Application And Mechanism Of Magnetic Targeted Fe₃O₄@PDA Labeled Mesenchymal Stem Cells In The Treatment Of Neuropathic Pain

Background:Neuropathic pain is pain caused by damage to the somatosensory nervous system or diseases,accounting for 20% to 25% of patients with chronic pain,and the prevalence rate may be as high as 7% to 8% in the general population.The prominent symptoms of patients with neuropathic pain are spontaneous pain,hyperalgesia and pain allodynia.The pain sensation of the patients was extremely strong,the pain score was significantly higher than that of other chronic pain people,and the quality of life decreased significantly.Although neuropathic pain is very common,patients usually cannot get enough pain relief from current drugs,and the treatment of neuropathic pain is still a challenge.In the last two decades,stem cell transplantation has been considered as a possible treatment.At present,many kinds of stem cells,such as bone marrow mesenchymal stem cells(MSCs),human amniotic fluid MSCs,adipose derived stem cells and ?-aminobutyric acid intermediate neuron progenitor cells,have been used in preclinical research.The results show that stem cells have strong therapeutic potential in the treatment of neuropathic pain and can reduce the typical manifestations of neuropathic pain,such as hyperalgesia and allodynia.A small number of clinical trials of stem cells in the treatment of neuropathic pain have been carried out with good results.Although the therapeutic effect of stem cells on neuropathic pain is exciting,stem cells are still a huge challenge in effectively delivering them to the target site.In the preclinical experiment of stem cells in the treatment of neuropathic pain,the cells are automatically targeted to the spinal cord or the corresponding injured area mainly by intrathecal injection or caudal vein injection.Studies have shown that most of the stem cells are trapped in the lungs after caudal vein injection.As an invasive method of drug administration,intrathecal injection has many clinical contraindications and the risk of infection.Therefore,how to recruit stem cells into the spinal cord is an important problem to limit the application of stem cells in the treatment of neuropathic pain.At this time,magnetic targeting technology provides us with an attractive possibility.Here,we used Fe₃O₄ nanoparticles as the core,PDA as the shell of Fe₃O₄@PDA composite ultraparticles and human umbilical cord MSCs to establish a magnet targeted therapy system.We labeled human umbilical cord MSCs with Fe₃O₄@PDA in vitro to make the stem cells magnetic due to Fe₃O₄@PDA loading.Then,the obtained Fe₃O₄@PDAlabeled stem cells are injected into the animal through the tail vein,and guided by the magnetic field.Therefore,a large number of MSCs can be delivered to the segments of spinal cord pain-related response,so as to make full use of the repair function of MSCs.It makes the curative effect of stem cells on neuropathic pain faster and better,and provides a new attempt for the stem cell treatment strategy of neuropathic pain.Methods:1.Umbilical cord MSCs were isolated and cultured,and Fe₃O₄@PDA nanoparticles were prepared by thermal decomposition and emulsification,and their morphology was characterized by transmission electron microscope.2.The optimal material concentration for labeling MSCs was determined by hemolysis test,CCK-8 method and Prussian blue staining.The characterization,ability of differentiation and proliferation of MSCs labeled with Fe₃O₄@PDA nanoparticles were verified by flow cytometry,adipogenic and osteogenic differentiation induction of stem cells and CCK-8 method.3.The magnetic effects of Fe₃O₄@PDA nanoparticles and Fe₃O₄@PDA labeled MSCs were tested in vitro.4.The model of chronic sciatic nerve compression injury(CCI)was established in rats,and the possible toxicity of stem cells and Fe₃O₄@PDA nanoparticles to the growth and development of rats were evaluated by recording body weight and organ weight,and the main organs were stained with HE.5.Animal fluorescence imaging technique was used to track the distribution of stem cells in rats.6.The of mechanical paw withdraw threshold and the thermal paw withdraw latency were measured to evaluate the behavioral pain response of rats,the expression of cFos(molecular level index of pain)in spinal cord was evaluated by immunohistochemical staining and PCR,and the changes of myelin sheath in rat spinal cord were evaluated by Luxol fast blue staining.7.The changes of serum interleukin-6(IL-6),interleukin-10(IL-10)and transforming growth factor-?(TGF-?)in rats were measured by ELISA,and the peripheral antiinflammatory effect of magnetically targeted Fe₃O₄@PDA-labeled MSCs was evaluated.8.Immunofluorescence staining and PCR technique were used to detect the changes of glial fibrillary acidic protein(GFAP,astrocyte marker)and ionized calcium binding adapter molecule-1(IBA-1,microglial marker)in rat spinal cord,and to explore the relationship between stem cells and central glial cell activation.Results:1.We obtained high purity umbilical cord MSCs and synthesized stable Fe₃O₄@PDA nanoparticles.2.When the concentration of labeled MSCs was 50 ? g/ml,the labeled cells showed good labeling rate,good blood compatibility and strong cell viability.The stem cell characterization and differentiation ability of MSCs labeled with 50 ?g/ml Fe₃O₄@PDA nanoparticles were not affected,and Fe₃O₄@PDA did not inhibit the proliferation of stem cells.3.The magnetic effects of Fe₃O₄@PDA nanoparticles and Fe₃O₄@PDA labeled MSCs were verified in vitro.4.In the course of treatment,the body weight and organ coefficient of rats in each group were not statistically significant,but the results of HE staining showed that the microstructure of the main circulatory organs of rats was normal and there was no toxic damage.5.The results of animal fluorescence imaging showed that under the action of magnets,the homing of Fe₃O₄@PDA-labeled MSCs to the spinal cord was accelerated,and the number of MSCs gathered in the spinal cord was more.6.The mechanical paw withdraw threshold and the observation of rat hindfoot showed that magnetically targeted Fe₃O₄@PDA labeled MSCs decreased mechanical hypersensitivity,curl and protection of hindfoot,and magnetically targeted Fe₃O₄@PDA labeled MSCs decreased the expression of c-Fos and demyelination in spinal cord.7.Serum ELISA showed that stem cells decreased the content of IL-6 in serum,and Fe₃O₄@PDA-labeled MSCs did not produce significant changes in inflammatory factors in peripheral blood.8.Immunofluorescence staining and PCR results showed that CCI operation significantly increased the expression of GFAP and IBA1,while stem cells could reduce the expression of GFAP and IBA-1.Magnetically targeted Fe₃O₄@PDA nanoparticles labeled MSCs significantly decreased the expression of GFAP and IBA-1 induced by CCI operation on the first day after injection,and the inhibitory effect was the most significant.Conclusion:1.We successfully obtained Fe₃O₄@PDA nanoparticles and internalized them by MSCs.The characteristics of MSCs labeled with Fe₃O₄@PDA are not changed,and they are magnetic,and their movement can be affected by external magnetic field.2.MSCs labeled with Fe₃O₄@PDA were injected into animals through tail vein.Under the synergistic action of magnetic field and Fe₃O₄@PDA,the analgesic effect of neuropathic pain was good.Stem cells gather more and faster in the segment(L4-L6)of spinal cord pain-related response,which solves the problem of low efficiency of stem cell recruitment to the spinal cord,thus making full use of the repair function of stem cells,and produces a good therapeutic effect in the early stage after cell injection.3.Magnetic targeted Fe₃O₄@PDA nanoparticles labeled MSCs can achieve analgesic effect on neuropathic pain by inhibiting the activation of spinal microglia and astrocytes in the early stage.