Construction Of Intracellular Zinc(Ⅱ)-regulating Nanomedicine And Basic Research For The Application

Metal ions are widely involved in many biological processes,including metabolic regulation and enzyme activity regulation.And metal ions play critical roles in cell activity.Any alteration of the metal content balance could induce a series of intracellular responses,even cell death.Ion-Interference Therapy(ⅡT)is a new strategy for disease treatment,which utilizes bioactive nanomaterials to exogenously import or endogenously consume metal ions for regulating metal ion homeostasis in the disease site,thus changing the physiological characteristics of diseased cells.Studies have shown that calcium,iron,and silver-mediated ⅡT play an important role in the field of anti-tumor or anti-bacterial.While,zinc(Ⅱ),as a key coenzyme,has rarely been thoroughly reported.Therefore,In the study,malignant tumors and drug-resistant bacteria were used as models,and a series of zinc(Ⅱ)-regulating nanomaterials were constructed for exploring the application of zinc-regulating strategies in the field of anti-tumor or anti-bacterial.The main contents are as follows:1.Stimulus-responsive zinc overloading nanodrugs induce systemic energy depletion in tumorsStarvation therapy has gained considerable attention for the clinical management of tumors in recent years.It is well-known that starvation treatment can inhibit tumors growth via ingredient consumption or blood vessel occlusion.However,incomplete and nonspecific energy blockade that leads to self-adaptive regulation of tumor cells and serious side effects on normal tissues restrict their clinical application.Here,a novel paradigm of starvation therapy is proposed to synergize the“zinc(Ⅱ)interference”-mediated glycolysis inhibition and zinc(Ⅱ)-activating glucose uptake blockade.Firstly,to obtain obvious intracellular zinc(Ⅱ)interference effect,Hyaluronic acid(HA)-modified ZIF-8 nanoparticles(HA/ZIF-8)with acidic-mediated zinc(Ⅱ)release characteristic were selected as potential positive zinc(Ⅱ)-regulating nanomaterials,which is expected to efficiently induce zinc overloading in tumor cells.We found that HA/ZIF-8 could induce abrupt intracellular Zinc(Ⅱ)elevation preferentially in melanoma cells with the assistance of HA-mediated tumor-specific targeting,and then achieve effective glycolysis blockade through“Zn(Ⅱ)interference”-triggered decrease of Nicotinamide adenine dinucleotide(NAD~+)and inactivation of glyceraldehyde-3-phosphate dehydrogenase(GAPDH).The result of Zinc(Ⅱ)content in the cells showed that,after 2 h treatment with HA/ZIF-8,the zinc(Ⅱ)content of malignant melanoma cells increased from 0.89μg/10~6 cells to 7.89μg/10~6 cells,while the zinc(Ⅱ)content of normal melanocytes only varied from 2.65μg/10~6 cells to 5.1μg/10~6 cells.In addition,the results further reflected that the initial zinc(Ⅱ)level in malignant melanoma cells was only 1/3 times that of normal melanoma cells,which attributed to a large amount of zinc(Ⅱ)efflux at the early stage of cancer.It is worth mentioning that the lower zinc(Ⅱ)content of malignant melanoma cells increased the susceptibility to intracellular zinc(Ⅱ)fluctuations.The monitoring results of glycolysis pathway products showed that HA/ZIF-8 could specifically block 71.0%NAD~+,leading to the inactivation of 64.6%GAPDH,the reduction of adenosine triphosphate(ATP)by 39.0%.Due to the negligible zinc(Ⅱ)fluctuation,HA/ZIF-8 only reduced ATP by 8.9%in normal melanocytes.Meanwhile,zinc(Ⅱ)chelator TPEN effectively alleviated the decrease of NAD~+,the inactivation of GAPDH,and the reduction of ATP in malignant melanoma cells,demonstrating that intracellular zinc(Ⅱ)overloading is the main cause of tumor glycolysis inhibition.These results suggested that HA/ZIF-8specifically blocks tumor energy mediated by zinc(Ⅱ)overloading.However,single glycolysis blockade often leads to the adaptive upregulation of the glucose demand,promoting the upregulation of glucose transporters 1(GLUT1)expression in tumors,increasing glucose uptake flux,and then triggering limited energy blockade.To achieve complete energy exhaustion of tumor cells,we designed zinc(Ⅱ)-activating DNAzymes for specifically cleaving GLUT1 m RNA(GD).Loading GD into HA/ZIF-8 constructed a zinc(Ⅱ)-overloading nanomedicine with tumor targeting,dual-responsive release property,and self-activated gene silencing for tumor systemic energy exhaustion.The nanodrug achieves"top-down"tumor energy blockade through the synergistic effect of zinc(Ⅱ)overloading-mediated glycolysis inhibition and zinc(Ⅱ)-initiated GLUT1 m RNA degradation.The characteristics of the formulation showed that HA/ZIF-8@GD has uniform morphology,dispersion,the mean size of117.0 nm,and an average Zeta potential of-19.7 m V.In vitro release results showed that HA/ZIF-8@GD could efficiently release zinc(Ⅱ)and DNAzymes under the double stimulation of hyaluronidase(HAase)and acidic p H.In vitro catalytic shearing results showed that the release of zinc(Ⅱ)from HA/ZIF-8@GD was sufficient to initiate the catalytic shearing activity of DNAzymes.Cell uptake experiments showed that HA/ZIF-8@GD pinpointed malignant melanoma cells via the CD44-mediated endocytosis pathway.Benefit from these,HA/ZIF-8@GD resulted in 50.1%GLUT1m RNA degradation,53.0%GLUT1 protein down-regulation and 63%glucose uptake blockade under the catalysis of excessive zinc(Ⅱ),which confirmed that HA/ZIF-8@GD successfully blocked the main energy source of tumor cells through zinc(Ⅱ)overloading-mediated self-activated gene silencing therapy.Lastly,due to the synergistic effect of zinc(Ⅱ)loading-mediated glycolysis inhibition and glucose uptake blockade,compared with control group and HA/ZIF-8 group,ATP content in HA/ZIF-8@GD group was reduced by 60.7%and 27.9%,respectively,thus promoting the apoptosis of malignant melanoma cells with the apoptosis rate up to 89.3%.The result showed that HA/ZIF-8@GD has excellent anti-tumor ability,which is attributed to the zinc(Ⅱ)overloading-mediated“top-down”energy blockade strategy.In vivo studies further demonstrated that HA/ZIF-8@GD could specifically target tumor and then effectively inhibit the tumor proliferation in malignant melanoma-bearing mice without systemic side effects,and tumor inhibition rate is up to 80.8%.Above all,this work provided a novel anti-tumor strategy based on Zinc(Ⅱ)Ion-Interference Therapy.2.Zinc(Ⅱ)deprivation-type adjuvant-like biomimetic nanomedicine combat New Delhi Metallo-β-lactamases-1(NDM-1)producing drug-resistance bacteria infectionsIn addition to malignancies,antibiotic resistance of bacteria is a growing global public-health problem.Especially,NDM-1-producing Enterobacteriaceae with wide drug resistance spectrum and high fatality rate of infection,has been listed as one of the most important pathogens in urgent need of antibiotic development by WHO.Considering the existing antibiotic failure combined with new antibiotics void,alternative strategies are urgently required to combat such pathogens.Antibiotic-adjuvant combination therapy is a promising tactic for tackling antimicrobial resistance.However,lacking effective but safe adjuvants and the off-target toxicity of drugs remain the major challenges for combined therapy.Previous studies documented that NDM-1 is a zinc(Ⅱ)-dependent periplasmic enzyme.It is expected to solve the above problems by searching for bioactive nanomaterial with zinc-depriving function and ensuring its targeted use.Therefore,based on the“old material-new application”strategy,we firstly found that polyamide-amine dendritic polymer(PAMAM)can be used as a highly efficient complexing agent of zinc(Ⅱ),and construct nano delivery system with zinc(Ⅱ)deprivation function for inactivating NDM-1,thus preventing NDM-1-producing Escherichia coli from hydrolyzing meropenem(MEM,a class of typical carbapenems).In addition,benefiting from the“proton sponge”effect under the weakly acidic microenvironment,PAMAM could penetrate bacterial membrane by inducing the formation of nanoscale holes in lipid membrane,promoting the intracellular targeting of PAMAM to NDM-1.Real-time hydrolysis of meropenem results showed that PAMAM prevented 74.1%of meropenem from hydrolyzation,which attributed to the dual effect of zinc(Ⅱ)deprivation mediated NDM-1 inactivation and protonation-induced membrane barrier-breaking.To ensure targeted use of PAMAM and meropenem,a Zinc(Ⅱ)deprivation-type adjuvant-like biomimetic nanomedicine(PMVs@MEM@PAMAM)was developed by ultrasonically storing meropenem-loaded polyamidoamine dendrimer(MEM@PAMAM)into platelet membrane vesicles(PMVs),which integrated potent zinc(Ⅱ)deprivation,high drugs loading yields,pathogen targeting and site-specific drug release properties.Leveraging natural pathogen affinity of PMVs and protonation effect of PAMAM,the nanovesicles precisely deliver PAMAM and meropenem to bacterial infection sites,simultaneously achieving inflammatory microenvironment-powered drugs rapid release.The characteristics of the formulation showed that PMVs@MEM@PAMAM had a uniform and spherical structure with a unilamellar membrane coating,and high drug loading yield.The average loading rates of meropenem and PAMAM were 36.8%and 18.8%,respectively.In vitro targeting results showed that thanks to bacterial adhesion proteins on platelet membranes,PMVs could target and adhere to bacterial surfaces,thus increasing the accumulation of meropenem and PAMAM at bacterial infectious sites.In vitro drug release results showed that,under the weak acidification of bacterial infection microenvironment,PMVs@MEM@PAMAM could rapidly and synchronously release PAMAM and meropenem with the aid of protonation effect of PAMAM.The release ratio of PAMAM and meropenem was about 81.1%and 79.9 in p H 5.5 PBS within 4 h.The result proved the site-specific drug burst release ability of PMVs@MEM@PAMAM,which laid a foundation for the efficient use of drugs in bacterial infection sites.In vitro antimicrobial results showed that the nanovesicles boosted meropenem efficacy against clinical six strains producing NDM-1 and six strains producing NDM-5.In vivo targeting experiments demonstrated that PMVs@MEM@PAMAM could specifically accumulate at bacterial infection sites,and release drugs in response to the infectious microenvironment.In vivo pharmacokinetic studies showed that the blood clearance rate(CL)in the PMVs@MEM@PAMAM group was nearly 22 times lower than that in the meropenem group,indicating that the platelet membrane biomimetic strategy prolonged the blood circulation time of meropenem.In vivo antibacterial results reflected that PMVs@MEM@PAMAM could effectively reduce the bacterial load of infected tissues in mice with pneumonia and sepsis,and improve the survival rate of mice to 100%,without obvious side effects after treatment.In a word,we developed a nanoscale adjuvant via zinc(Ⅱ)-depriving strategy,which provided the theoretical and experimental basis for developing a new class of antibiotic adjuvants.In addition,we offered a potential therapeutic platform by integrating polymer-antibiotic combination approach with biomimetic nanotechnology for precise NDM-producing bacteria treatment,which provided a novel idea for new resistance reversal strategy development.