Construction Of Broad-spectrum ROS-eliminating Nanoparticles For Prevention Of Inflammation And Oxidative Stress Related Diseases

Inflammation is a natural and protective response to different inciting stimuli.Historically,corticosteroids and non-steroidal anti-inflammatory drugs are widely used for the treatment of inflammatory diseases,but they may cause numerous side effects.It has been well-recognized that inflammation is intimately linked to reactive ox ygen species?ROS?which can lead to the imbalance of oxidative stress or induce localized tissue injury.Despite their great potential of numerous antioxidants and radical scavengers for pharmacotherapy of numerous diseases associated with inflammation and oxidative stress,many challenges remain for their clinical translation,largely due to limited reactive oxygen species?ROS?-scavenging capability and nonspecific distribution.To circumvent the above-mentioned limitations,herein we designed a functional material based on Tempol and phenylboronic acid pinacol ester simultaneously conjugated?-cyclodextrin?abbreviated as TPCD?,which is capable of eliminating a broad spectrum of ROS.Thus developed material may be easily produced into antioxidant and anti-inflammatory nanoparticles.Consistently,the newly engineered nanotherapy TPCD NP showed superior efficacies in five murine models of acute,chronic inflammation and drug-induced organ toxicity.Moreover,TPCD NP can significantly inhibit oxidative stress at the disease sites,which is more efficacious than the corresponding nanotherapies with relative narrow-spectrum ROS eliminating capability.Methods1.Synthesis and characterization of TPCDSpecifically,Tpl was activated by CDI.The obtained CDI-activated Tpl was dissolved in DMSO,into which?-CD and DMAP were added.The obtained mixture was stirred finally to give TCD.According to the above-mentioned methods,CDI-activated PBAP was synthesized.Subsequently,CDI-activated PBAP,DMAP,and TCD were co-dissolved.The reaction was carried out and obtained TPCD.¹H NMR spectra,matrix-assisted laser desorption/ionization time-of-flight?MALDI-TOF?mass spectrometry,fourier transform infrared?FT-IR?spectra,UV-visible spectroscopy and electron paramagnetic resonance?EPR?spectroscopy were recorded to characterize TPCD.2.Hydrolysis of TPCD in the presence of hydrogen peroxideAfter TPCD was incubated with H₂O₂,the hydrolyzed products were collected by lyophilization and characterized by ¹H NMR spectroscopy and MALDI-TOF mass spectrometry.TPCD was mixed with different concentration of H₂O₂,and then the hydrolyzed samples were analyzed with a high-performance liquid chromatography?HPLC?system.3.Elimination of different ROSDPPH·was used to quantify the radical scavenging capability of TPCD.TPCD were incubated with an excess amount of superoxide anion and H₂O₂.The remaining superoxide anion and H₂O₂ were measured by the Superoxide Anion Free Radical Detection Kit and Hydrogen Peroxide Detection Kit,respectively.The hypochlorite scavenging capability by TPCD was determined by using a luminescent nanoprobe developed in our previous study.Based on the similar methods,the broad-spectrum ROS scavenging capability of different materials?including TPCD,TCD,and PCD?was quantified and compared.4.Fabrication and characterization of nanoparticles based on TPCDIn brief,lecithin and DSPE-PEG were dissolved in deionized water.Then,the methanol solution containing TPCD was added.Finally,TPCD NP was obtained by freeze-drying.The size and size distribution profiles of NPs were measured using a Malvern Zetasizer NanoZS instrument.The morphology of NPs was observed with transmission electron microscopy?TEM?.Scanning electron microscopy?SEM?was also employed to observe the morphology of TPCD NP.5.ROS-responsive hydrolysis of TPCD NPs.For in vitro hydrolysis tests,TPCD NPs were incubated in various concentrations of H₂O₂.At predetermined time points,the transmittance values of NP-containing aqueous solutions were measured.The hydrolysis degree was calculated.To characterize the H₂O₂-concentration dependent change in size and morphology of TPCD NP,TPCD NP was separately incubated in different concentration of H₂O₂.Then the morphology,particle size distribution profiles,and zeta-potenial values of the corresponding samples were characterized by TEM and DLS.6.In vitro cellular uptakeRAW264.7 macrophage cells were incubated with Cy5/TPCD NP for different periods of time.LysoTracker Green?75 nM?was added and cells were fixed with 4%paraformaldehyde,and stained with DAPI.Confocal laser scanning microscopy?CLSM?observation was conducted.The fluorescence intensity of Cy5/TPCD NP in RAW264.7cells was determined via fluorescence activated cell sorting?FACS?.Through similar procedures,the dose-dependent internalization profile was examined after cells were incubated with varied doses of Cy5/TPCD NP for 2 h.7.In vitro anti-apoptosis activity of TPCD NP in macrophagesThe anti-apoptosis activity of TPCD NP in RAW264.7 macrophage cells was analysed by APC Annexin V Apoptosis Detection Kit with PI.According to the similar procedures,PCD NP,TCD,high and normal dose of Tpl,and combined use of Tpl/HMP were served as different therapy controls.8.Treatment of carrageen-induced paw edema in miceAcute inflammation in the paw of Sprague Dawley rats was induced by intradermal?i.d.?injection of 2 wt%carrageen in the paw.TPCD NP at 1.0 mg/kg,TPCD NP at 5.0mg/kg,Tpl,or saline was separately injected at 30 min after carrageen stimulation.The paw volume was measured before and after carrageen administration.9.Treatment of peritonitis in micePeritonitis in male BALB/c mice was induced by i.p.injection of zymosan.In each group,mice were i.p.administered with various doses of TPCD NP?0.1 or 1.0 mg/kg?or free Tpl.The levels of myeloperoxidase?MPO?,H₂O₂,tumor necrosis factor-??TNF-??,and interleukin-1??IL-1??in the supernatant were quantified by ELISA assay.Following exactly the similar protocols,therapeutic effects of PCD NP,TCD,10-fold Tpl,and Tpl/HMP were also investigated in a seperated study.10.Therapeutic effects of TPCD NP in mice with LPS-induced acute lung injuryTo induce acute lung injury?ALI?,LPS was administered by intratracheal?i.t.?inoculation.At 1 h after stimulation with LPS,TPCD NP at 0.1 or 1.0 mg/kg and Tpl was separately administered by i.v.injection.At 11 h after different treatments,mice were euthanized.Subsequently,neutrophil count was detected by flow cytometry.In addition,the levels of MPO,H₂O₂,TNF-?,and IL-1?in the supernatant were assessed by ELISA.The severity of pulmonary edema was evaluated.Histological sections were also made and stained with H&E.To evaluate the efficacies of PCD NP,TCD,10-fold Tpl,and Tpl/HMP,another cohort of studies were also conducted according to the above-mentioned procedures.11.Targeted treatment of asthma in mice with TPCD NPTo induce asthma,female BALB/c mice were induced with OVA/LPS.At 30 min before the challenge with OVA on days 14 and 17,TPCD NP?at 0.1 or 1.0 mg/kg?or free Tpl was separately administered by i.v.injection.On day 19,mice were euthanized and bronchoalveolar lavage fluid was collected immediately.Flow cytometric analysis of the neutrophil counts and the levels of MPO,H₂O₂,TNF-?,IL-1?,and IL-17 in the bronchoalveolar lavage fluid were analyzed.In a separate study,lungs were embedded in paraffin and the prepared histological sections were stained with H&E.12.Therapeutic effects of TPCD NP in mice with APAP-induced hepatotoxicity and renal injuryMale C57BL/6 mice were were i.p.injected with APAP at 200 mg/kg.At 6 h after stimulation with APAP,TPCD NP?at 0.1 or 1.0 mg/kg?or free Tpl was separately administered by i.v.injection.At 12 h after different treatments,mice were euthanized.Blood samples were collected for hematological analysis.A part of liver tissues was fixed for histopathological analysis.The remaining livers were homogenized and the resulting supernatants were collected for quantification of the levels of H₂O₂,TNF-?,and IL-1?by ELISA.In addition,the neutrophil counts and MPO levels in livers were analyzed by immunofluorescence.Finally,in vivo therapeutic benefits of TPCD NP were compared with different control therapies,based on the similar therapeutic procedures as mentioned above.13.Preliminary safety evaluation on TPCD NPA human hepatocellular liver carcinoma HepG2 cell and RAW264.7 mouse macrophage cells were cultured with TPCD NPs to test the cell cytotoxicity of TPCD NP.The 2%erythrocyte suspension was mixed with TPCD NPs to detect the hemolysis.Mice were intravenously?i.v.?or intraperitoneally?i.p.?administered with TPCD NP at 1000 mg/kg.After different treatments,mice were weighed at defined time points.At day30,animals were euthanized,and blood samples were collected for hematological analysis.Major organs were isolated,weighed,and histological sections were prepared and stained with hematoxylin and eosin?H&E?.Results1.TPCD was successfully synthesized based on Tempol and phenylboronic acid pinacol ester simultaneously conjugated?-cyclodextrin.Under oxidative conditions,TPCD can be thoroughly hydrolyzed into water-soluble products.Quantification of the hydrolyzed products by HPLC indicated that approximately 2 Tpl and 5 PBAP units were covalently linked to each?-CD molecule.In addition,TPCD was capable of eliminating a broad spectrum of reactive species.2.TPCD NPs were prepared by a modified nanoprecipitation/self-assembly method.The hydrolysis rate and degree of TPCD NP was positively correlated to the H₂O₂concentration,but displayed negligible hydrolysis in PBS without H₂O₂.3.TPCD NP can be rapidly and efficiently internalized by macrophages.Flow cytometric and CLSM analysis revealed both dose-dependent and time-dependent endocytosis.TPCD NP can significantly inhibit oxidative stress-mediated cell apoptosis by effectively scavenging ROS,which was more effective than nanotherapies with narrow-spectrum ROS-eliminating capacity.4.In the mouse model of paw edema,it was demonstrated that the degrees of paw edema were significantly inhibited by treatment with 5.0 mg/kg of TPCD NP.Of note,therapy with 5.0 mg/kg TPCD NP was more effective than the same dose of free Tpl and1.0 mg/kg of NPs.5.The levels of pro-inflammatory cytokines and ROS in peritonitis mice were effectively decreased by local treatment with TPCD NP.Treatment with free Tpl at the same dose of 1.0 mg/kg TPCD NP showed efficacy comparable to that of 0.1 mg/kg TPCD NP.It was also demonstrated that TPCD NP is more efficacious than the corresponding nanotherapies with relative narrow-spectrum ROS eliminating capability.6.TPCD NP can effectively target to injured lungs in mice.The lung wet/dry weight ratio could be decreased after treatment with TPCD NP.Also,the level of pro-inflammatory cytokines and ROS were remarkably reduced after treatment with TPCD NP,particularly at 1.0 mg/kg.In addition,treatment with TPCD NP significantly suppressed neutrophil infiltration as well as the histological abnormalities in H&E-stained sections.Moreover,therapeutic effects of TPCD NP were also compared with those of PCD NP,TCD,high-dose Tpl,and Tpl/HMP in the ALI mice.7.TPCD NP can also serve as an effective nanotherapy for the treatment of asthma which was more effective than the same dose of Tpl,with respect to reducing typical pro-inflammatory cytokines and the MPO level and neutrophil count in lavage fluid.In addition,TPCD NP still showed superior anti-inflammatory and oxidative stress effects compared with relative narrow-spectrum ROS eliminating groups in the asthma mice.8.Treatment with TPCD NP decreased the level of biomarker in a dose-dependent manner.The pro-inflammatory cytokines and the levels of ROS in liver were reduced as compared to those of the model group.Treatment of APAP-challenged mice with TPCD NP achieved more desirable protective effects,in comparison to other formulations.9.During a preliminary safety studies,the cytotoxicity of TPCD NP in RAW264.7and HepG2 cells was negligible.Subsequently,the degree of hemolysis was less than 1%even at a relative high concentration of 2 mg/mL TPCD NP.No significant changes in body weight occurred after i.p.and i.v.administration at 1000 mg/kg of TPCD NP.The organ index values of typical major organs of TPCD NP-treated mice were comparable to those of normal animals.Typical hematological parameters and biomarker were in normal levels for TPCD NP-treated mice.Likewise,examination on H&E-stained sections of major organs from TPCD NP-administered mice indicated that there were no pathological patterns.Conclusions1.We developed a SOD/catalase-mimetic material capable of eliminating a broad spectrum of ROS,which can be facilely synthesized by conjugating two functional moieties onto a cyclodextrin scaffold.This pharmacologically active material TPCD can be easily processed into effective antioxidant and anti-inflammatory nanotherapies.2.Cellularly,a TPCD-derived nanotherapy protected macrophages from oxidative stress-induced apoptosis,after rapidly and efficiently internalization into cells and scavenging ROS.3.In several murine models of inflammatory diseases including paw edema,peritonitis,and acute lung injury,the nanotherapy TPCD NP effectively attenuated oxidative stress and inhibited inflammatory responses in diseased tissues or organs.In addition,TPCD NP was able to serve as a nano-antidote to significantly inhibit drug-induced liver and/or kidney injury,by eliminating overproduced ROS.In all these cases,treatment with TPCD NP afforded more beneficial outcomes than the corresponding control small-molecule drug.4.Preliminary in vivo studies demonstrated that TPCD NP can serve as a safe nanotherapy.Consequently,TPCD NP deserves further development as an efficacious and safe nanotherapy for treatment of numerous diseases associated with inflammation and oxidative stress.The pharmacological activities of TPCD NP may be further enhanced by surface engineering with functional moieties targeting diseased tissues or mitochondrial oxidative stress,which can also be achieved by packaging with other anti-inflammatory therapeutics.