| Albumin-binding drugs are widely used in tumor therapy because of their good biocompatibility,stability,tumor targeting and long half-life in vivo.However,the effect of chemotherapy alone is limited,and long-term use will cause drug resistance and reduce treatment effect.With the rapid advancement of medical technology,many new and effective tumor therapies such as photodynamic therapy,photothermal therapy,anti-angiogenesis therapy,and immunotherapy have emerged.What’s more,the treatment of tumor has developed from a single therapy to a combination of therapies to achieve the synergy effect of "1+1>2".Therefore,it is of research significance to construct an albumin nano-delivery system for tumor synergistic therapy.Modifications to albumin are predominantly chemical,which often lead to uneven products,organic solvent residues,and toxic side effects.In this study,the genetic level bio-modeling of human serum albumin was carried out by recombinant DNA technology for preparing a homogeneous fusion protein,and a recombinant albumin fusion protein nano-delivery system was constructed to verify its potential possibility as a chemo-photodynamic and chemo-anti-angiogenesis combination therapy platform.The main contents were the following:(1)Recombinant human serum albumin fusion protein HSA-18His(HH18)was expressed through Pichia pastoris system,and the photosensitizer chlorin e6(Ce6)was loaded to by the intermolecular disulfide of HH18 to prepare HH18@Ce6.Then,docetaxel(DTX)was loaded in HH18@Ce6 due to the hydrophobic of 18 His for self-assembling into HH18@CD nanoparticles.The particle size of HH18@CD NPs was about 190 nm,and the drug loading rates of Ce6 and DTX were 3.45% and 7.35%,respectively.At in vitro drug release experiments,in the absence of 10 m M GSH or p H changing,the accumulated release rate of ce6 and DTX both reached 70% at 72 h,proving that drug release was redox responsive and p H responsive.Selecting 4T1 cell as the cell model for cytotoxic experiments,the cell survival rates of 50 μg/m L HH18@CD NPs without laser and with laser irradiation were 45.15% and 4.99%,respectively,indicating that laser irradiation significantly enhanced the killing effect on 4T1 cells.It also proved that chemo-photodynamic combination therapy had a synergistic tumor killing effect.Meanwhile,cell migration experiments also showed that the laser irradiation group could effectively inhibit the migration of 4T1 cells.At the same time,comparing with the laser-free group in CLSM assay,the laser group could produce a large number of reactive oxygen in the cytoplasm,which owned good phototoxicity indicating that HH18@CD NPs had the potential as a chemo-photodynamic combination therapy platform.(2)The fusion protein HSA-VEGF165b(HV165b)composed of human serum albumin and anti-angiogenic factor VEGF165 b was designed and expressed by using albumin fusion technology.VEGF165 is overexpressed in a variety of malignant tumors and can induce tumor vascular proliferation and tumor metastasis,while VEGF165 b can inhibit VEGF165-induced pathological vascular proliferation and has no effect on normal neovascularization.The MTT assay of HV165 b on human umbilical vein endothelial vascular cells(HUVEC)showed that the administration of HV165 b alone had no proliferative or inhibitory effect on HUVEC,but in the case of 50 ng/m L of VEGF165,100 μg/m L of HV165 b could significantly inhibit the proliferation of HUVEC,and the inhibition rate could reach to 50%.Also,HV165 b could significantly inhibit VEGF165-mediated HUVEC cell migration proved by cell migration assay,which indicated that HV165 b retained the activity of VEGF165 b,and had the potential as a chemo-anti-angiogenesis combination therapy platform.The doxorubicin(DOX)was loaded with HV165 b by reducing and self-assembling method to form HV165b@DOX NPs.The drug loading rate of HV165b@DOX NPs was7.85%,and in vitro drug release experiment showed that the cumulative drug release rate reached 78% at 72 h in 10 m M GSH solution,indicating the good redox responsive release of DOX.According to that VEGF165 was in high expression in malignant tumors,human breast cancer cells MDA-MB-231 were selected as the model cell.The cellular uptake assay showed that HV165b@DOX NPs could be rapidly took in MDA-MB-231 cells and released DOX slowly.What’s more,even after co-incubation with cells for 8 h,there was DOX in the nucleus,indicating that HV165b@DOX NPs could sustainably release DOX and improved the bioavailability of DOX.Therefore,HV165b@DOX NPs killed tumor cells through DOX on the one hand,and inhibited the proliferation and migration of vascular endothelial cells through HV165 b on the other hand,so as to achieve the effect of chemo-anti-angiogenesis combination therapy. |
PDF Full Text Request