| BackgroundLung cancer is one of the common malignant tumors in human society.The incidence of lung cancer in China is increasing at a rate of 26.9%per year,which continues to rise,accounting for 40%of the world's lung cancer incidence.As of 2017,the annual death toll of lung cancer in China reached 700,000.The number of cases has risen to 800,000 cases per year,so its harmfulness has intensified.Among all lung cancer cases,non-small cell lung cancer(NSCLC)is the major subgroup(85%-90%)and is associated with high recurrence rates and high mortality(1).Gefitinib(Gnb),a first-generation reversible epidermal growth factor receptor tyrosine kinase inhibitor(EGFR TKI),has been proved to provide clinical benefit for EGFR-mutant NSCLC patients(2).For patients with EGFR mutations,median progression-free survival(PFS)can be achieved for 9-13 months with the treatment of EGFR TKI,while median PFS for platinum-based chemotherapy regimens is 4-6 months.There is a significant difference.For treatment remission rate,the TKI group can be as high as 83%,while the chemotherapy group is only 36%,and the TKI group has obvious clinical advantages(3,4).However,most of patients initially sensitive to Gnb will develop acquired resistance(AR)within 6-12 months from the beginning of treatment,which will lead to treatment failure(5,6).EGFR drug resistance is associated with:(1)reduction of drug activation,or enhancement of intracellular drug detoxification;(2)activation of drug transporters in the cell,drug excretion;(3)disruption of the cell cycle or the cell death process being limited;(4)the drug target is altered and the repairability of the target is enhanced.The anti-tumor activity of gefitinib does not depend on the expression level of EGFR.EGFR levels are also not a criterion for the efficacy of gefitinib treatment.EGFR cells with high or low expression levels may be sensitive to gefitinib or may also develop drug resistance(7),so intracellular drug concentration plays an important role in EGFR TKI efficacy(8).Gefitinib's own metabolism and increase of drug excretion can both lead to reduction for the dose of gefitinib in the cell;while,on the contrary,if the intracellular gefitinib concentration is increased,both the EGFR autophosphorylation and activation of MAPK and PI3K/Akt/mTOR signaling pathways would be inhibited,cell proliferation would be naturally reduced.In this way,the drug efficacy would be surely enhanced(9,10).With the application of molecular targeted drug therapy in the treatment of lung cancer,the drug resistance has become an urgent problem that affects the clinical application of the drug.Problems of how to solve the local distribution of molecular targeted drugs in tumors and insufficient cumulative concentration,reduce drug resistance of anti-tumor molecular targeted drugs,and improve drug efficacy have become important issues in clinical researches(11,12).In recent years,with the advancement of basic sciences such as materials technology and bio-medicine,the application research of nano science and technology in the field of life and health has also developed rapidly,especially in the field of cancer treatment.By combining the advantages of basic disciplines such as materials science,chemistry,engineering,and bio-medicine,covering a variety of tumor-related cells,proteins,enzymes,etc.,and being characterized with its unique physical and chemical properties,Nanotechnology has achieved the therapies of modern oncology(13).Different from the traditional drug delivery systems,Nano drug-loaded delivery system is a novel drug delivery system that acquires drug concentration advantages at the tumor site through active or passive delivery mechanisms(14).Its unique properties include small volume,large surface volume ratio,strong surface modification,long packaged drugs,long cycle time,easy penetration of cell membranes,and strong site specificity(15),which are expected to set up new visions,new ideas and new methods for cancer drug delivery system.In recent years,researches on nano drug-loading delivery systems covered some basic materials such as polymers,micelles,dendrimers,liposomes,proteins,etc.(16-18).Because of different functions for these materials,nano drug-loading systems perform differently in the aspects of targeting,cycle time,intracellular permeability,and sensitivity to stimuli.However,research on the combined application of nano-targeted drug-loading systems and molecular-targeted drugs has not been reported.Whether or not the combination could synergistically overcome drug resistance is still a proposition for further study.Compared with normal tissues,tumor tissue is characteristic in richness of blood vessels,width in blood vessel wall gap,poor structural integrity,and lack of lymphatic reflux,in that point,the drug can stay within the tumor.This phenomenon is called high permeability and retention effect of solid tumor tissue(Enhanced permeability and retention effect,EPR effect)(19).Nanomaterials,with its particle sizes being within 3 and 200 nm,through the means of high permeability and retention effect of the tumor,could reach the tumor site,realize the tumor by passive targeting of the drug,get enrichment in tumor tissue and improve the concentration of drug in the tumor tissue with distribution reduction in normal tissues(20).In addition,on the basis of differences for biochemical characteristics such as microenvironment in which tumor and normal tissues always exist and cell surface receptors,nano-carriers with bio-responsiveness could be designed for the purpose of achieving targeted therapy for tumors(21-23).Great numbers of experiments have proved that attachment of targeting tumor cells and tissues with antibodies,small molecules and polymers on the surface of nanoparticles could significantly improve the biocompatibility of drugs and the ability to actively target tumors with reduction of toxicity to normal tissues(24,25).As a new type of nano drug delivery system,Micelle is a high molecular substance composed with hydrophilic and hydrophobic segments.Polymeric micelles can be used both as solubilization and a drug carrier(26,27),which can achieve improvement of drug stability and efficacy,release delay,reduction of toxicity,and targeting properties(28).Compared with liposomes,the block copolymer has the advantages of higher load capacity,stronger stability,longer cycle time,being easier to be modified and a wider range of application(29,30).Polymer micelles,for the reason of their unique characteristics such as small size and hydrophilic shells,could avoid the uptake of mononuclear phagocytic system(MPS)and renal excretion with high moleculars,became an effective passive targeting system.Ligands such as small molecule organic molecules,DNA/RNA aptamers,peptides,carbohydrates,and monoclonal antibodies can attach to the surface of the micelle,this could not only increase the accumulation of tumor sites,but also increase the uptake of drugs to cancer cells through the receptor-mediated endocytosis.(31-33).For the advantage of its ability to control the release drugs,The PH-sensitive co-delivery drugs has become a hot topic in recent years(34,35).PH-sensitive co-delivery anticancer drugs can easily control the delivery of drugs by simply utilizing the tumor micro-environment and the endocytic or lysosomal acidic environment with the result of reduction in toxic effects and improvement in therapeutic effects(36).PH-sensitive co-delivery chemotherapy drug in the tumor micro-environment is stable in blood circulation.After the drug's reaching the acidic environment of the tumor tissue,the outer shell with the pH sensitive component will fall off,the functional core will be exposed and assimilated by the tumor cell.All of these could increase the concentration of effective drug in the cellular and improve the anti-tumor effects.(37).In this paper,a mannose-labeled PLGA-based nanocarrier M-NP-Gnb was designed and prepared,which provides a new idea for the potential clinical application of molecularly targeted nanomedicine.In this research,PLGA was designed to form a hydrophobic core in a mixed micelle and PEG a shell,mannose and PLGA-PEG polymer block are conjugated to the outer surface of the nanoparticle.The nanocarrier formed in this way could be infiltrated into the tumor tissue with the EPR effect,increasing the concentration of the drug in the tumor tissue while reducing its presence in normal tissues.Compared with nano drugs linked with traditional chemistry,the preparation for this nano-drug which makes the host-guest complexes as its physical cross-linking points is much simpler,the chemical composition is more clear,and it is more conducive to the clinical application of nano-drugs.It is expected to become a kind of accurate nano medicine for large-scale preparation and application.Part I Preparation and Characterization of Mannose-conjugated PH-sensitive Nanocarriers for Targeted Delivery of GefitinibObjectiveThis study was to design a novel method for preparation of mannose-conjugated PH-sensitive nanocarriers for targeted delivery of gefitinib and to make assessment for its relevant performanceMethods1.Being based on PLGA-PEG block copolymers,functionalized nanomicelles were synthesized.The PLGA-PEG-NH2 and mPEG-polyhistidine were chemically synthesized to self-assemble into a nano-mixed micelle with gefitinib(Gnb).Among them,PLGA forms a hydrophobic core in the mixed micelle,and PEG forms a shell of micelles.2.The amine group in the PL-PEG-NH2 of D-mannose will be combined and present on the outer surface of the nano particle.3.Parameters such as particle size,size distribution,and zeta potential surface charge of nanomicelles will be evaluated by using dynamic light scattering(DLS)and parameters changes will be detected with pH changes so as to make assessment for pH sensitivity.4.The morphology of nanomicelles was observed by transmission electron microscope(TEM).5.Drug release was simulated in vitro;the drug encapsulation efficiency and drug release rate of the nanomicelle delivery system were analyzed by means of high performance liquid chromatography.Result1.M-NP-Gnb nanomicelles have a particle size of 165 nm,and the size distribution of M-NP-Gnb micelles is narrow and uniform,and the Zeta potential is-18.5 mV.Under alkaline conditions(pH=8.5)and under neutral conditions(pH=7.4),the size of the nanomicelles is smaller,while under acidic conditions(pH=6.5,pH=5.5),the size and the distribution of particle size.become larger,indicating that M-NP-Gnb is acidic responsive.2.Study on the vitro drug release found that M-NP-Gnb exhibits a high encapsulation efficiency of being>95%,a drug loading of being up to 24.7%,and a pH-sensitive release profile.After approximately 24 hours of sustained release,approximately 60%of the drug is released under acidic conditions(pH 5.5),while 35%of the drug is released under neutral conditions(pH 7.4).After 48 hours of sustained release,the drug of the nanomicelles under acidic conditions was nearly released away,and under neutral conditions,only 48%of the drug was released.ConclusionsIn this part a mannose-modified polymer nanomicelle M-NP-Gnb with targeted delivery of gefitinib was successfully synthesized.The size of the nanomicelle is<200 nm,which is suitable for being an antitumor drug carrier.The nanomicelle M-NP-Gnb has a good release rate in vitro and a high drug encapsulation efficiency.In addition,it was proved that the nanomicelle had the feature of acid responsiveness.Under acidic conditions,the particle size of the micelles began to increase,swell and dissociate,thereby control the drug in the carrier.Since the tumor microenvironment is an acidic microenvironment,the nanomicelle can get enrichment and release the drug around the tumor site,and reduce the drug release at the normal tissue,from which it could be concluded that the nano drug-loaded micelle has a good application prospect in the field of cancer treatment.Part II:Research on Targeting Effect and Anti-tumor Effect of Mannose-conjugated PH-sensitive Targeted Delivery of Gefitinib Nanocarrier SystemPurposeMake study on active targeting of mannose-conjugated PH-sensitive targeted gefitinib nanocarrier system to lung cancer cells;and make analysis on the mannose-conjugated PH-sensitive targeted delivery of gefitinib nanocarrier system for targeted drugs of anti-tumor effectMethod1.Determine the viability of cells with the method of MTT;2.Make analysis on the uptake of lung cancer cells in the nano drug-loading system by means of flow cytometry and immunofluorescence techniques;3.Assessment on toxicity of PH-sensitive targeted delivery of gefitinib nanocarrier system loaded with Gnb to lung cancer cells by employing MTT;4.Apoptosis effects were measured and evaluated by flow cytometry through the means of Annexin-V/PI double staining assay.Result1.There was no significant difference in cellular uptake for NP-Gnb and M-NP-Gnb in CHO cells(without expression of lectin receptor).In lung cancer A549 cells(expressing lectin receptor),the uptake of M-NP-Gnb was significantly higher than that of NP-Gnb after 2 hours of incubation.2.Uptake experiment results showed that the uptake capacity of mannose-labeled M-NP-Gnb nanomicelles was significantly higher than that of NP-Gnb nanomicelles,and the difference was statistically significant(P<0.05).3.Con-focal microscopy with laser scanning showed that rhodamine-labeled M-NP-Gnb co-localized with lysosomal lysotracker Green,while NP-Gnb was not co-localized with lysotracker Green.4.MTT assay showed time-dependent and concentration-dependent cytotoxic effects of Gnb(free and encapsulated)in lung cancer A549 cells.After 24 hours of incubation,M-NP-Gnb had an IC 50 value of 0.85 ?g/ml,NP-Gnb be 2.35 ?g/ml,and free Gnb be 4.12 ?g/ml.5.Apoptosis experiments showed that compared with free Gnb and NP-Gnb,M-NP-Gnb significantly induced apoptosis in A549 cells,the difference was statistically significant(P<0.05).ConclusionsNanomicelles can significantly enhance the interaction with lung cancer cells and thereby promote the uptake of gefitinib by lung cancer cells.At the same time,mannose-conjugated PH-sensitive targeted delivery of gefitinib nanocarrier system can significantly increase the effect of gefitinib on apoptosis of lung cancer cells.Part ? Study on the Vivo Targeting and Anti-tumor Effects of Mannose-conjugated PH-sensitive Targeted Delivery of Gefitinib NanocarriersPurposeMake study on the active targeting for lung cancer cells by mannose-binding PH-sensitive targeted delivery of gefitinib nanocarrier,and make analysis on the anti-tumor effect of the drug by the mannose-conjugated PH-sensitive targeted delivery of gefitinib nanocarrier.Method1.Establishment of a xenograft model of lung cancer in nude mice;2.Analysis on changes of metabolic dynamics and biological distribution for drugs in tumor-bearing mice by means of high-performance liquid chromatography;3.The therapeutic effect of nano drug-loaded delivery system on lung cancer xenografts in nude mice.4.The anti-proliferative effect of the nano drug-loaded delivery system was analyzed by immunohistochemistry.5.TUNEL staining was used to detect the level of apoptosis in tumor tissues.Result1.In vivo pharmacokinetic analysis showed that free Gnb would be cleared away through systemic circulation immediately within 4-6 hours,while NP-Gnb and M-NP-Gnb could extend Gnb retention time in the body to 24 hours.The half-life cycle of Gnb encapsulated in nanomicelles will increase by about 5 folds than that of free Gnb.2.Analysis on drug concentration in each tissue after administration showed that M-NP-Gnb was preferentially localized to tumor tissues.And the concentration of Gnb delivered by the M-NP-Gnb nano drug delivery system in the tumor tissue increased significantly compared with that delivered by the free drug.3.Experiment results in vivo tumor model showed that compared with those in NP-Gnb and free Gnb injection group(P<0.01),the tumors in M-NP-Gnb injection group showed the signs of slower growth and smaller volume and significant lower quality.4.The results of immunohistochemistry showed that the proliferation rate of tumor tissue in M-NP-Gnb injection group was significantly lower than that in NP-Gnb and free Gnb injection group.5.TUNEL staining results showed that the apoptosis index of tumor tissue in M-NP-Gnb injection group was significantly higher than that in NP-Gnb and free Gnb injection group.ConclusionsThis study found that nanomicelles can promote the accumulation of its contained drugs in lung cancer tissues and thereby improve the anti-tumor effects of molecular-targeted drugs.At the same time,nanomicellles could reduce the concentration of drugs in normal tissues and reduce the toxic side effects of the drug for normal organs to some extent.In the aspect of inhibiting tumors,nanomicelles can make effective inhibition for the growth of tumor-bearing volume and promotion for apoptosis of tumor cells with the least degree of toxic side effects.In conclusion,the nano drug-loading system with targeted-drugs has extensive and promising application prospect in tumor clinical aspects. |
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