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.

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.

Co-administration of Transportan Peptide Enhances the Cellular Entry of Liposomes in the Bystander Manner Both In Vitro and In Vivo.

Liposomes have been widely used as a drug delivery vector. One way to further improve its therapeutic efficacy is to increase the cell entry efficiency. Covalent conjugation with cell-penetrating peptides (CPPs) and other types of ligands has been the mainstream strategy to tackle this issue. Although efficient, it requires additional chemical modifications on liposomes, which is undesirable for clinical translation. Our previous study showed that the transportan (TP) peptide, an amphiphilic CPP, was able to increase the cellular uptake of co-administered, but not covalently coupled, metallic nanoparticles (NPs). Termed bystander uptake, this process represents a simpler method to increase the cell entry of NPs without chemical modifications. Here, we extended our efforts to liposomes. Our results showed that co-administration with the TP peptide improved the internalization of liposome into a variety of cell lines in vitro. This effect was also observed in primary cells, ex vivo tumor slices, and in vivo tumor tissues. On the other hand, this peptide-assisted liposome internalization did not apply to cationic CPPs, which were the main inducers for bystander uptake in previous studies. We also found that TP-assisted bystander uptake of liposome is receptor dependent, and its activity is more sensitive to the inhibitors of the macropinocytosis pathway, underlining the potential cell entry mechanism. Overall, our study provides a simple strategy based on TP co-administration to increase the cell entry of liposomes, which may open up new avenues to apply TP peptides in nanotherapeutics.

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.

Impact of Subthalamic Deep Brain Stimulation on Hyposmia in Patients With Parkinson's Disease Is Influenced by Constipation and Dysbiosis of Microbiota.

Background: Non-motor symptoms in PD usually arise at very early stage and vary during the whole disease progression. Deep brain stimulation (DBS) is considered as a highly efficient treatment option for PD's motor function. However, the effect of DBS on NMS, especially hyposmia, has not been fully understood and the deep connection between different NMS such as hyposmia and constipation is still unknown. Objective: The objective of this study was to evaluate the therapeutic effect of DBS on hyposmia in PD patients with or without constipation and find potential factors which might influence the efficacy. Methods: A retrospective analysis of 65 PD patients accepted STN-DBS operation in Qilu Hospital during 2019-2020 were conducted to evaluate the exact therapeutic effect of DBS on hyposmia in PD. Sub-group analyses about the relationship between hyposmia and constipation were carried out. Analysis of flora in nasal mucosa was also conducted to evaluate the abundance and variety in different PD groups. Results: Our study showed that DBS had clearly improved olfactory function in Parkinson patients (P = 0.012) and subgroup analysis found that PD patients with constipation have lower olfactory function scores (25.27 ± 3.44 vs. 33.90 ± 6.633, p = 0.014) and worse improvement after DBS operation (ΔTDI 12.11 ± 3.2 vs. 8.78 ± 2.91, p = 0.0072). Analysis of flora indicated the obvious discrepancy on olfactory function scores and degree of improvement might be related to the abundance and dysbiosis of microbiota. Conclusion: In summary, this article presents a study on PD with hyposmia and constipation after DBS operation, explored the relationship between different NMS and offer a potential explanation on why PD patients with constipation usually have worse olfactory function for the less abundance and variety of microbiota.

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.

Macropinocytosis as a cell entry route for peptide-functionalized and bystander nanoparticles.

Endocytic pathways provide the primary route for therapeutic and diagnostic nanoparticles (NPs) to enter cells and subcellular compartments. A better understanding of these cell entry processes will not only aid in nanomaterial applications but also broaden our knowledge of cell biology. Among the endocytic routes, macropinocytosis has unique characteristics for engulfing NPs and other large cargo, yet its molecular machinery and involvement in NP uptake are far less characterized relative to other pathways. In this review, we summarize the current knowledge on the macropinocytic machinery, and its involvement in NP internalization. Particularly, we differentiate ligand (specifically peptide)-functionalized and unfunctionalized NPs (bystander NPs). While most of previous research focused on ligand-functionalized NPs, we showcase here a synergistic effect between these two NP types during their cell entry through receptor-mediated macropinocytosis. The regulation of NP uptake efficiency by extracellular amino acids is also highlighted in the context of interconnections between macropinocytosis and metabolic signaling. These discussions may fuel future research interests in improving NP internalization through this pathway, and open a new avenue to study the interplay among endocytosis, metabolism and nanomedicine.

Protective effects of a traditional Chinese herbal formula Jiang-Xian HuGan on Concanavalin A-induced mouse hepatitis via NF-κB and Nrf2 signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiang-Xian HuGan (JXHG) formulated by five natural products including Freshwater clam (Corbicula fluminea), Curcuma longa L., Ligustrum lucidum, Eclipta prostrata (L.) L. and Paeonia lactiflora Pall., has exhibited a great hepatoprotective effect. AIM OF THIS STUDY: We investigated the effect of JXHG on concanavalin A (ConA)-induced acute live injury in mice, and to elucidate its underlying molecular mechanisms. MATERIALS AND METHODS: Jiangkanling Capsule (900 mg/kg), low-dose JXHG (LJXHG, 700 mg/kg), high-dose JXHG (HJXHG, 1400 mg/kg) were administered to mice by oral gavage daily for 20 days prior to a single intravenous injection of ConA (20 mg/kg). Liver injury was evaluated by measuring the serum levels of enzymes and cytokines as well as liver histological analysis. We also measured the hepatic expression of cytokines at mRNA levels and the proteins related to NF-κB and Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathways. RESULT: Our results showed that JXHG pretreatment significantly alleviated ConA-induced live injury as evidenced by decreased serum levels of glutamic-pyruvic transaminase (ALT) and glutamic oxalacetic transaminase (AST), and reduced hepatocyte apoptosis and mortality. Furthermore, JXHG was able to significantly reduce the serum levels of proinflammatory cytokines, down-regulate the mRNA expression of interleukin-6 (IL-6) and interferon-γ (IFN-γ), and up-regulate IL-10 as well as superoxide-dimutase-1 (SOD1), glutathione reductase (GSR) and Glutathione peroxidase 2 (GPX2) mRNA in the liver tissues after Con A injection. In addition, JXHG pretreatment dramatically suppressed the phosphorylation of NF-κB p65 (p65), increased Nrf2 expression, and decreased the expression ratio of cleaved caspase-3/caspase-3 in liver tissues. CONCLUSION: These results suggest that JXHG protects against ConA-induced acute live injury through inhibiting NF-κB mediated inflammatory pathway and promoting Nrf2 mediated anti-oxidative stress signaling pathway.

SNX10 mediates alcohol-induced liver injury and steatosis by regulating the activation of chaperone-mediated autophagy.

BACKGROUND & AIMS: Alcoholic liver disease (ALD) is a major cause of morbidity and mortality worldwide. However, the cellular defense mechanisms underlying ALD are not well understood. Recent studies highlighted the involvement of chaperone-mediated autophagy (CMA) in regulating hepatic lipid metabolism. Sorting nexin (SNX)-10 has a regulatory function in endolysosomal trafficking and stabilisation. Here, we investigated the roles of SNX10 in CMA activation and in the pathogenesis of alcohol-induced liver injury and steatosis. METHODS: Snx10 knockout (Snx10 KO) mice and their wild-type (WT) littermates fed either the Lieber-DeCarli liquid alcohol diet or a control liquid diet, and primary cultured WT and Snx10 KO hepatocytes stimulated with ethanol, were used as in vivo and in vitro ALD models, respectively. Activation of CMA, liver injury parameters, inflammatory cytokines, oxidative stress and lipid metabolism were measured. RESULTS: Compared with WT littermates, Snx10 KO mice exhibited a significant amelioration in ethanol-induced liver injury and hepatic steatosis. Both in vivo and in vitro studies showed that SNX10 deficiency upregulated lysosome-associated membrane protein type 2A (LAMP-2A) expression and CMA activation, which could be reversed by SNX10 overexpression in vitro. LAMP-2A interference confirmed that the upregulation of Nrf2 and AMPK signalling pathways induced by SNX10 deficiency relied on CMA activation. Pull-down assays revealed an interaction between SNX10 and cathepsin A (CTSA), a key enzyme involved in LAMP-2A degradation. Deficiency in SNX10 inhibited CTSA maturation and increased the stability of LAMP-2A, resulting in an increase in CMA activity. CONCLUSIONS: SNX10 controls CMA activity by mediating CTSA maturation, and, thus, has an essential role in alcohol-induced liver injury and steatosis. Our results provide evidence for SNX10 as a potential promising therapeutic target for preventing or ameliorating liver injury in ALD. LAY SUMMARY: Alcoholic liver disease is a major cause of morbidity and mortality worldwide. Recent studies highlight the involvement of chaperone-mediated autophagy (CMA) in regulating hepatic lipid metabolism. Our study reveals that deficiency of sorting nexin (SNX) 10 increases the stability of LAMP-2A by inhibiting cathepsin A maturation, resulting in the increase of CMA activity and, thus, alleviates alcohol-induced liver injury and steatosis.