A Preliminary Study On The Construction Of Biomimetic Platinum Liposome And Its Inhibition To Lymphoma With Fungal Infection

Lymphoma is a hematologic malignant disease.Both the disease itself and the means of treatment can easily lead to immunosuppression（IR）in the patient.Usually,lymphoma results in impairment of immune functions in such patients,and invasive pulmonary fungal infection（IPI）is a common complication.Most patients with lymphoma complicated by cryptococcal infection are critical cases,with a high fatality rate and expensive medical costs.Patients are treated simultaneously with antineoplastic drugs and antifungal drugs,which are expensive in medical expenses and have high toxic side effects.Patients are treated simultaneously with antineoplastic drugs and antifungal drugs,which are expensive in medical expenses and have high toxic side effects.Clinical treatment the standard drug for IPI is amphotericin B,which have severe nephrotoxicity and not suitable for immunocompromised lymphoma patients.Platinum drugs are widely used in the treatment of malignant tumors because of their simple structure and curative effect.Cisplatin,Carboplatin,and Oxaliplatin are common bivalent Pt（II）drugs that exert anticancer efficacy by inhibiting cancer cell DNA replication.Pt（II）link two ligands in axial direction can converted to Pt（Ⅳ）.Pt（Ⅳ）is low toxic,Pt（Ⅳ）can be reduced to toxic Pt（II）by glutathione and ascorbate to exert antitumor effects.Meanwhile,functional axial ligands may confer or tune a range of biochemical properties to platinum based drugs.Currently,platinum drugs have been confirmed to have activity against some pathogenic microorganisms,and previous studies have found that platinum drugs have antifungal potential.Modification of platinum drugs by functional axial ligands,which retain antitumor activity while improving their antifungal ability,it can provide a new idea of“one drug-two effects”for lymphoma with fungal infection.Cell membrane biomimetic drug delivery system to accomplish drug delivery is to increase the accumulation of platinum drugs in the focal site.Encapsulation of drug carriers using endogenous cell membranes,so that drugs can evade recognition versus clearance by the reticuloendothelial system（RES）while sharing the biological functions of the cell membrane with the inner core.Based on the characteristic of specific attachment of macrophage with lymphoma and pathogenic fungi,it was proposed to further encapsulate with macrophage membrane on the basis of constructing platinum drug liposomes to increase the biocompatibility,platinum drugs can achieve the aim of“one membrane-two targets”in this way.Invasive pulmonary fungal infection（IPFI）is the leading cause of IPI.Cryptococcus neoformans belongs to the major conditional pathogenic fungi of IPFI.Malignant lymphoma patients treated with high-dose corticosteroids and long-term immunosuppressive agents are susceptible to Cryptococcus neoformans.Therefore,we chose Cryptococcus as the experimental fungus with a preference for several structurally similar Pt（Ⅳ）synthesized in the previous period.Investigation of lymphoma with Cryptococcus infection by construction of biomimetic platinum drug liposomes.This paper is divided into three parts.The first part is the design and characterization of the preparation.Firstly,several new tetravalent platinum complexes with similar structures are designed together and the fat-soluble tetravalent platinum--Pt（Ⅳ）₁,which has the best comprehensive efficacy.The analysis found that the antibacterial mechanism of Pt（Ⅳ）₁was similar to the cisplatin bacteriostasis in the report,Pt（Ⅳ）₁further inhibited the growth of Cryptococcus by binding the functional protein Prp8 to attenuate its ability to cut RNA,which became more sensitive to the drug when Prp8 was low expressed and vice versa.PEG-LIP was prepared by thin-film hydration method,RAW-PEG-LIP was prepared by encapsulating extracted macrophage membrane on the surface of liposomes using physical extrusion effect of liposome extruder,comparing the physicochemical properties between them,it was found that the particle size of RAW-PEG-LIP increased and the potential decreased.By observing the morphology of the formulations by TEM,PEG-LIP exhibited a round sphere shape,while RAW-PEG-LIP presented an obvious shell-core structure.In the fluorescence co-localization images,the preparations of the RAW-PEG-LIP group were largely overlapped with the cell membrane,which further verified the integrity of the encapsulated liposomes by the cell membrane.The expression of important functional proteins on the cell membrane was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblot analysis.The gel electrophoresis experiment mainly compares the difference of protein bands between the membrane of macrophage and the liposomes before and after coating,which proves that the coating process of a physical extrusion does not cause the loss of a large number of proteins on the membrane of macrophage.The expression ofα₄protein and MAC-1 protein on the cell membrane of macrophages was detected by protein immunoblot analysis,which is crucial for macrophages to recognize Cryptococcus and lymphoma cells.The encapsulation rate and stability of different experimental groups all met the experimental requirements,and the sustained release performance of the RAW-PEG-LIP group was the best through in vitro release experiments.The second part is to investigate the long-cycle function and targeting effect of the liposomes.All in vivo experiments needed to construct corresponding animal models in advance:（1）Mouse model of cryptococcal lung infection;（2）Mouse model of subcutaneous tumor-bearing;（3）Tumor-bearing mouse model of cryptococcal infection.After modeling and comparison,it was found that IPFI infection in tumor-bearing mice would aggravate the symptoms of a single disease,suggesting that the growth of the malignant tumor and fungal infection interact to form a vicious cycle.Comparing the uptake of different liposomes by normal macrophages with the quantitative analysis of flow cytometry by qualitative observation with fluorescence microscopy,combined with the results of pharmacokinetic analysis,proved that the RAW-PEG-LIP group had some long circulation function.The study of single targeting of agents in vitro and in vivo is mainly divided into agents targeting Cryptococcus and agents targeting tumor cells.Firstly,the results of the in vitro fungal uptake assay showed that the uptake of cryptococci into raw-peg-lip was significantly higher than that of peg-lip,and this significant difference disappeared after blocking using the Mac-1 antibody,indicating that raw-peg-lip can achieve in vitro targeting of cryptococci through the Mac-1 protein.In vivo targeting experiments included live ex vivo imaging with fluorescence colocalization analysis in the lungs,and the study found that RAW-PEG-LIP would accumulate in the infected lung tissue,indirectly prolonging its circulation in vivo.It was found that the uptake of RAW-PEG-LIP in lymphoma cells was significantly higher than that of PEG-LIP.The Blocked lymphoma uptake experiment used either VCAM-1 or ICAM-1 antibodies as the Blocked group.Lymphoma cell uptake experiments incubating VCAM-1 or ICAM-1antibody as a blocked group showed that both antibodies could affect RAW-PEG-LIP uptake by lymphoma cells,in which the cellular uptake was significantly inhibited by incubating ICAM-1 antibody group,indicating that the latter plays a more functional role in the process of specific recognition.Combined with the results of in vivo imaging and tumor site fluorescence co-localization of mice,it was proved that RAW-PEG-LIP has a certain tumor-targeting ability.The distribution of different preparations in the tissues of tumor-bearing mice infected with Cryptococcus was investigated to analyze the comprehensive targeting effect of the preparations.The results showed that the distribution of RAW-PEG-LIP in the lungs and tumor sites of model mice was significantly higher than that of PEG-LIP.In conclusion,the encapsulation of macrophage cell membrane can enhance the targeting effect of liposomes on Cryptococcus and lymphoma cells in vitro and in vivo,and the targeting evaluation of this part of the preparation is also the experimental basis for subsequent pharmacological studies.The third part is the evaluation of drug efficacy and safety.The evaluation of the efficacy was divided into two aspects:single antibacterial and anti-tumor effects and comprehensive efficacy.Firstly,the antibacterial activity in vitro and in vivo of the preparation was compared by microplate dilution,fungal loading,and lung tissue fluorescence section test,and then the antitumor activity in vitro and in vivo of the preparation was compared with tumor size by MTT method.The results showed that RAW-PEG-LIP/Pt（Ⅳ）₁group had significantly better bacteriostatic and anti-tumor effects than PEG-LIP/Pt（Ⅳ）₁group.The lymphoma mice infected with Cryptococcus lung were selected for MRI,PAS staining,and survival cycle to determine the comprehensive efficacy of the preparation.MRI analysis showed that the tumor size of the RAW-PEG-LIP/Pt（Ⅳ）₁group was significantly reduced after treatment,and there was no obvious MRI infection signal in the infected lungs.PAS staining showed that RAW-PEG-LIP/Pt（Ⅳ）₁group had the least amount of residual fungi in lung.At the same time,we found that the survival cycle of mice treated with RAW-PEG-LIP/Pt（Ⅳ）₁was significantly prolonged.Finally,the safety of the preparation was determined by the pathological analysis of serum sections.The drug-carrying liposomes used for treatment had no obvious toxicity to mice,and the serum biochemical indexes of mice were in the normal range and the pathological sections showed no abnormality.In conclusion,this project successfully constructed a macrophage-coated liposomal drug delivery system by using liposome to encapsulate a novel lipid-soluble platinum prodrug.Through this bionic drug delivery system,the biological function of macrophage-specific recognition of lymphoma and Cryptococcus was transferred to drug-loaded liposomes,achieving the effect of one drug two treatment and one membrane two targets in patients.Meanwhile,the better biocompatibility of drug-loaded liposomes after camouflaged by macrophage membrane can extend the time of drug in vivo circulation by evading the recognition by the reticuloendothelial system.This study completed the targeted delivery of a novel tetravalent platinum complex,providing a new idea for the research of cell membrane-targeted drug delivery system and the design of tetravalent platinum drugs,which has a broad application prospect in the treatment of immunosuppressed people with IPFI infection.