Receptor-mediated Endocytosis Research Articles

Progesterone induces meiosis through two obligate co-receptors with PLA2 activity.

The steroid hormone progesterone (P4) regulates multiple aspects of reproductive and metabolic physiology. Classical P4 signaling operates through nuclear receptors that regulate transcription. In addition, P4 signals through membrane P4 receptors (mPRs) in a rapid nongenomic modality. Despite the established physiological importance of P4 nongenomic signaling, the details of its signal transduction cascade remain elusive. Here, using Xenopus oocyte maturation as a well-established physiological readout of nongenomic P4 signaling, we identify the lipid hydrolase ABHD2 (α/β hydrolase domain-containing protein 2) as an essential mPRβ co-receptor to trigger meiosis. We show using functional assays coupled to unbiased and targeted cell-based lipidomics that ABHD2 possesses a phospholipase A2 (PLA2) activity that requires mPRβ. This PLA2 activity bifurcates P4 signaling by inducing clathrin-dependent endocytosis of mPRβ, resulting in the production of lipid messengers that are G-protein coupled receptor agonists. Therefore, P4 drives meiosis by inducing an ABHD2 PLA2 activity that requires both mPRβ and ABHD2 as obligate co-receptors.

Uptake of iron from ferrous fumarate can be mediated by clathrin-dependent endocytosis in Hutu-80 cells

Iron uptake in the intestinal epithelium is associated with transport of ferrous iron via the DMT1 transporter (SLC11a2; NRAMP2). In later years, uptake of iron from complex sources, such as nanoparticles, has been found to be mediated through endocytosis. Here we propose that iron from the simple salt ferrous fumarate, a common iron supplement, can be absorbed by clathrin-mediated endocytosis. We used siRNA to silence DMT1 transporter expression, pharmacological inhibition of endocytosis, and Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) to show that iron uptake from ferrous fumarate can be mediated by both transport via DMT1 and by clathrin-dependent endocytosis in Hutu-80 cells. Iron uptake (ferritin L) from ferrous fumarate (0.5 mM, 24 h) in DMT1 silenced cells was significantly decreased (60% ± 11%) in comparison to iron controls while a 1-h dose of ferrous fumarate (0.5 mM) significantly decreased ferritin L formation in the presence of the clathrin inhibitor chlorpromazine (61% ± 10%, in post-confluent cells and 37% ± 9% in non-confluent cells). A pilot showed a similar trend for Ferritin (H) levels (confluent cells) and for total cellular iron load (non-confluent cells). ToF-SIMS analysis revealed diminished membrane-associated iron load in endocytosis-inhibited ferrous fumarate treated cells. The reported results support a clathrin-mediated endocytosis mechanism for uptake of iron from ferrous fumarate in addition to iron uptake by DMT1. More studies are needed to understand what determines which uptake mechanism are employed and to which extent.

Receptor-ligand interactions for optimized endocytosis in targeted therapies.

Receptor-mediated endocytosis plays a crucial role in the success of numerous therapies and remains central to advancing drug development. This process begins with ligand binding to specific receptors, triggering the internalization and intracellular trafficking of receptor-ligand complexes. These complexes are subsequently directed into distinct routes, either toward lysosomal degradation or recycling to the cell surface, with implications for therapeutic outcomes. This review examines receptor-ligand interactions as key modulators of endocytosis, emphasizing their role in shaping therapeutic design and efficacy. Advances in selecting receptor-ligand pairs and engineering ligands with optimized properties have enabled precise control over internalization, endosomal sorting, and trafficking, providing tailored solutions for diverse therapeutic applications. Leveraging these insights, strategies such as RNA-based therapies, antibody-drug conjugates (ADCs), and targeted protein degradation (TPD) platforms have been refined to selectively avoid or promote lysosomal degradation, thereby enhancing therapeutic efficacy. By bridging fundamental mechanisms of receptor-mediated endocytosis with innovative therapeutic approaches, this review offers a framework for advancing precision medicine.

C1orf115 interacts with clathrin adaptors to undergo endocytosis and induces ABCA1 to promote enteric cholesterol efflux

C1orf115 has been identified in high-throughput screens as a regulator of multidrug resistance possibly mediated through an interaction with ATP-dependent membrane transporter ABCB1. Here we show that C1orf115 not only shares structural similarities with FACI/C11orf86 to interact with clathrin adaptors to undergo endocytosis, but also induces ABCA1 transcription to promote cholesterol efflux. C1orf115 consists of an N-terminal intrinsically disordered region and a C-terminal α-helix. Its α-helix binds to phosphoinositides, and mediates C1orf115 localization to the plasma membrane, nucleolus and nuclear speckles. An acidic dileucine-like motif “ExxxIL” within C1orf115 binds with the AP2 complex and mediates its localization to clathrin-coating pits. The positively charged amphipathic α-helix undergoes acetylation, which redistributes C1orf115 from the plasma membrane and nucleolus to nuclear speckles. C1orf115 is widely expressed and most abundant in the small intestine. The ability of C1orf115 in clathrin-mediated endocytosis is required for its regulation of drug resistance, which is modulated by acetylation. RNA-seq analysis reveals that C1orf115 induces intestinal transcription of another ATP-dependent transporter ABCA1 and consequently promotes ABCA1-mediated cholesterol efflux in enterocytes. Our study provides mechanistic insight into how C1orf115 modulates drug resistance and cholesterol efflux through clathrin-mediated endocytosis and ABCA1 expression.

Saponin-based adjuvant uptake and induction of antigen cross-presentation by CD11b+ dendritic cells and macrophages

Saponin-based adjuvants (SBAs) distinguish themselves as vaccine adjuvants by instigating a potent activation of CD8+ T cells. Previously, we discovered SBA’s ability to induce cross-presentation in dendritic cells (DCs) leading to CD8+ T cell activation. Moreover, the MHCIIloCD11bhi bone marrow-derived DC (BMDC) subset was identified to be the most responsive DC subset to SBA treatment. To further investigate SBA’s mode of action, labeling of SBAs was optimized with the fluorescent dye SP-DiIC18(3). Efficient uptake of SBAs occurs specifically by MHCIIloCD11bhi BMDCs and bone marrow-derived macrophages (BMDMs) in vitro and cDC2s and macrophages ex vivo. Furthermore, SBAs are primarily taken up by clathrin-mediated endocytosis and uptake induces lipid bodies and antigen translocation to the cytosol in MHCIIloCD11bhi BMDCs and BMDMs. Importantly, BMDMs treated with SBAs exhibit cross-presentation leading to potent CD8+ T cells activation. Our findings explain the potency of SBAs as vaccine adjuvants and contribute to vaccine development.

DOP033 Pre-Clinical Development of SL-325, a High Affinity DR3 Blocking Antibody, for Durable Blockade of the DR3/TL1A Axis in Inflammatory Bowel Disease

Abstract Background TL1A blocking antibodies have demonstrated promising clinical remission rates in patients with inflammatory bowel disease (Sands B et al, NEJM 2024). TL1A promotes inflammation through binding to a single receptor, DR3. There are no known signaling ligands for DR3 aside from TL1A. Whereas TL1A is transiently expressed by antigen presenting and endothelial cells at sites of active inflammation in the small and large intestine, DR3 is constitutively expressed by lymphoid cells in the peripheral blood, and throughout the GI tract at both inflamed and adjacent non-inflamed tissue. Methods A human DR3 blocking antibody, SL-325, was produced from stably transfected CHO cells and purified using an affinity chromatography capture step, followed by two polishing steps. Sequence equivalents of tulisokibart and RO7790121 were produced by transient transfection and affinity purification. The safety, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of SL-325 were evaluated in a GLP toxicology study in cynomolgus macaques over a 6-week in-life period followed by a 4-week recovery at Charles River Laboratories. Results SL-325 binding affinity to human DR3 was measured at 1.3 pM. SL-325 bound to both monomeric and trimeric human DR3 prevented recombinant human TL1A binding with an approximately ~10X improvement in potency in comparison to sequence equivalents of tulisokibart or RO7790121. The DR3 epitope bound by SL-325 did not trigger receptor-mediated endocytosis, and no evidence of SL-325 mediated DR3 agonism was observed in the absence or presence of T cell receptor activation of primary human lymphocytes from healthy donors, or donors with Crohn’s Disease or Ulcerative Colitis. Naïve cynomolgus macaques received 3 doses of intravenous SL-325 (vehicle, 1 mg/kg, 10 mg/kg or 100 mg/kg dose groups), each given two weeks apart, and no evidence of toxicity, organ dysfunction or changes to complete blood counts or metabolic panels were observed. Full and durable DR3 receptor occupancy (RO) was observed in peripheral blood lymphocytes at doses of 1 mg/kg and higher within two hours of infusion and throughout the 14 day inter-dose interval. Serum exposure of free SL-325 increased in a dose proportional manner. No evidence of residual DR3 agonist activity was observed. Conclusion These results indicate SL-325 is a high-affinity DR3 blocking antibody with no evidence of toxicity or residual agonism in cynomolgus macaques, and with an RO/PK profile suggestive of extended dosing intervals that will be studied in an upcoming Phase 1 clinical trial. References Sands BE, Feagan BG, Peyrin-Biroulet L, Danese S, Rubin DT, Laurent O, Luo A, Nguyen DD, Lu J, Yen M, Leszczyszyn J, Kempinski R, McGovern DPB, Ma C, Ritter TE, Targan S. Phase 2 Trial of Anti-TL1A Monoclonal Antobidy Tulisokibart for Ulcerative Colitis. NEJM 2024;391(12):1119-1129

RNASEK interacting with PEDV structural proteins facilitates virus entry via clathrin-mediated endocytosis.

Porcine epidemic diarrhea virus (PEDV), as a type of Alphacoronavirus causing acute diarrhea and high death rate among sucking piglets, poses great financial damage to the swine industry. Nevertheless, the molecular mechanism whereby PEDV enters host cells is unclear, limiting the development of PED vaccines and anti-PEDV agents. The present study found that the host protein ribonuclease kappa (RNASEK) was regulated by USF2, a transcription factor, and facilitated the PEDV replication. RNASEK was identified as a novel binding partner of PEDV, which interacted with a spike (S), envelope (E), and membrane (M) proteins on PEDV virion surfaces to increase the uptake not for attachment of PEDV virions. PEDV enters cells through the endocytosis pathways. RNASEK knockdown or RNASEK knockout assay revealed that through clathrin-mediated endocytosis (CME), RNASEK promoted the internalization of PEDV virions. Clathrin and the adaptor protein EPS15 only interacted with PEDV E protein, demonstrating that the RNASEK could target more virions through interaction with PEDV S, E, and M proteins to clathrin and EPS15 proteins rather than merely interacting with PEDV E protein to mediate the PEDV entry through CME. Moreover, our findings suggest that RNASEK, a newly identified host-entry factor, facilitates PEDV internalization by increasing the interaction of PEDV virions and EPS15-clathrin complex and may also provide a potential target for anti-PEDV therapies.IMPORTANCEPEDV is the causative pathogen of porcine diarrhea, which is a highly infectious acute intestinal condition, that poses significant economic damage to the swine industry. However, the existing PED vaccines fail to provide adequate protection for piglets against PEDV infection. Although PEDV replication in cells has been widely described, the mechanisms beneath PEDV entry of the host cells are incompletely understood. In this study, we showed that RNASEK, regulated by the transcription factor USF2, is a new host factor increasing PEDV infection in LLC-PK1 cells. RNASEK can bind to multiple structural proteins of PEDV (S, E, and M proteins), therefore increasing the interaction between PEDV virions, clathrin, and EPS15 to promote PEDV virion entry. Apart from unraveling the entry mechanisms of PEDV, our findings also contributed to facilitating the development of anti-PEDV agents and PED vaccines.

Synergistic activity of Pitstop-2 and 1,6-hexanediol in aggressive human lung cancer cells

Metastatic cancer cells undergo metabolic reprogramming, which involves changes in the metabolic fluxes, including endocytosis, nucleocytoplasmic transport, and mitochondrial metabolism, to satisfy their massive demands for energy, cell division, and proliferation compared to normal cells. We have previously demonstrated the ability of two different types of compounds to interfere with linchpins of metabolic reprogramming, Pitstop-2 and 1,6-hexanediol (1,6-HD). 1,6-HD disrupts glycolysis enzymes and mitochondrial function, enhancing reactive oxygen species production and reducing cellular ATP levels, while Pitstop-2 impedes clathrin-mediated endocytosis and small GTPases activity. Besides, both compounds interfere with the integrity of nuclear pore complexes, the gatekeepers for all nucleocytoplasmic transport. Herein, we investigate the possible synergistic effects of both compounds on lowly, highly metastatic, and erlotinib-resistant non-small cell lung cancer. We observe a synergistic cytotoxic effect on erlotinib-resistant cells. Moreover, motility assays show that the compounds combination significantly impedes the motility of all cells. Drug safety and tolerability assessments were validated using the in vivo model organism Caenorhabditis elegans, where fairly high doses showed negligible impact on survival, development, or behavioral parameters. Our findings propose that the 1,6-HD and Pitstop-2 combination may usher in the design of potent strategies for treating advanced lung cancer.

A forward genetic screen identifies potassium channel essentiality in SHH medulloblastoma maintenance.

Distinguishing tumor maintenance genes from initiation, progression, and passenger genes is critical for developing effective therapies. We employed a functional genomic approach using the Lazy Piggy transposon to identify tumor maintenance genes invivo and applied this to sonic hedgehog (SHH) medulloblastoma (MB). Combining Lazy Piggy screening in mice and transcriptomic profiling of human MB, we identified the voltage-gated potassium channel KCNB2 as a candidate maintenance driver. KCNB2 governs cell volume of MB-propagating cells (MPCs), with KCNB2 depletion causing osmotic swelling, decreased plasma membrane tension, and elevated endocytic internalization of epidermal growth factor receptor (EGFR), thereby mitigating proliferation of MPCs to ultimately impair MB growth. KCNB2 is largely dispensable for mouse development and KCNB2 knockout synergizes with anti-SHH therapy in treating MB. These results demonstrate the utility of the Lazy Piggy functional genomic approach in identifying cancer maintenance drivers and elucidate a mechanism by which potassium homeostasis integrates biomechanical and biochemical signaling to promote MB aggression.

Deltacoronavirus HKU11, HKU13, PDCoV (HKU15) and HKU17 spike pseudoviruses enter avian DF-1 cells via clathrin-mediated endocytosis in a Rab5-, Rab7- and pH-dependent manner

Porcine deltacoronavirus (PDCoV), also known as HKU15, is a swine enteropathogenic virus that is believed to have originated in birds. PDCoV belongs to the genus Deltacoronavirus (DCoV), the members of which have mostly been identified in diverse avian species. We recently reported that chicken or porcine aminopeptidase N (APN), the major cellular receptor for PDCoV, can mediate cellular entry via three pseudotyped retroviruses displaying spike proteins from three avian DCoVs (HKU11, HKU13, and HKU17). In the present work, to better understand how avian-origin CoVs may be transmitted to pigs, we investigated the unknown DCoV entry pathway in avian cells. We show that clathrin-mediated endocytosis is involved in the entry of these DCoV pseudoviruses into chicken-origin DF-1 cells. Pseudovirus entry was suppressed by means of pharmacological inhibitors, dominant-negative mutants, and siRNAs targeting various cellular proteins and signalling molecules, suggesting that PDCoV and avian DCoV pseudovirus entry into DF-1 cells depends on clathrin, dynamin-2, cathepsins and a low-pH environment but is independent of caveolae and macropinocytosis. Furthermore, we found that DCoV pseudovirus entry was linked to Rab5- and Rab7-dependent pathways. This is the first report demonstrating that these DCoVs utilize clathrin-mediated endocytosis pathways to enter avian-origin cells, providing new insights into interspecies transmission of DCoVs.

Binding Kinetics, Bias, Receptor Internalization and Effects on Insulin Secretion in vitro and in vivo of a Novel GLP-1R/GIPR Dual Agonist, HISHS-2001.

The use of incretin analogues has emerged in recent years as an effective approach to achieve both enhanced insulin secretion and weight loss in type 2 diabetes (T2D) patients. Agonists which bind and stimulate multiple receptors have shown particular promise. However, off target effects, including nausea and diarrhoea, remain a complication of using these agents, and modified versions with optimized pharmacological profiles and/or biased signaling at the cognate receptors are increasingly sought. Here, we describe the synthesis and properties of a molecule which binds to both glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptors (GLP-1R and GIPR) to enhance insulin secretion. HISHS-2001 shows increased affinity at the GLP-1R, as well as a tendency towards reduced internalization and recycling at this receptor versus FDA-approved dual GLP-1R/GIPR agonist tirzepatide. HISHS-2001 also displayed significantly greater bias towards cAMP generation versus β-arrestin 2 recruitment compared to tirzepatide. In contrast, Gαs recruitment was lower versus tirzepatide at the GLP-1R, but higher at the GIPR. Administered to obese hyperglycaemic db/db mice, HISHS-2001 increased circulating insulin whilst lowering body weight and HbA1c with similar efficacy to tirzepatide at substantially lower doses. Thus, HISHS-2001 represents a novel dual receptor agonist with an improved pharmacological profile.

Botulinum Toxin: A Comprehensive Review of Its Molecular Architecture and Mechanistic Action.

Botulinum toxin (BoNT), the most potent substance known to humans, likely evolved not to kill but to serve other biological purposes. While its use in cosmetic applications is well known, its medical utility has become increasingly significant due to the intricacies of its structure and function. The toxin's structural complexity enables it to target specific cellular processes with remarkable precision, making it an invaluable tool in both basic and applied biomedical research. BoNT's potency stems from its unique structural features, which include domains responsible for receptor recognition, membrane binding, internalization, and enzymatic cleavage. This division of labor within the toxin's structure allows it to specifically recognize and interact with synaptic proteins, leading to precise cleavage at targeted sites within neurons. The toxin's mechanism of action involves a multi-step process: recognition, binding, and catalysis, ultimately blocking neurotransmitter release by cleaving proteins like SNAP-25, VAMP, and syntaxin. This disruption in synaptic vesicle fusion causes paralysis, typically in peripheral neurons. However, emerging evidence suggests that BoNT also affects the central nervous system (CNS), influencing presynaptic functions and distant neuronal systems. The evolutionary history of BoNT reveals that its neurotoxic properties likely provided a selective advantage in certain ecological contexts. Interestingly, the very features that make BoNT a potent toxin also enable its therapeutic applications, offering precision in treating neurological disorders like dystonia, spasticity, and chronic pain. In this review, we highlight the toxin's structural, functional, and evolutionary aspects, explore its clinical uses, and identify key research gaps, such as BoNT's central effects and its long-term cellular impact. A clear understanding of these aspects could facilitate the representation of BoNT as a unique scientific paradigm for studying neuronal processes and developing targeted therapeutic strategies.

Triple-Negative Breast Cancer Aptamer-Targeting Porous Silicon Nanocarrier.

Common treatment approaches for triple-negative breast cancer (TNBC) are associated with severe side effects due to the unfavorable biodistribution profile of potent chemotherapeutics. Here, we explored the potential of TNBC-targeting aptamer-decorated porous silicon nanoparticles (pSiNPs) as targeted nanocarriers for TNBC. A "salt-aging" strategy was employed to fabricate a TNBC-targeting aptamer functionalized pSiNP that was highly colloidally stable. Doxorubicin (Dox) was efficiently loaded into nanoparticles (179 ± 5 μg/mg of pSiNP) and experienced pH-dependent release kinetics. Further experiments highlighted that clathrin-mediated endocytosis was the primary route that aptamer-pSiNP conjugates take to enter the endolysosomal compartment of the MCF10Ca1h TNBC cells. A time-interval colocalization study shows the accumulation of an aptamer-decorated pSiNP conjugate in the lysosomes of TNBC cells, unlike for antibody-decorated pSiNPs, leading to particle-induced lysosomal swelling and membrane destabilization. Dox-loaded aptamer-pSiNPs efficiently reduced the viability of the TNBC cells (11.8 ± 1.5%) compared to nontargeted nanoparticles (58.2 ± 8.8%) while the developed system showed a low level of toxicity in healthy cells, both in vitro and in vivo. These findings have laid the foundation for further investigating the potential of aptamer-pSiNP conjugates as a targeted treatment strategy in preclinical TNBC models.

Exploring the potential of ferulic acid-loaded nanostructured lipid carriers: angiotensin inhibition via docking, formulation and pharmacokinetic and pharmacodynamics studies.

Ferulic acid (FA) is a natural phenolic compound that has been documented for its antioxidant properties and potential in managing hypertension. However, its use is limited due to poor solubility and permeability (BCS Class IV classification). To overcome this, nanostructured lipid carriers (NLCs) of FA were developed using the emulsification probe sonication technique, with formulation optimized through Box-Behnken design. The optimized FA-NLCs (F12) demonstrated a particle size of 103.4 nm, zeta potential of -43.6 mV, polydispersity index of 0.531, and entrapment efficiency of 88.9%. Key Findings of the research manifested, that during in-vitro release studies, FA-NLCs showed sustained release action (40.34% over 24 h) compared to plain FA (103.13% in 4 h). Pharmacokinetics of FA-NLC suggested that increased Cmax by 2.6-fold, AUC by 1.9-fold, and half-life significantly (p < .001), also Pharmacodynamics revealed that FA-NLCs reduced blood pressure more effectively (39.9 mmHg vs. 30.8 mmHg for plain FA; p < .001). Furthermore, FA-NLC was showing successful intestinal uptake through lymphatic absorption via clathrin-mediated endocytosis, bypassing first-pass metabolism, hence showed enhancement in bioavailability, Thus the study concluded that FA-NLCs significantly improve therapeutic efficacy and sustained blood pressure reduction compared to plain FA.

Human parietal epithelial cells as Trojan horses in albumin overload

Parietal Epithelial Cells (PECs) activation and proliferation are common to several distinct forms of glomerulopathies. Due to several stimuli, PECs can change to a progenitor (CD24+ and CD133/2+) or a pro-sclerotic (CD44+) phenotype. In addition, PECs, which are constantly exposed to filtered albumin, are known to be involved in albumin internalization, but how this mechanism occurs is unknown. We hypothesized that PECs can transport albumin via receptor-mediated endocytosis and that albumin overload may affect the state of PECs. Conditionally immortalized human PECs (hPECs) were incubated with different albumin concentrations at different times. Albumin internalization studies were performed. Protein expression was assessed using In-Cell Western and immunofluorescence. Cell morphology was analyzed by phase-contrast microscopy and F-actin staining. We demonstrate that hPECs internalize albumin via receptor-mediated mechanisms. Under albumin stimulation, megalin, cubilin, ClC-5, CD133/2, CD24, and CD44 were upregulated. The increase of pERK1/2, the upregulation of ROCK1, ROCK2, caspase -3, -6, and -7, and the morphological changes associated with loss of F-actin fibers indicated that inflammation, proliferation and apoptosis mechanisms had been activated. Our results demonstrate that long-term exposure to high doses of albumin induces up-regulation of molecules involved in the tubular protein uptake machinery and suggest that albumin overload is able to trigger a regenerative process as well as an activation state which might lead in vivo to glomerular crescent formation.

Live cell imaging of plant infection provides new insight into the biology of pathogenesis by the rice blast fungus Magnaporthe oryzae.

Magnaporthe oryzae is the causal agent of rice blast, one of the most serious diseases affecting rice cultivation around the world. During plant infection, M. oryzae forms a specialised infection structure called an appressorium. The appressorium forms in response to the hydrophobic leaf surface and relies on multiple signalling pathways, including a MAP kinase phosphorelay and cAMP-dependent signalling, integrated with cell cycle control and autophagic cell death of the conidium. Together, these pathways regulate appressorium morphogenesis.The appressorium generates enormous turgor, applied as mechanical force to breach the rice cuticle. Re-polarisation of the appressorium requires a turgor-dependent sensor kinase which senses when a critical threshold of turgor has been reached to initiate septin-dependent re-polarisation of the appressorium and plant infection. Invasive growth then requires differential expression and secretion of a large repertoire of effector proteins secreted by distinct secretory pathways depending on their destination, which is also governed by codon usage and tRNA thiolation. Cytoplasmic effectors require an unconventional Golgi-independent secretory pathway and evidence suggests that clathrin-mediated endocytosis is necessary for their delivery into plant cells. The blast fungus then develops a transpressorium, a specific invasion structure used to move from cell-to-cell using pit field sites containing plasmodesmata, to facilitate its spread in plant tissue. This is controlled by the same MAP kinase signalling pathway as appressorium development and requires septin-dependent hyphal constriction. Recent progress in understanding the mechanisms of rice infection by this devastating pathogen using live cell imaging procedures are presented.

Cell migration signaling through the EGFR-VAV2-Rac1 pathway is sustained in endosomes.

Ligand binding to EGFR activates Rho family GTPases, triggering actin cytoskeleton reorganization, cell migration and invasion. Activated EGFR is also rapidly endocytosed but the role of EGFR endocytosis in cell motility is poorly understood. Hence, we used live-cell microscopy imaging to demonstrate that endogenous fluorescently labeled VAV2, a guanine nucleotide exchange factor for Rho GTPases, is co-endocytosed with EGFR in genome-edited human oral squamous cell carcinoma (HSC3) cells, an in vitro model for head-and-neck cancer where VAV2 is known to promote metastasis and is associated with poor prognosis. Chemotactic migration of HSC3 cells toward an EGF gradient is found to require both VAV2 and clathrin-mediated endocytosis. Moreover, sustained activation of Rac1, a Rho family GTPase promoting cell migration and a major substrate of VAV2, also depends on clathrin. Endogenous fluorescently labeled Rac1 localizes to EGFR-containing endosomes. Altogether, our findings suggest that signaling through the EGFR-VAV2-Rac1 pathway persists in endosomes and that this endosomal signaling is required for EGFR-driven cell migration.

An environment-responsive platform based on acid-resistant metal organic framework for efficient oral insulin delivery

Oral insulin delivery is considered a revolutionary alternative to daily subcutaneous injections in terms of compliance and convenience. However, significant challenges remain in terms of inactivation in gastrointestinal environment and limited permeation across the intestinal epithelium. Herein, we used acid-resistant metal-organic framework (PCN-222) to load insulin and modified the exterior with sodium dodecyl sulfate (SDS) to achieve efficient oral insulin delivery. The PCN-222 nanocarrier with ordered mesoporous cage structure and suitable pore size achieved a high insulin loading of 75 %. The SDS on the surface of nanocarrier reduces its hydrophilicity while reversibly altering cell morphology and increasing epithelial cell permeability, thereby promoting intestinal epithelial absorption. The constructed particle (I@P@S) was encapsulated in sodium alginate (SA) microspheres to protect it from gastric acid degradation and releases it upon entry into the intestinal tract. Through an uptake pathway dominated by clathrin-mediated endocytosis, the released I@P@S realized efficient intestinal permeability and controlled insulin release under physiological conditions due to the phosphate sensitivity of PCN-222, leading to an in vivo bioavailability of 12.9 %. This work provides a valuable reference for the design of oral insulin delivery systems.

Anionic polymer coating for enhanced delivery of Cas9 mRNA and sgRNA nanoplexes.

Polymeric carriers have long been recognized as some of the most effective and promising systems for nucleic acid delivery. In this study, we utilized an anionic di-block co-polymer, PEG-PLE, to enhance the performance of lipid-modified PEI (C14-PEI) nanoplexes for delivering Cas9 mRNA and sgRNA targeting KRAS G12S mutations in lung cancer cells. Our results demonstrated that PEG-PLE, when combined with C14-PEI at a weight-to-weight ratio of 0.2, produced nanoplexes with a size of approximately 140 nm, a polydispersity index (PDI) of 0.08, and a zeta potential of around -1 mV. The PEG-PLE/C14-PEI nanoplexes at this ratio were observed to be both non-cytotoxic and effective in encapsulating Cas9 mRNA and sgRNA. Confocal microscopy imaging revealed efficient endosomal escape and intracellular distribution of the RNAs. Uptake pathway inhibition studies indicated that the internalization of PEG-PLE/C14-PEI primarily involves scavenger receptors and clathrin-mediated endocytosis. Compared to C14-PEI formulations, PEG-PLE/C14-PEI demonstrated a significant increase in luciferase mRNA expression and gene editing efficiency, as confirmed by T7EI and ddPCR, in A549 cells. Sanger sequencing identified insertions and/or deletions around the PAM sequence, with a total of 69% indels observed. Post-transfection, the KRAS-ERK pathway was downregulated, resulting in significant increases in cell apoptosis and inhibition of cell migration. Taken together, this study reveals a new and promising formulation for CRISPR delivery as potential lung cancer treatment.

Recent advances in biotin-based therapeutic agents for cancer therapy.

Biotin receptors, as biomarkers for cancer cells, are overexpressed in various tumor types. Compared to other vitamin receptors, such as folate receptors and vitamin B12 receptors, biotin receptor-based targeting strategies exhibit superior specificity and broader potential in treating aggressive cancers, including ovarian cancer, leukemia, colon cancer, breast cancer, kidney cancer, and lung cancer. These strategies promote biotin transport via receptor-mediated endocytosis, which is triggered upon ligand binding. Biotin, as the ligand of the biotin receptor, can be conjugated to anti-cancer drugs to form targeted therapies that bind to receptors overexpressed on tumor cells, thus increasing drug uptake. Despite these advantages, many candidate drugs have progressed slowly and remain in the preclinical stage, impeding clinical translation. This is mainly due to the effects of various conjugation methods and drug formulations on their functionality and efficacy. Therefore, developing novel biotin-based therapeutics is crucial. The innovation of this strategy lies in its multifunctionality-researchers can use different conjugation methods to design and synthesize these drugs, enabling precise targeting of various tumor types while minimizing toxicity to normal cells. These drugs include small-molecule-biotin conjugates (SMBCs) and nano-biotin conjugates (NBCs). This dual-platform approach represents a significant advancement in targeted therapy, offering unprecedented flexibility in drug design and delivery. Compared to chemotherapy drugs and traditional delivery systems, biotin-based drugs with tumor-specific targeting demonstrate enhanced targeting, improved efficacy, and reduced toxicity. This review examines strategies and applications for enhancing the delivery of chemotherapy drugs to cancer cells, highlighting the need for high-quality conjugates and strategies. It not only summarizes the latest progress but also provides key insights into how this emerging field could revolutionize personalized cancer treatment, especially in the context of precision medicine. Additionally, it offers perspectives on future research directions in this field.