Immunomodulatory Effect Of ARA290 Microneedles On Painful Diabetic Neuropathy And Its Mechanism

Background:Diabetic peripheral neuropathy is the most common complication of diabetes,with about 1/4 patients experiencing stubborn pain symptoms,namely painful diabetic neuropathy（PDN）.PDN is an important factor of anxiety and depression in diabetic patients,seriously affecting the patients’quality of life.Traditional treatment modalities mainly rely on nerve-nurturing and the symptomatic treatment with antalgic and antidepressant drugs.However,the slow onset of effect,unbearable side-effects and poor efficacy usually resulted in poor compliance,leading to the development of diabetes foot,amputation or even death.The pathogenesis of PDN is complex.It is caused by both peripheral neuropathy and the abnormal central nervous system conduction pathways.Recent studies have shown that microglia play a key role in the initiation and maintenance of PDN.Microglia are immune cells in the central nervous system,which monitor and regulate the local microenvironment.Based on the characteristics of stimulations,they can be activated and polarized to M1（pro-inflammatory）type and M2（anti-inflammatory）type.They regulate the normal homeostasis of the central nervous system.In PDN,the M1 polarization of microglia is hyperactive,and the M2 polarization is impaired.Such imbalance plays the key role in the development of PDN.It causes continuous amplification of inflammatory signals and induces central sensitization,that maintain and exacerbate the neuropathological pain.It is reported that erythropoietin（EPO）is produced locally in the stressed tissue and interact with the EPOR-βcommon Receptor complex,also known as the innate repair receptor（IRR）,by paracrine or autocrine.Microglia IRR activation mediated M1polarization inhibition and M2 polarization enhancement.However,it requires higher local EPO concentrations to activate the IRRs,and the hematopoietic activity of EPO may cause thrombosis and stroke risks.ARA290 is an 11-amino acid peptide that mimics the structure of the helix B in EPO.It selectively activates IRR but dose not have hematopoietic activity.Its biosafety is good.It has the effect of regulating glial cell polarization,inhibiting inflammatory response,anti-neuronal apoptosis,and promoting axon regeneration.The peptide can also pass through the blood-brain barrier,but the plasma half-life is extremely short.The half-life of the plasma is only 2 min.The pharmacokinetic defects and the bioavailability of ARA290 can be improved by multidisciplinary means of engineering and medicine.Microneedles（MNs）are arrays of several to hundreds of micron-scale fine needles that penetrate the corneum and epidermis and deliver the drug to the dermis.The drug passes through the capillary into the body circulation,avoiding the first pass elimination effect.Their tip diameters are below 20μm,which does not stimulate the nerves.With the advantages including pain-free,minimally invasive,and convenient,MNs can improve patient compliance,especially suitable for chronic disease treatment.Soluble microneedles are manufactured with safe,biodegradable polymers.After piercing into the skin,the drugs are slowly released during the dissolution,swelling and degradation of the polymers.They do not produce sharp biohazard wastes and can achieve sustained drug release.Based on the above background,a polymeric soluble microneedle delivery system of ARA290 is proposed and manufactured.Its therapeutic effect on PDN rats and the mechanism of microglia immune regulation are investigated.Materials and methods:1.20 male Sprague Dawley rats were divided into two groups.Three for normal-control（NC）group and 17 for PDN model construction.The rats were fed with high fat diet for 8weeks for insulin resistance induction,then given streptozocin（STZ）at a dose of 35 mg/kg after fasting 12 h by a single intraperitoneal injection.Mechanical withdrawal threshold（MWT）and thermal withdrawal latency（TWL）is tested every other week.Four weeks after the STZ injection,those with fasting blood-glucose（FBG）higher than 16.7 mmol/L and MWL lower than 5 g are regarded as PDN rats.The weight,FBG,MWT and TWL were monitored.Rats were sacrificed respectively on the fourth and eighth weekend for histological examination.Immunofluorescent staining was applied to the lumbar spinal cord for microglia polarization and TNF-αexpression analyses.Nissl’s staining was applied to dorsal root ganglions（DRG）s for morphology analysis of neurons.Luxolfastblue（LFB）staining was applied to sciatic nerve to display the myelin sheath.Skin of the foot plantar was also immunofluorescent stained for the intraepidermal nerve fiber density（IENFD）analyses.2.Soluble polymeric MNs loaded with ARA290 were made by micromoulding method.The needle tips were made of Ca Cl₂ crosslinked sodium alginate and carboxymethyl cellulose and the substrates were made of polyvinyl pyrrolidone and polyvinyl alcohol.The MN patches were examined and photographed under optical microscope.The mechanical property was analyzed with Parafilm M^? puncture test.Rodamin B was used as a simulated drug to investigate the in vitro release of the drug.The MNs was applied to rat skin to evaluate the puncture ability.Biocompatibility of microneedle materials was detected by NIH-3T3 cell live/dead staining and CCK-8 experiments.The skin irritation was detected on volunteers’arms.3.Nine PDN model rats were randomly divided into three groups:model control group（PDN）,ARA290 injection group（PT）and ARA290-loaded soluble MN group（MN）.PT group was given ARA290（60μg/kg）a subcutaneous injection once a week;the MN group was given a skin patch containing ARA290 microneedles,once a week;The PDN group was given an equivalent amount of PBS buffer subcutaneous injection once a week.Measurements of MWT and TWL were applied before weekly treatment administration.Immunofluorescence staining was used to determine the polarization status of microglia in the dorsal horns of spinal cords,TNF-αexpression of spinal cord and DRGs in rats in the fourth weekend.Nissl’s staining was applied to DRGs for morphology analysis of neurons.LFB staining was applied to sciatic nerve to display the myelin sheath.Skin of the foot plantar was also immunofluorescent stained for the intraepidermal nerve fiber density IENFD and growth associated protein-43 analyses.Results:1.The symptoms of allodynia in PDN rats appeared within two to four weeks after the formation of type II diabetes mellitus.The symptoms were stable at the fourth week and lasted for more than eight weeks without any natural relief.After four weeks of diabetes,M1polarization of microglia was significantly increased,while M2 polarization was not significantly changed.TNF-αexpression of spinal cord was increased.Large neurons of DRG were decreased and myelinated fibers of sciatic nerve were thinner and demyelinated.IENFD of foot planta was decreased.The peripheral neuropathy increases with the prolongation of the disease.2.ARA290-loaded soluble MNs have good biocompatibility and mechanical properties.In vitro release curve showed that the polymer microneedles released more than 90%of the drug within three days and almost completely released on the seventh day.The cross-linked polymer MN tips encapsulated the drugs and was implanted into the skin.The drugs released slowly as swelling and degradation occur.Soluble polymeric MNs can be used for the transdermal drug delivery and sustained release of drugs.It is less irritating to the skin and does not cause pain.3.In the central nervous system,ARA290-loaded MNs can significantly inhibit M1polarization and promote M2 polarization of microglia in the dorsal horn of spinal cord.In the peripheral nervous system,it inhibited inflammation,improved the morphology of DRG neurons,relieved sciatic nerve demyelination,and promoted regeneration of nerve fibers in the epidermis.In PDN rats,the allodynia symptoms were alleviated and the diabetic neuropathy was improved.4.Compared with subcutaneous injection,ARA290 MNs have significant advantages in inhibiting allodynia,promoting the function of M2-type microglia and promoting cutaneous nerve ingrowth.Conclusions:The ARA290-loaded soluble polymer MNs relieved the allodynia and improved diabetic neuropathy in PDN rats.Its protective mechanism is probably related to microglia immune regulation,inhibition of neuroinflammation and promotion of axon growth.