Assembly Of Bio-inspired "AND" Logic Gate Platform For Tumor-specific Drug Delivery On Endometrial Cancer

BackgroudTumour-targeted nanomaterials have led to significant innovation in drug delivery strategies,which have been actively developed for overcoming typical limitations of conventional free drugs,such as lack of selectivity,non-specific targeting,and significant side effects.Stimuli-responsive nanocarriers is a new tumor-targeting moiety and has been favored by the majority of researchers in recent years,the nanocarriers could realize the targeted release of drugs in the tumor area in response to external stimuli and internal stimuli,which has the advantages of reducing toxicity and increasing efficiency.Specific biological factors in tumor microenvironment,such as low pH,enzymes,ATP,redox-potential etc,could be specific triggers for controlled drug release.However,despite single biomarker or multi responsive systems have been further designed for accelerating the offloading of therapeutic drugs,which always lead to adverse off-target drug leakage on the way activated by each stimulus in complex biological milieu.Therefore,it is crucial to develop a delivery system with with simple synthesis,good biocompatibility and strong targeted release,to achieve higher tumor-accumulation of the drug,reducing the off-target toxicity of the free drug and maximizing treatment efficacy.Inspired by the Boolean logic idea,"AND" logic gate-based systems are favored for sequence-activated drug release in pathological sites with desired specificity,and the drug can be released only by the joint action of two stimulating factors at the same time to avoid the early leakage of the drug.Despite promising and effective,subjected to the inherent constraints on material composition and vehicle geometry,the uniqueness of each previously reported "AND" logic gate-based platform invariably necessitates a brand new material design that is generally not synthetically tractable.Some study have constructed nanocarrier that through a "AND" logic gate to control release of drug,which have the characteristic of tumor targeted intelligent release,but suffer from the disadvantages of complex synthesis,poor stability,etc.Endometrial cancer(EC)is the most common gynecological cancer in developed countries.With the improvement of living conditions in China,the number of obese and diabetic patients has increased,and the incidence of EC has gradually increased in recent years.Doxorubicin(DOX),are widely used in clinical practice and have good therapeutic efficiency for EC,nevertheless,the clinical utility of DOX is limited due to its marked toxicity.Therefore,how to reduce the toxicity of DOX,which has major clinical significance to treat endometrial cancer.Thus,there is a clinical need to develop nanoparticles with loading of DOX,and nanoparticles with the construction of "AND" logic gate-based platform presents some advantages,such as biocompatibility,low toxicity and side effects.Gelatin NPs possess several advantages compared to other materials,including good biocompatibility,easy accessibility,low toxicity of degradation products,good stability in vivo and vitro.The DOX molecule can be coupled to the surface of Gelatin NPs through aldimine condensation to form DG NPs,which has the characteristics of releasing DOX in response to matrix metalloproteinase-2/9(MMP 2/9,also known as gelatinase)Cryptobiosis also known as "secret life"(e.g.,tardigrades),which can withstand stressful and often lethal environmental conditions.This cytoprotective strategy commonly involves the formation of tough biomolecular sheaths on cellular structures to enter a dormant state for survival from outside threats and subsequent revival to proliferate by breaking the protective shells apart when the environment becomes favorable.Inspired from this fascinating cryptobiotic mechanism,chemically forming a durable shell on nanoparticles to enhance the stability of drugs in tissue circulation and reduce the early leakage of drugs.Owing to the broad-spectrum adhesion of polyphenols and the coordination and chelation characteristics of metal ions,Tannic acid(TA)and Fe3+ can self-assemble into shells on the surfaces of various objects with different shapes,forming metal-phenolic networks(MPN),which has good pH and ATP responsive to release DOX.Therefore,using the characteristics of high ATP concentration,low pH and high expression of MMP-2/9 to construct an "AND" logic gate-based platform for controlled release of drug.As a proof-of-concept study,an assembly of pH/ATP "AND" enzyme logic gate platform was successfully developed here to realize tumor-specific drug delivery.After conjugating DOX molecules on the surface of Gelatin NPs through typical aldimine condensation,conformal TA-Fe? shell was subsequently deposited on the surface of DG NPs to form DG@TA-Fe? NPs for realizing "AND" logic gate-controlled drug delivery in tumor regions.By preventing the contact between DG NPs and enzyme with durable TA-Fe? shells in healthy tissues,the obtained DG@TA-Fe? NPs were expected to exhibit a cryptobiotic behavior with negligible premature drug leakage.Once arrived at tumor regions,the mild acidic pH or upregulated ATP in the tumor microenvironment would firstly unlock the TA-Fe?shells and then the subsequently exposed inner DG cores could be hydrolysed by overexpressed MMP 2/9,in solid tumor regions to release cytotoxic DOX.In addition to this "AND" logic gate controlled DOX delivery in tumor,the good photothermal conversion activity of TA-Fe? complex would endow DG@TA-Fe? NPs with synergistic photothermal-chemo cell-killing ability under near-infrared(NIR)light irradiation.This nanoparticles of "AND" logic gate platform with microenvironment-triggered drug release may provide an efficient strategy to treat endometrial cancer cells and transplanted tumor through photothermal combined chemotherapy.PART 1 Fabrication and characterization of DG@TA-Fe? NPsObjective:As known to all,the TA-Fe? shell has good pH and ATP responsive to release drug,and the gelatin NPs has the characteristics of releasing drug in response to MMP 2/9.After conjugating DOX molecules on the surface of Gelatin NPs through typical aldimine condensation,conformal TA-Fe? shell was subsequently deposited on the surface of DG NPs to form DG@TA-Fe? NPs for realizing "AND" logic gate-controlled drug delivery in tumor regions.The drug loading of DOX was detected and the morphology,hydrodynamic diameters,ultraviolet-visible-near-infrared light absorption spectra and Zeta potential of the Nanoparticles were characterized.Methods:Monodispersed gelatin NPs were firstly developed from a well reported two-step desolvation process and subsequently crosslinked by GTA molecules.After mixing the DOX HC1 aqueous solution with GTA solution of various feeding mass ratios for 2 h at room temperature,the gelatin NPs were then quickly added into the aldehydated DOX suspension and stirred vigorously for further 2 h.The DG NPs could be successfully collected and the optimum feed ratio was calculated.The cryptobiotic DG@TA-Fe? NPs were developed by subsequently depositing TA and Fe? molecules on the surface of DG NPs to form conformal TA-Fe? shell via polyphenol-metal coordination chemistry.Hydrodynamic diameters,zeta potential of as-prepared,ultraviolet-visible-near-infrared light absorption spectra,fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy of NPs were measured.The morphologies of NPs were observed by scanning electron microscope and transmission electron microscope.Results:Considering the delivery advantage of NPs with small size,optimized DG NPs with a feeding weight ratio of 1:50 of DOX versus GTA were thus chosen for further study.The characteristic absorption peak at 276 nm of TA(assigned to the p-system of the benzene ring)was shifted and splited into two absorption peaks at 235 and 315 nm,further suggesting the formation of TA-Fe? complex.The zeta potential of NPs was reversed from positive DG NPs(+15.9 mV)to negative DG@TA NPs(-13.7 mV)and DG@TA-Fe? NPs(-24.8 mV),demonstrating the good packaging effect of TA-Fe? shell with a slight increase in hydrodynamic diameter distribution.As seen from TEM and SEM images,the obtained DG@TA-Fe? NPs exhibited monodispersed spherical morphologies with rough surfaces consisted of TA-Fe?complex which were confirmed by the element mapping results.The existence of Fe,gelatin and DOX were confirmed by element distribution patterns.There are multi-functional groups in all DG@TA-Fe? NPs samples.were confirmed by Fourier transform infrared spectroscopy.Among them,the infrared absorption peak at 3326-3357cm-1 is the stretching vibration peak of O-H bond,which is the characteristic performance of TA containing a large number of phenolic hydroxyl groups.The characteristic absorption peak of Fe2+can be seen in the XPS picture,which confirms the existence of TA-Fe? complex,because the part of Fe3+was convert to Fe2+by the reducibility of TA.Conclusions:The cryptobiotic DG@TA-Fe? NPs were successfully developed and were confirmed by hydrodynamic diameters,zeta potential,ultraviolet-visible-near-infrared light absorption spectra,fourier transform infrared spectroscopy,X-ray photoelectron spectroscopy and morphologies of NPs.PART 2 The study of release mechanism of "AND" logic gate platform and photothermal effect of DG@TA-Fe? NPsObjective:Inspired by the Boolean logic idea,"OR" logic gate-based systems was constructed by pH and ATP response,which can maximize the release of prodrug in tumor sites.Due to the uneven distribution of pH in the tumor site,single pH response release is insufficient.On this basis,the "AND" logic gate-based platform was constructed between pH/ATP response and MMP-2/9 response to minimize drug leakage.In this study,the "AND" logic gate release mechanism of DG@TA-Fe? NPs was verified and its photothermal performance was evaluated.Methods:To investigate the storage stability of as-prepared DG@TA-Fe? NPs.Hdrodynamic diameter and absorbance of DG@TA-Fe? NPs were examined in various mediums with different pH value.To study the "AND" logic gate release mechanism of DG@TA-Fe? NPs,a typical 1,10-phenanthroline chromogenic method was utilized to test the ATP/pH sensitive disassembly of TA-Fe? shell with various concentrations of ATP and pH values.The released amount of DOX was also determined by incubating the sealed DG@TA-Fe? NPs with different stimuli(ATP,acidic pH,gelatinase).Human Umbilical Vein Endothelial Cells(HUVECs)were used to investigate the cytotoxicity of DG@TA-Fe? NPs by a standard MTT assay.For evaluating the hemocompatibility,gradient concentrations of DG@TA-Fe? NPs were incubated with diluted healthy rabbit blood to calculate the relative percent hemolysis.Photothermal warming effects,photothermal conversion efficiency and photothermal stability of DG@TA-Fe? NPs were observed and calculated.Finally,the cancer cells were irradiated by an NIR laser,the intracellular uptake of DOX with various treatments was imaged by inverted fluorescence microscope.Results:Due to the hydrophilic and negative charged surface,no significant change in hydrodynamic diameter and absorbance of DG@TA-Fe? NPs could be observed in various mediums including DI water,PBS and RPMI-1640 cell culture medium with a pH value of 7.4.In contrast,the absorbance of DG@TA-Fe? NPs solution could be drastically decreased after incubating in acidic mediums(pH 5.0 and 6.0).Despite containing chemotherapeutic DOX molecules,the relative viability of HUVECs could reach 83.7±3.1%after 24 h incubation with 600 mg/mL of DG@TA-Fe? NPs.All NPs incubated groups exhibited negligible percent hemolysis which were comparable to negative control.DG NPs exhibits good stability and non-thermo responsive DOX release performance.As expected,as the stability constants of TA-Fe? decreased when the solution became more acidic,nearly 47.8%and 84.3%of iron could be disassembled from protective TA-Fe? shells for 12 h when DG@TA-Fe? NPs were incubated in acidic PBS with a pH value of 6.0 and 5.0,respectively.In sharp contrast,only 9.8%of iron was released at pH 7.4.The amount of iron released from DG@TA-Fe? NPs was almost negligible in the absence of ATP,while it increased as not only the prolonged time but also elevated ATP dosage.Particularly,nearly 68.4%and 75.6%of iron could be destabilized from TA-Fe? shells when mixing with 1.0 mg/mL of ATP for 4 and 12 h,respectively.Upon addition of gelatinase(mimic MMP 2/9 in tumor region)for 12 h,conjugated DOX could be quickly liberated from DG NPs which is over 6-fold increase versus the system without enzyme.Although either acidic pH or ATP could unlock the TA-Fe? shells,nearly no conjugated DOX molecules would be released without the assistance of gelatinase.Equally,it seems also impossible for gelatinase to directly cut off DOX molecules from DG@TA-Fe?NPs due to the hinder effect of intact TA-Fe? shells.Moreover,to mimic the coexistence of acidic pH,upregulated ATP level,gelatinase,that result in facilitated cytotoxic DOX release from DG@TA-Fe? NPs.As expected,DG@TA-Fe? NPs possessed good photothermal conversion capacity derived from their photosensitive TA-Fe? shells with both NPs dosage and power density of incident NIR light dependent manners.For instance,exposing DG@TA-Fe? NPs solution to NIR light for 3 min would elevated the solution temperature from 25? to 58?,respectively,while only 0.8 ? temperature rise was observed for DI water.In addition,the conversion efficiency of DG@TA-Fe? NPs was 36.1%.Conclusions:Thus,all of the above data indicated the good biocompatibility of as-prepared DG@TA-Fe? NPs which was essential for further biomedical application.The "AND" logic gate-based platform of DG@TA-Fe? NPs was constructed between pH/ATP response and MMP-2/9 response to minimize drug leakage.The DG@TA-Fe? NPs have considerable photothermal stability and conversion efficiency.PART 3 The study of DG@TA-Fe? NPs combined with photothermal therapy on endometrial cancerObjective:DOX plays a major therapeutic role for endometrial cancer,but be limited in clinical applications due to high toxicity lately.It is important that how to reduce DOX toxicity for endometrial cancer treatment.The combined strategy of photothermal therapy and chemotherapy can maximize anti-tumor effects,investigate the release characteristics of "AND" logic gate-based platform and photothermal effects of DG@TA-Fe? NPs.To investigate the therapeutic effect of DG@TA-Fe?NPs combined with photothermal therapy on the transplanted EC tumor.Methods:MTT method was used to detect the killing ability of DG@TA-Fe? NPs Combined with photothermal therapy for endometrial cancer Ishikawa cells.They were divided into three groups:DG@TA-Fe? NPs+NIR group,DG@TA-Fe?NPs+gelatinase group and DG@TA-Fe? NPs+gelatinase+NIR group.Establishment of a nude model of endometrial carcinoma xenograft with Ishikawa cell line.The tumor-bearing mice were randomly divided into six groups when the tumor volume achieved to?50 mm3.They were divided into PBS+NIR group;Gelatin@TA-Fe?NPs group,free DOX group;DG@TA-Fe? NPs group;Gelatin@TA-Fe? NPs+NIR group;DG@TA-Fe? NPs+NIR group.Then,tumors were selectively illuminated with/without NIR laser after intravenous injection.After various treatments,the tumor volume and body weight of mice were recorded every other day as well as the characterizations of typical H&E histological and TUNEL immunofluorescent staining of tumor slices.Results:DG@TA-Fe? NPs+gelatinase+NIR group had better effect than other groups,which had higher cytotoxicity and showed better killing effect on Ishikawa cells,and the therapeutic effect increased with the increase of concentration and time.The tumors treated with PBS+NIR laser and Gelatin@TA-Fe? NPs showed fast proliferating behaviors within 16 days,was also shown slight tumor inhibition effect due to poor tumor accumulation and the low dosage used here.For the mice treated with Gelatin@TA-Fe? NPs+NIR laser and DG@TA-Fe? NPs,notable tumor inhibition but subsequent recurrence occurred due to insufficient generated heat and dosage of released DOX in this study.Satisfactorily,nearly completely tumor inhibition and no recurrence occurred in the following 16 days were achieved in DG@TA-Fe? NPs+NIR laser group,As expected,DG@TA-Fe? NPs+NIR laser group present the most serious cell destruction and TUNEL signals in tumor cells owing to the synergistic photothermal-chemo effect.Moreover,the characterizations of body weight and major organ histological analysis suggested that no significant side effects.Conclusions:DG@TA-Fe? NPs can effectively kill endometrial cancer cells and inhibit the growth of endometrial cancer xenografts under 808 nm near-infrared light irradiation.