Research On The Construction Of Stem Cell Membrane Derived Biomimetic Liposome And Its Application In Acute Lung Injury Therapy

Acute lung injury(ALI)is a respiratory syndrome characterized by acute,diffuse alveolar damage,with a fatality rate of 30%-60%.Currently,there is still lacking an effective treatment for ALI.Anti-inflammatory therapy is essential to prevent further progression of ALI and protect lung tissue.Glucocorticoid(GC),such as methylprednisolone(MP),is the most clinically used anti-inflammatory drugs.However,its application in ALI treatment is limited due to the systemic adverse reactions caused by large doses or frequent administration of MP.A targeted pulmonary inflammation delivery system can overcome this limitation with providing a safe and effective MP delivery strategy for the anti-inflammation treatment of ALI.In this study,we hybridized mesenchymal stem cell(MSC)membranes with liposome,named MSCsome,to achieve efficient targeted drug delivery in inflamed lung to against pulmonary inflammation.Due to the preservation of targeting related proteins of MSC,MSCsome showed good ability of inflamed lung targeting ability.MP loaded MSCsome shows efficient and safe anti-inflammatory efficacy in lipopolysaccharide(LPS)induced lung inflammation.This study is divided into four parts.In the first part,MSCsome was constructed with the characteristic’s description.To obtained the hybridized biomimetic liposome,extracted MSC membrane and prepared liposome membrane were fused through ultrasonic.Results showed that MSCsome(117.0 nm.)exhibited a similar morphology with liposomes(112.5 nm).Compared with the zeta potential of liposome(-3.4 m V),the zeta potential of MSCsome(-7.9 m V)was decreased,which was closer to the zeta potential of MSC membrane(-10.5 m V).The results of FTIR,Western Blotting and SDS-PAGE results showed that the prepared MSCsome retained protein components are similar to MSC membrane.After standing at 4 ℃ for 7 days,the particle size of MSCsome did not change significantly.The encapsulation rate(84.9%)and cumulative release rate(53.3% at p H = 4.5 and 48.4% at p H = 7.4 respectively)of MP were qualified by HPLC analysis.It is proved that the hybridized liposomes prepared by the reported method can contain and release MP.A series of MSCsome formulation with different ratio of MSC membrane to phospholipid membrane(P:L)were screened in terms of particle average degree,membrane fusion,drug loading and hemolysis rate.The CLSM results showed that the green fluorescence signal of MSC membrane and the red fluorescence signal of liposome were highly overlapped in MSCsome(P:L = 1:4),which confirmed the successful fusion of cell membrane and liposome.The PDI of MSCsome(P:L = 1:4)was 0.3,with the encapsulation rate of was 85.2% and the drug loading rate was 5.7%.The hemolysis rate in rabbit blood was 4.6%.Thus,MSCsome(P:L = 1:4)was determined as the optimal prescription for subsequent experiments.The second part investigated the inflammatory targeting ability of MSCsome.TNF-α-injured HUVEC cells were used as a model of inflamed endothelium in vitro.The efficient penetration of MSCsome on the inflamed endothelium was investigated in transwell assay,and the morphology and membrane composition of the carrier after passing through the inflamed endothelium were observed.It turns out that MSCsome trend to passing through the inflamed endothelium and retains liposome-like and membrane hybridized morphology.Further exploration revealed that CXCR4 in MSCsome can binding with SDF-1α and promote MSCsome passing through the endothelial barrier in higher SDF-1α concentration.An ALI mouse model induced by LPS was constructed.Fluorescence labeled MSCsome was injected into ALI model mice,and revealed an inflammation targeting biodistribution in injured lung.Furthermore,MSCsome showed a long retention in lung tissue within 72 h after systemic injection.These results demonstrate the inflammation targeting ability of MSCsome.MP loaded MSCsome(MP-MSCsome)was intravenously injected into ALI model mice.The same result was repeated after MP-MSCsome injection.The third part verifies the anti-inflammatory effect of MP-MSCsome.MPMSCsome was systematically administered to ALI model mice.After 48 h treatment,samples were collected.The therapeutic effect of ALI was evaluated with the investigation of inflammation level,barrier damage degree and histopathology.The results showed that MP-MSCsome treated animals had lower levels of lung inflammatory factors,less pulmonary edema and protein fluid penetration than free MP and MP-liposome groups.In the H&E-stained pulmonary sections,a more health alveolar and less inflammatory cells infiltration were showed.These results prove that MP-MSCsome has a better anti-inflammatory effect on ALI,which reflect the advantage of inflammatory targeted delivery in the application of ALI antiinflammatory therapy.The fourth part evaluates the safety of MSCsome and MP-MSCsome.Firstly,the safety of MSCsome on related cells was investigated in vitro.The effects of different MSCsome concentrations on the viability of immune cells,lung epithelial cells and vascular endothelial cells were investigated.MSCsome-induced inflammation was evaluated by detecting the concentration changes of inflammatory cytokines secreted by immune cells after co-incubation with MSCsome.Experimental results showed that MSCsome did not impact cell viability or cause inflammation.The healthy mice were then injected with a therapeutic dose of MSCsome.The main organ construction and blood routine of the mice were analyzed.No obvious damage was shown in results.Both in vivo and in vitro studies have demonstrated the safety of MSCsome.In addition,to evaluate the safety of the treatment regimen,MP-MSCsome was systematically injected into ALI mice,and the changes in body weight,liver and kidney function were investigated 48 h later.The results showed that no adverse reaction was caused including weight gain,liver,and kidney impact.In conclusion,based on the inflammatory homing ability of MSC,this paper designed a hybridized liposome for inflammation targeting drug delivery.This membrane hybridized system not only retains the characteristics of high drug loading of liposome,but also reveals the high inflammatory targeting ability of MSC membrane.The lung-specific distribution of MP achieves superior inflammatory inhibition.No systemic adverse reactions were observed during the use.This research provides new insights into targeted anti-inflammatory drug delivery for ALI.