Construction And Evaluation Of A Reactive Oxygen Species(ROS)-scavenging Nanoparticle And Its Protection Capacity Against Drug-induced Tissue Damage

It is reported that numerous people suffer from drug side effects every year,and some of which are serious adverse reactions.For example,the use of non-steroidal anti-inflammatory drugs（NSAIDs）,may cause gastrointestinal damage,such as gastrointestinal ulcer,erosion,bleeding or perforation.Similarly,long-term use or overdose of acetaminophen（APAP）,can cause liver and kidney damage,leading to abnormal liver function.Extensive studies showed that tissue damage will produce large amounts of reactive oxygen species（ROS）,leading to increased oxidative stress level,imbalanced of oxidation and antioxidant ability,as well as consequent peroxidation and further aggravate tissue damage.Therefore,it is a new way to reduce the level of oxidative stress and eliminate free radicals in tissue injury.However,the traditional preparatio n for oral delivery of small molecule drugs often leads to tissue nonspecific drug distribution,whose half-life is short in vivo.Consequently the prevention effect is not ideal in most cases,and may cause new adverse reactions.Recent studies showed that nanomedicines can realize specific distribution in the damaged site,effectively increase the preventive effects of drug-induced tissue injury,and reduce side effects,due to their special nanometer size effect.However,most of the nano drug delivery systems studied in this field have some problems in degradation and metabolism in vivo,which affect the safety of long-term use.To solve the above problems,we constructed a ROS responsive and hydrogen peroxide（H₂O₂）-scavenging nanoparticle（NP）loaded with 4-hydroxyl Tempol（Tpl）,a radical scavenger.The NP could be targeted to the injured site with elevated oxidative stress.Under exposure to ROS,NP achieved the targeted therapeutic effect on drug induced tissue injury during hydrolysis and released of Tpl.Methods1.Synthesis and characterization of a H₂O₂ scavenging materialIn brief,9.44 mmol N,N’-carbonyldiimidazole（CDI）and carbonyl imidazole 4.72 mmol 4-hydroxymethylphenylboronic acid pinacol ester（PBAP）were dissolved in anhydrous dichloromethane（DCM）.The reaction mixture was stirred magnetically for 1 h at 25°C.The transparent solution was washed 3 times using deionized water and saturated NaCl solution,respectively,and dry in Na₂SO₄ for 30 min.CDI activated PBAP（CDI-PBAP）was obtained by drying the filtrate.1.52 g CDI-PBAP was dissolved in dimethyl sulfoxide（DMSO）at 25°C,into which 250 mg β-cyclodextrin（β-CD）and 0.8 g 4-dimethylaminopyridine（DMAP）was added.After overnight reaction,the solution was precipitated from deionized water and washed 3 times,and the final aqueous solution was lyophilized to obtain the H₂O₂-scavenging β-CD material（Ox-bCD）.The structure of Ox-bCD was characterized by Fourier transform infrared spectroscopy（FTIR）and nuclear magnetic resonance spectroscopy（NMR）.2.Preparation of blank NPLecithin and distearoyl phosphoethanolamine polyethylene glycol（DSPE-PEG2000）were dissolved in anhydrous ethanol and dispersed in water,and stirred at 65°C for 1h.Ox-bCD dissolved in anhydrous methanol was added to the solution at room temperature.After 2 h of reaction,the solution was centrifuged at 10000 rpm for 10 min to obtain blank NPs.The particle size and surface potential were measured,and the morphology of the NP was characterized by transmission electron microscopy（TEM）.3.Evaluation of hydrolytic properties of blank NPsSpecifically,5 mg NP was added to phosphate buffer solution（PBS）with different concentrations of H₂O₂,then the transmittance at 500 nm was measured by a UV-visible Spectrophotometer.The hydrolysis degree was then calculated.4.Preparation of a ROS-scavenging NPLecithin and DSPE-PEG2000 were dissolved in anhydrous ethanol and dispersed in water,and stirred at 65°C for 1 h.Ox-bCD and Tpl dissolved in anhydrous methanol were added to the solution at room temperature.After 2 h of reaction,the solution was centrifuged at 10000 rpm for 10 min to obtain Tpl-loaded NP（Tpl-NP）.The particle size and zeta-potential were measured,and the morphology of the NP was observed by TEM.High performance liquid chromatography（HPLC）was used to measure the Tpl content in Tpl-NPs.5.The in vitro Tpl release profile of Tpl-NPThe solutions with pH 1.2 or pH 7.4 were used to simulate the microenvironment of the gastrointestinal tract.Tpl-NP was placed in the simulation environment to obtain the release behavior at a specific time point.The release of Tpl was measured by HPLC.6.Determination of in vitro ROS-scavenging capacity of Tpl-NP（or Ox-bCD）To this end,30 m M H₂O₂ solution was used to prepare Tpl-NP（or Ox-b CD）solutions at 0.064,0.125,0.25,0.5,1,2,and 4 mg/m L.The remaining H₂O₂ was determined after 48 h of incubation at 37°C.Besides,above Tpl-NP（or Ox-bCD）solutions were tested in accordance with the anti-superoxide anion free radical kit instructions,to demine the superoxide anion-eliminating capability.7.Study on the distribution of orally administered ROS scavenging NPs in mice with gastrointestinal injury induced by NSAIDsLecithin and DSPE-PEG2000 were dissolved in anhydrous ethanol and dispersed in water,and stirred at 65°C for 1 h.Ox-bCD and Cyanine7.5 NHS ester（Cy7.5）dissolved in anhydrous methanol were added to the solution at room temperature.After 2 h of incubation,the solution was centrifuged at 10000 rpm for 10 min to obtain Cy7.5-loaded NP（Cy7.5-NP）.The particle size and zeta-potential were measured,and the morphology of the NP was characterized by TEM.After oral administration of Cy7.5-NP,mice were sacrificed at different time points.Tissues including stomach,jejunum,ileum,and colon were dissected.The fluorescence intensity was observed and counted by an in vivo imaging system.8.Pharmacodynamic evaluation of orally administered ROS-scavenging NP for the treatment of gastrointestinal injury induced by NSAIDsMale Balb/c mice were fasted for 12 h,and then indomethacin（IND）was oral administrated to build a model of gastrointestinal injury mediated by NSAIDs.The mice were administered with saline,Tpl,NP,Tpl-NP,hydrotalcite respectively at 1 h before.After 12 h,stomach,jejunum and ileum were dissected for hematoxylin eosin（H&E）staining.The contents of catalase（CAT）,hydrogen peroxide（H₂O₂）,malondialdehyde（MDA）,myeloperoxidase（MPO）,IL-1β,IL-10,TNF-α in the homogenate of stomach,jejunum and ileum were determined.9.Evaluation of the survival rate of orally delivered ROS-scavenging NP in NSAID induced gastrointestinal injuryMale Balb/c mice were treated with normal saline,Tpl,NP,Tpl-NP,hydrotalcite respectively.After 1 h,oral administration of indomethacin was performed.In this way for 10 days,the state of survival was observed.10.Preliminary pharmacodynamic evaluation of intravenously injected of ROS-scavenging NP on APAP-induced liver injuryMale C57BL/6J mice were fasted for 15-18 h.Then intraperitoneal injection of acetaminophen（APAP）was conducted to establish the model of acute liver injury.After 6h,saline or Tpl-NPs intravenously injected.And 12 h after treatment,the sectiond of liver were stained with H&E.The contents of H₂O₂,MDA,MPO,IL-1β,IL-10,TNF-α in liver homogenate were determined.11.Preliminary in vitro safety evaluation of ROS-scavenging NPMouse macrophage RAW264.7 cells were co-incubated with serum medium containing NPs.The cell viability was measured by MTT after 12 or 24 h,which was implanted in the orifice plate.12.In vitro hemolysis tests of ROS-scavenging NPThe red blood cells of male rats were extracted and mixed with different concentrations of Tpl-NP.The samples were placed at room temperature for 2 h,centrifuged and then added to the plate with 96 holes for determination of hemoglobin.13.Preliminary in vivo safety evaluation of orally ROS-scavenging NPsMice were administrated with a single dose of Tpl-NP of at 2.5 or 5.0 g/kg by oral gavage.The body weight was recorded every two days.After two weeks,whole blood was taken for blood test.The blood serum was collected for analysis of renal and liver functions.Typical major organs and tissues such as stomach,duodenum,jejunum and ileum were isolated for calculation of organ index and pathological analysis based on H&E stained sections.Results1.The carrier material with H₂O₂-eliminating capability was obtained,and the results showed that there were 5 PBAP units on β-CD.2.Based on the nano-precipitation method,blank NPs and Tpl-loaded NP were successfully obtained.The particle size weas 136.9 and 146.1 nm,and the zeta-potential was-30.9 and-24.7m V,respectively.The drug loading content rate of Tpl-NP was about 15%.3.ROS-scavenging NP almost did not hydrolyze in PBS,In solutions containing different concentrations of H₂O₂,the hydrolysis rate was different.With the increase of H₂O₂ concentration,the hydrolysis rate increased.Under the condition simulating gastrointestinal pH,release of ROS scavenging NPs in the acidic medium at pH 1.2 was slow,but the release was faster in the presence of p H 7.4 and H₂O₂.4.ROS scavenging capacity test showed that the H₂O₂ clearance of Tpl-NP（Ox-bCD）was linear,and the activity of superoxide anion resistance curve was linear in a certain range.5.In vivo imaging results showed that after oral administration of Cy7.5-NP,the concentration of Cy7.5 in the gastrointestinal injury model group was longer than that in the normal group,and the fluorescence intensity was stronger.6.Results of pharmacodynamic experiments showed that after oral administration of Tpl-NP,the oxidation levels of stomach,jejunum and ileum decreased,and the survival rate were increased.Tissue sections by H&E staining revealed that tissue damage were reduced.7.In the liver injury model,intravenous injection of Tpl-NP could effectively treat liver injury induced by APAP.8.The preliminary safety results showed that Tpl-NP had no obvious toxicity on RAW264.7 macrophages,and no hemolysis was observed.There was no change in hematological parameters,renal function,and liver function after administration of ROS-scavenging NP at a high dose.Examination on H&E stained sections showed no pathological changes in organs,stomach,duodenum,jejunum and ileum.Conclusion1.By means of covalent bonding of PBAP onto β-CD,the carrier material of Ox-b CD,which has ROS responsiveness and H₂O₂ scavenging ability,was obtained.2.The modified nanoprecipitation/self-assembly method was successfully used to prepare blank NP and Tpl-NP loaded with a radical scavenger.Thus obtained NP had good morphology,narrow size distribution,and ROS responsiveness.In vitro experiments showed that Tpl-NP could effectively remove H₂O₂ and superoxide anion.3.After oral or intravenous administration,Tpl-NP was able to target the site of tissue injury with increased oxidative stress,and slowly release Tpl to the site of injury and prolong the retention time in the targeted site.4.During consuming the H₂O₂ in the local tissue,released Tpl eliminated free radical,which played an important role in the protection and treatment of gastrointestinal injury and liver injury.5.ROS-scavenging NP showed less toxic to cells,and had no effect on hematological parameters,liver function and renal function after oral administration.No hemolysis was observed after intravenous injection.In summary,through construction of a ROS-responsive and H₂O₂-scavenging carrier material an loading a free radical scavenger Tpl,ROS scavenging Tpl-NPs were synthesized.Thus fabricated NP could be targeted to sites of tissue injury,prolong the retention time of a free radical scavenger.Accordingly,this is expected to become a new kind of nanomedicine of antagonize side effects to NSAIDs or APAP.