Nanosystems For The Delivery Of Biomacromolecular Drugs And Anti-Inflammation Therapy

Inflammation is a natural defense response of human body which plays a crucial role in protecting tissues from injury and infection.However,inflammation that does not subside in a timely manner can cause damage to host tissue and organ function and develops into inflammatory diseases,such as myocardial ischemia-reperfusion injury(MIRI),abdominal aortic aneurysm(AAA),inflammatory bowel disease,and rheumatoid arthritis,etc.Nowadays,in addition to the traditional small molecular anti-inflammatory drugs such as non-steroid anti-inflammatory drugs and glucocorticoids,emerging biomacromolecular drugs such as nucleic acid and protein offer new hope for the treatment of inflammatory diseases,as they have the advantages of high specific pharmacological activity,low toxicity,and low side effects.However,biomacromolecular drugs generally suffer from defects such as easy degradation in vivo,poor biofilm penetration,and poor targeting,which severely limit their therapeutic efficacy.In recent years,nanomaterial-based biomacromolecular drug delivery systems have developed rapidly,which effectively prolong the half-life of drugs in vivo,improve the biofilm penetration efficiency and improve the bioavailability of drugs.However,there are still many challenges for biomacromolecule nanodelivery systems.On one hand,multiple physiological barriers in vivo severely hinder the enrichment and penetration nanomedicines into the lesion tissues and further cellular uptake,and most of the existing biomacromolecular nanodelivery systems can hardly overcome multiple physiological barriers simultaneously.On the othe hand,the complex immunomodulatory mechanisms and pro-inflammatory factor networks of inflammatory diseases make single-target therapeutic strategies inadequate to achieve the expected therapeutic efficacy.In order to address these key scientific issues,this paper designs and prepares a variety of biomacromolecular drug nanodelivery systems to overcome multiple physiological barriers for in vivo delivery of biomacromolecular drugs and to coordinate the inflammatory pathological environment from multi-dimension,thereby alleviating excessive inflammatory responses and treating inflammatory diseases.Firstly,to overcome the cellular transefection barrier,a spherical α-helical cationic polypeptide with membrane-penetrating ability was designed to ensure efficient transmembrane delivery of small interference RNA(siRNA)into cardiomyocytes and modulate the inflammatory response after MIRI via intracardial injection.Then,in order to enhance circulation stability of the cationic gene carrie and apply it for systemic adimistration,a circulation stable,inflammation-targeting and reactive oxygen species(ROS)ultrasensitive drug/siRNA co-delivery system was designed to synergistically regulate the recruitment and infiltration of neutrophils after MIRI and to efficiently suppress the inflammatory response in myocardial tissue.Subsequently,to further enhance the penetration of siRNA vectors in the lesion tissue(inflammatory vascular tunica media),fluorinated cationic nanocapsules were designed for targeting delivery of siRNA to vascular smooth muscle cells(VSMCs)in AAA.Finally,considering the complex immunomodulatory machanisms and pro-inflammatory factors network in the inflammatory environment,biomimetic nanodecoys based on macrophage membranes were designed for inflammation-targeting delivery of membrane proteins such as pro-inflammatory factor receptors and co-stimulatory molecules(PD-L1)for the clearance of multiple pro-inflammatory factors and PD-1,which in turn modulates the inflammatory response and CD4+T-cell activity,enabling multi-targeted anti-inflammatory and immunomodulatory therapy for autoimmune diseases.The main research of this thesis is as follows.Chapter 1 provides an overview of inflammatory diseases,introducing therapeutic options for inflammatory diseases,physiological barriers that impede drug delivery,types,design,and functionalized modification strategies of nanodelivery carriers,and anti-inflammatory effects in vitro and in vivo.In order to achieve efficient intracellular delivery and lysosomal escape of siRNA,a spherical α-helical cationic polypeptide(DPP)with membrane penetration ability was constructed in Chapter 2 by ring-opening polymerization of y-(4alkynyloxybenzyl)-L-glutamic acid-N-carboxyanhydride and side chain guanidine modification using a third-generation dendrimer as initiator.Compared with linear αhelical polypeptide(LPP),DPP possessed stronger nucleic acid binding ability and membrane penetrating activity due to its multivalent structure and higher charge density.Subsequently,DPP formed stable nanocomplexes with E2F1 siRNA(siE2F1)by electrostatic adsorption,which significantly enhanced the uptake of siE2F1 by cardiomyocytes and efficientively escaped from endosome/lysosome,thus exerting efficient E2F1 silencing efficiency(72.8%),significantly higher than that of LPP(50.9%)and commercial transfection reagent 25k PEI(39.3%).In addition,downregulation of E2F1 expression significantly reduced intracellular ROS level in myocardial cells,which in turn inhibited cell apoptosis caused by oxidative stress.After intracardial injection of DPP/siE2F1 nanocomplexes in MIRI rats,E2F1 expression in myocardial tissues was effectively silenced and the silencing efficiency reached 67.4%,and the expression of both pro-inflammatory factors IL-6 and TNF-αwere significantly reduced.Meanwhile,the cardiac function of MIRI rats was effectively restored,myocardial tissue damage was significantly reduced,and the myocardial infarct area,cardiac fibrosis area,and myocardial apoptosis rate were decreased to 13.6%,12.3%and 12.8%,respectively.Due to the poor circulation stability,cationic carriers are easily cleared by reticuloendothelial cells,cannot be used for intravenous drug delivery,and suffer from lack of focal targeting.To address this problem,in Chapter 3,an endothelial cell-targeted and ROS-ultraresponsive cationic polymer(RPPT)was designed for delivery of VCAM-1 siRNA(siVCAM-1).Firstly,low-molecular-weight polyethyleneimine(PEI)was cross-linked via ditelluride bond and then was sequentially modified with polyethylene glycol(PEG)and cRGD to prepare the ROS responsive and inflammatory endothelial cell-targeting RPPT.RPPT was coated on the surface of dexamethasone(DXM)-loaded poly(lactic-co-glycolic acid)(PLGA)nanoparticles by electrostatic adsorption,followed by further condensation of siVCAM-1,thus constructing a nanocomplex co-loaded with siVCAM-1 and DXM.Due to ROS ultraresponsiveness of ditelluride moiety,RPPT could be degraded to low-molecular-weight fragments after treatment with low concentration of H2O2(100μM),thus promoting siRNA release.PEG can resist serum protein adsorption,while cRGD can bind to integrin αvβ3 overexpressed on the surface of inflammatory endothelial cells,so the nanocomplexes can prolong the blood circulation time and actively target the MIRI-damaged heart after intravenous injection.Upon entry into the inflamed endothelial cells,RPPT was degraded by intracellular ROS,promoting siVCAM-1 release and downregulating VCAM-1 expression.Thus,neutrophil adhesion to the vascular endothelium was effectively inhibited.Meanwhile,DXM inhibited the release of pro-inflammatory factor TNF-α from endothelial cells,which in turn reduced neutrophil migration to the injured heart.Ultimately,the synergistic effect of siVCAM-1 and DXM resulted in a significant decrease in the proportion of neutrophils in the injured myocardium(2.9%),which was lower than that in groups of single delivering siVCAM-1(11.3%)and DXM(4.4%).In addition,cardiac function in MIRI rats was almost restored after treatment with nanocomplexes.Myocardial infarct size,myocardial fibrosis,and myocardial apoptosis rates were decreased by 82.0%,75.1%and 72.7%,respectively.The dense extracellular matrix(ECM)is the major barrier to the penetration of nanomedicines into tissues.For example,abnormally deposited ECM severely hinders the penetration of nanomedicines in the vascular tunica media of AAA lesions,which in turn reduces the effective uptake of nanomedicines by VSMCs.To overcome this tissue penetration barrier,a reductionresponsive,lesion-vessel-targeted,small-sized,vascular tunica media-penetrated,and fluorinated polymer nanocapsule was designed in Chapter 4 for the encapsulation and systemic delivery of ALDH2 siRNA(siALDH2)to promote phenotypic conversion of VSMCs and modulate the inflammatory environment of the vascular wall for anti-inflammatory therapy of AAA.Firstly,siALDH2 was encapsulated by in situ radical polymerization of multiple monomers on the surface of siALDH2 and sequential modification with PEG and type Ⅳ collagen targeting peptides,which contributed to the formation of fluorinated nanocapsules(PFSsA NCs)with small size(～20 nm)and low positive surface charge(-12 mV).After intravenous injection,PEG helped the nanocapsules to avoid serum protein adsorption effectively,thus enabling long blood circulation.Subsequently,the nanocapsules actively identified exposed type Ⅳ collagen at the damaged vasculature via type Ⅳ collagen-targeting peptide and enriched to the AAA lesioned vasculature.The hydrophobic and lipophobic properties of fluorinated hydrocarbon chains allowed PFSsA NCs to effectively prevent the adhesion of multiple proteins and polysaccharides in the ECM,while the small size facilitated the diffusion of nanocapsules in the ECM,so that PFSsA NCs efficiently penetrated the vascular tunica media to a depth of 75 μm,whereas the unfluorinated nanocapsules(PNSsA NCs)could only stay on the inner surface of the vessel.Upon entering VSMCs,PFSsA NCs were dissociated by reduction and released siALDH2,which silences ALDH2 expression and upregulates a-SMA and SM22α expression through ALDH2-MAX-myocardin pathway,thus causing VSMCs switching to contractile phenotype.The expression of cell surface chemokine receptors was reduced on the cell surface of contractile-phenotyped VSMCs,thus leading to the reduction of inflammatory cells infiltration and ultimately reducing the inflammatory response and its resulting vascular tissue damage.In the CaCl2-induced AAA rat model,the vascular diameter growth rate in AAA rats treated with PFSsA NCs was 118.3%,which was significantly lower than that in PBS group(152.6%),PNSsA NCs group(135.2%),and FSsA NCs group(fluorinated and non-targeted,140.4%).In addition,medial elastic fiber damage,vessel wall calcification,and vessel wall integrity were all significantly improved.Since the inflammatory response involves complex immunomodulatory mechanisms and multiple pro-inflammatory factors,anti-inflammatory therapy targeting a single target is often not as effective as desired.To address these problems,in Chapter 5,biomimetic nanoparticles based on macrophage membrane were designed to deliver membrane proteins such PD-L1 and multiple proinflammatory factors receptors for multi-target disruption of inflammatory cascade response in autoimmune diseases.Firstly,RAW 264.7 cells were treated with IFN-y to obtain macrophage membrane(PRM)highly expressing PD-L1,pro-inflammatory factor receptors,and adhesion molecule receptors.Subsequently,the PRM was coated onto PLGA nanoparticles by sonication to construct nanodecoys(PRM NDs).The cell membrane coating helped PRM NDs to camouflage as endogenous cells,which contributes to effective evasion of RPM NDs from reticuloendothelial system capture after intravenous administration,resulting in long circulation in vivo(half-life up to 12.04 h).Adhesion molecule receptors can bind to intercellular adhesion molecules overexpressed on the surface of inflammatory cells,thus PRM NDs with high surface expression of adhesion molecule receptors can efficiently target inflammatory tissues.Subsequently,PRM NDs inhibited cell activation caused by pro-inflammatory factors by efficiently scavenging multiple free pro-inflammatory factors(TNF-α,IL-6,IL-1β,IFN-y).Furthermore,PRM NDs effectively suppressed CD4+T cell activation by scavenging free PD-1(sPD-1)or binding to PD-1 on the surface of CD4+T cells(mPD-1),thereby restoring immune tolerance.In the Zymosan A-induced arthritis mouse model,PRM NDs treatment caused a significant reduction in knee swelling,significantly reduced the proportion of pro-inflammatory CD4+T cells(Thl and Th17),inhibited bone loss,bone structure destruction,and inflammatory cell infiltration in the osteoarthritic joints,and ultimately effectively restored motor function in mice.In a mouse model of dextran sulfate induced ulcerative colitis,PRM NDs treatment effectively inhibited colonic shortening and weight loss,and maintained the integrity of intestinal epithelial tissue.More importantly,compared with TNF-α monoclonal antibody,PRM NDs showed superior anti-inflammatory and immunosuppressive efficacy in both of these two animal models,indicating that PRM NDs have promising applications in the treatment of inflammatory diseases.Chapter 6 summarized the work of this thesis and provided an outlook on future research directions.In summary,this thesis designed a series of biomacromolecular drug delivery systems to overcome multiple physiological barriers in vivo for improving the efficiency of nucleic acid and protein drug delivery,providing suggestions for design of biomacromolecular drug delivery carrier.Meanwhile,this study also explored the application of multi-target therapy in the inflammatory disease treatment by consideration of the complex immune regulatory mechanism and pro-inflammatory factor network in inflammatory diseases,which provides new strageties for inflammatory diseases therapy.