| Background: Rheumatoid arthritis(RA),gouty arthritis(GA)and osteoarthritis(OA)are three common aseptic joint inflammations,each of which affects tens of millions of people.RA and GA are inflammatory joint diseases caused by different factors.Given the fact that IL-1β is reported to play pivotal roles in the two inflammatory mechanisms,biologics targeting IL-1β(such as artificial recombinant IL-1Ra)have shown excellent anti-inflammatory effects.However,their clinical applications are limited by problems such as short half-life and low bioavailability.Therefore,researches on the above problems are of great significance for the treatment of RA and GA.OA is mainly characterized by cartilage degeneration and defect.Cartilage repair has always been a challenge facing human beings.Although the emergence of various functional materials has greatly improved the effect of cartilage repair,it is still difficult to achieve ideal tissue integration.It is reported that improving the tissue adhesion property of repair materials is expected to promote cartilage tissue integration.Elastin-like polypeptides(ELP)has good biocompatibility and flexible structural modification.It has shown certain advantages in the field of biofunctional materials such as drug modification and bio-adhesive research,which may help solve the above problems.Based on the above background,the following researches were carried out in this subject.Objective: To prepare long-acting IL-1Ra nano-biologics to improve the half-life and bioavailability of IL-1Ra and explore their therapeutic effects on RA and GA;to prepare new bio-adhesive materials and investigate their therapeutic effects in promoting cartilage repair.Methods and Results: 1.Preparation and characterization of long-acting IL-1Ra nano-formulations.A fusion amino sequence consisting of positively charged elastin-like polypeptides(ELP),named as K72,and IL-1Ra was constructed and expressed in E.coli.Then the chimera protein IL-1Ra-K72 was successfully prepared,and it exhibited positively charged property in a neutrol environment through Zeta potential test.It was subsequently combined with PEG& chondroitin sulfate(CS)respectively via electrostatic interaction and hydrophobic force to form two nano-particles,denoted as IL-1Ra-K72-PEG and IL-1Ra-K72-CS.Each kind of nanoparticle showed a uniform morphology under TEM observation.The hydrated size of the two kinds of nanoparticles measured by DLS method is ～200 nm respectively.In vitro drug release test,the two kinds of nanoparticles showed excellent sustained release ability.No obvious cytotoxicity was observed in both of the nanoparticles when co-cultured with BMSC cells in the concentration range of 0-800μg/mL respectively.2.Experimental researches in treating RA rat models using IL-1Ra-K72-PEG and IL-1Ra-K72-CS.The IL-1β signal blocking effect was characterized via cell experiments.Each of the two nano-biologics showed a significant inhibitory effect on the proliferation of IL-1β-dependent RPMI1788 cells.The ability of RA-FLS to secrete IL-1β downstream factors(PGE2 and MMP3)was also obviously inhibited.No significant difference was observed in the inhibitory effect of IL-1Ra-K72 and IL-1Ra.Pharmacokinetic tests in adult female SD rats exhibited that the half-life of IL-1Ra-K72-PEG reached up to(33.4±2.79)h,and the half-life of IL-1Ra-K72-CS also reached to(20.2±2.09)h while that of IL-1Ra was only(6.7±1.86)h.The in vivo bioavailability of each nano-biologics was 7 times that of IL-1Ra.In in vivo detections,we observed the therapeutical effect through investigating the changes in the degree of swelling of the rat paws,the levels of inflammatory factors(such as IL-1β,TNF-α,PGE2 and MMP3),histopathological changes and immunohistochemistry results.It was found that both of two nanomedicines showed better therapeutic effects than IL-1Ra group.At the same time,we found that therapeutical outcomes in IL-1Ra-K72-CS treatment group were significantly better than those in the IL-1Ra-K72-PEG treatment group.3.Experimental researches in treating GA rat models using IL-1Ra-K72-PEG nano-biologics.The secretion of PGE2 and MMP3 of FLS cells under the stimulation of IL-1β was also obviously inhibited.Pharmacokinetic tests in adult male SD rats exhibited that the half-life of IL-1Ra-K72-PEG reached up to(27.16±2.55)h,which was 4 times that of IL-1Ra.After a therapeutic observation during 72 hours in in vivo tests,we investigated the changes in the peremeters of the GA rat ankle joints,the levels of inflammatory factors(such as IL-1β,TNF-α,IL-6),histopathological changes and immunohistochemistry difference.The results showed that anti-inflammatory effect in IL-1Ra-K72-PEG treated groups was significantly better than that of IL-1Ra or indomethacin solely treated group.4.Preparation of supramolecular assembled bio-adhesives and its application on cartilage repair research.A novel bio-adhesive(KPC adhesive)was prepared using K72,PEG and CS via supramolecular assembly.The non-covalent formulation was proved by FTIR test.A loose and porous internal structure of KPC bio-adhesive was observed by SEM.In the bonding test on porcine skin and cartilage substrates,the shear bonding strength of KPC reached up to(33.11±2.26)KPa,which was better than the tissue adhesion strength of some reported commercial fibrin sealants.The sustained release of CS from KPC bio-adhesive lasting for more than 11 days was observed in in vitro release test.The adhesive showed excellent cell compatibility when co-cultured with A549 cells and BMSCs respectively.The expression of type II collagen and GAG in BMSCs was significantly promoted by KPC,especially in the group of cooperating with TGF-β1.KPC adhesive was implanted into the cartilage defect lesion established at the rat femoral trochlear site.After eight weeks,the morphological repair and histopathological outcomes was assessed.The KPC bio-adhesive showed significant better repair effect than other groups.And cartilage tissue integration was achieved to a great extent.Conclusions: 1.The method of positively charged ELP modification and non-covalent nano-assembly maintains the anti-inflammatory activity of IL-1Ra and improves the half-life and bioavailability of the biologics.This method offers references for researches of other protein drugs.2.The long-acting IL-1Ra nano-formulation can achieve effective treatment of RA and GA through few administrations,which may offer new candidate therapies for treatments of RA and GA as well as other IL-1β involved inflammatory diseases.3.In the treatment of RA,CS may cooperate with IL-1Ra to provide overlaid effects of anti-inflammation and cartilage protection.The specific mechanism needs to be further clarified.4.Supramolecular assembled bio-adhesive KPC has ultra-strong adhesive properties and it is promising to be used as medical adhesives in clinical applications.5.Adhesive repair materials can promote cartilage tissue integration to a certain extent,which is of great benefit for cartilage repair.This provides new insights for the development of cartilage repair materials and the treatment of OA.6.Given the excellent biocompatibility and easy-to-be-modified capability of ELP,it is of great potential in studies of biological functional materials and protein drugs. |
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