Study On Formulation,Evaluation And Application In Pharmacodynamics For Non-small Cell Lung Cancer Of Liposomal Co-delivery System Of Vinorelbine And Cisplatin

Lung cancer is the most frequently diagnosed cancer associated with a highly metastatic and poor prognosis,especially in men,and remains the main cause of cancer incidence and mortality worldwide,accounting for 18.4%of total cancer deaths.Approximately 85%of lung cancer is non-small cell lung cancer(NSCLC),which is extremely difficult to treat and has a very low survival rate?Combination therapy is a common method in oncology,and is an effective strategy to improve the therapeutic effect and overcome the toxic and side effects of delivered drugs,while reducing the dose of each drug,and treating multiple targets.Vinorelbine(NVB)plus cisplatin(CDDP),also called NP,has been recommended as a common drug combination therapy,and is a first-line treatment for many cancers.Moreover,adjuvant NP is an accepted standard chemotherapy for patients at initial stage II and stage IIIA NSCLC.NP demonstrated significant improvements in median relapse-free survival,median overall survival(OS),and an improvement of 15%in 5-years survival compared with completely resected early-stage NSCLC.However,in addition to the toxicity typically associated with monotherapy,the use of NVB plus CDDP is associated with toxicity.Therefore,to alleviate the difficulties of combination chemotherapy,it is necessary to develop an ideal drug delivery system to improve the therapeutic effect of delivered drugs while reducing their side effects.Recently,drug delivery systems using nanotechnology have shown great potential in the safety and effectiveness of tumor treatment.Common nano delivery systems include:nanoparticles,polymer micelles,liposomes,nanoemulsions and nanocapsules.Polymer entrapped drugs enhance drug solubility and prevent interaction with the immune system,as well as reduce the side effects of chemotherapy.The nano size of polymeric micelles(<30 nm)is favorable for easy accumulation at the tumor site,and biodegradable polymers demonstrate good clearance from the body,contributing to their safety.Liposomes can also help encapsulated drugs to avoid chemical inactivation and enzymatic degradation,and thereby preserve the potency of drugs until they are delivered to target tissues via the enhanced permeability and retention(EPR)effect.The development of PEG-liposomes permits for the avoidance of rapid capture and enables a prolonged circulation time in the blood.PEG-liposomes allow for prolonged retention and passive targeting to tumor tissues,and the appropriate particle size distribution of polymer micelles makes it easy for them to accumulate at the tumor site and avoid clearance through renal excretionThis paper is mainly divided into the following four parts:In the first part,the formulation of NVB polymer micelles(NVB-PM)was prepared by grafting NVB with poly(-L-glutamic acid)polyethylene glycol sodium(PLG-g-PEG_5K)through electrostatic interaction.NVB-PM shows spherical with particle size of 24.97±0.305 nm,PDI of 0.307±0.0196 and packing rate of 85.62±2.37%.CDDP and PLG-G-PEG5K were complexed to form stable CDDP polymer micelles(CDDP-PM).The diameter of CDDP-PM was 14.87±0.404 nm,PDI was 0.349±0.0232,and the encapsulation rate was 95.58±1.58%.Both NVB-PM and CDDP-PM had suitable particle size distribution,high encapsulation rate and good stability.Meanwhile,CDDP-PM also improved the solubility of CDDP,which provided a basis for the preparation of co-loaded liposomes.Besides,the HPLC method for the determination of NVB and CDDP was established.The two drugs showed a good linear relationship under the concentration determination.The methods are accurate and reliable because of specificity,good precision and accuracy,high stability,satisfactory recovery results.The second part is about the formulation,preparation,quality evaluation,in vitro release,stability and in vitro cytotoxicity evaluation of CoNP-lip.According to chou-Talalay equation,the optimal ratio of two drugs is 2:1 to achieve synergistic cytotoxic effect in a NSCLC cell line,such as A549.Lipid E-80,cholesterol,DPPG and DSPE-PEG₂₀₀₀(52:32:14:2,molar ratio)were selected by reverse evaporation method to prepare CoNP-lip.The particle size of CoNP-lip was 162.97±9.06 nm,PDI was0.175±0.0405,and zeta potential was-14.02±0.23 m V,indicating that the preparation had uniform particle size and was suitable for intravenous injection.The morphology of liposome was spherically spherical with bimolecular layer,and the particle size was consistent with the results of Marvin particle size analyzer.The encapsulation rate of NVB and CDDP in CoNP-lip was 52.61±2.21%and 59.63±1.53%,respectively,and the drug loading was 3.72±0.15%and 2.14±0.054%,respectively,which were close to the optimal combination ratio.In vitro stability showed that the structure and encapsulation rate of CoNP-lip remained stable during the measurement period.The release behavior of CoNP-lip in PBS solution and 10%plasma was investigated in vitro.The experimental results showed that CoNP-lip had obvious sustained release effect without sudden release.Cytotoxicity of CoNP-lip,NP-sol and blank liposome on A549and LLC cells were evaluated.The results showed that blank liposome had no significant cytotoxicity,while CoNP-lip and NP-sol had toxic effects on A549 and LLC cells.In the third part,the in vivo analysis methods of NVB and CDDP were established to study the pharmacokinetics of CoNP-lip in SD rats.The content of NVB in vivo was determined by UPLC/MS.The method showed that linearity,specificity,precision and recovery rate was accurate and reliable.The content of CDDP in rats was quantitatively analyzed by AAS.Under the measured concentration,the linear relationship was good,the precision and accuracy were high,and the results were accurate and reliable.The pharmacokinetic parameters of CoNP-lip and NP-sol in rats showed that c CoNP-lip had better pharmacokinetic parameters and in vivo.The AUC_(0-?),C_max,T_1/2and MRT_(0-?)of the CoNP-lip were much higher than those of the NP-sol,while the CLz and Vz were observably lower.It indicated that compared with NP-sol,CoNP-lip maintained a longer blood circulation time.The packaging of the liposomes could protect the CoNP-lip from RES recognition and avoid phagocytosis,which would protect the CoNP-lip from rapid removal from the blood circulation.In the fourth part,a tumor bearing model of LLC-C57BL/6 mice were established to investigate the antitumor effect of CoNP-lip and intratumor drug uptake.The results showed that CoNP-lip had better anti-tumor effect than drug mixed solution and single drug liposome,and the tumor inhibition rate was 70.29%.During the administration,the CoNP-lip group suffered the least weight loss and gradually recovered to their initial body weight after the second injection,indicating that the CoNP-lip group was well tolerated at the experimental dose and had less systemic toxicity than the drug mixed solution.The contents of two drugs in tumors were measured.The contents of NVB and CDDP in the CoNP-lip group and the single drug liposome group were both higher than those in the solution group at 1h and 48h,indicating that the accumulation of NVB and CDDP in tumors could be increased by passive targeting of CoNP-lip group.