Construction And Evaluation Of A Protein Drug Delivery System With Targeting And Real-time Tracing Functions Prepared By Coaxial Electrostatic Spray Technology

Recombinant human interleukin-2(rhIL-2)is a kind of protein drug used in the treatment of lung cancer.At present,a series of clinical studies show that rhIL-2 has a certain therapeutic effect on all kinds of cancer cells.Because of the disadvantages such as poor stability,short half-life,and difficult evaluation of targeting efficiency and so on,the protein was encapsulated in the microspheres with sustained release effect instead of the injection.The traditional preparation technology of sustained-release microspheres needs severe reaction conditions and addition of surfactants such as spray drying method,super boundary drying method,which will lead to burst release and low stability.Electrostatic spraying technology can avoid the problem of reduction of protein activity and sudden release phenomenon by one-step process,it may reduce the toxic effect and improve the interval time of drug delivery.Therefore,the coaxial electrostatic spray method is used to prepare core-shell structure rhIL-2 microspheres by one-step technology,the chitosan was used as core carrier to bind the fluorescent quantum dots and protein drug covalent conjugates,and sodium hyaluronate was used as the shell carrier material.The core-shell structure rhIL-2 microspheres may be targeted to the lung tumor to achieve the role of local treatment and its targeting can be evaluated by in vivo imaging.This paper is divided into five parts,as follows: Part ?:ReviewThis part briefly summarizes the application of coaxial electrostatic spray technology in the preparation of protein drug delivery system microspheres.Then it discusses the optical properties and the application of quantum dots in targeting tracers,and the coupling methods of quantum dots with protein.The active targeting application of sodium hyaluronate as an CD44 specific receptor on the surface of cancer cells was also introduced.Finally,the application of recombinant human interleukin-2 in antitumor research progress are summarized.Part ?: Preparation and characterization of quantum dots-rhIL-2 conjugatesIn this part,the particle size and morphology of QDs were characterized.Then,the coupling process of QDs and rhIL-2 was optimized by indirect method,including the molar ratio of QDs to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(EDC),n-hydroxythiosuccinimide(NHS)and rhIL-2.The optimal formulation is: the ratio of QDs to EDC is 1:1000,the ratio of EDC to NHS is 1:1,the ratio of QDs to rhIL-2 is 1:2,the reaction environment is a borate buffer with pH of 7.4,and the reaction time is 2 hours.The rhIL-2-QDs protein complexes were characterized by dynamic light scattering(DLS)and agarose gel electrophoresis.Finally,the protein bioactivity and QDs optical properties of the conjugates were verified by the Circular Dichroism and the Fluorescence Microscopy.Part ?: Preparation and characterization of nuclear layer microspheres by electrostatic sprayIn this part,Bovine serum albumin(BSA)is used as a model drug to optimize the technological parameters for preparing the core layer microspheres by single axis electrostatic spray.According to the size and morphology of the prepared microspheres,a series of parameters are optimized,including the concentration of the carrier,the magnitude of the positive voltage,the diameter of the pinhole and the velocity of the spray.The optimized formulation and technology were as following: the spray voltage was 25 kV,the chitosan concentration was 1%,the PVA concentration was 1%,the injection speed was 0.02 mm/min,the pinhole diameter was 0.67 mm.The nuclear layer microspheres have proper shape,the particle size distribution was uniform between 0.5 ?m-2.0 ?m,and the encapsulation efficiency of the microspheres was 83.47±2.69% and the drug loading was 20.23±3.18 ?g/mg.The results indicate that the drug is well encapsulated in chitosan microspheres,and the fluorescence properties of the microspheres are good under fluorescence microscope,which is conducive to the target tracking research of the microspheres in the later research.Part ?: Preparation and characterization of Core-shell structure microspheres by coaxial electrostatic sprayIn this part,albumin(BSA)is used as a model drug to optimize the process parameters for the preparation of core-shell structure microspheres by coaxial electrostatic spraying.The factors including carrier concentration,positive voltage,pinhole diameter and spray velocity were investigated.The best formulation and technology were: spray voltage 25 kV,receiving distance 21 cm,nuclear layer solution velocity 0.02 mm/min,shell solution flow rate 0.04 mm/min.The scanning electron microscope picture shows the successful preparation of the core-shell microspheres.The diameter of the coaxial microspheres was about 1.2 ?m-1.5 ?m investigated by the laser particle size meter,which was suitable for passive lung targeting.Furthermore,rhIL-2 was used to reappear the optimal formulation process and MTT method was used to evaluate the safety of the coaxial microspheres.The results showed that the cell survival rate reached 90% when the concentration of the coaxial microspheres reached 7.5 mg/ml,which proved that the safety of the microspheres was good.And the biological activity was evaluated by test the lung cancer cell inhibition rate,the results showed that the bioactivity of the microspheres was about 90% of recombinant IL-2 injection.In this part,the real-time tracking performance of the core-shell structure microspheres were investigated by animal experiments.According to the results of in vivo imaging test,it was observed that the fluorescence quantum dots in the core-shell structure microspheres were mainly concentrated in the lung cancer site,while the distribution of fluorescence quantum dots in the control group was only a small amount,which indicated that the coaxial microspheres had the effect of active target tracing.