| Objective: Osteoarthritis(OA)is a disabling joint disease associated with chronic inflammation.Macrophages,the major component of the mononuclear phagocyte system,play a central role in inflammation and host defense in the immune system.The polarization of macrophages plays the key role in inflammatory microenvironment of joint which is a therapeutic target for OA treatment.Herein,we report a boronatestabilized polyphenol-poloxamer assembled nanoparticle(PPNP)as a drug delivery system with a strong ROS-response and scavenging ability for macrophages modulate in OA therapy.This study also provided a novel platform for inflammation disease associated with high oxidative stress.Methods: In this study,the nanoparticle was fabricated using an organic solventmediated assembly approach.The hydrodynamic size of the nanoparticles was measured by dynamic light scattering(DLS)instrument.The morphology of the nanoparticles was obtained using a transmission electron microscope(TEM)instrument.In order to check the time-course stability of DEX@PPNP in different media.The DEX@PPNP solution was mixed with different media(PBS,water).The particle sizes of nanomedicine were measured by DLS to evaluate the stability of nanomedicine in vitro.To determine the drug release behavior of DEX@PPNP,DEX@PPNP filled into a dialysis bag were suspended in buffer with 0 μM H2O2/p H 7.2(environment of a normal joint)or 50 μM H2O2/p H 6.5(environment of an OA joint).The concentration of the DEX was determined using HPLC.To evaluate the ROSresponsive ability of DEX@PPNP,the size of DEX@PPNP cultivated with different concentrations of H2O2 solution were measured in a predetermined time.RAW264.7 cell line was cultured for cytotoxicity test,hydrogen peroxide assay and the NOscavenging ability of DEX@PPNP.Immunofluorescence staining assay was used to evaluate the protein expressions of IL-6 and IL-10.The m RNA expression of macrophage markers,including CD80 and Arg-1 were measured by using q RT-PCR.Then the OA mice were induced by MIA.Body weight,clinical arthritis scores,claw circumference,and kinematics score were recorded throughout the whole treatment period.To assess inflammatory progression during treatment,positron emission tomography(Micro-PET)imaging was performed on OA mice with 68Ga-RGD to determine the level of angiogenesis in vivo.The structure of knees in OA mice were also examined by microscopic computed tomography(Micro-CT)on day 36 after MIA injection.Histological examination of ankle joints after treatment was performed using H&E and Safranin-O fast green staining.Inflammatory cytokines were also examined to evaluate the macrophage polarization in the joints by ELISA analysis.Results: The nanoparticle was fabricated using an organic solvent-mediated assembly approach to obtain high drug loading efficacy.The transmission electron microscopy(TEM)image and dynamic light scattering(DLS)results revealed that the DEX@PPNP have a rice ball-like shape with a hydrodynamic size of 190 nm and the zeta potential was-20.3 ± 0.665 m V.DEX@PPNP was stable both in water and PBS,as there is a slight change in size in these solutions as long as 48 h.After removing the unloaded free drug from DEX@PPNP,the drug loading efficiency and drug encapsulation efficiencies were 11.2% and 12.9%,respectively,as determined by high performance liquid chromatography(HPLC).In the inflammatory joint stimulation microenvironment,the majority of DEX release(85%)in the first 8 hours with a fast release rate.The Sizes of DEX@PPNP were changed after cultivated with H2O2,indicating that the DEX@PPNP had good ROS-responsive ability.In vitro cell experiments,the cell viability was not significantly affected by DEX@PPNP incubation for 24 h.DEX@PPNP was significantly inhibited NO production in LPSactivated RAW264.7 macrophages at a same concentration.The similar results can also be found in ROS scavenging assay analyzed by FACS and fluorescent microscopy.The fluorescence images taken by CLSM.After DEX@PPNP treatment,IL-6 levels decreased and IL-10 levels increased remarkably.Compared to the non-treated cells,DEX@PPNP could significantly decrease the expression of CD80(M1 marker)while increase the Arg-1(M2 marker).These suggesting the successful re-polarization of LPS-activated RAW264.7 macrophages from M1 to M2.After confirming the high anti-inflammation efficiency of DEX@PPNP in vitro,the in vivo OA treatment was performed.In this study,the OA mice were induced by MIA with KM mice for six days.The body weight of DEX@PPNP treated OA mice was similar to that of normal mice during all the treatment time.For clinical arthritis scores,claw circumference,and kinematics score,all the DEX@PPNP treated mice were similar to normal mice.The OA joint uptake of 68Ga-RGD of mice treated by DEX@PPNP reached 0.88 %ID/g at day 36,which is close to the normal group and significantly lower than the OA joint uptake(p<0.001).The number of trabecular and bone mineral density in the DEX@PPNP group were similar to the normal group,and higher than in the saline,DEX or PPNP groups.H&E and Safranin-O fast green staining shown that after local treatment with DEX@PPNP,the surface integrity was not disrupted and the superficial layer of the cartilage was smooth,similar to that of normal mice.ELISA analysis of knee joints showed a significantly decrease of pro-inflammatory cytokines IL-6 and TNF-α levels in the DEX@PPNP group,compared to those in the saline group while the anti-inflammation cytokine IL-10 of DEX@PPNP group increased.These results demonstrate the good anti-inflammation activity of DEX@PPNP local treatment.No detectable change was observed from the H&E stained of heart,liver,spleen,lung and kidney after the treatment of DEX@PPNP.In addition,the similar body weight change of DEX@PPNP compared with normal mice.Consequently,DEX@PPNP present as a highly safe treatment for OA therapy.Conclusions: We successfully fabricated a boronate-stabilized polyphenol-poloxamer assembled dexamethasone nanodrug.With ROS scavenging ability and ROSresponsive drug release behavior,the nanodrug enables efficient repolarization of macrophages for antiinflammation.Angiogenesis,cartilage degradation and OA progression were remarkably reduced in the OA mice,demonstrating the excellent therapeutic efficient of this nanodrug in OA treatment.This study also provided a novel platform for inflammation disease associated with high oxidative stress. |
