| Interferon(IFN)is a kind of glycoprotein cytokines with broad-spectrum anti-tumor,antiviral activity and immune enhancement,and is one of the most important anti-tumor biological products in clinical practice.Recombinant human interferon?-2b(rh IFN?-2b)has the same biological activity as natural human IFN?-2b,which can be combined with other drugs to treat small cell or non-small cell lung cancer continuously.Like most protein macromolecular drugs,the half-life of rhIFN?-2b is short,pharmacological activity is not specific,and the preparation conditions and release environment all significantly influence its stability,then limit its wide application.In this paper,we use ion-exchange and electrostatic self-assembly technique to prepare carboxymethyl Chitosan microspheres(CS-rhIFN?-2b-CMCS-MS)to improve protein stability,stable the release rate of rhIFN?-2b,and target to the lung passively.The drug delivery system can be widely used in protein.This paper is divided into five sections as shown in the following.Part ?:ReviewThis part mainly introduced the research progress on physicochemical properties,anti-lung cancer activity and research direction of model drug(rhIFN?-2b).Common carriers,preparation methods and difficulties of biodegradable microspheres of protein were also summarized.Finally,the application and mechanism of Carboxymethyl Chitosan(CMCS)ion exchange matrix in biological macromolecular drug delivery system are introduced.Part ?:Preparation and characterization of carboxymethyl chitosan porous microspheresThe blank CMCS-MS was prepared by emulsification-chemical crosslinking method.The appearance,particle size and porosity of the microspheres were used as the main indexes to optimize CMCS-MS preparation process.Based on single factor investigation experiment,the optimal compositions were presented as follows:2%CMCS saline solution:atolein=1:8(v/v),2%Tween 80 of the water phase(w/v),and 2%Span 80 of oil phase(w/v),ethyl acetate:water phase=2:5(v/v),crosslinking agent:CMCS(w/w)=1:5,emulsifying speed 900 rpm for 1 h,and crosslinking for 3 h.After reaction,the microspheres were washed and extracted by n-hexane,ethanol and deionized water for 3 times separately.Finally,microsphere aqueous dispersion was centrifuged and freeze-dried,yellow powder microspheres were obtained.CMCS-MS have round appearance,multiple pores,and the maximum pore size is 200~300 nm based on SEM.More than75%of CMCS-MS diameters are located in 5~15?m,which is easily blocked by pulmonary capillary.FTIR proves the formation of crosslinked microspheres.The porosity of microspheres was(30.28±1.14)%,ion exchange capacity was(9.97±0.07)mmol/g,and zeta potential in each solution was negative,proved that it has the ability to attract and contain positive drugs.In addition,the dissociation behavior of the exchangeable group(carboxyl group)on the microsphere was clarified by using the potentiometric titration curve,and the ion exchange working range of the microsphere was determined to be higher than p H4.3.CMCS-MS was pH and ionic responsive,the swelling of CMCS-MS in pH7.4 PBS and 0.9%NaCl was significantly higher than the pH1.2gastric juice simulation environment.CMCS-MS has good biodegradability,thermal stability and biocompatibility.Part ?:Preparation of BSA-carboxymethyl chitosan microspheresIn this section,Brandford method was established to determine the content of protein drugs.The standard curve was established and validated,which proved that the method was accurate and reliable to meet the requirements of methodology.BSA-CMCS-MS were prepared by column method using blank CMCS-MS as the matrix.The penetration rate of ion exchange column was fixed as 0.1,buffer solution,pH,ionic strength,initial drug concentration,reaction temperature,cross sectional area of the column and flow rate of the solution were screened.The final preparation process was determined as follows:dynamic glass exchange column(16 mm×20 mm),ratio of column height to diameter 7:1,temperature(25±0.5)?,5 mg/mL BSA solution,0.02mol/L pH4.6 HAc-NaAc buffer solution,flow rate of 0.2 m L/min for about 12.5 minutes before stopping the loading process,the column was washed with deionized water.BSA-CMCS-MS were then obtained by freeze-drying after the solution was exhausted.In order to slow down the release,CS-BSA-CMCS-MS was further prepared by the electrostatic self-assembly technique.The encapsulation efficiency of CS-BSA-CMCS-MS was(84.39±6.19)%.The results of in vitro release and DSC showed that BSA and CMCS-MS were mainly combined with ionic bonds.After self-assembly,the release mechanism was transformed to membrane diffusion.The sustained release could be maintained for at least 24 hours and the accumulative release amount in pH7.4PBS(including 0.02%Tween80)was 86.78%.The results of CD and SDS-PAGE electrophoresis showed that there was no significant change in the secondary structure and purity of BSA.Part ?:Preparation of rhIFN?-2b-carboxymethyl chitosan microspheresOn the basis the optimal prescription of part three,rh IFN?-2b-CMCS-MS were prepared by column method.The penetration rate of ion exchange column was fixed as 0.1,and the effect of pH on the drug loadig was investigated.In order to slow down the release,CS-rhIFN?-2b-CMCS-MS was further prepared by the electrostatic self-assembly technique.The encapsulation efficiency was(89.91±0.387)%.The results of in vitro release showed that rhIFN?-2b and CMCS-MS were mainly combined with ionic bonds.After self-assembly,the release mechanism was transformed to membrane diffusion.The sustained release could be maintained for at least 24 hours and the accumulative release amount in pH7.4 PBS(including 0.02%Tween80)was 83.89%.The results of CD and SDS-PAGE electrophoresis showed that there was no significant change in the secondary structure and purity of rh IFN?-2b.Results of inhibition rate of A549 cell proliferation evaluated by MTT assay showed that the release solution of microspheres for 24 h retained more than 91.98%of the antitumor activity of the original solution,which provided the first evidence of pharmacobiological activity of CS-rhIFN?-2b-CMCS-MS.Part ?:Study on pharmacokinetics and tissue distribution in miceIn this part,an ELISA method was established to determine the content of rhIFN?-2b in plasma and tissue samples of mice.The linear relationship was good in the range of 7.5~240 pg/m L,and the standard curve equation was Y=143.82X-15.45 r~2=0.9968.In vivo pharmacokinetic experimental results showed that the concentration of rhIFN?-2b solution could only be maintained for 6 hours,while the microspheres group was nearly 48 hours,indicating the microspheres had obvious sustained release effect.Finally,the distribution of rh IFN?-2b microspheres suspension and solution in mice was studied.It is proved that microspheres can increase the concentration of drug in lung. |
PDF Full Text Request