| Lymphoma is one of the fastest rising malignancies wordwide.Lymphoma patients usually have granulocytosis and low immune capacity,and the immune capacity of patients will be further reduced by factors such as high-dose chemotherapy or immunosuppressive therapy before stem cell transplantation,so it is common for lymphoma patients to be complicated with fungal infections（such as Candida albicans,Cryptococcus,etc.）At present,the main clinical drugs are doxorubicin,bleomycin,cisplatin,etc.,which have great toxic and side effects.At the same time,after the complication of fungal infection,they should be combined with antifungal drugs for treatment,and multi-drug combination will further increase drug toxicity.Itraconazole（ITZ）as a clinical commonly used classic resistance to fungal infection,compared with other antibacterial drugs（amphotericin B）the side effects is smaller,especially the recent studies have found that itraconazole for non-small cell lung cancer,prostate cancer,basal cell carcinoma,medulloblastoma tumors have inhibitory effect,and some has entered the stage of clinical trials.Therefore itraconazole is expected to be a multiagent agent for the treatment of tumor and its associated fungal infection.However,the physical and chemical properties of itraconazole are poor,and the usual delivery system has poor encapsulation efficiency and poor stability,as well as lack of targeting to tumors and fungi.Based on this,a biomimetic micelle of itraconazole was designed in this project.Its construction idea is:based on the interaction between tea polyphenol analogs（EGA）and ITZ,PEG-EGA was used as drug carrier to self-assemble with ITZ,and a biomimetic micelle with good stability and high encapsulation rate was formed.Based on the presence of functional proteins that interact with lymphoma and cryptococcus on the macrophage cell membrane,we further encapsulated the macrophage cell membrane on the basis of biomimetic micelles to construct a targeted drug delivery system,the preliminarily relevant evaluation and pharmacodynamics study on the drug delivery system were carried out.This paper is divided into three parts.In the first part,a bionic drug delivery system with itraconazole nanomicelles wrapped in macrophage membrane was constructed,and characterize its physical and chemical properties and biological characteristics.First,PEG-EGA was synthesized,and the results of MALDI-TOF MS spectrum proved that the molecular weight of the synthesized polymer was consistent with the expected results.The interaction between EGA and ITZ was used to form nano-micelles.The critical micelle concentration（CMC）of micelles determined by surface tension method was1.52×10-5 mol/L-1.The macrophage cell membrane was wrapped on the surface of the nano-micelles by physical extrusion method.The particle size and Zeta potential of the nano-micelles were measured by Malvan laser particle size analyzer.It was found that the particle size increased,and the Zeta potential of the nano-micelles changed from-3.8 m V to-22.4 m V,which was basically consistent with the potential of the macrophage cell membrane,suggesting that the encapsulation of the macrophage cell membrane may be realized.A ring structure was observed on the outside of the circular nano-micelles by transmission electron microscopy,which proved that the cell membrane was well wrapped on the surface of the nano-micelles.The differences of proteins on isolated macrophage cell membranes and encapsulated nano-micelles were investigated by sodium dodecyl sulphate polyacrylamide gel electrophoresis.The results showed that the protein bands of the two groups were basically the same,indicating that the proteins on the cell membrane were not damaged in the process physical extrusion.Western blotting also detected SIRP-α,Dectin-1 andα4 proteins,which laid a foundation for the transfer of the related biological functions of macrophage cell membrane recognition of tumor cells and cryptococcus to nano-micelles.The fluorescence co-localization also showed that the cell membrane was intact on the surface of the nano-micelles.A method for the determination of itraconazole was established by HPLC.It was found that the encapsulation rate of the formulation reached more than 80%.After continuous monitoring for 72 hours,it was found that the encapsulation rate and particle size of the preparation had no significant change,showing a good stability.In the in vitro release experiments,the cumulative release rate of the drug coated by macrophage cell membrane was about two thirds of that of the control group,showing a better sustained release performance.In the first part,a bionic drug delivery system containing macrophage cell membrane and itraconazole nanomicelles was successfully constructed.In the second part,its long cycle function and targeting function was evaluated.Uptake of the formulation by the reticuloendothelial system was simulated by the uptake of normal macrophages.The results of fluorescence microscopy and flow cytometry both showed that the encapsulation of cell membrane could effectively reduce the uptake of nanoparticles by macrophages,which showed a good long-term circulation effect in vitro.An in vivo analysis method of itraconazole was established.Pharmacokinetic experiments showed that the half-life of nano-micelles coated by macrophage cell membrane was about twice that of ordinary nano-micelles,which significantly extended the drug’s circulation time in vivo.A20 cells were used as lymphoma model cells and Cryptococcus as model fungi.The uptake of preparation by A20 cells and Cryptococcus showed that the uptake of nano-micelles wrapped by macrophage cell membrane was significantly higher than that of ordinary nano-micelles,which proved that the nano-micelles wrapped by macrophage cell membrane had the targeting ability to both A20 cells and Cryptococcus.After the inhibition of the relevant antibodies,the uptake of the coated nano-micelles by A20 cells and Cryptococcus was significantly lower than that of the unblocked group,indicating that the antibodies on the macrophage membrane played a key role in the specific recognition.Subsequently,a subcutaneous tumor-bearing mouse model was constructed,and in vivo and in vitro imaging experiments were conducted to prove that the nanoscale micelles wrapped in the macrophage cell membrane had the ability to target the tumor sites of the tumor-bearing mice.Considering that the natural way for cryptococcal infection of the lungs is to enter the body through the respiratory tract,this study constructed a mouse pulmonary infection model by nasal drip of cryptococcus liquid.In vivo imaging and fluorescence co-location images were used to demonstrate that nano-micelles coated with macrophage cell membrane targeted to lung tissues infected with fungi.Subsequently,a subcutaneous tumor-bearing mouse model with cryptococcus infection was constructed.The distribution of itraconazole in rat brain,heart,liver,spleen,lung and kidney tissues and tumour site also showed that nano-micelles wrapped by macrophage cell membrane would accumulate in the lungs and tumour site again confirming the tumour targeting function and lung targeting of the formulation in vivo.In the third part of this paper,the efficacy and safety of the bionic drug delivery system of macrophage membrane coated with itraconazole nano-micelles were preliminarily evaluated.Firstly,the IC50 value of the preparation to A20 cells was 0.5μg/m L in vitro by MTT assay.The minimum inhibitory concentration（MIC）of free ITZ against Cryptococcus was 0.25μg/m L,while the MIC of MNC-ITZ was 0.125μg/m L,indicating that PEG-EGA could reduce the MIC of ITZ when it was used as a carrier to form micelles.The minimum inhibitory concentration was 0.125μg/m L by microplate dilution assay.It was confirmed that the preparation had good anti-tumor and anti-bacterial properties in vitro.Subsequently,the tumor inhibition effect of the preparation was investigated in the subcutaneous tumor-bearing mouse model,and the results showed that the body weight of the RAW-MNC-ITZ group remained normal and the tumor inhibition effect was the best.The model mice were dissected on the third day and the sixth day after administration,and the fungal count was performed on the plate of lung tissue homogenate.It was found that the therapeutic effect of nano-micelles coated with macrophage cell membrane was significantly better than that of the control group.These results indicate that the nano-micelles wrapped in macrophage membranes also have good anti-tumor and anti-bacterial efficacy in vivo.Finally,the safety of the formulation in vivo was preliminarily investigated.The serum biochemical indexes of AST,ALT,CREA and UREA were all within the normal range,and there was no abnormality in pathological sections,indicating that the formulation had no obvious toxicity to the body.In summary,this project used the natural product PEG-EGA as a drug carrier to integrate the biological functions of cell membrane,and successfully constructed a bionic micelle preparation of itraconazole.Various in vivo and in vitro experiments have confirmed that the micelle has active targeting for lymphoma and pulmonary infection caused by cryptococcus,and has good long-term circulation function,which is better than the MNC-ITZ group,and has a good application prospect in the treatment of lymphoma and its complicated fungal infection. |
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