Construction Of PH-responsive CS Microspheres/SA-ADH-OHA Hydrogel Composite System And Study On The Killing Effect Of Loaded Drugs On Melanoma

Chemotherapy is one of the main methods of cancer treatment.Chemotherapy drugs are usually administered orally or intravenously,leading to the poor drug targeting and high toxic side effects.The local drug delivery systems can effectively avoid systemic drug transportation,improve targeting and reduce drug side effects.Among them,hydrogel local delivery system have attracted wide attention due to its advantages such as the similar structure of extracellular matrix,injectability,degradability,and the ability to continously release drugs inside tumors.In order to adapt the demand for drug release in specific tumor microenvironment,the environment-responsive hydrogel drug delivery system uses external stimuli(p H,light and temperature,etc.)to control drug release,which is more conducive to improving the efficiency of drug delivery and therapeutic effect.Studies have shown that hypoxia,inflammation and immunosuppression are the main characteristics of tumor microenvironment.Due to hypoxia,high metabolism and accumulation of acidic substances in the tumor site,the p H in the tumor site is lower than that in normal tissues.Moreover,the stimulation of inflammatory mediators can maintain tumor growth and invasion,release various cytokines,recruit inflammatory cells to the tumor site,and amplify the inflammatory effect.Therefore,p H-responsive hydrogels targeting the special microenvironment of tumors,with anti-inflammatory and anti-proliferative combination chemotherapeutics,is a new idea for tumor targeted therapy.Among the p H-responsive hydrogels,the hydrogels based on Schiff base reaction of aldehyde group and amino group tend to hydrolyze and fracture in acidic environment,resulting in the collapse of hydrogel network structure,which is widely used to regulate and control the diffusion and release of drugs in hydrogels.Sodium alginate(SA)and Hyaluronic acid(HA)are natural polysaccharide polymers with good biological safety and degradability,which are widely used in the field of drug delivery.However,both SA and HA only contain carboxyl group and hydroxyl group,so chemical modification is usually required to abtain corresponding aldehyde group and amino group to construct p H-responsive hydrogels through Schiff base reaction.However,the application of SA and HA in hydrogel delivery system is limited,because both SA and HA are hydrophilic materials,which have a low loading rate for hydrophobic drugs and are prone to drug burst release.Microsphere is tiny spherical particles with a particle size of 0.1-1000μm,which can deliver drugs by dispersing drugs into the material.At present,microsphere drug delivery system has been widely used in the field of biological tissue engineering and drug delivery.Studies have shown that CS microspheres can be used as delivery carriers for hydrophobic drugs(curcumin,ibuprofen,etc.).In addition,encapsulating the drug-carrying microspheres into hydrogels can improve the loading rate of hydrophobic drugs and reduce drug burst release of hydrogel delivery system.Accordingly,this study aims to construct a p H-responsive hydrogel with SA and HA based on Schiff base,and load CS microspheres in the hydrogel to develop a microspheres/hydrogel composite drug delivery system with good biocompatibility,stable mechanical performance and sustainable drug release.Then,the anti-proliferative drug Doxorubicin hydrochloride(DOX)and anti-inflammatory drug Paeonol(Pae)were encapsulated in the composite system to study the anti-tumor effect in the mouse melanoma transplantation model.This study will provide a new idea for the delivery of chemotherapeutic drugs.ObjectiveThis project aims to obtain SA-ADH polymer(modified by Dionyl hydrazine adipate(ADH))and Oxidation of hyaluronic acid(OHA)(treated with sodium periodate),then construct SA-ADH-OHA injectable hydrogel.Different hydrogels were prepared by fixing the concentration of SA-ADH and adjusting the content of OHA.The best proportion of injectable SA-ADH-OHA hydrogel was screened by testing the gelation time,moisture content,stability,mechanical strength and biocompatibility of the hydrogel.Then CS microspheres loaded with DOX and Pae by emulsification were dispersed into the hydrogel to prepare the CS microspheres/SA-ADH-OHA hydrogel composite system.The surface morphology,stability,biocompatibility and drug release of the composite system were analyzed.Finally,CS microspheres/SA-ADH-OHA hydrogel with Pae and DOX were injected into melanoma xenografts by in situ injection.The therapeutic effect of the microsphere/hydrogel composite delivery system on melanoma in vivo and the potential anti-tumor mechanism of DOX and Pae were preliminarily studied at molecular level.MethodPart 1:Preparation and characterization of SA-ADH-OHA hydrogelThe ADH was modified to the carboxyl group of SA by the EDC/NHS coupling system to obtain SA-ADH polymer with amino group.OHA polymers with aldehyde groups was obtained by oxidation of HA with sodium periodate.The modification results of the two polymers were detected by ~1H NMR.The hydrogels were prepared by Schiff base reaction with 0.5,0.75 and 1%OHA and 0.5%SA-ADH at the volume ratio of 1:1.Then the gelatin time was detected by the inversion.Water content was analyzed by freeze-drying method.The stability of the hydrogel was studied by wet weight method.Degradability was detected enzymolysis approach.The cell compatibility was analyzed by CCK-8 analysis.The blood compatibility was studied by hemolysis method.The stability and mechanical strength were detected by rotational rheology instrument.And the internal structure was observed by the scanning electron microscope.According to the results,the appropriate ratio of hydrogels was selected for subsequent experiments.Part 2:Preparation and characterization of CS microspheres/SA-ADH-OHA hydrogel composite system1.The killing effect of DOX combined with Pae on melanoma B16 cellsThe CCK-8 method was used to detect the effect of Pae or/and DOX on the survival of B16 cells.The scratch test was used to detect the effect of DOX combined with Pae on the migration ability of B16 cells.The plate cloning method was used to detect the effect of DOX combined with Pae on the proliferation of B16 cells.2.Preparation and characterization of drug-loaded CS microspheresChitosan microspheres were prepared by emulsification method using 10%sodium tripolyphosphate(STPP)as crosslinking agent.Pae dissolved in absolute ethanol was added in chitosan containing DOX to prepare drug-loaded microspheres.The encapsulation efficiency of DOX and Pae was detected by ultraviolet spectrophotometry.The morphology and size of microspheres were detected by scanning electron microscope.The blood compatibility of microspheres was detected by hemolysis method.3.Preparation and characterization of CS microspheres/SA-ADH-OHA hydrogel composite systemCS microspheres was dispersed in the preliquid of SA-ADH-OHA hydrogel to prepare drug-loaded microspheres/hydrogel composite system.The mechanical strength of the composite system was detected by the rotational rheometer.The microstructure was detected by scanning electron microscope.The biological safety was detected by CCK-8,the Live/Dead stainning,hemolysis test,and subcutaneous histocompatibility test in mice.The release of DOX and Pae in vitro was detected by the rotating basket method and ultraviolet spectrophotometry.Part III:Anti-tumor effect of drug-loaded CS microspheres/SA-ADH-OHA hydrogel compound system on mouse melanoma35 C57 mice were subcutaneously injected with melanoma B16 cells to establish the C57 transplanted tumor model.When the transplanted tumors growed to 100mm~3,C57 mice were randomly divided into 7 groups(Blank control group,blank microspheres/hydrogel group,Pae microspheres/hydrogel group,DOX microspheres/hydrogel group,free DOX and Pae group,DOX and Pae microspheres,DOX and Pae microspheres/hydrogel group)for in situ treatment.The long and short diameter of the transplanted tumor and the weight of C57 mice were measured every two days,then the tumor growth curve was drawn.After 12 days,C57 mice were sacrificed and the transplanted tumors were stripped and weighed,and the tumor volume and growth inhibition rate were calculated.The apoptosis of cells in transplanted tumor tissues in each group was examined by TUNEL method.The protein expression of Bax and Bcl-2 in tumor tissues was detected by Western blot.The expression of Ki67,CD206 and i NOS in transplanted tumor tissues was detected by immunofluorescence.The morphology changes of transplanted tumors and different tissues were analyzed by HE staining.Research resultPart 1:Preparation and characterization of SA-ADH-OHA hydrogelThe concentration of SA-ADH was maintained at 0.5%,and the OHA concentration was adjusted to 0.5,0.75,and 1%to prepare three SA-ADH-OHA hydrogels with different ratios.The three hydrogels are translucent with plasticity.SEM results showed that the hydrogels have a loose and porous network structure.With the increase of OHA concentration,the gelation time was decreased,the water content was gradually reduced,the degradation rate became slower,the elastic modulus was increased,and the stability was enhanced.In addition,the three hydrogels have good cell biocompatibility and blood capacitive.According to the gel forming time,stability and mechanical properties,the hydrogel with an OHA concentration of 0.5%was finally selected for subsequent experiments.Part 2:Preparation and characterization of CS microspheres/SA-ADH-OHA hydrogel composite system1.The killing effect of DOX combined with Pae on melanoma B16 cellsCCK-8 results showed that DOX combined with Pae had a synergistic inhibitory effect on the growth of melanoma B16 cells.Compared with DOX or Pae alone,the combined drug had a more significant inhibitory effect on the proliferation and migration of B16 cells.2.Preparation and characterization of drug-loaded CS microspheresCS microspheres were prepared by emulsification method.SEM results showed that the microspheres were round in appearance and the average particle size was 0.1-1μm.The hemolysis experiment confirmed that CS microspheres had good biocompatibility.The encapsulation rate of DOX was 65%and the encapsulation rate of Pae was 40.7%detected by UV spectrophotometer.3.Preparation and characterization of CS microspheres/SA-ADH-OHA hydrogel composite systemCS microspheres were dispersed in the hydrogel precursor to prepare a microspheres/hydrogel composite system.The SEM results show that the microspheres can be evenly dispersed inside the hydrogel.The rotation rheometer test showed that the addition of microspheres can improve the mechanical strength of the hydrogel.The results of cell compatibility,blood compatibility and histocompatibility showed that the microspheres/hydrogels composite system had good biocompatibility.The in vitro drug release results showed that the release of DOX and Pae from CS microspheres/SA-ADH-OHA hydrogel compound system in 12 h under p H 6.0 was 18.97%and 9.73%higher than that under neutral p H 7.4.The release amount of DOX from compound system was 10.35%lower than that of SA-ADH-OHA hydrogel in 12 h under the condition of p H 6.0.These results indicated that the compound system is p H-responsive and can alleviate the sudden release of drugs from SA-ADH-OHA hydrogel to a certain extent.Part III:Anti-tumor effect of drug-loaded CS microspheres/SA-ADH-OHA hydrogel composite system on melanoma in miceThe transplanted tumor experiments showed that the tumors of the combined drug group were significantly smaller than the single drug group.Compared with the control group,the blank microspheres/hydrogel group and the Pae and DOX microspheres group,the microspheres/hydrogel composite system loaded DOX and Pae can significantly inhibit the growth of transplanted tumors.And the expression of Ki67 and Bax in the transplanted tumor tissues was significantly increased,the expression of Bcl-2 was significantly reduced,and the number of apoptosis cells was increased.The HE staining results of mouse heart,liver,spleen,lung,and kidney tissues showed that the DOX and Pae microspheres/hydrogel composite system had no obvious side effects on mice.Immunofluorescence results showed that DOX combined with Pae treatment increased the expression of M1 macrophage-specific protein i NOS and decreased the expression of M2 macrophage-specific protein CD206 in the tumor tissues.Conclusion1.SA-ADH-OHA injectable hydrogels have stable porous and loose internal structure,high moisture content and stability,appropriate gelatin time fro requirement,small elastic modulus,large deformation,strong plasticity,and good biocompatibility.Therefore,the hydrogel can provide an ideal supporting environment for microsphere loading and drug release in vivo.2.CS microspheres have a high loading rate for DOX and Pae,and can be uniformly dispersed in the SA-ADH-OHA hydrogel to prepare CS microspheres/SA-ADH-OHA hydrogel composite system.The microspheres/hydrogels system have good biocompatibility and p H responsiveness,and can reduce the drug burst release phenomenon of SA-ADH-OHA hydrogels.The results showed that the p H-responsive drug loading microspheres/hydrogel composite system was successfully prepared,which realized the effective encapsulation of hydrophobic drugs and the continuous controlled release of drugs.3.The results of in vivo experiments showed that the microspheres/hydrogel composite system loaded with Pae and DOX had the most significant inhibitory effect on the growth of transplanted tumor,and had no obvious toxic and side effects.Immunofluorescence detection results showed that DOX combined with Pae could inhibit the growth of melanoma by promoting the increase of M1 type macrophages.These indicated that CS microspheres/SA-ADH-OHA hydrogel composite system can effectively deliver drugs in vivo,reduce drug toxicity,and has a good application potential.