| rhINF?-2b is an interferon product with relatively mature preparation technology.Like most protein drugs,IFN?-2b has a relatively short serum half-life(2~6 h),low treatment index,large fluctuations in blood drug levels and rapid proteolysis,which would affect its efficacy.Moreover,due to its short residence time in vivo,unmodified IFN?-2b needs to be administered in multiple doses during the treatment process,which is easy to cause adverse reactions.At present,the serum half-life of IFN?-2b is mainly prolonged through the chemical coupling of polyethylene glycol and the delivery systems of nanoparticles and microparticles.The microenvironment produced by common preparation methods will damage the activity of protein drugs in the process of durg loading,formation and drying of microcarriers.This project innovatively applies reactive electrostatic spray technology and ion-responsive drug-loading technology to protein macromolecule drug delivery system to produce recombinant interferon a-2b mesoporous lung targeted sustained-release microspheres.The preparation process is simple and the yield is high,which is suitable for the industrial production of protein preparations,and provides a new direction for the research of protein-peptide drug sustained-release preparations.The paper is divided into the following five parts:Part I: ReviewThis chapter first provides a brief overview of the macromolecular protein drug-interferon and its formulation development.Secondly,it briefly outlines the overview and application of the new preparation technology-electrostatic spray technology.And then there is a brief introduction of the application of ion exchange technology on drug loading.Finally,a summary of the carrier-sodium hyaluronate and its application in drug delivery systems are reviewed.Part II:Preparation and characterization of sodium hyaluronate mesoporous microspheresIn this chapter,sodium hyaluronate mesoporous microspheres(SHMM)were prepared by electrostatic spray technology,which solved the problem of the double helix structure of sodium hyaluronate in the electrostatic spray process.Firstly,single factor test was used to investigate solvent,the concentration of sodium hyaluronate and polyethylene oxide,the flow rate,the voltage and needle inner diameter,so as to determine the best formulation of sodium hyaluronate microspheres with good shape and a particle size that meets the requirements of passive targetingto lung.In addition,the concentration of the porogen and drying temperature were investigated by single factor experiment to obtain SHMM with obvious pores.The three batches of SHMM had a porosity of(20.30±1.51)% and showed negative potential in water,which proved that they had a good ability to combine with positively charged protein drugs,thus providing feasibility for later ion exchange drug loading.Part III: Preparation and Characterization of Crosslinked Sodium Hyaluronate Mesoporous MicrospheresIn order to solve the problem that sodium hyaluronate cross-linked mesoporous microspheres(SHCMM)dissolves in the process of cross-linking and drug loading in an aqueous environment,which will destroy its physical form,this chapter uses reactive electrostatic spray method to prepare SHCMM by one step,so that the cross-linking reaction simultaneously occurs with electrostatic spraying.Using 1,4-butanediol diglycidyl ether(BDDE)as the cross-linking agent and the hydroxyl group of sodium hyaluronate as the cross-linking target,an orthogonal test was used to determine the best prescription process for cross-linked microspheres.SHCMM had good morphology and the particle size distribution of the microspheres ranged from 3 to 6?m.SHCMM under the pH of drug loading had a good ability to bind positively charged protein drugs,which laid the experimental foundation for the later ion exchange drug loading.The optimized cross-linked microspheres were characterized by FTIR and DSC to verify the cross-linking reaction between BDDE and SH.This chapter establishes a gas chromatography method with specificity and good precision to determine the content of residual crosslinking agent in crosslinked microspheres,and the results showed that the residual BDDE content of SHCMM meets the regulations.The MTT method was used to evaluate the biocompatibility of SHCMM,and the results showed that the biocompatibility was good.Part IV: Preparation and characterization of recombinant human interferon ?-2b crosslinked mesoporous microspheresIn this chapter,sodium hyaluronate cross-linked mesoporous microspheres(SHCMM)are used as a special biodegradable ion exchange material,and the drug is loaded dynamically.Using dynamic drug loading ion exchange technology to prepare recombinant human interferon ?-2b crosslinked mesoporous microspheres(rhIFN?-2b-SHCMM)with high activity and high encapsulation rate.This chapter establishes the Coomassie Brilliant Blue G-250 staining methodto determine the content and in vitro release of protein drugs in drug-loaded microspheres,and the results meet the methodological requirements.Taking the drug loading capacity as the evaluation index,the effects of the concentration of the injection solution,the pH and the flow rate of the injection solution on the ion-exchange drug loading were investigated by orthogonal test,so as to determine the best prescription process for preparing rhIFN?-2b-SHCMM.RhIFN?-2b-SHCMM had good morphology;the particle size distribution was 3~6 ?m;the Zeta potential was(-12.25±1.19)mV;and the encapsulation rate was(89.09±2.37)%.In vitro release experiments show that rhIFN?-2b-SHCMM has a good sustained release effect,with a cumulative release of(86.26±1.29)% at 24 hours.The model fitting results showed that the in vitro release process conforms to the particle diffusion(ion exchange)release mechanism.In addition,the results of differential scanning calorimetry verified that the drug loading mode was not a physical mixture but an ion exchange combination.The results of circular dichroism spectroscopy and SDS-PAGE gel electrophoresis showed that the secondary structure of rhIFN?-2b in rhIFN?-2b-SHCMM release solution did not change significantly after ion-exchange drug loading and in vitro release.MTT experiment was used to evaluate the biological activity of rhIFN?-2b-SHCMM against lung cancer.The extract of rhIFN?-2b-SHCMM maintained more than 90% of the biological activity of the original solution,which initially proved to maintain high biological activity.Part V: Study on pharmacokinetics and tissue distribution of self-made drug-loaded microspheresIn this chapter,a double antibody enzyme-linked immunoassay(ELISA)is established to determine the concentration of recombinant human interferon ?-2b in mouse serum and tissues.This methodology meets the determination requirements of in vivo analysis.The in vivo pharmacokinetic results show that the elimination half-life and average residence time of rhIFN?-2b-SHCMM are significantly longer than that of the original solution,which shows that rhIFN?-2b-SHCMM prepared by electrostatic spray combined with ion exchange technology has a significant sustained-release effect.The tissue distribution results show that compared with rhIFN?-2b solution,the distribution of rhIFN?-2b-SHCMM in the lung is significantly increased after absorption by tail vein injection.The concentration of rhIFN?-2b-SHCMM in the lung is1.77 times that of rhIFN?-2b solution,which shows that rhIFN?-2b-SHCMM has certain lung targeting. |
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