Preparation Of Cuttlefish Ink-derived Melanin Nanoparticles/sodium Alginate Hydrogel And Its Therapeutic Effect On Rats With Osteoarthritis

Osteoarthritis(OA)has a complex pathogenesis and is a whole joint disease involving structural changes such as joint cartilage,synovium,tendons,ligaments and surrounding muscles.Osteoarthritis is a common disease in pets and has attracted attention with the increasing number of pets in China.At present,there is no radical cure for osteoarthritis,and the clinical treatment strategy is mainly symptomatic treatment(such as analgesic and anti-inflammatory,joint nutrition supplementation,etc.).Therefore,there is an urgent need to explore a new strategy for treating osteoarthritis.Squid ink particles and alginate are natural materials,squid ink is extracted from squid ink sac,recent studies have shown that it has a strong ability to clear reactive oxygen species(ROS);Alginate is a naturally occurring ingredient extracted from kelp or sargassum of brown algae and is a widely used polymer material.During the normal physiological metabolism of the body,reactive oxygen species(ROS)are continuously produced,including hydrogen peroxide(H2O2),hydroxyl radicals(OH-),superoxide anion radicals(O2-)etc.Under normal circumstances,the production and scavenging of free radicals in the body are in dynamic balance,but under the interference of external adverse factors,the imbalance between oxidation and oxidation in the body leads to excessive accumulation of ROS.Pyroptosis is an inflammatory cell death that can release IL-1β and IL-18 into the external environment,and excessive ROS in the joint cavity of osteoarthritis patients will promote inflammatory Pyroptosis of macrophages,aggravate the inflammatory microenvironment of the joint cavity,and thus cause continuous damage to joint tissue cells,which may be one of the reasons why osteoarthritis is difficult to be effectively controlled.Chondrocytes are the only cell type in joint tissue,and excess ROS in the joint cavity of osteoarthritis patients will promote chondrocytes aging,resulting in a breakdown of the balance of extracellular matrix synthesis and decomposition,which will directly destroy joint cartilage tissue affects the normal physiological function of joints.Therefore,an effective strategy for the treatment of osteoarthritis is:by removing ROS,reducing macrophage pyroptosis,improving the joint cavity microenvironment,thereby preventing it from causing continuous damage to joint tissue cells,and reducing chondrocytes aging by removing ROS,thereby protecting joint cartilage tissue and maintaining normal physiological functions of joints.In this study,Cuttlefish ink-derived melanin nanoparticles/sodium alginate hydrogels(CIMNPs/Alg)were first prepared,then the effects of CIMNPs/Alg hydrogels on macrophage pyroptosis and chondrocytes senescence were studied in vitro,and finally the therapeutic effect of CIMNPs/Alg hydrogels on osteoarthritis rats was studied in vivo.The specific content and results are as follows:1.Preparation and characterization of Cuttlefish ink-derived melanin nanoparticles/sodium alginate hydrogelCuttlefish ink-derived melanin nanoparticles(CIMNPs)were first prepared by centrifugation and passed by scanning electron microscope(SEM)and transmission electron m icroscope,TEM)was characterized and the results showed that CIMNPs were homogeneous spherical particles of uniform size around 150 nm.Then,in this experiment,squid ink nanoparticles/sodium alginate hydrogels(CIMNPs/Alg)were prepared by Ca2+ crosslinking,and characterized by camera and SEM,and the results showed that CIMNPs/Alg hydrogels showed black uniform gels from the macroscopic perspective and loose and porous morphology from the microscopic perspective,and CIMNPs were encapsulated in Alg hydrogels.Finally,the ability of CIMNPs/Alg hydrogels to remove ROS was studied by antioxidant capacity experiment,and the results showed that CIMNPs/Alg hydrogels had good ROS scavenging ability and showed concentration-dependence.The degradation of CIMNPs/Alg hydrogels in vivo was studied by subcutaneous injection experiments,and the results showed that CIMNPs/Alg hydrogels had good degradability.2.Cuttlefish ink-derived melanin nanoparticles/sodium alginate hydrogel alleviates macrophage pyroptosis by removing ROSFirstly,the safe dose range of CIMNPs/Alg hydrogels for macrophages was studied by MTT experiments,and the results showed that CIMNPs/Alg hydrogels had a good safety profile for macrophages,and finally CIMNPs/Alg hydrogels with CIMNPs concentration of 200 μg/mL were selected for subsequent experiments.Then,the scavenging ability of CIMNPs/Alg hydrogels on macrophage ROS was studied by confocal/flow cytometry,and the results showed that CIMNPs/Alg hydrogels could significantly reduce macrophage ROS levels.Finally,this experiment investigated whether CIMNPs/Alg hydrogels can reduce the pyroptosis of macrophages through SEM and Western blot,which showed that CIMNPs/Alg hydrogels could reduce the formation of pores on the surface of macrophage membranes,and Western blot results showed that CIMNPs/Alg hydrogels could reduce the expression of macrophage NLPR3/Caspase-1 pyrosis pathway protein.In summary,we believe that CIMNPs/Alg hydrogels can alleviate pyroptosis of macrophages by clearing ROS.3.Cuttlefish ink-derived melanin nanoparticles/sodium alginate hydrogel alleviates chondrocytes senescence by removing ROSFirstly,the safe dose range of CIMNPs/Alg hydrogels on chondrocytes was studied by MTT experiment,and the results showed that CIMNPs/Alg hydrogels had a good safety profile for chondriocytes,and finally CIMNPs/Alg hydrogels with CIMNPs concentration of 200 μg/mL were selected for subsequent experiments.Then,the clearance ability of CIMNPs/Alg hydrogels on chondrocytes ROS was studied by confocal study,and the results showed that CIMNPs/Alg hydrogels could significantly reduce the ROS level of chondrocytes.Finally,Western blot results showed that TBHP(50 μM)could successfully induce the establishment of chondrocytes aging model,and Western blot results showed that CIMNPs/Alg hydrogel could reduce the expression of chondrocytes senescence-related proteins.In summary,we believe that CIMNPs/Alg hydrogels can reduce the aging of chondrocytes by removing ROS.4.Therapeutic effect of Cuttlefish ink-derived melanin nanoparticles/sodium alginate hydrogel on osteoarthritis ratsIn this experiment,arat model of osteoarthritis was established by the modified Hul th method,and CIMNPs/Alg hydrogel was injected into the rat joint cavity for treatment.The safety of CIMNPs/Alg hydrogels on rats was studied by monitoring the trend of rat body weight during treatment,and the experimental results showed that the body weight of rats showed an upward trend during treatment,indicating that CIMNPs/Alg hydrogels had a good safety profile for rats.By observing the joint photos and joint pathology sections of rats after treatment,the therapeutic effect of CIMNPs/Alg hydrogel on osteoarthritis rats was studied,and the experimental results showed that CIMNPs/Alg hydrogel had a good therapeutic effect on rat osteoarthritis models.