SGLT2 inhibitor empagliflozin promotes revascularization in diabetic mouse hindlimb ischemia by inhibiting ferroptosis.

Gliflozins are known as SGLT2 inhibitors, which are used to treat diabetic patients by inhibiting glucose reabsorption in kidney proximal tubules. Recent studies show that gliflozins may exert other effects independent of SGLT2 pathways. In this study we investigated their effects on skeletal muscle cell viability and paracrine function, which were crucial for promoting revascularization in diabetic hindlimb ischemia (HLI). We showed that treatment with empagliflozin (0.1-40 µM) dose-dependently increased high glucose (25 mM)-impaired viability of skeletal muscle C2C12 cells. Canagliflozin, dapagliflozin, ertugliflozin, ipragliflozin and tofogliflozin exerted similar protective effects on skeletal muscle cells cultured under the hyperglycemic condition. Transcriptomic analysis revealed an enrichment of pathways related to ferroptosis in empagliflozin-treated C2C12 cells. We further demonstrated that empagliflozin and other gliflozins (10 µM) restored GPX4 expression in high glucose-treated C2C12 cells, thereby suppressing ferroptosis and promoting cell viability. Empagliflozin (10 µM) also markedly enhanced the proliferation and migration of blood vessel-forming cells by promoting paracrine function of skeletal muscle C2C12 cells. In diabetic HLI mice, injection of empagliflozin into the gastrocnemius muscle of the left hindlimb (10 mg/kg, every 3 days for 21 days) significantly enhanced revascularization and blood perfusion recovery. Collectively, these results reveal a novel effect of empagliflozin, a clinical hypoglycemic gliflozin drug, in inhibiting ferroptosis and enhancing skeletal muscle cell survival and paracrine function under hyperglycemic condition via restoring the expression of GPX4. This study highlights the potential of intramuscular injection of empagliflozin for treating diabetic HLI.

p52-ZER6: a determinant of tumor cell sensitivity to MDM2-p53 binding inhibitors.

Targeting MDM2-p53 interaction has emerged as a promising antitumor therapeutic strategy. Several MDM2-p53 inhibitors have advanced into clinical trials, but results are not favorable. The lack of appropriate biomarkers for selecting patients has been assumed as the critical reason for this failure. We previously identified ZER6 isoform p52-ZER6 as an oncogene upregulated in tumor tissues. In this study we investigated whether p52-ZER6 acted as a blocker of MDM2-p53 binding inhibitors, and whether p52-ZER6 could be used as a biomarker of MDM2-p53 binding inhibitors. In p53 wild-type colorectal carcinoma HCT116, hepatocarcinoma HepG2 and breast cancer MCF-7 cells, overexpression of p52-ZER6 enhanced MDM2-p53 binding and promoted p53 ubiquitination/proteasomal degradation. Furthermore, overexpression of p52-ZER6 in the tumor cells dose-dependently reduced their sensitivity to both nutlin and non-nutlin class MDM2-p53 binding inhibitors. We showed that p52-ZER6 restored tumor cell viability, which was suppressed by nutlin-3, through restoring their proliferation potential while suppressing their apoptotic rate, suggesting that MDM2-p53 binding inhibitors might not be effective for patients with high p52-ZER6 levels. We found that nutlin-3 treatment or p52-ZER6 knockdown alone promoted the accumulation of p53 protein in the tumor cells, and their combinatorial treatment significantly increased the accumulation of p53 protein. In HCT116 cell xenograft nude mouse model, administration of shp52-ZER6 combined with an MDM2-p53 binding inhibitor nutlin-3 exerted synergistic antitumor response. In conclusion, this study reveals that p52-ZER6 might be a potential biomarker for determining patients appropriate for MDM2-p53 binding inhibition-based antitumor therapy, and demonstrates the potential of combinatorial therapy using MDM2-p53 binding inhibitors and p52-ZER6 inhibition.

Unspliced XBP1 contributes to cholesterol biosynthesis and tumorigenesis by stabilizing SREBP2 in hepatocellular carcinoma.

Cholesterol biosynthesis plays a critical role in rapidly proliferating tumor cells. X-box binding protein 1 (XBP1), which was first characterized as a basic leucine zipper-type transcription factor, exists in an unspliced (XBP1-u) and spliced (XBP1-s) form. Recent studies showed that unspliced XBP1 (XBP1-u) has unique biological functions independent from XBP1-s and could promote tumorigenesis; however, whether it is involved in tumor metabolic reprogramming remains unknown. Herein, we found that XBP1-u promotes tumor growth by enhancing cholesterol biosynthesis in hepatocellular carcinoma (HCC) cells. Specifically, XBP1-u colocalizes with sterol regulatory element-binding protein 2 (SREBP2) and inhibits its ubiquitination/proteasomal degradation. The ensuing stabilization of SREBP2 activates the transcription of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), a rate-limiting enzyme in cholesterol biosynthesis. We subsequently show that the XBP1-u/SREBP2/HMGCR axis is crucial for enhancing cholesterol biosynthesis and lipid accumulation as well as tumorigenesis in HCC cells. Taken together, these findings reveal a novel function of XBP1-u in promoting tumorigenesis through increased cholesterol biosynthesis in hepatocarcinoma cells. Hence, XBP1-u might be a potential target for anti-tumor therapeutic strategies that focus on cholesterol metabolism in HCC.

Development of aptamer-based inhibitors for CRISPR/Cas system.

The occurrence of accidental mutations or deletions caused by genome editing with CRISPR/Cas9 system remains a critical unsolved problem of the technology. Blocking excess or prolonged Cas9 activity in cells is considered as one means of solving this problem. Here, we report the development of an inhibitory DNA aptamer against Cas9 by means of in vitro selection (systematic evolution of ligands by exponential enrichment) and subsequent screening with an in vitro cleavage assay. The inhibitory aptamer could bind to Cas9 at low nanomolar affinity and partially form a duplex with CRISPR RNA, contributing to its inhibitory activity. We also demonstrated that improving the inhibitory aptamer with locked nucleic acids efficiently suppressed Cas9-directed genome editing in cells and reduced off-target genome editing. The findings presented here might enable the development of safer and controllable genome editing for biomedical research and gene therapy.

Enhanced affinity of racemic phosphorothioate DNA with transcription factor SATB1 arising from diastereomer-specific hydrogen bonds and hydrophobic contacts.

Phosphorothioate modification is commonly introduced into therapeutic oligonucleotides, typically as a racemic mixture in which either of the two non-bridging phosphate oxygens is replaced by sulfur, which frequently increases affinities with proteins. Here, we used isothermal titration calorimetry and X-ray crystallography to investigate the thermodynamic and structural properties of the interaction between the primary DNA-binding domain (CUTr1) of transcription factor SATB1 and dodecamer DNAs with racemic phosphorothioate modifications at the six sites known to contact CUTr1 directly. For both the modified and unmodified DNAs, the binding reactions were enthalpy-driven at a moderate salt concentration (50 mM NaCl), while being entropy-driven at higher salt concentrations with reduced affinities. The phosphorothioate modifications lowered this susceptibility to salt, resulting in a significantly enhanced affinity at a higher salt concentration (200 mM NaCl), although only some DNA molecular species remained interacting with CUTr1. This was explained by unequal populations of the two diastereomers in the crystal structure of the complex of CUTr1 and the phosphorothioate-modified DNA. The preferred diastereomer formed more hydrogen bonds with the oxygen atoms and/or more hydrophobic contacts with the sulfur atoms than the other, revealing the origins of the enhanced affinity.

The transcription factor PBX3 promotes tumor cell growth through transcriptional suppression of the tumor suppressor p53.

Pre-B-cell leukemia transcription factor 3 (PBX3) is a member of the PBX family and contains a highly conserved homologous domain. PBX3 is involved in the progression of gastric cancer, colorectal cancer, and prostate cancer; however, the detailed mechanism by which it promotes tumor growth remains to be elucidated. Here, we found that PBX3 silencing induces the expression of the cell cycle regulator p21, leading to an increase in colorectal cancer (CRC) cell apoptosis as well as suppression of proliferation and colony formation. Furthermore, we found that PBX3 is highly expressed in clinical CRC patients, in whom p21 expression is aberrantly low. We found that the regulation of p21 transcription by PBX3 occurs through the upstream regulator of p21, the tumor suppressor p53, as PBX3 binds to the p53 promoter and suppresses its transcriptional activity. Finally, we revealed that PBX3 regulates tumor growth through regulation of the p53/p21 axis. Taken together, our results not only describe a novel mechanism regarding PBX3-mediated regulation of tumor growth but also provide new insights into the regulatory mechanism of the tumor suppressor p53.

Comparative Glycomic Analysis of Exosome Subpopulations Derived from Pancreatic Cancer Cell Lines.

Extracellular vesicles such as exosomes are generally covered with an array of glycans, which are controlled by the host-cell glyco-synthetic machinery, similar to secreted and membrane glycoproteins. Several exosome subpopulations classified by their tetraspanin expression have been investigated in the context of diseases. However, a comparative analysis of their glycomics has never been attempted. Herein, we report a method for the comparative glycomic analysis of exosome subpopulations among pancreatic cancer cell lines. Glycomic profiles were obtained for extracellular vesicles, secreted glycoproteins, and membrane glycoproteins from eight cell lines. Statistical analyses revealed high populations of PHA-L-binding proteins in the vesicles. The surfaces of extracellular vesicles were labeled with Cy3 and captured by magnetic beads with antibodies against tetraspanins (CD9, CD63, and CD81). The coprecipitated vesicles were lysed and subjected to a lectin microarray analysis. A hierarchical clustering analysis using 19 glycomic profiles confirmed that most subpopulations, except CD81-positive exosomes, could be distinguished according to the host-cell species. Principal component analysis and subsequent lectin-affinity capturing of intact exosomes highlighted that CD81-positive exosomes preferentially expressed not PHA-L- but LEL-binding proteins on their surfaces. These data suggested that exosomal glycomics depended on the host-cell type and subpopulation.

Neurogenic differentiation factor 1 promotes colorectal cancer cell proliferation and tumorigenesis by suppressing the p53/p21 axis.

Neurogenic differentiation factor 1 (NeuroD1) is a transcription factor critical for promoting neuronal differentiation and maturation. NeuroD1 is involved in neuroblastoma and medulloblastoma; however, its molecular mechanism in promoting tumorigenesis remains unclear. Furthermore, the role of NeuroD1 in non-neural malignancies has not been widely characterized. Here, we found that NeuroD1 is highly expressed in colorectal cancer. NeuroD1-silencing induces the expression of p21, a master regulator of the cell cycle, leading to G2 -M phase arrest and suppression of colorectal cancer cell proliferation as well as colony formation potential. Moreover, NeuroD1-mediated regulation of p21 expression occurs in a p53-dependent manner. Through chromatin immunoprecipitation and point mutation analysis in the predicted NeuroD1 binding site of the p53 promoter, we found that NeuroD1 directly binds to the p53 promoter and suppresses its transcription, resulting in increased p53 expression in NeuroD1-silenced colorectal cancer cells. Finally, xenograft experiments demonstrated that NeuroD1-silencing suppresses colorectal cancer cell tumorigenesis potential by modulating p53 expression. These findings reveal NeuroD1 as a novel regulator of the p53/p21 axis, underscoring its importance in promoting non-neural malignancies. Furthermore, this study provides insight into the transcriptional regulation of p53.

Incorporation, intracellular trafficking and processing of extracellular heparanase by mast cells: Involvement of syndecan-4-dependent pathway.

We investigated the fate of proheparanase added to the culture media of mast cells. A recombinant protein mimicking proheparanase was continuously internalized into mastocytoma cells as well as bone marrow- and peritoneal cell-derived mast cells. Internalized heparanase molecules were accumulated in granules and a significant portion was released by stimulation with ionomycin, indicating that the internalized heparanase was sorted into secretory granules. The pro-form heparanase was processed into a mature and an active form inside the cells, in which intracellular heparin was fragmented by the mature enzyme. The internalization was substantially inhibited by addition of heparin and heparan sulfate to the culture medium, suggesting that glycosaminoglycan is involved in the uptake pathway. Out of four syndecans, expression of syndecan-3 and syndecan-4, especially cell surface syndecan-4, was detected in the mastocytoma cells. Two knockdown clones transfected with a shRNA expression vector targeting the syndecan-4 gene took up significantly lower amounts of heparanase than mock cells. We propose that some exogenous substances like proheparanase can be incorporated into mast cell granules via a glycosaminoglycan-mediated, especially syndecan-4-dependent, uptake pathway.

Analysis of ATP and AMP binding to a DNA aptamer and its imidazole-tethered derivatives by surface plasmon resonance.

Imidazole was tethered to the C5 position of thymine in an ATP-binding DNA aptamer with two types of linkers, and the affinities of each aptamer for ATP and AMP were determined by surface plasmon resonance measurements. The imidazole-tethered aptamers exhibited higher affinity for ATP, almost independently of the linker structure or the modification site.

Synergistic cooperation of MDM2 and E2F1 contributes to TAp73 transcriptional activity.

TAp73, a structural homologue of p53, plays an important role in tumorigenesis. E2F1 had been reported as a transcriptional regulator of TAp73, however, the detailed mechanism remains to be elucidated. Here we reported that MDM2-silencing reduced the activities of the TAp73 promoters and the endogenous TAp73 expression level significantly; while MDM2 overexpression upregulated them. We further revealed that the regulation of TAp73 transcriptional activity occurs as a synergistic effect of MDM2 and E2F1, most probably through their physical interaction in the nuclei. Furthermore, we also suggested that MDM2 might be involved in DNA damage-induced TAp73 transcriptional activity. Finally, we elucidated that MDM2-silencing reduced the proliferation rate of colon carcinoma cells regardless of the p53 status. Our data show a synergistic effect of MDM2 and E2F1 on TAp73 transcriptional activity, suggesting a novel regulation pathway of TAp73.

YY2/BUB3 Axis promotes SAC Hyperactivation and Inhibits Colorectal Cancer Progression via Regulating Chromosomal Instability.

Spindle assembly checkpoint (SAC) is a crucial safeguard mechanism of mitosis fidelity that ensures equal division of duplicated chromosomes to the two progeny cells. Impaired SAC can lead to chromosomal instability (CIN), a well-recognized hallmark of cancer that facilitates tumor progression; paradoxically, high CIN levels are associated with better therapeutic response and prognosis. However, the mechanism by which CIN determines tumor cell survival and therapeutic response remains poorly understood. Here, using a cross-omics approach, YY2 is identified as a mitotic regulator that promotes SAC activity by activating the transcription of budding uninhibited by benzimidazole 3 (BUB3), a component of SAC. While both conditions induce CIN, a defect in YY2/SAC activity enhances mitosis and tumor growth. Meanwhile, hyperactivation of SAC mediated by YY2/BUB3 triggers a delay in mitosis and suppresses growth. Furthermore, it is revealed that YY2/BUB3-mediated excessive CIN causes higher cell death rates and drug sensitivity, whereas residual tumor cells that survived DNA damage-based therapy have moderate CIN and increased drug resistance. These results provide insights into the role of SAC activity and CIN levels in influencing tumor cell survival and drug response, as well as suggest a novel anti-tumor therapeutic strategy that combines SAC activity modulators and DNA-damage agents.

Correction: Histone Demethylase JMJD2B Functions as a Co-Factor of Estrogen Receptor in Breast Cancer Proliferation and Mammary Gland Development.

[This corrects the article DOI: 10.1371/journal.pone.0017830.].

p52-ZER6/IGF1R axis maintains cancer stem cell population to promote cancer progression by enhancing pro-survival mitophagy.

Cancer stem cells (CSCs), which are distinct subpopulations of tumor cells, have a substantially higher tumor-initiating capacity and are closely related to poor clinical outcomes. Damage to organelles can trigger CSC pool exhaustion; however, the underlying mechanisms are poorly understood. ZER6 is a zinc-finger protein with two isoforms possessing different amino termini: p52-ZER6 and p71-ZER6. Since their discovery, almost no study reported on their biological and pathological functions. Herein, we found that p52-ZER6 was crucial for CSC population maintenance; p52-ZER6-knocking down almost abolished the tumor initiation capability. Through transcriptomic analyses together with in vitro and in vivo studies, we identified insulin like growth factor 1 receptor (IGF1R) as the transcriptional target of p52-ZER6 that mediated p52-ZER6 regulation of CSC by promoting pro-survival mitophagy. Moreover, this regulation of mitophagy-mediated CSC population maintenance is specific to p52-ZER6, as p71-ZER6 failed to exert the same effect, most possibly due to the presence of the HUB1 domain at its N-terminus. These results provide a new perspective on the regulatory pathway of pro-survival mitophagy in tumor cells and the molecular mechanism underlying p52-ZER6 oncogenic activity, suggesting that targeting p52-ZER6/IGF1R axis to induce CSC pool exhaustion may be a promising anti-tumor therapeutic strategy.

Enhanced cancer immunotherapy using STAT3-depleted dendritic cells with high Th1-inducing ability and resistance to cancer cell-derived inhibitory factors.

STAT3 signaling constitutes an important negative feedback mechanism for the maintenance of immune homeostasis, a suppressive signal for the Th1 immune response in murine macrophages, and a cancer immune evasion signal in various immune cells. The strategy for STAT3 signal inhibition should be considered, because these features could impede effective cancer immunotherapy. We have evaluated the effects of STAT3 inactivation in dendritic cells (DCs) on immune responses in mice and humans. DCs derived from LysMcre/STAT3(flox/flox) mice displayed higher cytokine production in response to TLR stimulation, activated T cells more efficiently, and were more resistant to the suppression of cytokine production by cancer-derived immunosuppressive factors compared with DCs from control littermates. Antitumor activities of STAT3-depleted and control DCs were compared by intratumoral administration of gp70 Ag peptide-pulsed DCs in the therapeutic MC38 tumor model. Intratumoral administration of STAT3-depleted DCs significantly inhibited MC38 tumor growth of both injected and nontreated remote tumors. The inhibition was accompanied by an increase in gp70-specific T cell response as well as in systemic Th1 immune response. STAT3-depleted human DCs with adenoviral STAT3 short hairpin RNA were also capable of producing more cytokines with TLR stimulation and more resistant to cancer-derived factors, and they induced tumor Ag-specific T cells more efficiently than control DCs. The identified role of DC STAT3 signaling in both in vivo therapeutic tumor models in mice and in vitro-specific T cell induction in humans indicates that STAT3-inactivated DCs may be a promising approach for cancer immunotherapy.

DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response.

Central to innate immunity is the sensing of pathogen-associated molecular patterns by cytosolic and membrane-associated receptors. In particular, DNA is a potent activator of immune responses during infection or tissue damage, and evidence indicates that, in addition to the membrane-associated Toll-like receptor 9, an unidentified cytosolic DNA sensor(s) can activate type I interferon (IFN) and other immune responses. Here we report on a candidate DNA sensor, previously named DLM-1 (also called Z-DNA binding protein 1 (ZBP1)), for which biological function had remained unknown; we now propose the alternative name DAI (DNA-dependent activator of IFN-regulatory factors). The artificial expression of otherwise IFN-inducible DAI (DLM-1/ZBP1) in mouse fibroblasts selectively enhances the DNA-mediated induction of type I IFN and other genes involved in innate immunity. On the other hand, RNA interference of messenger RNA for DAI (DLM-1/ZBP1) in cells inhibits this gene induction programme upon stimulation by DNA from various sources. Moreover, DAI (DLM-1/ZBP1) binds to double-stranded DNA and, by doing so, enhances its association with the IRF3 transcription factor and the TBK1 serine/threonine kinase. These observations underscore an integral role of DAI (DLM-1/ZBP1) in the DNA-mediated activation of innate immune responses, and may offer new insight into the signalling mechanisms underlying DNA-associated antimicrobial immunity and autoimmune disorders.

YY2-DRP1 Axis Regulates Mitochondrial Fission and Determines Cancer Stem Cell Asymmetric Division.

Cancer stem cells (CSCs) are associated with tumor progression, recurrence, and therapeutic resistance. To maintain their pool while promoting tumorigenesis, CSCs divide asymmetrically, producing a CSC and a highly proliferative, more differentiated transit-amplifying cell. Exhausting the CSC pool has been proposed as an effective antitumor strategy; however, the mechanism underlying CSC division remains poorly understood, thereby largely limiting its clinical application. Here, through cross-omics analysis, yin yang 2 (YY2) is identified as a novel negative regulator of CSC maintenance. It is shown that YY2 is downregulated in stem-like tumor spheres formed by hepatocarcinoma cells and in liver cancer, in which its expression is negatively correlated with disease progression and poor prognosis. Furthermore, it is revealed that YY2 overexpression suppressed liver CSC asymmetric division, leading to depletion of the CSC pool and decreased tumor-initiating capacity. Meanwhile, YY2 knock-out in stem-like tumor spheres caused enrichment in mitochondrial functions. Mechanistically, it is revealed that YY2 impaired mitochondrial fission, and consequently, liver CSC asymmetric division, by suppressing the transcription of dynamin-related protein 1. These results unravel a novel regulatory mechanism of mitochondrial dynamic-mediated CSCs asymmetric division and highlight the role of YY2 as a tumor suppressor and a therapeutic target in antitumor treatment.

YY2/PHGDH axis suppresses tumorigenesis by inhibiting tumor cell de novo serine biosynthesis.

Metabolic reprogramming is one of the key features of tumors facilitating their rapid proliferation and adaptation to harsh microenvironments. Yin Yang 2 (YY2) has recently been reported as a tumor suppressor downregulated in various types of tumors; however, the molecular mechanisms underlying its tumor-suppressive activity remain poorly understood. Furthermore, the involvement of YY2 in tumor cell metabolic reprogramming remains unclear. Herein, we aimed to elucidate the novel regulatory mechanism of YY2 in the suppression of tumorigenesis. Using transcriptomic analysis, we uncovered an unprecedented link between YY2 and tumor cell serine metabolism. YY2 alteration could negatively regulate the expression level of phosphoglycerate dehydrogenase (PHGDH), the first enzyme in the serine biosynthesis pathway, and consequently, tumor cell de novo serine biosynthesis. Mechanistically, we revealed that YY2 binds to the PHGDH promoter and suppresses its transcriptional activity. This, in turn, leads to decreased production of serine, nucleotides, and cellular reductants NADH and NADPH, which subsequently suppresses tumorigenic potential. These findings reveal a novel function of YY2 as a regulator of the serine metabolic pathway in tumor cells and provide new insights into its tumor suppressor activity. Furthermore, our findings suggest the potential of YY2 as a target for metabolic-based antitumor therapeutic strategies.

The p52-ZER6/G6PD axis alters aerobic glycolysis and promotes tumor progression by activating the pentose phosphate pathway.

Abnormal glucose metabolism is a highlight of tumor metabolic reprogramming and is closely related to the development of malignancies. p52-ZER6, a C2H2-type zinc finger protein, promotes cell proliferation and tumorigenesis. However, its role in the regulation of biological and pathological functions remains poorly understood. Here, we examined the role of p52-ZER6 in tumor cell metabolic reprogramming. Specifically, we demonstrated that p52-ZER6 promotes tumor glucose metabolic reprogramming by positively regulating the transcription of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway (PPP). By activating the PPP, p52-ZER6 was found to enhance the production of nucleotides and nicotinamide adenine dinucleotide phosphate, thereby providing tumor cells with the building blocks of ribonucleic acids and cellular reductants for reactive oxygen species scavenging, which subsequently promotes tumor cell proliferation and viability. Importantly, p52-ZER6 promoted PPP-mediated tumorigenesis in a p53-independent manner. Taken together, these findings reveal a novel role for p52-ZER6 in regulating G6PD transcription via a p53-independent process, ultimately resulting in tumor cell metabolic reprogramming and tumorigenesis. Our results suggest that p52-ZER6 is a potential target for the diagnosis and treatment of tumors and metabolic disorders.

Surface modulation of extracellular vesicles with cell-penetrating peptide-conjugated lipids for improvement of intracellular delivery to endothelial cells.

Exosomes (diameter 30-200 nm) are a subtype of extracellular vesicles secreted by cells containing DNA, microRNA (miRNA), and proteins. Exosomes are expected to be valuable as a means of delivering drugs or functional miRNAs in treatment of diseases. However, the delivery of exosomes is not sufficiently effective, even though exosomes have intrinsic delivery functions. Cell-penetrating peptides (CPPs) are short peptide families that facilitate cellular intake of molecules and vesicles. We previously reported that the modification of cells, and liposomes with CPP-conjugated-lipids, CPPs conjugated with poly (ethylene glycol)-conjugated phospholipids (PEG-lipid), that induce adhesion by CPPs, can be useful for cell-based assays and harvesting liposomes. In this study, we aimed to modulate the exosome surface using Tat peptide (YGRKKRRQRRR)-PEG-lipids to improve intracellular delivery to endothelial cells. We isolated and characterized exosomes from the medium of HEK 293 T cell cultures. Tat conjugated PEG-lipids with different spacer molecular weights and lipid types were incorporated into exosomes using fluorescein isothiocyanate labeling to optimize the number of Tat-PEG-lipids immobilized on the exosome surface. The exosomes modified with Tat-PEG-lipids were incubated with human umbilical vein endothelial cells (HUVECs) to study the interaction. Tat conjugated with 5 kDa PEG and C16 lipids incorporated on the exosome surface were highly detected inside HUVECs by flow cytometry. Fluorescence was negligible in HUVECs for control groups. Thus, Tat-PEG-lipids can be modified on the exosome surface, improving the intracellular delivery of exosomes.