| Osteoarthritis is a common degenerative disease,which is characterized by articular cartilage degeneration and synovial inflammation.It is a kind of disabling disease with a high incidence and seriously threatens human health and life.At present,there is no effective treatment for osteoarthritis.Clinically,drug therapy(oral or intra-articular injection)is mainly used to eliminate synovial inflammation and relieve the symptoms of osteoarthritis,but it cannot repair the cartilage damage of osteoarthritis.Recently,the gene therapy for osteoarthritis has received widespread attention,which can repair cartilage damage by regulating the expression of related genes.Moreover,compared with monotherapy,combined treatment can play a role in enhancing efficacy.Therefore,the combined application of gene and drug can provide a new idea for the treatment of osteoarthritis.Lornoxicam,a non-steroidal anti-inflammatory drug,can treat osteoarthritis by exerting anti-inflammatory and analgesic effects.MicroRNA-140 is a small non-coding RNA and can repair cartilage damage by regulating the expression of genes related to osteoarthritis.Thus,the combined application of lornoxicam and microRNA-140 can treat osteoarthritis by inhibiting inflammation and repairing cartilage damage.However,microRNA-140 is difficult to enter the cell and is easily degraded by nuclease,so it is still necessary to find a suitable microRNA delivery vector to overcome the obstacle of microRNA application.Gold nanoparticles are associative colloids of gold with a diameter of 1～100 nm.They have many excellent properties and are commonly used gene delivery vehicles.In this study,microRNA-140 loaded gold nanoparticles complex were firstly prepared and their properties and nuclease stability were inveatigated.Then,the in vitro safety,ability to enter cells and ability to regulate genes were characterized.Finally,the effect of combined application of lornoxicam and microRNA-140(successively injected into the joint cavity)in the treatment of osteoarthritis was verified by pharmacodynamic experiments in rats.The main research contents and results are as follows:1.Preparation,characterization and nuclease stability of AuNPs/miR-140 complex.First,gold nanoparticles(AuNPs)were prepared using the polyethyleneimine(PEI)reduction method.The UV-Vis absorption characteristic curve of AuNPs was used as evaluation index and the optimal conditions for preparing AuNPs were screened through single factor experiments.And the morphology,particle size,and zeta potential of AuNPs were characterized;Then,microRNA-140(miR-140)was connected to AuNPs by electrostatic adsorption to prepare gene-loaded gold nanoparticles(AuNPs/miR-140 complex).The particle size,zeta potential,and the ability of AuNPs to compress miR-140 were measured to optimize the weight ratio of AuNPs/miR-140 complex;Finally,to prove whether AuNPs/miR-140 could protect miR-140 from nuclease degradation,the stabilitiesof AuNPs/miR-140 and naked miR-140 in nucleases were investigated.The results showed that AuNPs were prepared successful using PEI reduction method.And the optimal preparation conditions were:PEI volume was 400 μL,reaction temperature was 60℃,and reaction time was 2 h.The prepared aqueous solution of AuNPs showed clear wine red.Microscopically,AuNPs were spherical particles with uniform size and good dispersion.The particle size was(22.31+4.41)nm,PDI was(0.25±0.03),and the zeta potential was(32.23± 1.16)mV;When the weight ratio(Au:miR-140)of AuNPs/miR-140 complex was 28.8:1,miR-140 could be fully compressed by AuNPs.And at this weight ratio,complex had the smallest particle size(27.04±2.59 nm)and suitable zeta potential(30.4± 1.93 mV).Thus,the optimal weight ratio of the complex was 28.8:1;The AuNPs/miR-140 complex could be stable for at least 24 h in the presence of nucleases.And it had significant meaning:when complex was used to treat osteoarthritis in vivo,this ensured that the complex could protect miR-140 from degradation and miR-140 could exert the therapeutic effect.2.In vitro evaluation of AuNPs/miR-140 complexFirst,chondrocytes were incubated in vitro,PEI was used as a positive control,and the chondrocyte toxicity of AuNPs/miR-140 complex was investigated by MTT method;Then,miR-140 was labeled with cy3,and fluorescence microscope and flow cytometry were used to investigate the ability of complex to enter cells;Finally,qPCR experiments were used to determine the relative expression levels of miR-140 and COL2A mRNA in the cells after transfection.The experimental results of the MTT method showed that the chondrocyte survival rates of the AuNPs/miR-140 group were greater than 90%at a concentration range of 3.125 to 200 nM,while the cell survival rate of the PEI/miR-140 group at 200 nM was only 78%.It revealed that AuNPs/miR-140 complex was less cytotoxic than PEI/miR-140 complex;The results of cell transfection experiments showed that the chondrocyte transfection rates of AuNPs/miR-140 and PEI/miR-140 were close to 100%,both of them could efficiently transfect miR-140 into chondrocytes.But the fluorescence intensity in the chondrocytes of the AuNPs/miR-140 group was weaker than that of the PEI/miR-140 group,demonstrating the ability of AuNPs to delivery miR-140 into cells was still slightly lower than that of PEI.The toxicity of PEI limited its further application.In general,AuNPs were safe and efficient gene delivery vectors;The results of qPCR experiments showed that the AuNPs/miR-140 complex could increase the expression of miR-140 in chondrocytes by 220 times and increase the expression of COL2A mRNA by 1.37 times,which had a positive significance for cartilage repair.3.Pharmacodynamic study of microRNA-140 combined with lornoxicam in the treatment of osteoarthritisFirst,a rat osteoarthritis model was established by injecting papain into joint cavity.And the knee joint width was measured to initially prove whether the rat osteoarthritis model was successfully established.Then,rats were grouped and administered.A was the normal control group(not modeled).The model rats were divided into five groups:B,C,D,E,and F.B was the osteoarthritis model group,C was the naked miR-140 group,D was the drug(Lnxc-sol)group,E was AuNPs/miR-140 complex group,and F was combination treatment group.Groups B were administered with saline by intra-articular injection;group C was injected with naked miR-140;group D was injected with Lnxc-sol and group E was injected with AuNPs/miR-140 complex.The above groups were injected once a week for six weeks.For group F,Lnxc-sol was injected into the articular cavity once a week for three consecutive weeks;from the fourth week,AuNPs/miR-140 complex was injected into the articular cavity once a week for three consecutive weeks.Finally,a series of processings were performed on rats to verify the degree of articular cartilage repair and inflammation improvement after the different treatments were given.The knee joint width and body weight of rats were measured during the experiment;The rats were sacrificed one week after the last administration,and the femoral ankle,tibial plateau and synovium of the rats were separated and observed macroscopically;The femoral ankle and tibial plateau were sliced,and HE staining and saffron O-fast green staining were performed.And microscopic observation of the stained cartilage slices and ManKin scoring were performed;The synovium was sliced and HE staining was performed.And microscopic observation of the stained synovium slices and histopathological score of synovium were performed.The experimental results showed that the body weights of the rats injected with papain were smaller than that of normal rats,but there were no significant difference in the body weights of the rats in each group after administration;After modeling,the knee joint widths increased from about 9 mm to about 10.1 mm.One week after the last administration,the average knee widths of group B and group C were 10.80 mm and 10.75 mm,respectively,and there were no significant difference between them.The average knee widths of the groups D,E and F were 9.72,9.81,and 9.58 mm,respectively,which were significantly smaller than the average knee width of group B;The macroscopical observation results suggested that the cartilage surface in group A was smooth and shiny.However,the cartilages in groups B and C were seriously damaged.and sufaces of them were gloomy and deep cracks were visible.The cartilage damages in groups D and E were repaired to some extent.Moreover,the cartilage surface in group F was almost same as that in group A and only small lesions were seen;HE staining of cartilage,saffron O-fast green staining,and ManKin score showed that cartilage was not damaged in group A,and the order of degrees of cartilage damages in the other groups was:group B≈C>D≥E>F;HE staining and histopathological score of the synovium showed that there was no synovium inflammation in group A,and the order of the severities of synovium inflammations in the other groups was:group B≈C>E>D≈F.The above results indicated that the rat osteoarthritis model was successfully established.Osteoarthritis could cause cartilage damage and synovium inflammation in rats.Injection of normal saline and naked miR-140 could not alleviate the symptoms of osteoarthritis in rats after modeling.However,Lnxc-sol and AuNPs/miR-140 could play a significant role in treating osteoarthritis,and the combined application of them was more effective.Combination therapy could play dual roles in eliminating inflammation and repairing cartilage damage,which had scientific research and application value.Conclusion:MicroRNA loaded gold nanoparticles were successfully prepared.The particle and size potentials of AuNPs/miR-140 complex were suitable and they had good stability in nucleases.In vitro cell experiments showed that gold nanoparticles were safe and efficient gene delivery vehicles.In vivo pharmacodynamic experiments showed that the combination treatment(Lnxc-sol and AuNPs/miR-140)played dual roles in eliminating inflammation and repairing cartilage damage,which could achieve the expected purpose of the experiment and had great social benefits and application value. |
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