| Malignant tumor has been one of the major public health problems that seriously threaten the quality of life in our country.Combining dominant chemotherapy drugs with other drugs is the focus of cancer treatment research in recent years.At present,common strategies for drug combination therapy included two or more kinds of chemotherapy drugs,chemotherapy drugs and cytokines,chemotherapy drugs and monoclonal antibody,chemotherapy drugs and genes,et al.Among of the above drugs,cytokines could not only play an important role in the growth,proliferation and differentiation of cells,but also directly induce tumor cell apoptosis or indirectly induce tumor cell apoptosis by regulating the immune.Cytokines which were produced and secreted by immune cells(such as mononuclear macrophages,natural killer cells,lymphocytes,etc.)or non-immune cells(such as vascular endothelial cells,epidermal cells,etc.),were proteins with high biological activity,good specificity and wide application.However,in addition to the cytokines IL-2 and IFN-a,which have made significant progress,most cytokines could show complex signaling pathways in tumor therapy.Moreover,the same cytokines could possess inhibitory and tumor-promoting effects at different stages of tumor developmentGranzyme B(GrB)is a serine protease mainly produced and secreted by natural killer cells and cytotoxic T lymphocytes.GrB is a direct "weapon" which could kill tumors in the last step of the immune system.Therefore,GrB could exhibit great potential for tumor treatment.However,positive GrB could bind to the negatively charged cell membrane in a non-specific way.Compared with systemic therapy,local therapy is an important treatment for solid tumors could achieve precise administration,increase the drug concentration at the tumor site,and significantly reduce systemic toxicity.Herein,GrB was applied by local administration in order to reduce systemic toxicity,and combined with a kind of broad-spectrum chemotherapy drug docetaxel(DTX)to achieve ideal antitumor efficacy.Thermosensitive hydrogel,one of the most common delivery systems for local drug delivery,possessed the following advantages:(1)forming a drug storage via local administration,and low systemic toxicity;(2)convenient administration and a variety of drugs loading;(3)sustained release.Nanocarriers incorporated hydrogels,such as micelles incorporated hydrogels and nanogel incorporated hydrogels,is a delivery strategy with good application prospects,which could not only integrate the advantages of different carriers,but also make up for their shortcomings.This project intends to construct nanocarriers-micelles for GrB and DTX loading,and incorporated the micelles into thermosensitive hydrogel via local injection for tumor treatment.Based on the above therapeutic concept,the project were designed as follows:(1)cytokine GrB and chemotherapy drug DTX were co-delivered and played synergistic anti-tumor efficacy via different mechanisms,in which GrB mainly promoted tumor cell apoptosis by caspase dependent way and DTX mainly inhibited tumor cell mitosis by promoting tubulin aggregation and suppress the depolymerization;(2)micelles incorporated hydrogel which possessed both pH sensitivity and thermosensitivity,was constructed as the co-delivery carrier for two drugs with different properties;(3)local administration was applied for tumor treatment to reduce systemic toxicity.Main methods and results in this study are listed as follows:1.Establishment of content determination methodDetermination method of DTX was established by HPLC,and determination method of CC was established by UV.Both drugs showed good linear relationships under determinated concentrations,and the precisions and recoveries could meet the requirements.2.Preparation and characterization of co-delivery carriersDTX-PIC micelles(DM)were prepared by thin film hydration method,and CC/DTX-PIC micelles(CDM)were prepared by electrostatic adsorption.CC/DTX-PIC micelles incorporated hydrogel(CDMH)was further formed through stirring under ice-bath.The physical and chemical properties such as particle size,potential,morphology and stability,were systematically characterized by particle size and potential analyzer,TEM,SEM,rheometer,etc.The results indicated that co-delivery carriers were successfully constructed.For the co-loaded micelles,particle size was 26.9 ±0.09 nm,zeta potential was-14.53 ±0.58 mV,and encapsulation efficiency(%)of DTX and CC were 70.84± 1.53%and 96.04±0.45%,respectively.When incorporated into micelles,hydrogel solution began gelation at 35 ?and showed no influence on co-loaded micelles.In vitro release results indicated that both drugs could be released more quickly and completely at pH 5.5 condition,while the release rate in micelles incorporated hydrogel was slow at both pH 7.4 and pH 6.5 conditions,which could realize sustained release in tumor tissues and quickly release in lysosomes of tumor cells.3.In vitro and in vivo evaluations of co-delivery carriersMelanoma was used as the model for in vitro and in vivo evaluations at the cell level(B16 cells)and animal level(melanoma bearing C57BL/6 mice).The deep penetration capacity,lysosomal escape ability,co-delivery efficiency,in vitro and in vivo antitumor efficacy and safety of co-delivery carriers were investigated,respectively.The results demonstrated that the co-delivery carriers could achieve deep penetration in tumor tissue and lysosomal escape in the tumor cells,high co-delivery efficiency in vivo or in vitro and superior antitumor efficacy in vivo or in vitro.Preliminary safety in vivo was good and the main organs of mice were not significantly damaged after local administration.In summary,based on the therapeutic concept of combining cytokines with chemotherapy drags,this project constructs the pH-temperature dual-responsive micelles incor:porated hydrogel to co-deliver GrB and DTX,which could synergistically inhibit the growth of tumor cells and enhance the antitumor efficacy via different mechanisms. |
PDF Full Text Request