Design And Properties Study Of Protein-based Intelligent Anti-tumor Drug Nano-delivery System

The development and treatment of cancer drugs has always been a research hotspot in the field of biomedicine.Although there have been clear breakthroughs in immune checkpoint inhibitor drugs in recent years,the therapeutic effect is still objectively dependent on increasing the dose.Most of the chemotherapeutic drugs widely used clinically have poor water solubility,low bioavailability,and large toxic and side effects,making it difficult to achieve the ideal effective therapeutic dose.Even for a targeted drug with a clear therapeutic effect,its kinetic process in the body only has the broad distribution of the drug and the targeted characteristics of the occurrence of the effect,and the therapeutic effect is also limited by the administered dose.In the current situation where the discovery and modification of new drug compounds is still difficult to significantly break through the high-dose administration,the improvement of this problem from the perspective of formulation materials has attracted widespread attention in the industry.Cases of using nanoparticles to encapsulate poorly soluble chemotherapeutic drugs to improve their solubility,increase the targeting of tumor tissues,and achieve"toxin reduction and efficiency enhancement"have been reported.However,these nano-drug delivery systems still have common problems such as low accumulation of chemotherapeutic drugs in target tissues,many influencing factors of drug release in target tissues,and limitations of chemical treatment alone.In this thesis,the body's own protein was used as the material to design and prepare a nano-drug carrier with good biocompatibility,strong targeting,and drug release with multiple responses to the tumor microenvironment,and evaluated the excipient performance of new preparations.The main findings are as follows:(1)Based on the report that high concentrations of matrix metalloproteinase 2(MMP-2)and hyaluronidase(HAase)were generally present in the extracellular matrix of the tumor microenvironment,the type I collagen and hyaluronic acid as the substrate of the two enzymes was designed and prepared as gated and targeted molecules on the surface of mesoporous silica nanoparticles.Taking doxorubicin(Dox),which has a clinically clear therapeutic effect,as an experimental example,a dual-enzyme-responsive nano-drug delivery system(FMSN-Dox-C2H)was prepared.The designed mesoporous silica nanoparticles had green fluorescence,while dox had red fluorescence,which could visually evaluate the functional response of FMSN-Dox-C2H nanoparticles.FMSN-Dox-C2H nanoparticles were160×80 nm cylinders with low albumin adsorption properties.The grafting rates of collagen and hyaluronic acid were 8%and 6%,respectively,and the drug loading was 9.9±0.3%.In vitro drug release experiments found that when MMP-2 and HAase were added to the FMSN-Dox-C2H group,the dox release rate after 72 h reached 70.3±7.5%,while the release rate of the control group without enzyme was only 7.06±2.8%,which achieved the design goals of using collagen and hyaluronic acid to block the particle pores,improved the stability of nanoparticles in body fluids and double enzyme responsed drug release.Taking the normal cell line Cos-7 which did not express MMP-2 and HAase and the liver cancer cell Hep G2with low expression of the two enzymes as a reference,it was found that FMSN-Dox-C2H nanoparticles had a dose-dependency of killing effect to the cervical cancer cell He La with high expression of the two enzymes.Laser confocal microscopy observed that the amount of dox diffused into He La cells was significantly higher than that of Hep G2 cells,suggested that the responsiveness of nanoparticles to dual enzymes met the design expectations for effective release of doxorubicin,and hyaluronic acid had an effect on the surface of He La cells.The targeting effect of the corresponding receptor CD₄₄ was also a possible explanation for this intracellular concentration difference.The in vivo experimental results of the He La cell tumor-bearing mouse CDX model of FMSN-Dox-C2H nanoparticles also verified the possibility of this explanation.(2)In view of the inability to degrade the mesoporous silica nanoparticles in vivo and the technical difficulties in quality control of the already marketed Abraxane,the paper used gene recombination technology to design with polyhistidines(His),MMP-2 restriction site and RGD peptide at both ends of human serum albumin(HSA)fusion gene sequence,realized the expression of a series of fusion proteins in the Pichia pastoris yeast system.The fusion albumin(3RGD-HSA-MMP-18His,RHMH18)with a molecular mass of 71796 Da containing 18His could be easily and controlled to self-assembly,and the formed nanoparticles were in a regular spherical shape with a diameter of about 100 nm.The RHMH18 nanoparticles prepared after encapsulating paclitaxel(PTX)had a drug loading of6.6±0.3%.The drug release rate of 72 h after adding MMP-2 and changing the p H could reach 68.7±4.2%,which was significantly higher than the release amount of the control group(6.2±0.9%),and the experiment found that RHMH18 nanoparticles had a quantity-dependment of drug release rate of histidine.Based on the expression of RGD receptor???₃-integrin,low-expressing breast cancer MCF-7 cells,high-expressing lung adenocarcinoma A549 cells,and MGC-803 cells were selected as test cells,and it was found that RHMH18 nanoparticles had obvious?_??₃ targeting and sensitivity to MMP-2.The in vivo experimental results of RHMH18 nanoparticles in CDX models of mice bearing MGC-803cell tumor and A549 cell tumor also verified the reliability of the in vitro experimental results,and it was observed that the in vivo half-life of PTX of the nanoparticle group could reach63.36 h,and the LD₅₀ was 58.5 mg/kg/day.The above results suggested that the design of RHMH18 nanoparticles could achieve the medicinal effects of mesoporous silica nanoparticles with type I collagen and hyaluronic acid as gated and targeted molecules,and clear source,diverse functions and single controllable quality.The self-assembled nanoparticles had uniform size and balanced drug loading,which were significantly better than type I collagen and hyaluronic acid with a large molecular mass range,and were beneficial to be used as excipients for the preparation of corresponding pharmaceutical preparations,in vivo experimental results were significantly improved the safety of the dose of PTX and the concentration of the drug at the tumor site which showed obvious therapeutic effects.Both RHMH18 nanoparticles and the Abraxane were used the self-assembly of the hydrophobic area on the surface of albumin to solve the difficult preparation problems of preparations of insoluble drugs,but the principle of action was different.The former was to encapsulate/release the drug through the hydrophilic and hydrophobic transformation of the poly-His fused to albumin under different p H conditions,and to target???₃-integrin through the RGD peptide to achieve a high degree of drug aggregation in tumor tissues which was theoretically superior to the latter's mechanism of intracellular drug release.In addition,the designed RHMH18 with poly-His had strong self-assembly capabilities,which clearly improved the latter's shortcomings of difficult quality control in production.(3)In order to improve the drug-carrying capacity of RHMH18 nanoparticles and the need for multi-functional synergistic treatment of tumors,RHMH18@Au D co-encapsulating gold nanoparticles and relatively polar docetaxel drug was designed and prepared,and its drug loading rate was 18.5±0.9%,particle size 80 nm.Based on the tumor treatment indications of docetaxel,ovarian cancer A2780 cells with high expression of RGD receptor???₃-integrin were selected as the test cells,and it was found that the RHMH18@Au D nanoparticles containing docetaxel had clear characteristics:tumor cell targeting,tumor microenvironment enzyme responsiveness,p H responsiveness,and photothermal effect.The system investigated the effects of light intensity,time,and p H of the solution on the photothermal efficiency of RHMH18@Au D nanoparticles,and found that the thermal effect could reach up to 56.8°C.The in vivo experimental results of the RHMH18@Au D nanoparticles containing docetaxel in the CDX model of A2780 cell tumor-bearing mice verified that the nanoparticles had the effect of chemical drug-photothermal synergistic treatment.The survival period evaluation of the model animals showed that the survival time of the synergistic treated animals was longer than that of the control group treated with chemical drugs,and the dosage could be reduced by half to achieve the tumor-inhibiting growth rate of chemical treatment alone.It was suggested that the designed RHMH18@Au had multiple functions,which could not only increase the drug loading level of nanoparticles,improve the loading ability of preparations,but also provided a means for combined tumor therapy.