Microneedle-Mediated Delivery Of Biomacromolecules For Rheumatoid Arthritis Treatment

Objective:Biomacromolecules are an important type of therapeutics for rheumatoid arthritis(RA)due to their great efficacy and safety.However,most biomacromolecules are administrated by injection,which causes pain,redness,risk of infection,and low compliance.Drug delivery strategies can be used to overcome these disadvantages for RA treatment.In this study,dissolvable microneedle(MN)loading etanercept(EN)and hydrogel MN loading DEK-targeting aptamer(DTA)were fabricated to investigate the potential of MN-based delivery of biomacromolecules therapy for RA treatment.Methods:First,hyaluronic acid(HA)was modified with Methacrylic anhydride to form modified HA(mHA)and the degree of methacrylation was measured using nuclear magnetic resonance spectroscopy.The dissolvable MN was fabricated using micromoulding method for the delivery of EN.The morphology and mechanical properties of MN were characterized using optical microscope,scanning electron microscope and texture analyzer.Transdermal insertion,skin recovery and solubility of MN were tested on mice skin.In vitro bioactivity of EN under ultraviolet(UV)light was analyzed by dynamic light scattering(DLS)and circular dichroism spectrum(CD).The drug content of MN was detected by measuring the fluorescent intensity of the SRB-EN mixture.In vivo evaluation of MN was studied on adjuvant-induced arthritis mice by detecting body weight,paw volume,cytokine,and histologic section.Based on dissolvable MN,the crosslink intensity was enhanced by prolonging the time of grafting reaction and UV crosslink to obtain hydrogel MN.Polyvinyl alcohol(PVA)and ethocel(EC)were used as the materials of the back layer.DTA was loaded on MN using dip coating or drop coating method.The morphology,solubility in water and drug release in agarose gel were studied to screen MN with a better performance.The morphology,mechanical properties,skin insertion,and skin recovery were investigated as described above.The Drug distribution on MN,drug release in pig skin and mice were studied using fluorescent labeling and confocal laser scanning microscopy.The bioactivity of chemical modified DTA before and after loading to MN was tested by gel electrophoresis.Results:HA was successfully modified with methacrylic anhydride.The degree of methacrylation was ranged from 20/%to 70%by adjusting reaction time.The dissolvable MN consisted of 225(15×15,1 cm×1 cm)conical needles.The height,base diameter,and spacing of needles were 800 μm,300 μm,and 600 μm respectively.The force-displacement curve showed that the failure force per needle was about 580 mN,which was sufficient for the skin insertion.Trypan blue staining image and H&E staining section showed that MN could effectively pierce into the dermis with a depth of 200 μm.The needles of MN dissolved rapidly within 1.5 h in the skin.The inserted skin recovered completely within 120 min after the removal of MN.The results of DLS and CD showed that UV radiation had little affection on the bioactivity of EN.The EN content of MN was about 42.72 μg.In vivo study showed that MN-mediated delivery of EN had similar efficacy to the subcutaneous injection and effectively inhibited paw inflammation,expression of cytokine,and cartilage damage in the joint.In the next work,Hydrogel MN was successfully prepared using mHA and EC.Hydrogel MN had similar properties to dissolvable MN in appearance,skin insertion and skin invasion.Aptamer was loaded to MN using drop coating method and mainly distributed on the tip and the surface of needles.Drug release study showed that SRB in MN could release into pig skin.Aptamers in MN rapidly release in mice skin within 1h.And about 92%of aptamer was released in 4 h.Finally,gel electrophoresis proved that aptamers had little degradation during the drug loading process.Conclusion:This study described the exploration on transdermal delivery of biomacromolecules by MN for RA therapy.The dissolvable MN constructed in this study can effectively load and transdermally deliver EN.MN-mediated delivery of EN has achieved significant efficacy in pharmacodynamics study,which proves the strategy is feasible.Then hydrogel MN inherited the great properties of dissolvable MN.The preparation of hydrogel MN can avoid drug waste and inactivation.Aptamers were loaded to MN by drop coating method with little degradation.MN can absorb body fluids and quickly release drugs in a concentration-dependent manner after applying on the skin.Though the therapeutic effect was not studied,hydrogel MN may have great potential in the delivery of aptamers for treating RA.