studied in nude mice owing to the limitation of the cell source,and the immunodeficiency characteristics of nude mice made it impossible to study the changes of lymphocytes in vivo.At the same time,it was found that the chemical bond between ADH and Rh would restrict the ability of Rh to produce ROS to some extent.Therefore,further studies were carried out on the basis.2.The construction and in vitro and in vivo study of docetaxel-based redox-responsive chondroitin sulfate-emodin-perfluorooctanoic acid polymeric nanoparticles(CS-cys-Rh-PFC)On the basis of the anti-tumor effect by the combined chemo-sonodynamic therapy in the first subject,the previously connecting arm of ADH was replaced with cys for higher grafting rate,and the Rh connection method was also changed,which could improve the SDT effect and increase the content of Rh as the sonosensitizer.Meanwhile,in order to improve the conversion efficiency of ROS and alleviate the hypoxia around tumor cells,the oxygen-carrying substances with good biocompatibility of PFC was introduced,and the subject is expected to realize a more ideal anti-tumor effect.First,Rh was connected to CS backbones via the cys bonds to construct an amphiphilic CS-cys-Rh polymer.The content of Rh in the polymers was determined by UV=vis spectrophotometry(UV-vis),and the values range between 0.46%and 11.54%.On this basis,different preparing methods of CS-cys-Rh,ethanol concentration for precipitation and precipitation time were all investigated.Subsequently,PFC was connected to CS-cys-by amide reaction with different feed ratios to construct a CS-cys-Rh-PFC polymer,and the corresponding CAC values ranged between 27.52 and 33.83 μg/mL.Besides,the nanoparticles distributed uniformly in spherical shape and the size varied between 131.1 and 149.6 nm,and the zeta potential was between-32 mV and-46 mV.The contents of Rh in all synthesized polymers varied in the range of 10.7%and 11.7%.After the DTX-loaded nanoparticles were prepared by the same ultrasonic-dialysis method,the maximum drug loading reached to 16.8%at the 10:3 feed ratio.The CS-cys-Rh-PFC nanoparticles(CRP-NPs)showed good redox-sensitive property.The DTT solution(20 mM)was prepared in order to simulate the reductive environment in the tumor,and the research discovered that the intramolecular disulfide bonds within CRP-NPs could be broken,damaging the nanoparticle structures into fragments.Thereby the encapsulated DTX and linked Rh molecules were quickly released.Compared with the low density of DTT solution and blank solution,the cumulative release rates and amount of Rh and DTX were significantly improved in 20 mM DTT solution,and the rate reach to 61.1%and 96.9%,respectively,after incubating for 72 h,which proved the benign redox-responsive property and stability of CRP-NPs.According to the measurement of dissolved oxygen content in the polymer solution in water,CRP-NPs showed better oxygen-carrying capacity with higher oxygen content of 17.68 mg/L than pure water(oxygen content of 11 mg/L),and the value increased with the richer of CRP concentration.Additional,CRP-NPs could significantly increase the singlet oxygen production when the polymers were placed under sufficient oxygen,which preliminarily indicated that the introduction of PFC was beneficial to the overall structure and function of CRP-NPs.In cell experiment part,CRP-NPs showed good cellular uptake ability to B16F10 melanoma cells,and the active targeting process was also mediated by the CD44 receptors.Besides,the nanoparticles showed good Golgi targeting ability compared with the free drug.The DTX/CRP-NPs exhibited benign cytotoxicity during the combined chemo-sonodynamic therapy,and the toxicity increased with the prolongation of ultrasound time.In a relatively short period of time,the CS-cys-Rh and CRP-NPs could all stimulate the ROS generation with the help of SDT,and the CRP-NPs could generate more ROS.Therefore,the introduction of PFC significantly increased the content of ROS,leading to the improved anti-tumor efficacy.According to the study of cell cycles,both CRP-NPs and DTX/CRP-NPs were found to increase the tumor cell numbers in G2/M phase,which was dominated by DTX.Therefore,the nanoparticles could inhibit the tumor cell mitosis favourably.At the same time,the changes of microtubule structure after drug intervention were observed,and the result showed that DTX/CRP-NPs could significantly promote the aggregation of microtubulin.While the synergistic treatment with SDT showed a very high tumor killing effect and promoted the cell apoptosis,therefore showed unconspicuous changes of microtubule structures.Both CRP-NPs and DTX/CRP-NPs could induce the calreticulin(CRT)eversion to the outside of tumor cell membranes,resulting in the occurrence of immune prototype cell death(ICD).The DTX/CRP-NPs could significantly increase the expression of apoptosis-related proteins of cleaved caspase-3 and cleaved caspase-9,increase the values of BAX/Bcl-2,and inhibit the expression of MMP9.After applying SDT,the apoptosis rate of tumor cells increased,and the value reached 57.37%for combined therapy of DTX/CRP-NPs and SDT.After the fluorescent substance of DiR was encapsulated into CRP-NPs and injected into B16F10 tumor-bearing mice,the in vivo imaging was observed at different time points.Later,the mice were dissected after injecting for 24 h,and all the major organs were collected,followed by quantitative analysis of the DiR fluorescence intensity.Similar to the first subject,the DiR/CRP-NPs exhibited longer circulating time in the body and better tumor targeting ability compared with free DiR.In addition,the pharmacokinetics of DTX/CRP-NPs was studied in SD rats.The results showed that DTX/CRP-NPs could significantly increase the distribution time of DTX in blood and reduce the drug clearance rate compared with the commercially available drug Taxotere(?),and the half-life of DTX increased from 2.1 h to 9.5 h.In the constructed tumor-bearing mice models,CRP-NPs+SDT could significantly increase the ROS production in tumor tissues compared with the blank control,and the oxygen molecules carried in the inner part of nanoparticles relieved the hypoxia situation in tumor tissues effectively,which inhibited cell migration.After excapsulating DTX,DTX/CRP-NPs exhibited a good anti-tumor effect in anti-tumor study in vivo.Compared with the 44%tumor-inhibiting rate of the CS-Rh+SDT group,the tumor suppression rate of DTX/CRP-NPs,DTX/CRP-NPs+SDT and Taxotere(?)groups were 66.9%,75.6%,and 47.6%,respectively.The H&E staining results of all organs showed that all the preparations were safe when applied in mice,and the H&E staining and TUNEL staining results of tumor tissues mutually confirmed the advantage of promoting the tumor apoptosis of DTX/CRP-NPs+SDT.In addition,by measuring the secretion of cytokines in the mice serum,the DTX/CRP-NPs+SDT could increase the expression of IL-6,TNF-α and IFN-γ in the whole body significantly,except for the IL-2 values.After dissection of mice,the drainage lymph nodes(TDLN)of mice were stained,and it was found that the combined treatment group could increase the proportion of mature dendritic cells(DCs)in lymph nodes.Further,the tumor tissues were ground followed by testing the expression of CD3+CD4+T cells and CD3+CD8+T cells,and DTX/CRP-NPs+SDT could significantly increase the expression of the two kinds T cells compared with the blank group.Similar results were obtained via the immunofluorescence staining result on tumor slices for observing T cells.The results proved that DTX/CRP-NPs could produce a strong systemic immune response with the assistant of ultrasound,thereby inhibiting the tumor growth effectively. |
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