Extracellular Targets Research Articles

Extracellular ATP targets Arabidopsis RIBONUCLEASE 1 to suppress mycotoxin stress-induced cell death.

Summary Extracellular ATP is a purinergic signal with important functions in regulating plant growth and stress‐adaptive responses, including programmed cell death. While signalling events proximate to receptor activation at the plasma membrane have been characterised, downstream protein targets and the mechanism of cell death activation/regulation are unknown.We designed a proteomic screen to identify ATP‐responsive proteins in Arabidopsis cell cultures exposed to mycotoxin stress via fumonisin B1 (FB1) application.Arabidopsis RIBONUCLEASE 1 (RNS1) was identified by the screen, and transgenic plants overexpressing native RNS1 showed greater susceptibility to FB1, while a gene knockout rns1 mutant and antisense RNS1 transgenic plants were resistant to FB1‐induced cell death. Native RNS1 complemented rns1 mutants and restored the cell death response to FB1, while a catalytically inactive version of the ribonuclease could not. The FB1 resistance of salicylic acid (SA)‐depleted nahG‐expressing plants was abolished by transformation with native RNS1, but not the catalytically dead version.The mechanism of FB1‐induced cell death is activation of RNS1‐dependent RNA cleavage, which is blocked by ATP via RNS1 suppression, or enhanced by SA through induction of RNS1 expression. Our study reveals RNS1 as a previously unknown convergence point of ATP and SA signalling in the regulation of stress‐induced cell death.

Endothelial Cell-Derived Extracellular Vesicles Target TLR4 via miRNA-326-3p to Regulate Skin Fibroblasts Senescence.

Backgrounds Skin aging could be regulated by the aberrant expression of microRNAs. In this manuscript, we explain that endothelial cell-derived extracellular vesicles could act as supporters to deliver exogenous miR-326-3p to accelerate skin fibroblasts senescence. Methods β-galactosidase senescence staining assay, Hoechst 33258 apoptosis staining assay, and Ki67 staining assay were used to evaluate the biological function of mouse skin fibroblasts. Real-time PCR was applied to assay miRNAs and mRNAs expressions. Western blot was used to detect TLR4 protein expression. The target gene of miRNA were identified using a double luciferase reporter assay. miR-326-3p mimic/inhibitor and siRNA-TLR4 can demonstrate a nonnegligible link between miR-326-3p-TLR4 and skin aging. Results In coculture experiment, senescence endothelial cells could promote the skin fibroblasts senescence and apoptosis via extracellular vesicles pathway. In contrast, miR-326-3p mimics accelerated senescence and apoptosis of skin fibroblasts, while miR-326-3p inhibitor could dramatically delay skin fibroblasts senescence and apoptosis. TLR4 was proved to be a miR-326-3p directly target gene via double luciferase assay. After skin fibroblasts transfected with siRNA-TLR4, cellular senescence and apoptosis were significantly increased. Furthermore, the skin tissues of aging mice were shown with overexpression of miR-326-3p and decrease of TLR4 gene and protein expression levels. Conclusions Endothelial cell-derived extracellular vesicles delivery of miR-326-3p was found to have a function in skin fibroblasts via target TLR4. Therefore, endothelial cell-derived extracellular vesicles in antiaging therapies might be a new treatment way for delaying skin aging.

Vertebrate Lonesome Kinase Phosphorylates EphB2 Receptors on an Extracellular Residue to Drive NMDA-Dependent Pain

EphB/ephrinB signaling plays a key role in synaptic function and is linked to several neuropathological conditions, including neuropathic pain. The EphB2 receptor tyrosine kinase is known to interact with N-Methyl-D-aspartate glutamate receptors (NMDAR), resulting in increased surface retention of NMDARs at the synapse and an enhancement of postsynaptic currents. As changes in NMDAR signaling have been heavily implicated in the development of both acute and chronic pain, targeting this receptor interaction may produce clinically relevant therapeutics. We have recently demonstrated that the phosphorylation of a tyrosine residue (Y504) on the extracellular domain of the EphB2 receptor is necessary and sufficient to induce its interaction with NMDARs and cause mechanical pain hypersensitivity. The kinase responsible for this phosphorylation is unknown. We hypothesized that Vertebrate Lonesome Kinase (VLK) may be the key effector. Since the phosphorylation site of interest is located on the extracellular face of the EphB2 receptor, a secreted tyrosine kinase like VLK is an ideal candidate. Our results provide strong support for our hypothesis that VLK drives pain via extracellular phosphorylation of EphBs. We show that intrathecal injection of VLK resulted in long-lasting mechanical hypersensitivity in mice. Additionally, the effects observed for VLK were dependent on kinase activity and entirely dependent upon NMDARs because they were blocked by AP-5. We also show evidence of VLK protein and mRNA expression in dorsal root ganglion and spinal cord neurons of both mice and humans. Our results provide evidence for the involvement of VLK in pain behaviors and spinal cord NMDAR signaling, and identify a completely unique extracellular kinase target for the alleviation of pain. Grant support from NIH grants NS111976 & NS115441.

Salmonella enterica serovar Typhimurium chitinases modulate the intestinal glycome and promote small intestinal invasion.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the leading causes of food-borne illnesses worldwide. To colonize the gastrointestinal tract, S. Typhimurium produces multiple virulence factors that facilitate cellular invasion. Chitinases have been recently emerging as virulence factors for various pathogenic bacterial species, and the S. Typhimurium genome contains two annotated chitinases: STM0018 (chiA) and STM0233. However, the role of these chitinases during S. Typhimurium pathogenesis is unknown. The putative chitinase STM0233 has not been studied previously, and only limited data exists on ChiA. Chitinases typically hydrolyze chitin polymers, which are absent in vertebrates. However, chiA expression was detected in infection models and purified ChiA cleaved carbohydrate subunits present on mammalian surface glycoproteins, indicating a role during pathogenesis. Here, we demonstrate that expression of chiA and STM0233 is upregulated in the mouse gut and that both chitinases facilitate epithelial cell adhesion and invasion. S. Typhimurium lacking both chitinases showed a 70% reduction in invasion of small intestinal epithelial cells in vitro. In a gastroenteritis mouse model, chitinase-deficient S. Typhimurium strains were also significantly attenuated in the invasion of small intestinal tissue. This reduced invasion resulted in significantly delayed S. Typhimurium dissemination to the spleen and the liver, but chitinases were not required for systemic survival. The invasion defect of the chitinase-deficient strain was rescued by the presence of wild-type S. Typhimurium, suggesting that chitinases are secreted. By analyzing N-linked glycans of small intestinal cells, we identified specific N-acetylglucosamine-containing glycans as potential extracellular targets of S. Typhimurium chitinases. This analysis also revealed a differential abundance of Lewis X/A-containing glycans that is likely a result of host cell modulation due to the detection of S. Typhimurium chitinases. Similar glycomic changes elicited by chitinase deficient strains indicate functional redundancy of the chitinases. Overall, our results demonstrate that S. Typhimurium chitinases contribute to intestinal adhesion and invasion through modulation of the host glycome.

Modern views on the metabolism and biological effects of vitaminD

VitaminD is a steroid hormone that plays a crucial role in maintaining normal bone condition and calcium homeostasis. In recent years, vitaminD has become a hot topic of endocrinological research, largelyDue to the COVID-19 pandemic and the likely correlation between hypovitaminosisD and a high risk of chronic lung disease and associated mortality. Recent studies have shown that vitaminD exhibits a complex multistage metabolism and acts as a hormone on many extracellular targets. This review examines some new intriguing and as yet unclear aspects of vitaminD metabolism, such as new concepts of enzyme regulation, new pleiotropic effects of vitaminD receptor activation (VDR), and epigenetic effects. The mechanisms of vitaminD synthesis in the skin, its metabolism in the hepatic cytochrome P450 system, catabolism, metabolites and transport, gene control and epigenetic modulation are considered inDetail. In addition to the well-known role of vitaminD in calcium and bone metabolism, it has many pleiotropic extraskeletal effects, including potent effects on the immune system, cardiovascular system, adipose tissue and glucose/lipid metabolism, muscle and more. Experimental studies have shown that VDRs are expressed by cancer cell lines. Recent studies have shown a link between low levels of vitaminD and almost all aspects of the metabolic syndrome, such as type 2 diabetes, fasting blood glucose, hypertension, dyslipidemia, obesity and insulin resistance. Several studies have focused on the role of vitaminD in adipose tissue biology. In particular, a negative correlation between vitaminD and leptin or resistin is shown, as well as an inverse correlation with adiponectin. Recent studies in vitaminD-deficient mice have shown impaired secretion of glucose-stimulated insulin by pancreatic islets. VitaminD is thought to play a role in the pathogenesis and progression of cancer, and vitaminD analogues can slow cancer progression and metastasis. It is concluded that vitaminD is a molecule with several endocrine, paracrine and autocrine effects on many tissues and organs, in addition to maintaining skeletal homeostasis. Research in this area, which aims to clarify the pleiotropy of many effects of vitaminD and its metabolites, continues.

Dissecting extracellular and intracellular distribution of nanoparticles and their contribution to therapeutic response by monochromatic ratiometric imaging

Efficient delivery of payload to intracellular targets has been identified as the central principle for nanomedicine development, while the extracellular targets are equally important for cancer treatment. Notably, the contribution of extracellularly distributed nanoparticles to therapeutic outcome is far from being understood. Herein, we develop a pH/light dual-responsive monochromatic ratiometric imaging nanoparticle (MRIN), which functions through sequentially lighting up the intracellular and extracellular fluorescence signals by acidic endocytic pH and near-infrared light. Enabled by MRIN nanotechnology, we accurately quantify the extracellular and intracellular distribution of nanoparticles in several tumor models, which account for 65–80% and 20–35% of total tumor exposure, respectively. Given that the majority of nanoparticles are trapped in extracellular regions, we successfully dissect the contribution of extracellularly distributed nanophotosensitizer to therapeutic efficacy, thereby maximize the treatment outcome. Our study provides key strategies to precisely quantify nanocarrier microdistribtion and engineer multifunctional nanomedicines for efficient theranostics.

Mechanism of Action of Tumorigenesis of Anticancer Molecules of Palm Oil Tocotrienols (Elaeis Guieensis Jacq.): A Systematic Review

Palm oil contains vitamin E which comprises two structurally similar compounds namely tocopherols and tocotrienols. For many years tocopherols considered the best therapy in curing disease, but in recent research advances, tocotrienol has been widely used as a powerful therapy in several diseases, including cancer. We prepared this article to review and analyze the role of tocotrienols from palm oil as anticancer molecules and their mechanism of action in the process of tumorigenesis based on the literature from PubMed, Scopus, Science Direct, and ProQuest databases published from 2011 to 2020, and according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Nineteen English articles and abstracts according to the inclusion criteria, systematically reviewed and compiled. The results showed that tocotrienol acts on intracellular and extracellular targets involved in cell signaling pathways and suppresses the growth of various malignancies, including breast, lung, prostate, ovarian, liver, brain, colon, pancreatic, and myeloma cancers. This literature review shows that the molecular mechanism of action of tocotrienols has several advantages, namely a positive effect on cell proliferation, apoptosis, angiogenesis, metastasis, and inflammation, which when working together can function as effective agents in cancer therapy. The tocotrienol-mediated therapeutic action mechanism could help clinicians to design new strategies for cancer treatment.

Mechanism of Action of Tumorigenesis of Anticancer Molecules of Palm Oil Tocotrienols (Elaeis Guieensis Jacq.): A Systematic Review

Palm oil contains vitamin E which comprises two structurally similar compounds namely tocopherols and tocotrienols. For many years tocopherols considered the best therapy in curing disease, but in recent research advances, tocotrienol has been widely used as a powerful therapy in several diseases, including cancer. We prepared this article to review and analyze the role of tocotrienols from palm oil as anticancer molecules and their mechanism of action in the process of tumorigenesis based on the literature from PubMed, Scopus, Science Direct, and ProQuest databases published from 2011 to 2020, and according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Nineteen English articles and abstracts according to the inclusion criteria, systematically reviewed and compiled. The results showed that tocotrienol acts on intracellular and extracellular targets involved in cell signaling pathways and suppresses the growth of various malignancies, including breast, lung, prostate, ovarian, liver, brain, colon, pancreatic, and myeloma cancers. This literature review shows that the molecular mechanism of action of tocotrienols has several advantages, namely a positive effect on cell proliferation, apoptosis, angiogenesis, metastasis, and inflammation, which when working together can function as effective agents in cancer therapy. The tocotrienol-mediated therapeutic action mechanism could help clinicians to design new strategies for cancer treatment.

Partial Magneto-Endosomalysis for Cytosolic Delivery of Antibodies.

Modulation of protein functions and interactions is the most direct and effective means to intervene in cellular processes and pathogenesis. The majority of the critical intracellular signaling pathways, however, are considered undruggable using small molecules. In this regard, antibodies are superior in structural and functional diversity and are significantly easier to raise compared to the screening of small molecules. Despite these advantages, the uses of antibodies in live cells (either as an imaging agent or as a therapeutic compound) are substantially undermined, only acting on extracellular targets. The inability of targeting intracellular proteins is because of a fundamental issue: antibodies enter cells through endocytosis where the vast majority are trapped in endosomes for degradation. Here, we report a nanoparticle self-assembly strategy enabling antibody endosomal escape. We demonstrate the intracellular bioavailability of antibodies and the preserved binding specificity to their cytosolic targets. This technology is simple and opens exciting opportunities for live-cell imaging, therapeutics development, and cell engineering.

Incorporation of Endosomolytic Peptides with Varying Disruption Mechanisms into EGFR-Targeted Protein Conjugates: The Effect on Intracellular Protein Delivery and EGFR Specificity in Breast Cancer Cells.

Intracellular delivery of protein therapeutics remains a significant challenge limiting the majority of clinically available protein drugs to extracellular targets. Strategies to deliver proteins to subcellular compartments have traditionally relied on cell-penetrating peptides, which can drive enhanced internalization but exhibit unreliable activity and are rarely able to target specific cells, leading to off-target effects. Moreover, few design rules exist regarding the relative efficacy of various endosomal escape strategies in proteins. Accordingly, we developed a simple fusion modification approach to incorporate endosomolytic peptides onto epidermal growth factor receptor (EGFR)-targeted protein conjugates and performed a systematic comparison of the endosomal escape efficacy, mechanism of action, and capacity to maintain EGFR-targeting specificity of conjugates modified with four different endosomolytic sequences of varying modes of action (Aurein 1.2, GALA, HA2, and L17E). Use of the recently developed Gal8-YFP assay indicated that the fusion of each endosomolytic peptide led to enhanced endosomal disruption. Additionally, the incorporation of each endosomolytic peptide increased the half-life of the internalized protein and lowered lysosomal colocalization, further supporting the membrane-disruptive capacity. Despite this, only EGFR-targeted conjugates modified with Aurein 1.2 or GALA maintained EGFR specificity. These results thus demonstrated that the choice of endosomal escape moiety can substantially affect targeting capability, cytotoxicity, and bioactivity and provided important new insights into endosomolytic peptide selection for the design of targeted protein delivery systems.

Photobiomodulation and Sports: Results of a Narrative Review.

Benefits of photobiomodulation (PBM) have been known for several decades. More recently, PBM applied in sports offers a special chance to support the modeling of the performance and recovery. Increasingly complex physical activities and fierce competition in the world of sports generate a state of psycho-emotional and physical stress that can induce chronic fatigue syndrome, failure in physical training, predisposition to muscle damage, physical and emotional exhaustion etc., for which PBM could be an excellent solution. To evaluate and identify all risk factors and the influence of PBM on health and performance in sport and for a better understanding of its effects, we did a search for “Photobiomodulation and Sports” on PubMed, to update the PBM science applied in sports, and we retained for analysis the articles published from 2014 to date. The term “PBM” is recent, and we did not include previous studies with “low level laser therapy” or “LLLT” before 2014. In the present research, PBM has been shown to have valuable protective and ergogenic effects in 25 human studies, being the key to success for high performance and recovery, facts supported also by 22 animal studies. PBM applied creatively and targeted depending on sport and size of the level of physical effort could perfectly modulate the mitochondrial activity and thus lead to remarkable improvements in performance. PBM with no conclusive results or without effects from this review (14 studies from a total of 39 on humans) was analyzed and we found the motivations of the authors from the perspective of multiple causes related to technological limitations, participants, the protocols for physical activity, the devices, techniques and PBM parameters. In the near future, dose–response experiments on physical activity should be designed and correlated with PBM dose–response studies, so that quantification of PBM parameters to allow the energy, metabolic, immune, and neuro-endocrine modulation, perfectly coupled with the level of training. There is an urgent need to continuously improve PBM devices, delivery methods, and protocols in new ingenious future sports trials. Latest innovations and nanotechnologies applied to perform intracellular signaling analysis, while examining extracellular targets, coupled with 3D and 4D sports motion analysis and other high-tech devices, can be a challenge to learn how to maximize PBM efficiency while achieving unprecedented sports performance and thus fulfilling the dream of millions of elite athletes.

Fighting the Fiber: Targeting Collagen in Lung Fibrosis.

Organ fibrosis is characterized by epithelial injury and aberrant tissue repair, where activated effector cells, mostly fibroblasts and myofibroblasts, excessively deposit collagen into the extracellular matrix. Fibrosis frequently results in organ failure and has been estimated to contribute to at least one-third of all global deaths. Also, lung fibrosis, in particular idiopathic pulmonary fibrosis (IPF), is a fatal disease with rising incidence worldwide. As current treatment options targeting fibrogenesis are insufficient, there is an urgent need for novel therapeutic strategies. During the last decade, several studies have proposed to target intra- and extracellular components of the collagen biosynthesis, maturation, and degradation machinery. This includes intra- and extracellular targets directly acting on collagen gene products, but also such that anabolize essential building blocks of collagen, in particular glycine (Gly) and proline (Pro) biosynthetic enzymes. Collagen, however, is a ubiquitous molecule in the body and fulfills essential functions as a macromolecular scaffold, growth factor reservoir, and receptor binding site in virtually every tissue. This review summarizes recent advances and future directions in this field. Evidence for the proposed therapeutic targets and where they currently stand in terms of clinical drug development for treatment of fibrotic disease is provided. The drug targets are furthermore discussed in light of 1) specificity for collagen biosynthesis, maturation, and degradation, and 2) specificity for disease-associated collagen. As therapeutic success and safety of these drugs may largely depend on targeted delivery, different strategies for specific delivery to the main effector cells and the extracellular matrix are discussed.

124P Effective silencing of PD-1 gene in human peripheral blood T-lymphocytes by self-deliverable siRNA

Like all monoclonal antibodies, inhibitors of immune checkpoints (ICIs) can only interact with cell surface receptors, but not with intracellular proteins, which can be a promising target for cancer immunotherapy. ICIs can only block the receptor (e.g., PD-1), but not decrease its level. In contrast, small interfering RNAs (siRNAs) trigger mRNA degradation resulting in protein synthesis inhibition. It is possible to regulate the levels of both intracellular and extracellular target proteins using siRNAs.

Avilar Therapeutics launches with $60 million to degrade extracellular targets

Avilar Therapeutics launches with $60 million to degrade extracellular targets

Extracellular vesicle-derived miR-320a targets ZC3H12B to inhibit tumorigenesis, invasion, and angiogenesis in ovarian cancer

Extracellular vesicles (EVs) play crucial roles in intercellular communication. miRNAs derived from EVs emerge as promising diagnostic indicators and therapeutic targets in a variety of malignancies. Tremendous studies have revealed the function of miRNAs derived from EVs in tumorigenesis, metastasis and other aspects. The mechanism of action of EV-derived miRNAs, however, in ovarian cancer remains largely unknown. In this study, EVs were enriched from the ovarian cancer cell lines. EVs as a whole could promote cell proliferation, invasion and new vasculature formation. However, the down-regulated EV-derived miR-320a was demonstrated to potentially suppress tumorigenesis, metastasis and angiogenesis. Moreover, EV-derived miR-320a has been proved to directly regulate a previously unknown target, ZC3H12B. An unreported role of ZC3H12B in promoting ovarian cancer cell proliferation has been elucidated and miR-320a could mediate the expression of ZC3H12B, thereby inhibiting the downstream response. As for the practical clinic values, lower expression of EV-derived miR-320a correlates with shorter survival period, indicating that EV-derived miR-320a may also serve as a prognostic biomarker in ovarian cancer. This research provides new insight into the molecular mechanism of EV-derived miR-320a in ovarian cancer and may provide new therapeutic and prognostic strategies for ovarian cancer treatment.

Synthetic Protein Circuits and Devices Based on Reversible Protein-Protein Interactions: An Overview.

Protein-protein interactions (PPIs) contribute to regulate many aspects of cell physiology and metabolism. Protein domains involved in PPIs are important building blocks for engineering genetic circuits through synthetic biology. These domains can be obtained from known proteins and rationally engineered to produce orthogonal scaffolds, or computationally designed de novo thanks to recent advances in structural biology and molecular dynamics prediction. Such circuits based on PPIs (or protein circuits) appear of particular interest, as they can directly affect transcriptional outputs, as well as induce behavioral/adaptational changes in cell metabolism, without the need for further protein synthesis. This last example was highlighted in recent works to enable the production of fast-responding circuits which can be exploited for biosensing and diagnostics. Notably, PPIs can also be engineered to develop new drugs able to bind specific intra- and extra-cellular targets. In this review, we summarize recent findings in the field of protein circuit design, with particular focus on the use of peptides as scaffolds to engineer these circuits.

Human Liver Stem Cell-Derived Extracellular Vesicles Target Hepatic Stellate Cells and Attenuate Their Pro-fibrotic Phenotype.

Liver fibrosis occurs in response to chronic liver injury and is characterized by an excessive deposition of extracellular matrix. Activated hepatic stellate cells are primarily responsible for this process. A possible strategy to counteract the development of hepatic fibrosis could be the reversion of the activated phenotype of hepatic stellate cells. Extracellular vesicles (EVs) are nanosized membrane vesicles involved in intercellular communication. Our previous studies have demonstrated that EVs derived from human liver stem cells (HLSCs), a multipotent population of adult stem cells of the liver with mesenchymal-like phenotype, exert in vivo anti-fibrotic activity in the liver. However, the mechanism of action of these EVs remains to be determined. We set up an in vitro model of hepatic fibrosis using a human hepatic stellate cell line (LX-2) activated by transforming growth factor-beta 1 (TGF-β1). Then, we investigated the effect of EVs obtained from HLSCs and from human bone marrow-derived mesenchymal stromal cells (MSCs) on activated LX-2. The incubation of activated LX-2 with HLSC-EVs reduced the expression level of alpha-smooth muscle actin (α-SMA). Conversely, MSC-derived EVs induced an increase in the expression of pro-fibrotic markers in activated LX-2. The analysis of the RNA cargo of HLSC-EVs revealed the presence of several miRNAs involved in the regulation of fibrosis and inflammation. Predictive target analysis indicated that several microRNAs (miRNAs) contained into HLSC-EVs could possibly target pro-fibrotic transcripts. In particular, we demonstrated that HLSC-EVs shuttled miR-146a-5p and that treatment with HLSC-EVs increased miR-146a-5p expression in LX-2. In conclusion, this study demonstrates that HLSC-EVs can attenuate the activated phenotype of hepatic stellate cells and that their biological effect may be mediated by the delivery of anti-fibrotic miRNAs, such as miR-146a-5p.

Efficient Delivery of Antibodies Intracellularly by Co-Assembly with Hexahistidine-Metal Assemblies (HmA).

PurposeThere has been a substantial global market for antibodies, which are based on extracellular targets. Binding intracellular targets by antibodies will bring new chances in antibody therapeutics and a huge market increase. We aim to evaluate the efficiency of a novel delivery system of His6-metal assembly (HmA) in delivering intracellular antibodies and biofunctions of delivered antibodies.MethodsIn this study, the physicochemical properties of HmA@Antibodies generated through co-assembling with antibodies and HmA were well characterized by dynamic light scatter. The cytotoxicity of HmA@Antibodies was investigated by Cell Counting Kit-8 (CCK-8). The endocytic kinetics and lysosome escape process of HmA@Antibodies were studied by flow cytometry and fluorescent staining imaging, respectively. Compared to the commercialized positive control, the intracellular delivery efficiency by HmA@Antibodies and biofunctions of delivered antibodies were evaluated by fluorescent imaging and CCK-8.ResultsVarious antibodies (IgG, anti-β-tubulin and anti-NPC) could co-assemble with HmA under a gentle condition, producing nano-sized (~150 nm) and positively charged (~+30 eV) HmA@Antibodies particles with narrow size distribution (PDI ~ 0.15). HmA displayed very low cytotoxicity to divers cells (DCs, HeLa, HCECs, and HRPE) even after 96 h for the feeding concentration ≤100 μg mL−1, and fast escape from endosomes. In the case of delivery IgG, the delivery efficiency into alive cells of HmA was better than a commercial protein delivery reagent (PULSin). For cases of the anti-β-tubulin and anti-NPC, HmA showed comparable delivery efficiency to their positive controls, but HmA with ability to deliver these antibodies into alive cells was still superior to positive controls delivering antibodies into dead cells through punching holes.ConclusionOur results indicate that this strategy is a feasible way to deliver various antibodies intracellularly while preserving their functions, which has great potential in various applications and treating many refractory diseases by intracellular antibody delivery.

Scalable Biosynthetic Production of Knotted Peptides Enables ADME and Thermodynamic Folding Studies.

Knotted peptides present a wealth of structurally diverse, biologically active molecules, with the inhibitor cystine knot/knottin class among the most ecologically common ones. Many of these natural products interact with extracellular targets such as voltage-gated ion channels with exquisite selectivity and potency, making them intriguing therapeutic modalities. Such compounds are often produced in low concentrations by intractable organisms, making structural and biological characterization challenging, which is frequently overcome by various expression strategies. Here, we sought to test a biosynthetic route for the expression and study of knotted peptides. We screened expression constructs for a biosynthesized knotted peptide to determine the most influential parameters for successful disulfide folding and used NMR spectroscopic fingerprinting to validate topological structures. We performed pharmacokinetic characterization, which indicated that the interlocking disulfide structure minimizes liabilities of linear peptide sequences, and propose a mechanism by which knotted peptides are cleared. We then developed an assay to monitor solution folding in real time, providing a strategy for studying the folding process during maturation, which provided direct evidence for the importance of backbone organization as the driving force for topology formation.

Enzymatically Transformable Polymersome-Based Nanotherapeutics to Eliminate Minimal Relapsable Cancer.

Prevention of metastatic and local-regional recurrence of cancer after surgery remains difficult. Targeting postsurgical premetastatic niche and microresiduals presents an excellent prospective opportunity but is often challenged by poor therapeutic delivery into minimal residual tumors. Here, an enzymatically transformable polymer-based nanotherapeutic approach is presented that exploits matrix metalloproteinase (MMP) overactivation in tumor-associated tissues to guide the codelivery of colchicine (microtubule-disrupting and anti-inflammatory agent) and marimastat (MMP inhibitor). The dePEGylation of polymersomes catalyzed by MMPs not only exposes the guanidine moiety to improve tissue/cell-targeting/retention to increase bioavailability, but also differentially releases marimastat and colchicine to engage their extracellular (MMPs) and intracellular (microtubules) targets of action, respectively. In primary tumors/overt metastases, the vasculature-specific targeting of nanotherapeutics can function synchronously with the enhanced permeability and retention effect to deter malignant progression of metastatic breast cancer. After the surgical removal of large primary tumors, nanotherapeutic agents are localized in the premetastatic niche and at the site of the postsurgical wound, disrupting the premetastatic microenvironment and eliminating microresiduals, which radically reduces metastatic and local-regional recurrence. The findings suggest that nanotherapeutics can safely widen the therapeutic window to resuscitate colchicine and MMP inhibitors for other inflammatory disorders.