hUC-MSCs therapy for Crohn's disease: efficacy in TNBS-induced colitis in rats and pilot clinical study.

BACKGROUND: The use of mesenchymal stem cells (MSCs) has recently emerged as a promising new therapeutic strategy for many diseases including perianal fistulizing Crohn's disease (CD). Whether hUC-MSCs can promote the healing of luminal ulcer in CD has not been studied so far. METHODS: The model of TNBS-induced colitis in rats was used to confirm the efficacy of hUC-MSCs in the treatment of CD. Then, seventeen CD patients refractory to or unsuitable for currently available therapies were enrolled and received once submucosal local injection through colonoscopy combined with once intravenous drip on the next day. All patients received a 24-week follow-up. Clinical and laboratory assessments were monitored at baseline, week 4, 8, 12, and 24. Endoscopic evaluations were conducted at baseline and week 12. Mucosal specimens were obtained at the margin of lesions by endoscopy biopsies and used for RNA sequencing. Two hUC-MSCs co-culture systems were established in vitro, one with the mucosa specimens and the other with M1 macrophages induced from THP1. The expressions of genes representing inflammation (TNFα, IL-6, and IL-1ß) and intestinal barrier function (ZO1, CLAUDIN1, and CDH1) were tested by RT-PCR. FINDINGS: hUC-MSCs treatment increased body weight and decreased disease activity index (DAI), colon macroscopic damage index (CMDI), and histopathological score (HPS) of rats with TNBS-induced colitis. The results of the clinical study also showed that this mode of hUC-MSCs application was associated with regression of intestinal ulceration. Eight patients (47%) got endoscopic responses (SES-CD improvement of ≥50% from baseline) and three patients (17.65%) got mucosal healing (SES-CD is zero), with a parallel improvement of clinical and laboratory parameters without serious adverse events. RNA sequencing showed hUC-MSCs therapy was associated with an upregulation of transcripts linked to intestinal epithelial barrier integrity and a downregulation of inflammatory signaling pathways in the intestinal mucosa, especially the TNF signaling pathway, IL-17 signaling pathway, and TLR signaling pathway. RNA expression of intestinal epithelial tight junction protein (ZO1, CLAUDIN1, and CDH1), and the RNA expression of major intestinal inflammatory factors in CD (IL-1ß, IL-6, and TNFα, p < 0.001 for all) were improved significantly. Moreover, hUC-MSCs could attenuate the polarization of M1 macrophage induced from THP1, thereby decreasing the mRNA expression of IL-1ß, IL-6, and TNFα significantly (p < 0.05 for all). TSG-6 expression was evaluated in hUC-MSCs culture supernatant after treatment with TNFα, IFNγ, and LPS for 48 h. And hUC-MSCs could inhibit the phosphorylation of JAK/STAT1 in the intestinal mucosa of CD patients. INTERPRETATION: hUC-MSCs transplantation alleviated TNBS-induced colitis in rats. In this pilot clinical study, preliminary data suggested that this approach to administering hUC-MSCs might have potential for clinical efficacy and manageable safety in treating refractory CD, potentially providing hope for better outcomes. No serious adverse events were observed. FUNDING: This work was funded by General Program of National Natural Science Foundation of China (Grant No. 82270639), the Scientific research project of Shanghai Municipal Health Committee (Grant No. 202240001), Specialty Feature Construction Project of Shanghai Pudong New Area Health Commission (Grant No. PWZzb2022-05), Shanghai East Hospital Youth Research and Cultivation Foundation program (Grant No. DFPY2022015), Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai, Technology Development Project of Pudong Science, Technology and Economic Commission of Shanghai (Grant No. PKJ2021-Y08), Key Disciplines Group Construction Project of Shanghai Pudong New Area Health Commission (Grant No. PWZxq2022-06), Medical discipline Construction Project of Pudong Health Committee of Shanghai (Grant No. PWYgf2021-02) and National Natural Science Foundation of China (Grant No. 82300604).

Peptide targeting improves the delivery and therapeutic index of glucocorticoids to treat rheumatoid arthritis.

Rheumatoid arthritis (RA) is a prevalent autoimmune disease characterized by excessive inflammation in the joints. Glucocorticoid drugs are used clinically to manage RA symptoms, while their dosage and duration need to be tightly controlled due to severe adverse effects. Using dexamethasone (DEX) as a model drug, we explored here whether peptide-guided delivery could increase the safety and therapeutic index of glucocorticoids for RA treatment. Using multiple murine RA models such as collagen-induced arthritis (CIA), we found that CRV, a macrophage-targeting peptide, can selectively home to the inflammatory synovium of RA joints upon intravenous injection. The expression of the CRV receptor, retinoid X receptor beta (RXRB), was also elevated in the inflammatory synovium, likely being the basis of CRV targeting. CRV-conjugated DEX increased the accumulation of DEX in the inflamed synovium but not in healthy organs of CIA mice. Therefore, CRV-DEX demonstrated a stronger efficacy to suppress synovial inflammation and alleviate cartilage/bone destruction. Meanwhile, CRV conjugation reduced immune-related adverse effects of DEX even after a long-term use. Last, we found that RXRB expression was significantly elevated in human patient samples, demonstrating the potential of clinical translation. Taken together, we provide a novel, peptide-targeted strategy to improve the therapeutic efficacy and safety of glucocorticoids for RA treatment.

Human umbilical cord mesenchymal stem cells attenuate diet-induced obesity and NASH-related fibrosis in mice.

Non-alcoholic steatohepatitis (NASH) is a progressive form of non-alcoholic fatty liver disease (NAFLD) that may progress to cirrhosis and hepatocellular carcinoma but has no available treatment. Mesenchymal stem cells (MSCs) have become increasingly prominent in cell therapy. Human umbilical cord MSCs (hUC-MSCs) are considered superior to other MSCs due to their strong immunomodulatory ability, ease of collection, low immune rejection, and no tumorigenicity. Though hUC-MSCs have received increasing attention in research, they have been rarely applied in any investigations or treatments of NASH and associated fibrosis. Therefore, this study evaluated the therapeutic efficacy of hUC-MSCs in C57BL/6 mice with diet-induced NASH. At week 32, mice were randomized into two groups: phosphate-buffered saline and MSCs, which were injected into the tail vein. At week 40, glucose metabolism was evaluated using glucose and insulin tolerance tests. NASH-related indicators were examined using various biological methods. hUC-MSC administration alleviated obesity, glucose metabolism, hepatic steatosis, inflammation, and fibrosis. Liver RNA-seq showed that the expression of the acyl-CoA thioesterase (ACOT) family members Acot1, Acot2, and Acot3 involved in fatty acid metabolism were altered. The cytochrome P450 (CYP) members Cyp4a10 and Cyp4a14, which are involved in the peroxisome proliferator-activator receptor (PPAR) signaling pathway, were significantly downregulated after hUC-MSC treatment. In conclusion, hUC-MSCs effectively reduced Western diet-induced obesity, NASH, and fibrosis in mice, partly by regulating lipid metabolism and the PPAR signaling pathway.

Cholesterol Depletion and Membrane Deformation by MeßCD and the Resultant Enhanced T Cell Killing.

Recent studies have demonstrated the crucial role of cholesterol (Chol) in regulating the mechanical properties and biological functions of cell membranes. Methyl-ß-cyclodextrin (MeßCD) is commonly utilized to modulate the Chol content in cell membranes, but there remains a lack of a comprehensive understanding. In this study, using a range of different techniques, we find that the optimal ratio of MeßCD to Chol for complete removal of Chol from a phosphocholine (PC)/Chol mixed membrane with a 1:1 mol ratio is 4.5:1, while the critical MeßCD-to-Chol ratio for membrane permeation falls within the range between 1.5 and 2.4. MeßCD at elevated concentrations induces the formation of fibrils or tubes from a PC membrane. Single lipid tracking reveals that removing Chol restores the diffusion of lipid molecules in the PC/Chol membrane to levels observed in pure PC membranes. Exposure to 5 mM MeßCD for 30 min effectively eliminates Chol from various cell lines, leading to an up to 8-fold enhancement in melittin cytotoxicity over Hela cells and an up to 3.5-fold augmentation of T cell cytotoxicity against B16F10-OVA cells. This study presents a diagram that delineates the concentration- and time-dependent distribution of MeßCD-induced Chol depletion and membrane deformation, which holds significant potential for modulating the mechanical properties of cellular membranes in prospective biomedical applications.

Asymmetric disturbance and permeabilization of bilayer membranes by 3-nm carbon dots.

Small-sized fluorescent carbon dots (CDs) are gaining increasing attention in the field of biomedical applications. The environmental and biological compatibility of positively charged CDs has been extensively investigated; however, the potential cytotoxicity caused by negatively and particularly neutrally charged small CDs has been significantly overlooked. In this study, we conducted a comprehensive investigation into the cellular membrane disruption effect of weakly negatively charged 3-nm CDs using a combination of various biophysical techniques. Our findings demonstrate that even at a low concentration of 0.5 µg mL-1, these CDs induce significant perturbations on the cellular membrane, resulting in increased membrane permeability due to asymmetric disruption of the bilayer structure. Furthermore, CDs exhibit distinct mechanisms at different concentrations, including prompt insertion into the bilayer at low concentrations (<20 µg mL-1) and a synergistic effect after a threshold time at high concentrations (e.g., 25-200 µg mL-1). Moreover, these CDs possess specific antibacterial properties against Acinetobacter baumannii (with a minimum inhibitory concentration of 50 µg mL-1) while showing minimal hemolytic or cytotoxic effects on mammalian cells. This study provides comprehensive insights into the biophysical aspects of cellular membrane toxicity caused by small weakly negatively charged CDs and contributes to assessing their potential biomedical applications.

Identification of an ALK-2 inhibitor as an agonist for intercellular exchange and tumor delivery of nanomaterial.

Inefficient extravasation and penetration in solid tissues hinder the clinical outcome of nanoparticles (NPs). Recent studies have shown that the extravasation and penetration of NPs in solid tumor was mostly achieved via an active transcellular route. For this transport process, numerous efforts have been devoted to elucidate the endocytosis and subcellular trafficking of NPs. However, how they exit from one cell and re-enter into neighboring ones (termed intercellular exchange) remains poorly understood. We previously developed cellular assays that exclusively quantify the intercellular exchange of NPs in vitro. Our study showed that a significant portion of NPs are transferred inside extracellular vesicles (EVs). Pharmacological inhibition of EV biogenesis significantly reduced the tumor accumulation and vascular penetration of both inorganic and organic NPs in vivo. Intrigued by this result, we performed here a manual chemical screen with our assay, which identified that LDN-214117 (an inhibitor for activin receptor-like kinase-2, ALK-2) is an agonist of NP intercellular exchange. We further showed that LDN-214117 regulates the intercellular exchange by increasing the EV biogenesis. Mechanistic investigation showed that LDN-214117 functions via BMP (bone morphogenetic protein)-MAPK (mitogen-activated protein kinase) signaling pathway to increase EV biogenesis. We further demonstrated that LDN-214117 treatment in vivo enhanced the tumor accumulation and vascular penetration of a variety of NPs in multiple tumor models, which improves their antitumor efficacy. Overall, we showcase here the identification of a novel chemical compound with our intercellular exchange assays to modulate EV biogenesis and EV-mediated transport, thus boosting up the delivery and therapeutic efficacy of nanomaterial.

Peptide-guided delivery improves the therapeutic efficacy and safety of glucocorticoid drugs for treating acute lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening conditions with excessive inflammation in the lung. Glucocorticoids had been widely used for ALI/ARDS, but their clinical benefit remains unclear. Here, we tackled the problem by conjugating prednisolone (PSL) with a targeting peptide termed CRV. Systemically administered CRV selectively homes to the inflamed lung of a murine ALI model, but not healthy organs or the lung of healthy mice. The expression of the CRV receptor, retinoid X receptor ß, was elevated in the lung of ALI mice and patients with interstitial lung diseases, which may be the basis of CRV targeting. We then covalently conjugated PSL and CRV with a reactive oxygen species (ROS)-responsive linker in the middle. While being intact in blood, the ROS linker was cleaved intracellularly to release PSL for action. In vitro, CRV-PSL showed an anti-inflammatory effect similar to that of PSL. In vivo, CRV conjugation increased the amount of PSL in the inflamed lung but reduced its accumulation in healthy organs. Accordingly, CRV-PSL significantly reduced lung injury and immune-related side effects elsewhere. Taken together, our peptide-based strategy for targeted delivery of glucocorticoids for ALI may have great potential for clinical translation.

Extracellular Vesicles Mediate the Intercellular Exchange of Nanoparticles.

To exert their therapeutic effects, nanoparticles (NPs) often need to travel into the tissues composed of multilayered cells. Accumulative evidence has revealed the crucial role of transcellular transport route (entry into one cell, exocytosis, and re-entry into another) in this process. While NP endocytosis and subcellular transport are intensively characterized, the exocytosis and re-entry steps are poorly understood, which becomes a barrier for NP delivery into complex tissues. Here, the authors term the exocytosis and re-entry steps together as intercellular exchange. A collagen-based three-dimension assay is developed to specifically quantify the intercellular exchange of NPs, and distinguish the contributions of several potential mechanisms. The authors show that NPs can be exocytosed freely or enclosed inside extracellular vesicles (EVs) for re-entry, while direct cell-cell contact is hardly involved. EVs account for a significant fraction of NP intercellular exchange, and its importance in NP transport is demonstrated in vitro and in vivo. While freely released NPs engage with the same receptors for re-entry, EV-enclosed ones bypass this dependence. These studies provide an easy and precise system to investigate the intercellular exchange stage of NP delivery, and shed the first light in the importance of EVs in NP transport between cells and into complex tissues.

Synergistic Entry of Individual Nanoparticles into Mammalian Cells Driven by Free Energy Decline and Regulated by Their Sizes.

Cell entry is one of the common prerequisites for nanomaterial applications. Despite extensive studies on a homogeneous group of nanoparticles (NPs), fewer studies have been performed when two or more types of NPs were coadministrated. We previously described a synergistic cell entry process for two heterogeneous groups of NPs, where NPs functionalized with TAT (transactivator of transcription) peptide (T-NPs) stimulate the cellular uptake of coadministered unfunctionalized NPs (bystander NPs, B-NPs). Here, we show that the synergistic cell entry of NPs is driven by free energy decline and depends on B-NP sizes. Simulations showed that when separately placed initially, two NPs first move toward each other instead of initiating cell entry individually. Only T-NP invokes an inward bending of membrane mimicking endocytosis, which attracts the nearby NPs into the same "vesicle". A two-phase free energy decline of the entire system occurred as two NPs get closer until contact, which is likely the thermodynamic driver for synergistic NP coentry. Experimentally, we found that T-NPs increase the apparent affinity of B-NPs to plasma membrane, suggesting that T-NPs help B-NPs "trapped" in the endocytic vesicles. Next, we varied the sizes of B-NPs and found that bystander activity peaks around 50 nm. Simulations also showed that the size of B-NPs influences the free energy decline, and thus the tendency and dynamics of NP coentry. These efforts provide a system to further understand the synergistic cell entry among individual NPs or multiple NP types on a biophysical basis and shed light on the future design of nanostructures for intracellular delivery.

iRGD-liposomes enhance tumor delivery and therapeutic efficacy of antisense oligonucleotide drugs against primary prostate cancer and bone metastasis.

Nucleotide-based drugs, such as antisense oligonucleotides (ASOs), have unique advantages in treating human diseases as they provide virtually unlimited ability to target any gene. However, their clinical translation faces many challenges, one of which is poor delivery to the target tissue in vivo. This problem is particularly evident in solid tumors. Here, we functionalized liposomes with a tumor-homing and -penetrating peptide, iRGD, as a carrier of an ASO against androgen receptor (AR) for prostate cancer treatment. The iRGD-liposomes exhibited a high loading efficiency of AR-ASO, and an efficient knockdown of AR gene products was achieved in vitro, including AR splice variants. In vivo, iRGD-liposomes significantly increased AR-ASO accumulation in the tumor tissue and decreased AR expression relative to free ASOs in prostate tumors established as subcutaneous xenografts. Similar results were obtained with intra-tibial xenografts modeling metastasis to bones, the predominant site of metastasis for prostate cancer. In treatment studies, iRGD-liposomes markedly improved the AR-ASO efficacy in suppressing the growth of both subcutaneous xenografts and intra-tibial xenografts. The inhibitory effect on tumor growth was also significantly prolonged by the delivery of the AR-ASO in the iRGD-liposomes. Meanwhile, iRGD-liposomes did not increase ASO accumulation or toxicity in healthy organs. Overall, we provide here a delivery system that can significantly increase ASO accumulation and efficacy in solid tumors. These benefits are achieved without significant side effects, providing a way to increase the antitumor efficacy of ASOs.

Membrane-curvature-mediated co-endocytosis of bystander and functional nanoparticles.

Efficient cellular uptake of nanoparticles (NPs) is necessary for the development of nanomedicine in biomedical applications. Recently, the coadministration of functionalized NPs (FNPs) was shown to stimulate the cellular uptake of nonfunctionalized NPs (termed bystander NPs, BNPs), which presents a new strategy to achieve synergistic delivery. However, a mechanistic understanding of the underlying mechanism is still lacking. In this work, the bystander uptake effect was investigated at the cell membrane level by combining the coarse-grained molecular dynamics, potential of mean force calculation and theoretical energy analysis methods. The membrane internalization efficiency of BNPs was enhanced by co-administered FNPs, and such activity depends on the affinity of both NPs to the membrane and the resultant membrane deformation. The membrane-curvature-mediated attraction and aggregation of NPs facilitated the membrane uptake of BNPs. Furthermore, quantitative suggestions were given to modulate the BNP internalization through controlling the FNP properties such as size, concentration and surface-ligand density. Our results provide insight into the molecular mechanism of the bystander uptake effect, and offer a practical guide to regulate the cellular internalization of NPs for targeted and efficient delivery to cells.

Transportan Peptide Stimulates the Nanomaterial Internalization into Mammalian Cells in the Bystander Manner through Macropinocytosis.

Covalent coupling with cell-penetrating peptides (CPPs) has been a common strategy to facilitate the cell entry of nanomaterial and other macromolecules. Though efficient, this strategy requires chemical modifications on nanomaterials, which is not always desired for their applications. Recent studies on a few cationic CPPs have revealed that they can stimulate the cellular uptake of nanoparticles (NPs) simply via co-administration (bystander manner), which bypasses the requirement of chemical modification. In this study, we investigated the other classes of CPPs and discovered that transportan (TP) peptide, an amphiphilic CPP, also exhibited such bystander activities. When simply co-administered, TP peptide enabled the cells to engulf a variety of NPs, as well as common solute tracers, while these payloads had little or no ability to enter the cells by themselves. This result was validated in vitro and ex vivo, and TP peptide showed no physical interaction with co-administered NPs (bystander cargo). We further explored the cell entry mechanism for TP peptide and its bystander cargo, and showed that it was mediated by a receptor-dependent macropinocytosis process. Together, our findings improve the understanding of TP-assisted cell entry, and open up a new avenue to apply this peptide for nanomaterial delivery.

Rapid chelator-free radiolabeling of quantum dots for in vivo imaging.

Most current nanoparticle-based PET tracers are radiolabeled through metal chelators conjugated on the nanoparticle surface. Metal chelation usually requires sophisticated optimization and may impact the physical or chemical properties of nanoparticles, which leads to the changes in their distribution and pharmacokinetics in vivo. A chelator-free radiolabeling approach is thus highly desirable. Here, we report that zinc sulfide (ZnS) quantum dots (QDs) can be rapidly radiolabeled with 68Ga or 64Cu through cation exchange without chelators. The radiolabeling was accomplished in times as short as 5 min at 37 °C in aqueous solution, yielding a high labeling efficiency and radiochemical purity for both isotopes. Surface functionalization with targeting peptides was also readily achieved to enable or enhance the cellular uptake of QDs. In vivo PET imaging showed that 64Cu-labeled QDs had a much higher tumor uptake (7.3% ID g-1) than 64Cu-DOTA in a murine cancer model. Overall, this study presents a QD-based platform to achieve convenient and chelator-free radiolabeling, and improve PET imaging of solid tumors.

Cellular internalization of bystander nanomaterial induced by TAT-nanoparticles and regulated by extracellular cysteine.

Entry into cells is necessary for many nanomaterial applications, and a common solution is to functionalize nanoparticles (NPs) with cell-penetrating ligands. Despite intensive studies on these functionalized NPs, little is known about their effect on cellular activities to engulf other cargo from the nearby environment. Here, we use NPs functionalized with TAT (transactivator of transcription) peptide (T-NPs) as an example to investigate their impact on cellular uptake of bystander cargo. We find that T-NP internalization enables cellular uptake of bystander NPs, but not common fluid markers, through a receptor-dependent macropinocytosis pathway. Moreover, the activity of this bystander uptake is stimulated by cysteine presence in the surrounding solution. The cargo selectivity and cysteine regulation are further demonstrated ex vivo and in vivo. These findings reveal another mechanism for NP entry into cells and open up an avenue of studying the interplay among endocytosis, amino acids, and nanomaterial delivery.

Tumor-specific macrophage targeting through recognition of retinoid X receptor beta.

Macrophages play important and diverse roles during cancer progression. However, cancer therapies based on macrophage modulation are lacking in tools that can recognize and deliver therapeutic payloads to macrophages in a tumor-specific manner. As a result, treatments tend to interfere with normal macrophage functions in healthy organs. We previously identified a macrophage-binding peptide, termed CRV. Here, we show that upon systemic administration into tumor-bearing mice, CRV selectively homes to tumors, extravasates, and preferentially binds to macrophages within. CRV exhibits a higher affinity for tumor macrophages than for other cells in tumors or for other macrophage types elsewhere in the body. We further identified and validated retinoid X receptor beta (RXRB) as the CRV receptor. Intriguingly, although it is known as a nuclear receptor, RXRB shows a prominent cell surface localization that is largely restricted to tumor macrophages. Systemic administration of anti-RXRB antibodies also results in tumor-selective binding to macrophages similar to CRV. Lastly, we demonstrate the ability of CRV to improve the delivery of nano-carriers into solid tumors and macrophages within. In summary, we describe here a novel cell surface marker and targeting tools for tumor macrophages that may aid in future development of macrophage-modulatory cancer therapies.

Fluorescent peptide highlights micronodules in murine hepatocellular carcinoma models and humans in vitro.

Early detection and clear delineation of microscopic lesions during surgery are critical to the prognosis and survival of patients with hepatocellular carcinoma (HCC), a devastating malignancy without effective treatments except for resection. Tools to specifically identify and differentiate micronodules from normal tissue in HCC patients can have a positive impact on survival. Here, we discovered a peptide that preferentially binds to HCC cells through phage display. Significant accumulation of the fluorescence-labeled peptide in tumor from ectopic and orthotopic HCC mice was observed within 2 hours of systemic injection. Contrast between tumor and surrounding liver is up to 6.5-fold, and useful contrast lasts for 30 hours. Micronodules (0.03 cm in diameter) in liver and lung can clearly be distinguished from normal tissue with this fluorescence-labeled peptide in orthotopic HCC mice and HCC patients. Compared to indocyanine green, a Food and Drug Administration-approved imaging contrast agent, an up to 8.7-fold higher differentiation ratio of tumor to fibrosis is achieved with this fluorescence-labeled peptide. Importantly, this peptide enables up to 10-fold differentiation between HCC and peritumoral tissue in human tissues and the complete removal of tumor in HCC mice with surgical navigation. No abnormalities in behavior or activity are observed after systemic treatment, indicating the absence of overt toxicity. The peptide is metabolized with a half-life of approximately 4 hours in serum. CONCLUSION: Our findings demonstrate that micronodules can be specifically differentiated with high sensitivity from surrounding tissue with this molecule, opening clinical possibilities for early detection and precise surgery of HCC. (Hepatology 2018).

Maintenance of cyclic GMP-AMP homeostasis by ENPP1 is involved in pseudorabies virus infection.

In a previous study, we demonstrated that porcine cyclic GMP-AMP (cGAMP) synthase (cGAS) catalyzes cGAMP production and is an important DNA sensor for the pseudorabies virus (PRV)-induced activation of interferon ß (IFN-ß). Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) has recently been identified as the hydrolase of cGAMP in rodents, but its role in porcine cells is not clear. Our recent study demonstrated that porcine ENPP1 is responsible for the homeostasis of cGAMP and is critical for PRV infection. Porcine ENPP1 mRNA is predominantly expressed in muscle. PRV infection was enhanced by ENPP1 overexpression and attenuated by silencing of ENPP1. During PRV infection, the activation of IFN-ß and NF-κB was reduced in ENPP1 overexpressed cells and promoted in ENPP1 knockdown cells. Investigation of the molecular mechanisms of ENPP1 during PRV infection showed that ENPP1 hydrolyzed cGAMP in PRV-infected or cGAMP-transfected cells and inhibited IRF3 phosphorylation, reducing IFN-ß secretion. These results, combined with those for porcine cGAS, demonstrate that ENPP1 acts coordinately with cGAS to maintain the reservoir of cGAMP and participates in PRV infection.

pH-responsive pHLIP (pH low insertion peptide) nanoclusters of superparamagnetic iron oxide nanoparticles as a tumor-selective MRI contrast agent.

Superparamagnetic iron oxide nanoparticles (SPION) are contrast agents used for noninvasive tumor magnetic resonance imaging (MRI). SPION with active targeting by tumor-specific ligands can effectively enhance the MRI sensitivity and specificity of tumors. However, the challenge remains when the tumor specific markers are yet to be determined, especially in the case of early tumor detection. In this study, the effectiveness of pH-responsive SPION via a pH low insertion peptide (pHLIP) to target tumor acidic microenvironments was investigated. Polylysine polymers were first successfully modified with pHLIP to have the pH-responsive capability. SPION pHLIP nanoclusters of 64, 82, 103, and 121nm size were then assembled by the pH-responsive polymers in a size-controlled manner. The pH-responsive SPION nanoclusters of the 64nm size exhibited the most effective pH-responsive retention in cells and tumor selective imaging in MRI. More importantly, the unique contrast enhancement of tumor inner core by the pH-responsive SPION in three different tumor models demonstrated the clinical potential to target tumor acidic microenvironment through pHLIP for tumor early detection and diagnosis by MRI. STATEMENT OF SIGNIFICANCE: Detection and diagnosis of tumors at early stage are critical for the improvement of the survival rate of cancer patients. However, the challenge remains when the tumor specific markers are yet to be determined, especially in early tumor detection. pH low insertion peptide (pHLIP) has been used as a specific ligand to target the tumor acidic microenvironment for tumors at early and metastatic stages. Superparamagnetic iron nanoparticles (SPION) are contrast enhancing agents used in the noninvasive magnetic resonance imaging for tumors. This research has demonstrated that pH-responsive pHLIP nanoclusters of SPION were able to target different tumors and facilitate the noninvasive diagnosis of tumors by MRI.

For Better or Worse, Iron Overload by Superparamagnetic Iron Oxide Nanoparticles as a MRI Contrast Agent for Chronic Liver Diseases.

Superparamagnetic iron oxide nanoparticles (SPIONs) have recently been used as an effective magnetic resonance imaging (MRI) contrast agent for the noninvasive diagnosis of chronic liver diseases including nonalcohol fatty liver diseases, nonalcohol steatohepatitis, and cirrhosis as well as liver tumors. However, the potential risk of the iron overload by SPIONs has been highly underestimated in chronic liver diseases. While most of SPIONs have been shown safe in the healthy group, significant toxicity potential by the iron overload has been revealed through immunotoxicity, lipid peroxidation, and fatty acid and cholesterol metabolism in cirrhosis as a high risk factor. As a result, the systems toxicology assessments of SPIONs are crucial in both healthy ones and chronic liver disease models to determine the margin of safety. In addition, the challenge of the iron overload by SPIONs requires better designed SPIONs as MRI contrast agents for chronic liver diseases such as the biodegradable nanocluster assembly with urine clearance.

Iron overload by Superparamagnetic Iron Oxide Nanoparticles is a High Risk Factor in Cirrhosis by a Systems Toxicology Assessment.

Superparamagnetic iron oxide nanoparticles (SPIONs) as a contrast agent have been widely used in magnetic resonance imaging for tumor diagnosis and theranostics. However, there has been safety concern of SPIONs with cirrhosis related to excess iron-induced oxidative stress. In this study, the impact of iron overload by SPIONs was assessed on a mouse cirrhosis model. A single dose of SPION injection at 0.5 or 5 mg Fe/kg in the cirrhosis group induced a septic shock response at 24 h with elevated serum levels of liver and kidney function markers and extended impacts over 14 days including high levels of serum cholesterols and persistent low serum iron level. In contrast, full restoration of liver functions was found in the normal group with the same dosages over time. Analysis with PCR array of the toxicity pathways revealed the high dose of SPIONs induced significant expression changes of a distinct subset of genes in the cirrhosis liver. All these results suggested that excess iron of the high dose of SPIONs might be a risk factor for cirrhosis because of the marked impacts of elevated lipid metabolism, disruption of iron homeostasis and possibly, aggravated loss of liver functions.