| Invasive fungal infection（IFI）refers to a disease in which fungus invades the human body,grows and reproduces in the blood,tissues,or organs,and causes inflammation and tissue damage.It is also called deep fungal infection or systemic fungal infection.In recent years,with the abuse of chemotherapeutics,immunosuppressants,and broad-spectrum antibiotics,the number of immunosuppressed patients has gradually increased,leading to an increasing number of invasive fungal infections.The high fatality rate of IFI is mainly related to the lack of effective antifungal therapy.At present,the drugs used to treat IFI are relatively limited,only polyenes（amphotericin B,AmB）,pyrimidine analogs（5-fluorocytosine）,and triazoles（fluconazole,itraconazole）.Because these drugs have different degrees of adverse reactions,their clinical application is limited.As one of the typical pathogenic fungi of invasive fungal infection,Cryptococcus neoformans（C.neoformans）is the main pathogen of cryptococcosis.It first enters the human body through the respiratory tract and causes lung infection.If the pathogen spreads to the central nervous system（CNS）,it will cause cryptococcal meningitis,causing extremely high morbidity and mortality.More seriously,as a facultative intracellular pathogen in lung infections,C.neoformans can not only escape the host’s phagocytosis,survive and replicate highly in phagocytes,but can also dissolve macrophages or pass through the phagosome extrusion process to escape from macrophages,leading to repeated attacks of disease.In short,Cryptococcus-infected macrophages are also one of the potential target cells.Therefore,the infection of multiple organs and multiple target cells represented by C.neoformans indicates that in addition to the need for further development of highly effective antifungal drugs,advanced delivery methods are used to assist drugs to penetrate various physiological and pathological barriers to achieving effective treatment is also important.A targeted delivery system（TDDS）refers to a drug delivery system that selectively delivers the drug to target tissues or organs through a carrier after the preparation is administered through a certain route,mainly with passive targeting and active targeting to achieve effective drug delivery to two targeted mechanisms.In passive targeting,the particle size of the preparation is the main factor affecting organ targeting.Under normal circumstances,preparations with larger particle diameters are easier to retain in certain tissues.While preparations with smaller particle diameters have better tissue penetration,they are often faced with achieving better tissue accumulation and permeability at the same time.Because of the above problems,researchers have proposed a large class of microenvironment-responsive TDDS in recent years.According to the characteristics of the disease microenvironment,the delivery system mainly uses microenvironmental stimulating factors such as enzymes,p H,glutathione（GSH）,reactive oxygen species（ROS）,hypoxia,and adenosine triphosphate（ATP）to achieve mutual conversion of the particle size of the preparation,It can not only increase the accumulation of the preparation in the lesion site but also penetrate deeply into the tissues and exert a good therapeutic effect.Active targeting uses the biological specific interaction of ligand-receptor binding to achieve targeted drug delivery.At present,the commonly used ligands mainly include surfactants,carbohydrates,folic acid,transferrin,and peptides.Some complex diseases often have more than one target cell.When there are multiple receptors,the delivery system usually needs to be modified with multiple ligands,which not only complicates the delivery system but also greatly reduces the druggability.Therefore,if the common receptors for the lesion site and the affected cells can be found,a single ligand can be modified to achieve the effect of"multiple birds with one stone".Through preliminary studies on the microenvironment of Cryptococcus infection,we found that matrix metalloproteinase 3（MMP-3）is highly expressed in the infectious microenvironment（IME）,which is designed to deliver the response to the infection microenvironment,the system laid the foundation.SPARC proteins in infected macrophages and lung and brain microvascular endothelial cells were also found to be significantly up-regulated,suggesting that SPARC may serve as a potentially shared receptor.Based on this,we designed and constructed a variable particle size albumin targeted delivery system based on infection microenvironment response for anti-C.neoformans infection.Since albumin is a good drug carrier material and a natural ligand of SPARC,albumin was selected as the carrier material and AmB was used as a model drug to prepare AmB-loaded bovine serum albumin nanoparticles（BSA NP/AmB）.To further assemble NPs into microspheres,we designed and synthesized a special dual-function linker.We first obtained a peptide（PNG）that can bind to albumin through phage display random peptide library screening,and then integrated it with the MMP-3 response peptide sequence（NFF-3）into a new peptide（PN）,and adopted SPR technology verified the affinity between PN polypeptide and albumin.Then through the maleimide reaction and 4-arm-PEG-Mal combined to form a special linker（PN-PEG）,the linker can not only ensure that the albumin nanoparticles are ordered to form microspheres and have good stability when the formulation is assembled.In the microenvironment of the lesion,it can realize the rapid response of the microspheres and transform into the form of nanoparticles.Finally,the linker and NP were co-incubated successfully to prepare a targeted delivery system of variable particle size albumin in response to the infection microenvironment.We expect that this responsive delivery system can quickly accumulate in the lungs through passive targeting,and then respond quickly to the up-regulated MMP-3 in the IME to break the connecting arms into nanoparticles,and further actively target cells or tissues that highly express SPARC,achieving multiple targeting and effective treatment.The main research contents are as follows:1.Construction of microenvironment-responsive albumin targeted delivery system（1）First screen albumin-binding peptides by phage display technology,and after three consecutive rounds of elutriation through the steps of coating,binding,elution,and amplification,phage single clones are selected for specific identification,and some can be successfully selected.A positive clone of the phage that specifically binds to albumin and four specific affinity peptide sequences were obtained.After analysis,the peptide with the highest repetition rate was selected for SPR and molecular docking technology to verify its affinity with albumin.It was found that the peptide PGNLALRPDSNS（PGN）had the highest affinity with albumin,with a KD value of2.55×10-7M,it was found through molecular docking.The peptide may form strong hydrogen bond interactions with the Asp111,Leu115,Lys114,Glu519,Lys523,Thr518,Glu424,and Arg458 amino acid residues of albumin.Then integrate it with the MMP-3responsive polypeptide（NFF-3,Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys（Dnp）-NH2）sequence into a new polypeptide（PN）,And react with 4-arm-PEG-Mal to synthesize bifunctional linker（PN-PEG）through maleimide reaction.（2）Next,prepare AmB-loaded albumin nanoparticles（BSA NP/AmB）by emulsification solvent evaporation method,detect their particle size by Malvern laser particle size analyzer,observe their morphology by scanning electron microscope,and measure the average of BSA NP/AmB The particle size is about 115nm,with good dispersibility;and its morphology is round and the size is uniform.A method for determining the content of amphotericin B was established by high-performance liquid chromatography,and the test showed that the encapsulation rate of the preparation reached more than 90%.Finally,the BSA NP/AmB and PN-PEG were co-incubated.By adjusting the ratio of BSA NP and PN-PEG,it was found that when the ratio of the two reached 4:1,the average particle size of BSA MTN/AmB was about 7μm,and When the ratio increases,the particle size no longer changes significantly,indicating that this condition is the best ratio and the particle size meets the requirements of later experiments.Through the laser particle size analyzer and scanning electron microscope,it is found that the dispersion is uniform,the shape is round,and the surface has a certain size of aperture.The gap left by the assembly can be seen,indicating that the nanoparticles have been successfully assembled into a microenvironment responsive white with a suitable particle size Targeted protein delivery system（BSA MTN/AmB）.（3）By investigating the stability and drug release performance of BSA NP/AmB and BSA MTN/AmB,it is found that both are stable at 4℃and compared with BSA NP/AmB,BSA MTN/AmB has a certain degree of slowness.This may be because BSA MTN/AmB is formed by the affinity aggregation of BSA NP/AmB and linker,so it has a certain degree of compactness,which slows down the release of the drug.2.In vitro responsiveness and targeting evaluation of the microenvironment-responsive albumin targeted delivery system（1）Based on the successful preparation of BSA MTN,we prepared the control preparation BSA MP by the same method with the scrambled peptides of the MMP-3response sequence,and at the same time investigated its degradation under the action of MMP-3.Through SEM morphological observation and particle size measurement results,it was found that BSA MTN was completely dissociated into BSA NP after co-incubation with MMP-3 for 2 hours,while the morphology of the control preparation BSA MP did not change significantly under this condition,indicating that BSA MTN effects MMP-3 can respond specifically.（2）To explore the potential targeting of albumin preparations to corresponding cells,we first used RAW264.7 infected with Cryptococcus intracellularly as model cells for in vitro experiments.Through cell uptake experiments,it was found that compared with normal cells,the uptake of albumin preparations by infected macrophages was significantly increased,and the flow quantification results also showed the same trend,and the uptake was about twice that of normal cells.Next,the cell surface proteins were analyzed by immunofluorescence and western blotting,and it was found that compared with normal cells,SPARC protein expression in infected macrophages was relatively high.The SPARC gene was further silenced by anti-SPARC antibody blocking protein and si RNA,and it was found that the uptake of albumin preparations by infected cells was significantly reduced after treatment,which verified that SPARC is a potential receptor,and the targeting mechanism of preparations is based on the specificity of albumin and SPARC.Combine.Fluorescence co-localization experiments proved that albumin preparations mainly rely on the endocytosis of clathrin to enter the cell,escape the lysosome,and transport more to the endoplasmic reticulum,which is consistent with the results reported in the literature.（3）Next,we constructed cryptococcal infection models of lung and brain microvascular endothelial cells to explore the potential targeting of albumin preparations to both.Through fluorescence imaging analysis,it was found that compared with normal mouse lung microvascular endothelial cells（PMVECs）,the uptake of albumin preparations by infected cells was significantly increased,and the flow quantification results also showed the same trend.Through immunofluorescence experiments,it was found that SPARC was also highly expressed on the cell surface of infected PMVECs,indicating that the preparation was targeted to infected PMVECs through SPARC mediation.Finally,the ability of the preparation to penetrate the blood-brain barrier was analyzed by laser confocal layer scanning,and it was found that compared with normal lung microvascular endothelial cells（b End3）cells,albumin preparations had stronger penetration in the blood-brain barrier（BBB）infection model.Permeability,flow cytometry results also show that the intake of albumin preparations by infected BBB is significantly increased.Western blot experiments also found that the infected b End3 cells highly expressed SPARC.The above results indicate that BSA MTN has good in vitro responsiveness,and the response albumin preparation can well target cryptococcal infected macrophages,brain,and lung microvascular endothelial cells.The targeting mechanism is based on albumin.The specific binding with SPARC once again verified that SPARC is a common receptor for several potential target cells.This result laid a certain foundation for the in vivo response and targeting of BSA MTN.3.In vivo responsiveness and targeted distribution of microenvironment-responsive albumin targeted delivery system（1）Based on in vitro experiments,further investigate the responsiveness and targeted distribution of the preparation in vivo.First,an in vivo analysis method of amphotericin B was established.Pharmacokinetic experiments showed that the half-life of BSA MTN was about twice that of ordinary albumin nanoparticles,which significantly extended the circulation time of the drug in the body.Considering that the natural way of cryptococcal infection is to enter the body through the respiratory tract,this project uses the nose drop method to construct a mouse cryptococcal infection model.In vivo imaging of small animals was used to evaluate the enrichment of BSA MTN in the lungs.The results showed that the accumulation of BSA MTN and BSA MP in the lungs of normal and infected mice was significantly higher than that of BSA NP,and in the in vitro imaging of infected mice,BSA MTN The accumulation in the brain is significantly higher than that of BSA MP,reflecting that BSA MTN responsively degrades into nanoparticles in infected mice and then penetrates the blood-brain barrier.The distribution of amphotericin B in mouse brain and lung tissues also showed the same targeting ability.（2）To investigate the expression of enzymes in the lungs of infected mice,we detected the expression of MMP-3 by immunofluorescence and western blotting.It was found that compared with normal mice,the expression of MMP-3 in the lungs of infected mice was significantly increased,and was significantly higher than that of MMP-2 and MMP-9,again verifying that MMP-3 is the microenvironmental stimulating factor of the response system.Further study its in vivo responsiveness,with BSA MP as a control,it was found that in infected mice,BSA MTN disintegrated rapidly in the lungs,no obvious microsphere morphology was observed within 8 hours and finally dispersed in the lung tissue.While BSA MP The metabolism is slow,and a large amount of it is still trapped in the pulmonary capillaries,indicating that BSA MTN has better enzyme responsiveness in infected mice.（3）To further explore the ability to target the lung,brain,and potential target cells of the main affected organs after BSA MTN response.First,analyze the fluorescence colocalization experiment of tissue slices.It is found that the green fluorescence of the preparation in the brain and lung slices overlaps with the red fluorescence of SPARC,indicating that the BSA MTN response has good active targeting to the lung and brain.And this targeting is based on the high expression of SPARC in infected mouse lung and cerebral vascular endothelial cells,which is consistent with the results of in vitro studies.Then we extracted and studied the infected mouse alveolar macrophages.It was found that SPARC was also highly expressed on the surface,and the green fluorescence of the preparation in the infected mouse macrophages was significantly higher than that of normal mice,indicating that the preparation can target potential target cells after responding.The above results indicate that BSA MTN also has good enzyme responsiveness in vivo,and this preparation not only has the passive lung targeting of micron-level preparations but also can be well targeted to the micro-environment after its response to disintegration in the infection microenvironment.Infects mouse lung,brain,and alveolar macrophages,showing good active targeting.It provides a good basis for follow-up treatment.4.Preliminary evaluation of the antifungal effect and safety of the microenvironment-responsive albumin targeted delivery systemFirstly,the antifungal effects of BSA NP/AmB,BSA MP/AmB,and BSA MTN/AmB were evaluated through the MIC experiment.Taking the crude drug as a control,it was found that BSA NP/AmB had the best antifungal effect,which was superior to the drug substance and other preparations.Next,a mouse cryptococcal infection model was established by the nasal drip method.The model mice were dissected on the third and seventh days after administration,and the lung and brain tissue homogenate were coated to count the fungi.The results showed that BSA MTN/AmB The therapeutic effect of the drug is significantly better than that of other preparation groups.In the MRI analysis images,it can be seen that the lungs of the mice treated with BSA MTN/AmB have almost no residual MRI infection signals,and the survival cycle is longer than that of the control mice,indicating that BSA MTN/AmB has better in vivo efficacy.Finally,the safety of the preparation in vivo was investigated.The results showed that the four serum biochemical indexes of alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,urea（UREA）,and creatinine（CREA）were all within the normal range.The slices were also normal,and the kidney function monitoring system of small animals in vivo did not observe obvious nephrotoxicity,indicating that the preparation had no obvious toxicity to the body.Conclusion:In summary,we designed and constructed variable-size albumin targeted delivery system based on infection microenvironment response for anti-C.neoformans infection.After intravenous injection,the delivery system first reaches the lungs through the blood circulation and is mechanically trapped by the pulmonary capillaries,so that the drug is concentrated in the lungs to achieve passive targeting.When it is broken into nanoparticles in the microenvironment of infection,By actively targeting through the blood vessel wall and targeting brain tissue and infected macrophages,better active targeting is achieved.The delivery system combines the advantages of active targeting and passive targeting,which not only realizes the effective enrichment of the preparation in the lesion site but also improves the ability of the preparation to penetrate the blood vessel wall and the affinity to the lesion site,realizing multiple targeting and effective treatment.Since the lung is the main organ for many pathogenic microorganisms to invade,the delivery system has broad application prospects in the treatment of lung diseases,especially diseases with intracellular infections. |
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