| Cancer is a type of disease that seriously plagues human survival and health,The pathogenesis is extremely complicated,and genetic,environmental and even dietary habits and psychological burdens play a very important role.In recent years,with the development of pharmacology,pharmacy,molecular biology and medical devices,cancer treatment has made continuous breakthroughs.However,unfortunately,many people still pay insufficient attention to cancer screening.Most cancers are in advanced stage at the time of initial diagnosis.Therefore,chemotherapy still dominates clinically and is an indispensable means of cancer control and treatment.However,due to the lack of selectivity in the systemic administration of chemotherapeutic drugs,they bring serious undesired side effects to the patient while achieving therapeutic goals.For this reason,the intratumoral injection mode of direct targeting of the lesion has gradually become an increasingly attractive route of administration.Although intratumoral injection can directly target the drug directly to the lesion,greatly improving the bioavailability of the drug,but after the intratumor injection of the common injection,as time goes by,a large amount of drug will leak into the systemic circulation through the interstitial space,resulting in the drug loss,the potential of chemotherapy is still not fully utilized.Therefore,in order to achieve high-efficiency and low-toxic drug use,it is imperative to change the pharmaceutical dosage form to have a sustained release effect and to be retained in the tumor site.Microspheres have been widely used as one of the delivery vehicles,and PLGA is a widely recognized carrier material and has been approved by the FDA.The choice of effective chemotherapy drugs is the most critical step in cancer treatment.As a broad-spectrum anticancer drug,irinotecan has a strong inhibitory effect on a variety of cancer cells.However,it must be converted to the active metabolite SN-38 under the metabolism of carboxylesterase to exert anticancer effects.Unfortunately,the conversion rate is only 2%to 8%.Being unable to be used efficiently can cause great waste to a certain extent.To this end,the high-efficiency metabolite SN-38 was directly selected as a model drug to make PLGA microspheres,which can maintain an effective high concentration in the tumor area for a long time,and is expected to cope with the above problems.In this study,SN-38 was used as the model drug and PLGA was used as the carrier material.The SN-38-PLGA-MS for intratumoral injection was prepared by O/W emulsification-solvent evaporation method.The research content mainly includes:Optimum preparation of SN-38-PLGA-MS and characterization of physical and chemical properties including appearance microscopic morphology and particle size distribution;In vivo pharmacokinetic study and leakage of SN-38-PLGA-MS at the tumor site;Pharmacodynamic study of intratumoral injection of SN-38-PLGA-MS for H22 liver cancer.Part ?:Preparation and physicochemical properties of SN-38-PLGA-MSFirstly,the method of determining the content of SN-38 in vitro was established by UV-Vis and its method was verified.Secondly,SN-38-PLGA-MS was prepared by emulsifying solvent volatilization method.The encapsulation efficiency and particle size were taken as the main indicators to investigate the single factors,and several factors with great influence were found and orthogonalized.The experimental design was further optimized to obtain the best formulation and process for preparing the microspheres.Then,carry out three batches of reproducibility tests to verify whether the prescription and process are reasonable;Different concentrations of mannitol were selected as lyoprotectants.The lyophilized powder was prepared by lyophilization method.The lyophilized powder was used to prepare SN-38-PLGA-MS freeze-dried powder.Finally,use DSC to verify whether SN-38-PLGA-MS is formed;At the same time,different methods were used to characterize and evaluate the physical and chemical properties of SN-38-PLGA-MS,such as appearance,microscopic morphology,particle size and distribution,encapsulation efficiency and drug loading.The results showed that SN-38 showed a good linear relationship between absorbance and concentration in the concentration range of 4.4?13.2 ?g/mL,which can be accurately used for the determination of in vitro content.Through single factor investigation,it was found that the process factors(shearing time and shearing speed)had little effect on the encapsulation efficiency and drug loading of the microspheres,but had a greater influence on the particle size of the microspheres.Therefore,according to the needle requirements and the previous investigation on intratumoral injection,the optimal process was determined by using the particle size as the index as:the shear rate was 5000 rpm and the shear time was 1 min.At the same time,the effects of prescription factors on the encapsulation efficiency and drug loading of microspheres were investigated,and then several factors with greater influence were selected to determine the best prescription by orthogonal test:The ratio of drug to fat is 1:10,the concentration of PVA in the aqueous phase is 1.5%,the volume ratio of oil to water is 1:3,the concentration of PVA in the dispersed phase is 0.5%,and the volume ratio of dispersed phase of the aqueous phase is 1:5.The average particle size of SN-38-PLGA-MS prepared under the optimal formulation and process conditions was(10.02±0.05)?m;the encapsulation efficiency was(81.91±1.91)%;the drug loading was(6.89±0.07)%.The reproducibility between batches is good.When the amount of lyoprotectant is 5%,the shape of the microspheres is loose and the reconstitution speed is fast.The appearance and microscopic morphology showed that the prepared microspheres were round and smooth,and the particle size distribution was relatively uniform,which could meet the requirements of intratumoral injection.Part ?:Pharmacokinetics of SN-38-PLGA-MS in mouse tumors siteFirstly,the method of SN-38 determination in mice was established by high performance liquid chromatography(HPLC)and its methodological investigation wascarried out.Secondly,H22-bearing mice were used as an animal model,and the SN-38 solubilizing solution was used as a control,and the SN-38-Sol and SN-38-PLGA-MS were injected intratumorally,by measuring the drug concentration in the tumor site of the mouse at different time points and the drug concentration in the plasma,the drug retention amount of the two formulations in the tumor and the amount of the drug leaking into the systemic circulation were compared and a drug-time curve was plotted;The relevant pharmacokinetic parameters were obtained by DAS 2.0,and the sustained release and long-acting effects of SN-38-PLGA-MS were investigated.The results showed that the concentration of SN-38 in mouse tumor tissues and plasma was determined by HPLC.The endogenous components had no effect on the determination at the selected wavelength.All the indexes were within the required range,and the obtained biological samples were stored.It is more stable and can fully meet the needs of its analytical methods;After intratumoral administration,the concentration of drug SN-38 in the SN-38-PLGA-MS group was significantly higher than that in the solution group at each time point,indicating that SN-38 significantly increased its target at the time of microsphere formation.The retention of the area,as well as the retention time has also been extended.Pharmacokinetic analysis data showed that SN-38 can effectively reduce the leakage of SN-38 into the blood after it is made into microspheres,thus reducing or even avoiding the systemic side effects.Part ?:Pharmacodynamic evaluation of SN-38-PLGA-MS for cancer treatmentFirstly,the H22 mice were used as animal models,and normal saline(NS)was used as a negative control.Intratumoral injection of NS,blank microspheres(Blank-PLGA-MS),SN-38 solubilization solution and SN-38-PLGA-MS were performed.The scaly caliper was used to record the tumor volume every two days,and the tumor volume-time relationship map was drawn.The relevant tumor inhibition parameters were calculated and the inhibition levels of each preparation were quantitatively evaluated.At the same time,the toxicity of SN-38-PLGA-MS was indirectly evaluated by recording changes in mouse body weight during the experimental period;Then,H&E staining analysis was performed on the tumor tissues and main organs(heart,liver,spleen,lung,kidney and skin)obtained at different time points,and the anti-tumor effect of SN-38-PLGA-MS was qualitatively evaluated;The in vivo safety of SN-38-PLGA-MS was evaluated by observing the sections of the main organs.The results showed that within the experimental period(18 days),the tumor volume of the Blank-PLGA-MS group and the NS group were close to each other(p>0.05),both showed a rapid growth trend and proliferated vigorously,indicating that the carrier material itself can not inhibit the growth of tumors.However,the tumors of SN-38-Sol group and SN-38-PLGA-MS group were inhibited to different degrees(p<0.01),and the latter inhibition effect was more obvious(p<0.001);Quantitative results showed that the TGI,DT,and SGR of the SN-38-PLGA-MS group were 1.42,1.97;and 0.5 times than that of the SN-38-Sol group,respectively,indicating that SN-38-PLGA-MSS can better inhibit the growth of tumor cells.The body weight of the SN-38-PLGA-MS group showed an upward trend,which proved that the microspheres were safe.H&E sections of mouse tumor cells showed that the SN-38-PLGA-MS group was more effective at the same dose.The H&E section of the main organs of the mice showed that there was no significant difference between the groups,indicating that SN-38-PLGA-MS had no obvious side effects on the main organs of the mice,and it was safe and had certain clinical application potential.In summary,this study prepared SN-38-PLGA-MS by emulsified solvent evaporation method with high encapsulation efficiency,high drug loading and moderate particle size,which can be injected intratumorally.The MS has a slow release effect,which can greatly prolong the retention time of the encapsulated SN-38 at the tumor site,and has a good anti-tumor effect.At the same time,it has good safety,no obvious side effects,and achieves the purpose of high-efficiency and low-toxicity,which lays a foundation for its clinical application. |
PDF Full Text Request