Temporal-spatial low shear stress induces heterogenous distribution of hematopoietic stem cell budding in zebrafish.

Hematopoietic stem/progenitor cells (HSPCs) originate from endothelial cells (ECs) localized on the ventral side of the dorsal aorta (DA), and hemodynamic parameters may suffer sharp changes in DA at HSPCs development stage for intersegmental vessel formation. However, the temporal-spatial shear stress parameters and biomechanics mechanisms of HSPC budding remain unknown. Here, we found that the hematopoietic endothelium (HE) in the aorta-gonad-mesonephros was heterogeneous; that is, HEs were mainly distributed at the ventral side of the vascular bifurcation in zebrafish embryos, which was found to show low shear stress (LSS) through numerical simulation analysis. Furthermore, HSPCs localized in the posterior somite of aorta-gonad-mesonephros with slow velocity. On the temporal scale, there was a slow velocity and LSS during HE budding from 36 h post-fertilization and decreased shear stress with drug expanded HSPC numbers. Mechanistically, matrix metalloproteinase (MMP) expression and macrophage chemotaxis were significantly increased in HEs by RNA-seq. After treatment with an MMP13 inhibitor, HSPCs were significantly reduced in both the aorta-gonad-mesonephros and caudal hematopoietic tissue in embryos. Our results show that HSPC budding is heterogeneous, and the mechanism is that physiological LSS controls the emergence of HSPCs by promoting the accumulation of macrophages and subsequent MMP expression.

Efficient and risk-reduced genome editing using double nicks enhanced by bacterial recombination factors in multiple species.

Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and 'cleaner' knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.

Functionalized nanoparticles with monocyte membranes and rapamycin achieve synergistic chemoimmunotherapy for reperfusion-induced injury in ischemic stroke.

BACKGROUND: Ischemic stroke is an acute and severe neurological disease, and reperfusion is an effective way to reverse brain damage after stroke. However, reperfusion causes secondary tissue damage induced by inflammatory responses, called ischemia/reperfusion (I/R) injury. Current therapeutic strategies that control inflammation to treat I/R are less than satisfactory. RESULTS: We report a kind of shield and sword nano-soldier functionalized nanoparticles (monocyte membranes-coated rapamycin nanoparticles, McM/RNPs) that can reduce inflammation and relieve I/R injury by blocking monocyte infiltration and inhibiting microglia proliferation. The fabricated McM/RNPs can actively target and bind to inflammatory endothelial cells, which inhibit the adhesion of monocytes to the endothelium, thus acting as a shield. Subsequently, McM/RNPs can penetrate the endothelium to reach the injury site, similar to a sword, and release the RAP drug to inhibit the proliferation of inflammatory cells. In a rat I/R injury model, McM/RNPs exhibited improved active homing to I/R injury areas and greatly ameliorated neuroscores and infarct volume. Importantly, in vivo animal studies revealed good safety for McM/RNPs treatment. CONCLUSION: The results demonstrated that the developed McM/RNPs may serve as an effective and safe nanovehicles for I/R injury therapy.

Coordinated histone modifications and chromatin reorganization in a single cell revealed by FRET biosensors.

The dramatic reorganization of chromatin during mitosis is perhaps one of the most fundamental of all cell processes. It remains unclear how epigenetic histone modifications, despite their crucial roles in regulating chromatin architectures, are dynamically coordinated with chromatin reorganization in controlling this process. We have developed and characterized biosensors with high sensitivity and specificity based on fluorescence resonance energy transfer (FRET). These biosensors were incorporated into nucleosomes to visualize histone H3 Lys-9 trimethylation (H3K9me3) and histone H3 Ser-10 phosphorylation (H3S10p) simultaneously in the same live cell. We observed an anticorrelated coupling in time between H3K9me3 and H3S10p in a single live cell during mitosis. A transient increase of H3S10p during mitosis is accompanied by a decrease of H3K9me3 that recovers before the restoration of H3S10p upon mitotic exit. We further showed that H3S10p is causatively critical for the decrease of H3K9me3 and the consequent reduction of heterochromatin structure, leading to the subsequent global chromatin reorganization and nuclear envelope dissolution as a cell enters mitosis. These results suggest a tight coupling of H3S10p and H3K9me3 dynamics in the regulation of heterochromatin dissolution before a global chromatin reorganization during mitosis.

SRGN, a new identified shear-stress-responsive gene in endothelial cells.

Endothelial cells (ECs) play an important role in the pathogenesis of cardiovascular disease, especially atherosclerosis (AS). The abnormal wall shear stress (WSS) which directly contacts with ECs is the key stimulating factor leading to AS. However, the underlying mechanism of ECs responding to WSS is still incompletely understood. This study aims to explore the novel mechano-sensitive genes and its potential mechanism in response to WSS in ECs by employing bioinformatics methods based on previously available high-throughput data from zebrafish embryos, both before and after blood flow formation. Six common differentially expressed genes (DEGs) (SRGN, SLC12A3, SLC25A4, PVALB1, ITGAE.2, zgc:198419) were selected out from two high-throughput datasets (GSE126617 and GSE20707) in the GEO database. Among them, SRGN was chosen for further verification through the in vitro shear stress loading experiments with human umbilical vein endothelial cells (HUVECs) and the in vivo partial ligation of carotid artery in mice. Our data indicated that low shear stress (LSS) could enhance the expression of SRGN via the PKA/CREB-dependent signaling pathway. The proportion of Ki67+ cells and the concentration of nitric oxide (NO) were high in SRGN high expression cells, suggesting that SRGN may be involved in the proliferation of HUVECs. Furthermore, in the partial ligation of the carotid artery mice model, we observed that the expression of SRGN was significantly increased in atherosclerotic plaques induced by abnormal shear stress. Taken together, this study demonstrated that SRGN is a key gene in the response of ECs to WSS and could be involved in AS.

Impairment of Cargo Transportation Caused by gbf1 Mutation Disrupts Vascular Integrity and Causes Hemorrhage in Zebrafish Embryos.

ADP-ribosylation factor GTPases are activated by guanine nucleotide exchange factors including Gbf1 (Golgi brefeldin A-resistant factor 1) and play important roles in regulating organelle structure and cargo-selective vesicle trafficking. However, the developmental role of Gbf1 in vertebrates remains elusive. In this study, we report the zebrafish mutant line tsu3994 that arises from N-ethyl-N-nitrosourea (ENU)-mediated mutagenesis and is characterized by prominent intracerebral and trunk hemorrhage. The mutant embryos develop hemorrhage accompanied by fewer pigments and shorter caudal fin at day 2 of development. The hemorrhage phenotype is caused by vascular breakage in a cell autonomous fashion. Positional cloning identifies a T → G nucleotide substitution in the 23rd exon of the gbf1 locus, resulting in a leucine → arginine substitution (L1246R) in the HDS2 domain. The mutant phenotype is mimicked by gbf1 knockouts and morphants, suggesting a nature of loss of function. Experimental results in mammalian cells show that the mutant form Gbf1(L1246R) is unable to be recruited to the Golgi apparatus and fails to activate Arf1 for recruiting COPI complex. The hemorrhage in tsu3994 mutants can be prevented partially and temporally by treating with the endoplasmic reticulum stress/apoptosis inhibitor tauroursodeoxycholic acid or by knocking down the proapoptotic gene baxb Therefore, endothelial endoplasmic reticulum stress and subsequent apoptosis induced by gbf1 deficiency may account for the vascular collapse and hemorrhage.

Novel and rapid osteoporosis model established in zebrafish using high iron stress.

Osteoporosis is a global public health concern and, it can result from numerous pathogenic mechanisms, many of which are closely related with age, nutritional disorders, endocrine imbalance, or adverse drug side effects presented by glucocorticoids, heparin, and anti-epileptics. Given its wide range etiologies, it is crucial to establish an animal model of osteoporosis for use in screening potential drugs quickly and effectively. Previous research has reported that an accumulation of elevated iron in the body is an independent risk factor for osteoporosis. As such, we sought to use both zebrafish larvae and adults to model an osteoporosis phenotype using high iron stress (FAC, ferric ammonium citrate). Skeletal staining results suggested that iron-overload caused a significant decrease in bone calcification as well as severe developmental cartilage defects. In addition, osteoblast and cartilage-specific mRNA expression levels were downregulated after exposure to a high-iron environment. Most importantly, we demonstrated in both larval and adult fish that high iron-induced osteogenic defects were significantly rescued using alendronate (AL), a drug known to be effective against to human osteoporosis. Even more, the repair effect of AL was achieved by facilitating osteoblast differentiation and targeting Bmp signaling. Taken together, our findings propose an rapid and effective osteoporosis model, which could be used widely for future osteoporosis drug screening.

Stress granules in atherosclerosis: Insights and therapeutic opportunities.

Atherosclerosis, a complex inflammatory and metabolic disorder, is the underlying cause of several life-threatening cardiovascular diseases. Stress granules (SG) are biomolecular condensates composed of proteins and mRNA that form in response to stress. Recent studies suggest a potential link between SG and atherosclerosis development. However, there remain gaps in understanding SG role in atherosclerosis development. Here we provide a thorough analysis of the role of SG in atherosclerosis, covering cellular stresses stimulation, core components, and regulatory genes in SG formation. Furthermore, we explore atherosclerosis induced factors such as inflammation, low or oscillatory shear stress (OSS), and oxidative stress (OS) may impact SG formation and then the development of atherosclerotic lesions. We have assessed how changes in SG dynamics impact pro-atherogenic processes like endothelial dysfunction, lipid metabolism, and immune cell recruitment in atherosclerosis. In summary, this review emphasizes the complex interplay between SG and atherosclerosis that could open innovative directions for targeted therapeutic strategies in preventing or treating atherosclerotic cardiovascular diseases.

The interplay between histone modifications and nuclear lamina in genome regulation.

Gene expression is regulated by chromatin architecture and epigenetic remodeling in cell homeostasis and pathologies. Histone modifications act as the key factors to modulate the chromatin accessibility. Different histone modifications are strongly associated with the localization of chromatin. Heterochromatin primarily localizes at the nuclear periphery, where it interacts with lamina proteins to suppress gene expression. In this review, we summarize the potential bridges that have regulatory functions of histone modifications in chromatin organization and transcriptional regulation at the nuclear periphery. We use lamina-associated domains (LADs) as examples to elucidate the biological roles of the interactions between histone modifications and nuclear lamina in cell differentiation and development. In the end, we highlight the technologies that are currently used to identify and visualize histone modifications and LADs, which could provide spatiotemporal information for understanding their regulatory functions in gene expression and discovering new targets for diseases.

Genetically Encoded Fluorescence Resonance Energy Transfer Biosensor for Live-Cell Visualization of Lamin A Phosphorylation at Serine 22.

Extensive phosphorylation at serine 22 (pSer22) on lamin A is the hallmark of cell mitosis, which contributes to the breakdown of nuclear envelope. In the interphase, pSer22 lamin A exists in low abundance and is involved in mechanotransduction, virus infection, and gene expression. Numerous evidences emerge to support lamin A regulation on cell function and fate by phosphorylation. However, live-cell imaging tools for visualizing the dynamics of pSer22 lamin A are yet to be established. Herein, we developed a novel lamin A phosphorylation sensor (LAPS) based on fluorescence resonance energy transfer (FRET) with high sensitivity and specificity. We observed the dynamic lamin A phosphorylation during the cell cycle progression in single living cells: the increase of pSer22 modification when cells entered the mitosis and recovered upon the mitosis exit. Our biosensor also showed the gradual reduction of pSer22 modification during cell adhesion and in response to hypotonic environment. By applying LAPS, we captured the propagation of pSer22 modification from inside to outside of the inner nuclear membrane, which further led to the breakdown of nuclear envelope. Meanwhile, we found the synchronous phosphorylation of pSer22 lamin A and H3S10ph at mitosis entry. Inhibition of Aurora B, the responsible kinase for H3S10ph, could shorten the mitotic period without obvious effect on the pSer22 modification level of lamin A. Thus, LAPS allows the spatiotemporal visualization of the lamin A pSer22, which will be useful for elucidating the molecular mechanisms underlying cell mitosis and mechanoresponsive processes.

Silk fibroin promotes H3K9me3 expression and chromatin reorganization to regulate endothelial cell proliferation.

Silk fibroin (SF), which is extensively utilized in tissue engineering and vascular grafts for enhancing vascular regeneration, has not been thoroughly investigated for its epigenetic effects on endothelial cells (EC). This study employed RNA sequencing analysis to evaluate the activation of histone modification regulatory genes in EC treated with SF. Subsequent investigations revealed elevated H3K9me3 levels in SF-treated EC, as evidenced by immunofluorescence and western blot analysis. The study utilized H2B-eGFP endothelial cells to demonstrate that SF treatment results in the accumulation of H2B-marked chromatin in the nuclear inner cavities of EC. Inhibition of H3K9me3 levels by a histone deacetylase inhibitor TSA decreased cell proliferation. Furthermore, the activation of the MAPK signaling pathway using chromium picolinate decreased the proliferative activity and H3K9me3 level in SF-treated EC. SF also appeared to enhance cell growth and proliferation by modulating the H3K9me3 level and reorganizing chromatin, particularly after oxidative stress induced by H2O2 treatment. In summary, these findings indicate that SF promotes EC proliferation by increasing the H3K9me3 level even under stress conditions.

Unravelling the antioxidant behaviour of self-assembly ß-Sheet in silk fibroin.

Local oxidative stress in diseases or injury severely hinders cell homeostasis and organ regeneration. Antioxidant therapy is an effective strategy for oxidative stress treatment. Biomaterials with good biocompatibility and reactive oxygen species (ROS) scavenging ability are good choices for antioxidant therapeutics. However, there are few natural biomaterials that are identified with both biocompatibility and strong antioxidant activity. Here, we show, for the first time, that silk fibroin (SF) is a strong antioxidant, which can eliminate ROS in both cells and zebrafish. We further demonstrate that the ß-sheet structures turn into a random coiled structure when SF is treated with hydrogen peroxide. The content of ß-sheet structures can be increased by heating, thus enhancing the antioxidation properties of SF. Therefore, SF can serve as a good antioxidant biomaterial for therapeutics, and its ß-sheet structure-based antioxidation mechanism provides a novel theoretical basis, which could be a new cue for more antioxidant biomaterial discovery and identification.

Coupled single-cell and bulk RNA-seq analysis reveals the engulfment role of endothelial cells in atherosclerosis.

The clearance of apoptotic cell debris, containing professional phagocytosis and non-professional phagocytosis, is essential for maintaining the homeostasis of healthy tissues. Here, we discovered that endothelial cells could engulf apoptotic cell debris in atherosclerotic plaque. Single-cell RNA sequencing (RNA-seq) has revealed a unique endothelial cell subpopulation in atherosclerosis, which was strongly associated with vascular injury-related pathways. Moreover, integrated analysis of three vascular injury-related RNA-seq datasets showed that the expression of scavenger receptor class B type 1 (SR-B1) was up-regulated and specifically enriched in the phagocytosis pathway under vascular injury circumstances. Single-cell RNA-seq and bulk RNA-seq indicate that SR-B1 was highly expressed in a unique endothelial cell subpopulation of mouse aorta and strongly associated with the reorganization of cellular adherent junctions and cytoskeleton which were necessary for phagocytosis. Furthermore, SR-B1 was strongly required for endothelial cells to engulf apoptotic cell debris in atherosclerotic plaque of both mouse and human aorta. Overall, this study demonstrated that apoptotic cell debris could be engulfed by endothelial cells through SR-B1 and associated with the reorganization of cellular adherent junctions and cytoskeleton.

Universal cell membrane camouflaged nano-prodrugs with right-side-out orientation adapting for positive pathological vascular remodeling in atherosclerosis.

A right-side-out orientated self-assembly of cell membrane-camouflaged nanotherapeutics is crucial for ensuring their biological functionality inherited from the source cells. In this study, a universal and spontaneous right-side-out coupling-driven ROS-responsive nanotherapeutic approach, based on the intrinsic affinity between phosphatidylserine (PS) on the inner leaflet and PS-targeted peptide modified nanoparticles, has been developed to target foam cells in atherosclerotic plaques. Considering the increased osteopontin (OPN) secretion from foam cells in plaques, a bioengineered cell membrane (OEM) with an overexpression of integrin α9ß1 is integrated with ROS-cleavable prodrugs, OEM-coated ETBNPs (OEM-ETBNPs), to enhance targeted drug delivery and on-demand drug release in the local lesion of atherosclerosis. Both in vitro and in vivo experimental results confirm that OEM-ETBNPs are able to inhibit cellular lipid uptake and simultaneously promote intracellular lipid efflux, regulating the positive cellular phenotypic conversion. This finding offers a versatile platform for the biomedical applications of universal cell membrane camouflaging biomimetic nanotechnology.

OxLDL stimulates Id1 nucleocytoplasmic shuttling in endothelial cell angiogenesis via PI3K pathway.

Angiogenesis plays remarkable roles in the development of atherosclerotic rupture plaques. However, its essential mechanism remains unclear. The purpose of the study was to investigate whether inhibitor of DNA binding-1 or inhibitor of differentiation 1 (Id1) promoted angiogenesis when exposed to oxidised low-density lipoprotein (oxLDL), and to determine the molecular mechanism involved. Using aortic ring assay and tube formation assay as a model system, a low concentration of oxLDL was found to induce angiogenic sprouting and capillary lumen formation of endothelial cell. But the Id1 expression was significantly upregulated by oxLDL at low and high concentrations. The Id1 was localised in the nuclei of the human umbilical vein endothelial cells in the control group and in the high-concentration oxLDL group. Id1 was translocated to the cytoplasm at low oxLDL concentrations. The nucleocytoplasmic shuttling at low oxLDL concentration was inhibited by treatment with the nuclear export inhibitor leptomycin B. Protein kinase A (PKA) inhibitor H89 promoted nuclear export of Id1, and phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 reduced the nuclear export of Id1. PI3K inhibition blocked oxLDL-induced angiogenesis. Low concentrations of oxLDL promoted angiogenic sprouting and capillary formation. And this process depends on nuclear export of Id1, which in turn is controlled by the PI3K pathway. This report presents a new link between oxLDL and Id1 localisation, and may provide a new insight into the interactions of ox-LDL and Id1 in the context of atherosclerosis.

Significance of rs1271572 in the estrogen receptor beta gene promoter and its correlation with breast cancer in a southwestern Chinese population.

BACKGROUND: To characterize single nucleotide polymorphisms (SNPs) within the promoter region of the estrogen receptor beta (ERß) gene and to analyze the association of ERß SNPs with susceptibility to breast cancer. Genotype frequencies of five SNPs (rs3020449, rs3020450, rs2987983, rs1271572 and rs1887994) in the promoter region of the ERß gene in 873 women with breast cancer, 645 women with fibroadenoma and 700 healthy women were determined using an allele-specific tetra-primer polymerase chain reaction (PCR). Kaplan-Meier survival analysis was performed to evaluate the association of selected rs1271572 with prognosis in breast cancer. Electrophoretic mobility-shift assays were conducted to explore the binding of SNP rs1271572 containing probes to transcriptional factor Ying Yang 1 (YY1). RESULTS: Women with the homozygous TT genotype of rs1271572 had a significantly higher risk in developing breast cancer. Breast cancer patients with the TT genotype of rs1271572 had lower five-year survival rates than those with other genotypes and were more likely to suffer brain metastases. The rs1271572 G→T SNP abrogated YY1 binding and reduced the transcription activity of the promoter 0 N in the ERß gene in vitro. CONCLUSIONS: TT genotype of rs1271572 is associated with increased risk for breast cancer in Chinese women and is associated with unfavored prognosis in Chinese breast cancer patients. TT genotype of rs1271572 inhibited expression of ERß gene by down regulating transcriptional activity of the promoter 0 N in the ERß gene. Our data revealed that the TT genotype of rs1271572 resulted in loss of the YY1 binding site and reduced the transcription activity of the promoter 0 N in the ERß gene.

[Effect of horizontal rotary culture on zebrafish vascular development].

With the development of space life science, a study on the influence of microgravity on organism has been an increasingly concerned topic. Lots of studies indicate that microgravity plays an important role in the early development of embryos. The vascular system as the first-function system of embryos provides an interesting topic for many researchers. However, those studies were mostly carried out in vitro by rotary cell culture system (RCCS), while few experiments were done in vivo. Using zebrafish as a model, this research investigated the effects of horizontal rotary culture on the vascular development in vivo. Zebrafish embryos at 24 hpf (hour post-fertilization) were selected and divided into two groups. One group was cultured by the shaker, and the other was cultured normally as the control. After 12 h, all the embryos were collected and detected. The phenotype of zebrafish was observed by stereo microscope. Then, the expression of vascular specific expression factor, flk1, flt4, and ephrinB2 was compared by RT-PCR, qPCR, and in situ hybridization, respectively. Cell apoptosis and proliferation in situ were observed using TUNEL assay and bromodeoxyuridine incorporation. The results demonstrated that horizontal rotary culture at 90 r/min decreased the hatching of embryos (10.3±0.41 vs. 0.0, P<0.05), accelerate the heart rate (223.5±2.32 vs. 185.0±3.23, P<0.05) and increased the content of melanin in zebrafish significantly. At the same time, we found some differences in the vascular system of zebrafish after horizontal rotary culture which caused a down regulation of flk1, flt4, and ephrinB2. On the other hand, horizontal rotary culture accelerated the apoptosis of cells in zebrafish, but showed no significance in proliferation. In conclusion, horizontal rotary culture has a significant influence on the vascular development in zebrafish.

Oscillatory shear stress-induced downregulation of TET1s injures vascular endothelial planar cell polarity by suppression of actin polymerization.

Vascular endothelial polarity induced by blood flow plays crucial roles in the development of atherosclerosis. Loss of endothelial polarity leads to an increase in permeability and leukocyte recruitment, which are crucial hallmarks of atherosclerotic initiation. Endothelial cells exhibit a morphological adaptation to hemodynamic shear stress and possesses planar cell polarity to the direction of blood flow. However, the mechanism of how hemodynamic shear stress regulates endothelial planar cell polarity has not been firmly established. Here, we found that TET1s, a short isoform of Tet methylcytosine dioxygenase 1, was a mediator in the regulation of the planar cell polarity in endothelial cells in response to hemodynamic shear stress. In the process, low expression of TET1s induced by oscillatory shear stress led to the endothelial planar polarity damage through inhibition of F-actin polymerization. TET1s can regulate demethylation level of the sFRP-1 promoter to alter the expression of sFRP-1, which affects the interaction of sFRP-1/Fzd4 and F-actin polymerization. Our study revealed the mechanism of how TET1s mediates endothelial planar cell polarity in response to hemodynamic shear stress and provides a new insight for the prevention of atherosclerosis.

Shear stress regulation of nanoparticle uptake in vascular endothelial cells.

Nanoparticles (NPs) hold tremendous targeting potential in cardiovascular disease and regenerative medicine, and exciting clinical applications are coming into light. Vascular endothelial cells (ECs) exposure to different magnitudes and patterns of shear stress (SS) generated by blood flow could engulf NPs in the blood. However, an unclear understanding of the role of SS on NP uptake is hindering the progress in improving the targeting of NP therapies. Here, the temporal and spatial distribution of SS in vascular ECs and the effect of different SS on NP uptake in ECs are highlighted. The mechanism of SS affecting NP uptake through regulating the cellular ROS level, endothelial glycocalyx and membrane fluidity is summarized, and the molecules containing clathrin and caveolin in the engulfment process are elucidated. SS targeting NPs are expected to overcome the current bottlenecks and change the field of targeting nanomedicine. This assessment on how SS affects the cell uptake of NPs and the marginalization of NPs in blood vessels could guide future research in cell biology and vascular targeting drugs.

Spontaneously Right-Side-Out-Orientated Coupling-Driven ROS-Sensitive Nanoparticles on Cell Membrane Inner Leaflet for Efficient Renovation in Vascular Endothelial Injury.

Biomimetic cell membrane camouflaged technology has drawn extensive attention as a feasible and efficient way to realize the biological functions of nanoparticles from the parent cells. As the burgeoning nanotherapeutic, the right-side-out orientation self-assembly and pathological dependent "on-demand" cargo release of cell membrane camouflaged nanocarriers remarkably limit further development for practical applications. In the present study, a spontaneously right-side-out-orientated coupling-driven ROS-sensitive nanotherapeutic has been constructed for target endothelial cells (ECs) repair through the synergistic effects of spontaneously right-side-out-orientated camouflaging. This condition results from the specific affinity between the intracellular domain of key transmembrane receptors band 3 on cell membrane inner leaflet and the corresponding P4.2 peptide-modified nanoparticles without the additional coextrusion. The "on-demand" cargo release results from the pathological ROS-cleavable prodrug. Particularly, the red blood cell camouflaged nanotherapeutics (RBC-LVTNPs) can enhance target drug delivery through low oscillatory shear stress (LSS) blood flow in the injured ECs lesion. Both in vitro and in vivo results collectively confirm that RBC-LVTNPs can restore the damaged ECs and function with the recovered vascular permeability and low inflammation microenvironment. The findings provide a powerful and universal approach for developing the biomimetic cell membrane camouflaged nanotechnology.