Construction Of Inhalable MRNA Nano-delivery System And Study In The Therapy Of Pulmonary Fibrosis

Idiopathic pulmonary fibrosis(IPF)is a progressive pulmonary interstitial disease.Its pathological features are characterized by the abnormal activation and proliferation of fibroblasts,which gradually mediate the excessive accumulation of lung interstitial matrix,excessive scarring of lung tissue,and progressively destroyed alveoli structure,eventually leading to impaired gas exchange,loss of lung function,respiratory failure,and death.The prognosis of the disease is poor,the mortality rate is high,and the median survival time of patients after diagnosis is only 3 to 5 years.Among the current clinical treatments for IPF,except for lung transplantation,which can reverse IPF,both drug therapy and non-drug therapy based on pulmonary rehabilitation training only slow down the progression of IPF,but lack the possibility of reversing the established fibrotic lesions.Lung transplantation has great therapeutic limitations due to the shortage of lung source and high cost.Therefore,a promising therapeutic strategy urgently needs to be developed to achieve the recovery of fibrotic lesions,the improvement of patient lung function and the effective prolongation of patient survival.Although the pathogenic factors and mechanisms of IPF are not fully understood,recent studies have shown that repeated exposure to traumatic stimuli can lead to abnormal function of alveolar epithelium,overreaction to wound healing,overactivation of myofibroblasts,and excessive deposition of extracellular matrix(ECM).Therefore,IPF is considered as a disease characterized by alveolar epithelial injury,type Ⅱ alveolar epithelial cell reduction or alveolar stem cell failure,and alveolar collapse.In recent years,among the domestic and foreign studies on the mitigation of IPF,cell therapy represented by mesenchymal stem cells and type Ⅱ alveolar epithelial cells,metabolic regulation therapy represented by metformin,antagonizing the whole process of action(synthesis,activation and binding to receptor)of the major pro-fibrotic factor TGF-β1,promoting the degradation of excessive ECM,anti-fibrotic cytokine therapy and hormone therapy were put forward to reduce the load of fibrosis,However,the main target is the progression of the disease rather than the recovery of the lesion in some studies,or the treatment drugs are not highly targeted,and the side effects are non-negligible.Considering that the main pathological features of IPF that causes the patient’s respiratory failure are the excessive accumulation of ECM and the invaded or destroyed alveoli.Combination therapy that promotes the degradation of scarred ECM to restore alveoli and their gas-exchange spaces between with blood vessels,and promotes alveolar epithelial remodeling could theoretically mediate the recovery of fibrotic lesions and improve the lung function of patients.Based on this,this study proposed a therapeutic mechanism for IPF--the combination of degradation and reconstruction.(1)Degradation of ECM:matrix metalloproteinases(MMPs)can degrade almost all kinds of protein components in ECM,but studies have shown that some MMP can promote fibrosis.For example,MMP2 is highly expressed in fibrotic lesions,which can destroy the alveolar epithelial basement membrane and contribute to scar invasion into the alveoli.MMP13 is a collagenase that mainly degrades type Ⅰ,Ⅱ and Ⅲ collagen without damaging the alveolar epithelial basement membrane,and has been proved to have the anti-fibrotic effect.Therefore,MMP 13 is selected as the delivery choice.In our work,MMP 13 is delivered in the form of mRNA,which has the advantages of safety and control compared with DNA drugs and protein drugs.However,several key issues must be considered in mRNA delivery:How to avoid the degradation of mRNA by nuclease?How to promote highly efficient uptake of mRNA by target cells?How to promote mRNA to escape endo/lysosome in cells and translate into target protein?In order to solve the above key issues,our study firstly proposed to use endogenous cationic ribosome protein as the carrier for condensing mRNA and subsequently cover the surface of mRNA-ribosome protein formulation with a polymer which can reverse charge and promote escape under the condition of pH5.5 to deliver MMP 13 mRNA.(2)Reconstruction of alveoli:studies have shown that a lot of lung distal epithelial stem cells are exhausted in IPF,and proliferative factors are needed to promote their regeneration.Keratinocyte growth factor(KGF),which specifically promotes the proliferation of epithelial cells but does not act on fibroblasts,has been shown to reduce or prevent bleomycin-induced pulmonary fibrosis in mice by overexpression of KGF or intratracheal infusion.Recombinant human KGF has been approved for the treatment of severe mucositis following chemotherapy.Therefore,KGF was selected in our study to reconstruct alveolar epithelium.However,KGF also has a pro-proliferative effect on normal alveolar epithelium,and untargeted administration has certain side effects,and even has the risk of carcinogenicity.In order to reduce this potential side effect,KGF was designed to be released in response to the overexpressed MMP2 in fibrotic lesions,so that it was only effective at the lesions.Main researches and results of the study are as follows:1.Preparation and characterization of co-loaded KGF and MMP13 mRNA nanoformulations(mMMP13@RPL32/P-KGF)In this work,the poly-L-lysine-derived polymer modified on the outer layer of nanoformulation that can realize charge reversal under the pH conditions of endo/lysosome was first synthesized.The pre-screened optimal 60S ribosomal protein L32(RPL32)and mMMP13 were prepared into mMMP13@RPL32 composite at the optimal mass ratio(RPL32:mMMP13=8)by electrostatic interaction,and then the composite was modified with cis-aconitic acid and bifunctional peptide-grafted polyL-lysine-derived polymers(P).A certain amount of KGF was grafted onto the surface of mMMP13@RPL32/P nanocomposite,and an appropriate amount of mPEG was grafted to buffer the shear force in the process of atomization.Thus,final mMMP13@RPL32/P-KGF nanoformulations were prepared for inhalation therapy.The properties of mMMP13@RPL32/P-KGF nanoformulations were preliminarily characterized by Malvern zetasizer and transmission electron microscope.The results showed that the changes of morphology,particle size and potential of mMMP13@RPL32/P-KGF nanoformulations before and after atomization(before atomization:261.6±2.558 nm,-3.63±0.26 mV;after atomization:251.2±5.093 nm,3.23±0.46 mV)were both not significant,and showed obvious morphological changes after incubation with MMP2 for 24 h and at pH 5.5.In vitro KGF release experiment result showed that the cumulative release rate of KGF in the condition of 50 nM MMP2(67.2%)was higher than that in the condition of 25 nM MMP2(43.1%)in 24 h,indicating that KGF has a good release ability in response to MMP2.2.In vitro delivery and safety evaluation of atomized mMMP13@RPL32/PKGF nanoformulationsUsing human type Ⅱ alveolar epithelial cell(AEC2)as cell model,the in vitro safety and epithelial regeneration ability of atomized mMMP13@RPL32/P-KGF nanoformulation were investigated by cytotoxicity test and pro-proliferation test respectively.The results showed that the nanoformulation had no significant toxicity to AEC2 and had a significant proliferation-promoted effect on AEC2 after pre-incubation with MMP2,indicating that the nanoformulation had good safety and potential to promote epithelial regeneration in vitro.Using TGF-β1-induced myofibroblasts and A549 cells as cell models,the results of in vitro cell uptake experiments showed that the atomized nanoformulation group with MMP2 pre-treating significantly enhanced the intracellular uptake signal of mRNA compared with the group without MMP2 pretreating(P<0.0001).Moreover,the intracellular uptake signal of mRNA in the 6 h group was stronger than those in the 3 h or 1 h group,indicating that the constructed nanoformulation could be well uptaken into cells under the action of RGD-integrin.In vitro endo/lysosome escape experiment results showed that comparing with the mRNA signal of 1 h-administrated group,mRNA escape signal from endo/lysosome of 6 hadministrated group was significantly increased.Enhanced green fluorescent protein mRNA(mEGFP)was used as a model mRNA to study the in vitro delivery efficiency of the delivery system.Confocal microscopy and flow cytometry showed that the atomized nanoformulation could deliver the mRNA well after MMP2 pre-treating,and the delivery efficiency was about 65%.ELISA results showed that MMP13 protein could be translated by delivered mMMP13 in vitro and remained elevated within 48 h.3.In vivo efficacy and safety evaluation of mMMP13@RPL32/P-KGF nanoformulationsC57BL/6J male mice with IPF induced by intratracheal infusion of bleomycin were used as animal models for in vivo evaluation.In vivo distribution results,the mFluc-formulated nanoformulation showed signal only in the lungs of mice after inhalation.Hematoxylin and Eosin(H&E)tissue staining results in in vivo safety evaluation showed that the constructed nanodelivery system had no significant toxicity to the heart,liver,spleen,and kidney of mice with IPF.In pharmacodynamic evaluation,tissue staining(H&E staining,masson staining,sirius red staining and α-SMA staining),hydroxyproline content and western blot results showed that mMMP13@RPL32/PKGF nanoformulation could significantly alleviate pulmonary fibrosis in mice.The markers of type I alveolar epithelial cells--aquaporin 5(AQP5)and type Ⅱ alveolar epithelial cells--surfactant protein C(SP-C)were severally stained by immunofluorescence staining.The results showed that mMMP13@RPL32/P-KGF nanoformulation could protect or regenerate the alveolar epithelium.In the pulmonary function test,the results of each indicator showed that the mMMP13@RPL32/P-KGF nanoformulation could protect or improve the lung function of mice with IPF induced by bleomycin.In the statistical experiment on the survival time of model mice in each group,comparing with the PBS-treated group,the mMMP13@RPL32/P-KGF nanoformulation group could significantly prolong the survival time of mice.In conclusion,based on the synergistic treatment idea of ablation and remodeling,this project proposed a combined delivery strategy of mMMP13 and KGF.In this study,a nano-delivery system co-loading with mMMP 13 and KGF was prepared by double functional modification using recombinant ribosomal protein as a carrier.The system has excellent biocompatibility and drug delivery capability.After aerosol inhalation,the nanoformulation carried by the droplets is deposited in the alveoli,and the outer layer of KGF is responsively released in fibrotic foci,which promotes the proliferation of alveolar epithelial cells.The nanocomposite of mMMP13 and recombinant ribosomal protein is uptaken by cells,and in situ production of MMP13 accelerates the degradation of extracellular matrix in the alveolar space which promotes the repair of fibrotic lesions.Small animal experiments show that the system can reduce the level of fibrosis in mouse lung tissue,synergistically restore alveolar integrity,and improve lung function in a bleomycin-induced IPF mouse model.