Receptor-mediated Endocytosis Research Articles

A Pioneer Review on Lactoferrin-Conjugated Extracellular Nanovesicles for Targeting Cellular Melanoma: Recent Advancements and Future Prospects.

Melanoma, a highly aggressive form of skin cancer, presents a formidable challenge in terms of treatment due to its propensity for metastasis and resistance to conventional therapies. The development of innovative nanocarriers for targeted drug delivery has opened new avenues in cancer therapy. Lactoferrin-conjugated extracellular nanovesicles (LF-EVs) have emerged as a promising vehicle in the targeted treatment of cellular melanoma, owing to their natural biocompatibility, enhanced bioavailability, and ability to traverse biological barriers effectively. This review synthesizes recent advancements in the use of LF-EVs as a novel drug delivery system for melanoma, emphasizing their unique capacity to enhance cellular uptake through LF's receptor-mediated endocytosis pathways. Key studies demonstrate that LF conjugation significantly increases the specificity of extracellular nanovesicles for melanoma cells, minimizes off-target effects, and promotes efficient intracellular drug release. Furthermore, we explore how LF-EVs interact with the tumor microenvironment, potentially inhibiting melanoma progression and metastasis while supporting antitumor immune responses. Future prospects in this field include optimizing LF conjugation techniques, improving the scalability of LF-EV production, and integrating multifunctional payloads to target drug resistance mechanisms. This review highlights the potential of LF-EVs to transform melanoma treatment strategies, bridging current gaps in therapeutic delivery and paving the way for personalized and less invasive melanoma therapies.

Third intracellular loop of HCMV US28 is necessary for signaling and viral reactivation.

The human cytomegalovirus (HCMV) encoded chemokine receptor US28 plays a critical role in viral pathogenesis, mediating several processes such as cellular migration, differentiation, transformation, and viral latency and reactivation. Despite significant research examining the signal transduction pathways utilized by US28, the precise mechanism by which US28 activates these pathways remains unclear. We performed a mutational analysis of US28 to identify signaling domains that are critical for functional activities. Our results indicate that specific residues within the third intracellular loop (ICL3) of US28 are major determinants of G-protein coupling and downstream signaling activity. Alanine substitutions at positions S218, K223, and R225 attenuated US28-mediated activation of MAPK and RhoA signal transduction pathways. Furthermore, we show that mutations at positions S218, K223, or R225 result in impaired coupling to multiple Gα isoforms. However, these substitutions did not affect US28 plasma membrane localization or the receptor internalization rate. Utilizing CD34+ HPC models, we demonstrate that attenuation of US28 signaling via mutation of residues within the ICL3 region results in an inability of the virus to efficiently reactivate from latency. These results were recapitulated in vivo, utilizing a humanized mouse model of HCMV infection. Together, our results provide new insights into the mechanism by which US28 manipulates host signaling networks to mediate viral latency and reactivation. The results reported here will guide the development of targeted therapies to prevent HCMV-associated disease.IMPORTANCEHuman cytomegalovirus (HCMV) is a β-herpesvirus that infects between 44% and 100% of the world population. Primary infection is typically asymptomatic and results in the establishment of latent infection within CD34+hematopoietic progenitor cells (HPCs). However, reactivation from latent infection remains a significant cause of morbidity and mortality in immunocompromised individuals. The viral chemokine receptor US28 influences various cellular processes crucial for viral latency and reactivation, yet the precise mechanism by which US28 functions remains unclear. Through mutational analysis, we identified key residues within the third intracellular loop (ICL3) of US28 that govern G-protein coupling, downstream signaling, and viral reactivation in vitro and in vivo. These findings offer novel insights into how US28 manipulates host signaling networks to regulate HCMV latency and reactivation and expand our understanding of HCMV pathogenesis.

Multiple mechanisms of allosteric regulation of the luteninizing hormone receptor

The regulatory effects of luteinizing hormone (LH) and chorionic gonadotropin (CG) are realized through the activation of the G-protein coupled LH/CG receptor (LH/CG-R). The result of this is the activation of various types of G proteins, which leads to stimulation (Gs) or inhibition (Gi) of the cAMP-dependent pathway and stimulation of calcium signaling (Gq/11, Gi), and the recruitment of β-arrestins, which prevent G protein signaling through receptor internalization and downregulation, but can also activate the mitogen-activated protein kinase cascade. Despite a certain similarity in the effects of LH and CG, there are differences between them both in efficiency and in the pattern of regulation of LH/CG-R. This is a consequence of differences in the affinity of LH and CG to the orthosteric site of the receptor, as well as differences at the level of allosteric regulation of the receptor, which is due to the presence of a C-terminal extension in the β-subunit of CG, including sites for O-glycosylation, and the variability of N-glycosylation of α- and β-subunits of gonadotropins. Moreover, the number of N-glycans, the degree of their branching and charge differ, which leads to different efficiency of activation of intracellular cascades, affecting the physiological response of the reproductive system to gonadotropins. Of great importance is the formation of homodi(oligo)meric complexes of LH/CG-R and its heterocomplexes with the follicle-stimulating hormone receptor, where protomers allosterically influence the efficiency of LH/CG-R activation and the bias of signal transduction. Taking into account the large number of allosteric sites in LH/CG-R, the development of low-molecular allosteric regulators is underway, including agonists based on thieno[2,3-d]-pyrimidine and peptides derived from the cytoplasmic loops of LH/CG-R. These regulators can become prototypes of drugs for correcting the functions of the reproductive system. This review is devoted to the analysis of data on the similarities and differences in the signaling and physiological effects of gonadotropins with LH activity, the role of allosteric mechanisms in this, and the prospects for creating allosteric regulators of LH/CG-R.

Myosin VI drives arrestin-independent internalization and signaling of GPCRs

G protein-coupled receptor (GPCR) endocytosis is canonically associated with β-arrestins. Here, we delineate a β-arrestin-independent endocytic pathway driven by the cytoskeletal motor, myosin VI. Myosin VI engages GIPC, an adaptor protein that binds a PDZ sequence motif present at the C-terminus of several GPCRs. Using the D2 dopamine receptor (D2R) as a prototype, we find that myosin VI regulates receptor endocytosis, spatiotemporal localization, and signaling. We find that access to the D2R C-tail for myosin VI-driven internalization is controlled by an interaction between the C-tail and the third intracellular loop of the receptor. Agonist efficacy, co-factors, and GIPC expression modulate this interaction to tune agonist trafficking. Myosin VI is differentially regulated by distinct GPCR C-tails, suggesting a mechanism to shape spatiotemporal signaling profiles in different ligand and physiological contexts. Our biophysical and structural insights may advance orthogonal therapeutic strategies for targeting GPCRs through cytoskeletal motor proteins.

Identification of cepharanthine as an effective inhibitor of African swine fever virus replication

ABSTRACT African swine fever virus (ASFV) causes highly contagious swine disease, African swine fever (ASF), thereby posing a severe socioeconomic threat to the global pig industry and underscoring that effective antiviral therapies are urgently required. To identify safe and efficient anti-ASFV compounds, a natural compound library was screened by performing an established cell-based ELISA in an ASFV-infected porcine alveolar macrophage (PAM) model. In total, 6 effective anti-ASFV compounds with low cytotoxicity were identified. Cepharanthine (CEP), a bisbenzylisoquinoline alkaloid, was the most potent inhibitor effect with an IC50 of 0.3223 μM. To further investigate the mechanism through which CEP inhibits ASFV replication, transcriptome profiles were generated in PAMs treated with CEP and/or infected with ASFV. ASFV infection dramatically altered immune response-associated gene expression. CEP treatment upregulated the expression of cholesterol biosynthesis-related genes, regardless of infection status. According to time-of-addition experiments, CEP primarily exerts its antiviral effect during the early stages of ASFV infection, specifically by inhibiting viral entry. Transcriptomic analysis suggested that CEP blocks ASFV entry through the clathrin-mediated endocytosis pathway by increasing EHD2 gene expression in macrophages. Disrupting EHD2 with small interfering RNA promoted ASFV entry into clathrin-positive vesicles. Finally, the protective effect of CEP in vivo was evaluated using ASFV-infected pigs. CEP could provide partial protection against ASFV infection, as indicated by an increase in survival time from 9.67 days to 16.67 days. Our findings imply that CEP exhibits potential antiviral activity against ASFV infection in PAMs, positioning it as a promising therapeutic strategy for ASF.

Bifunctional Oxaliplatin (IV) Prodrug Based pH-Sensitive PEGylated Liposomes for Synergistic Anticancer Action Against Triple Negative Breast cancer.

Triple negative breast cancer (TNBC) exhibits higher susceptibility towards oxaliplatin (OXA) due to a faulty DNA damage repair system. However, the unfavorable physicochemical properties and risk of toxicities limit the clinical utility of OXA. Therefore, to impart kinetic inertness, site-specific delivery, and multidrug action, an octahedral Pt(IV) prodrug was developed by using chlorambucil (CBL) as a choice of ligand. The combination of OXA and CBL exhibited synergistic anti-cancer action in TNBC cell lines. Further, to maximize tumor-specific delivery, intracellular accumulation, and in-vivo performance, the developed prodrug (OXA-CBL) was encapsulated in pH-sensitive PEGylated liposomes into (OXA-CBL/PEG-Liposomes). The fabricated liposomes had smaller particle size < 200nm and higher drug loading (~ 4.26 ± 0.18%). In-vitro release displayed pH-dependent sustained release for up to 48h. Cellular internalization revealed maximal uptake via clathrin-mediated endocytosis. The cytotoxicity assay showed reduced IC50 in the 4T1 (~ 1.559-fold) and MDA-MB-231 (~ 1.539-fold) cell lines than free OXA-CBL. In-vivo efficacy in 4T1-induced TNBC model revealed a marked increase in % tumor inhibition rate, while diminished % tumor burden in OXA-CBL/BSA-NPs treated animals. Toxicity assessment displayed no signs of systemic and hemolytic toxicity. Overall, delivery of Pt (IV) prodrug as a pH-sensitive PEGylated liposomes offers a safer and efficient system to manage TNBC.

TRPML-1 Dysfunction and Renal Tubulopathy in Mucolipidosis Type IV.

Loss of function mutations in the lysosomal channel TRPML-1 cause mucolipidosis type IV (MLIV), a rare lysosomal storage disease characterized by neurological defects, progressive vision loss, and achlorhydria. Recent reports have highlighted kidney disease and kidney failure in patients with MLIV during the second to third decade of life; however, the molecular mechanisms driving kidney dysfunction remain poorly understood. A cross-sectional review of medical records from 21 MLIV patients (ages 3-43 years) was conducted to assess kidney function impairment. Additionally, we examined the kidney phenotype of MLIV mice at various ages, along with human kidney cells silenced for TRPML-1 and primary tubular cells from wild-type and MLIV mice. Immunohistology and cell biology approaches were used to phenotype nephron structure, the endolysosomal compartment, and inflammation. Kidney function was assessed through proteomic analysis of mouse urine and in vivo renal filtration measurements. Of the 21 MLIV patients only adults were diagnosed with stage 2-3 chronic kidney disease. Laboratory abnormalities included decreased eGFR, higher levels BUN/Creatine in bloodand proteinuria. In MLIV mice, we observed significant alterations in endolysosomal morphology, function, and impaired autophagy in proximal and distal tubules. This led to the accumulation of megalin (LRP2) in the subapical region of proximal tubular cells, indicating a block in apical receptor-mediated endocytosis. In vivo and in vitro experiments confirmed reduced fluid-phase endocytosis and impaired uptake of ligands, including β-lactoglobulin, transferrin, and albumin in MLIV proximal tubular cells. Urine analysis revealed tubular proteinuria and enzymuria in mice with MLIV. Additionally, early-stage disease was marked by increased inflammatory markers, fibrosis, and activation of the pro-inflammatory transcription factor NF-κB, coinciding with endolysosomal defects. Importantly, AAV-mediated TRPML-1 gene delivery reversed key pathological phenotypes in Mucolipidosis type IV mice, underscoring TRPML-1's critical role in kidney function. Our findings link TRPML-1 dysfunction to the development of kidney disease in MLIV, providing new insights into its pathogenesis and potential therapeutic targets.

Pickering high internal phase emulsions stabilized by soy protein isolate/κ-carrageenan complex for enhanced stability, bioavailability, and absorption mechanisms of nobiletin

Nobiletin (NOB), a lipid-soluble polymethoxyflavone with potent antioxidant, antimicrobial, and anti-inflammatory properties, suffers from poor stability and pH sensitivity, limiting its bioavailability. In this study, Pickering high internal phase emulsions (HIPEs) stabilized by soy protein isolate (SPI) and κ-carrageenan (KC) were developed to encapsulate and protect NOB. The emulsions, containing a 75 % medium-chain triglyceride (MCT) volume fraction, were optimized by investigating the effects of pH and KC concentration on the key properties such as the creaming index, particle size, zeta potential, microstructure, and rheology. Results showed that under optimal conditions (pH 7 and 1.0 % KC), the SPI/KC HIPEs exhibited improved physicochemical properties. Furthermore, encapsulation of NOB in HIPEs significantly improved its stability against UV exposure, heat, and storage conditions. Additionally, simulated gastrointestinal digestion studies revealed that the SPI/KC HIPEs improved the digestion stability and bioaccessibility of NOB, with controlled release in the intestinal phase. Moreover, the SPI/KC HIPEs facilitated increased cellular uptake and bioavailability of NOB, with clathrin-mediated endocytosis and macropinocytosis as primary absorption pathways. The encapsulated NOB also showed enhanced inhibition of inflammatory markers, including NO, IL-6, and TNF-α. These findings suggested that SPI/KC HIPEs provided a promising delivery system for improving the bioavailability and bioactivity of hydrophobic compounds.

Monosaccharide coating modulate the intracellular trafficking of gold nanoparticles in dendritic cells

Dendritic cells (DCs) have emerged as a promising target for drug delivery and immune modulation due to their pivotal role in initiating the adaptive immune response. Gold nanoparticles (AuNPs) have garnered interest as a platform for targeted drug delivery due to their biocompatibility, low toxicity and precise control over size, morphology and surface functionalization. Our investigation aimed to elucidate the intracellular uptake and trafficking of AuNPs coated with different combinations of monosaccharides (mannose, galactose, and fucose) in DCs. We used 30 unique polymer-tethered monosaccharide combinations to coat 16 nm diameter spherical gold nanoparticles and investigated their effect on DCs phenotype, uptake, and intracellular trafficking.DCs internalised AuNPs coated with 100% fucose, 100% mannose, 90% mannose + 10% galactose, and 80% mannose + 20% galactose with highest efficiency. Flow cytometry analysis indicated that 100% fucose-coated AuNPs showed increased lysosomal and endosomal contents compared to other conditions and uncoated AuNPs. Imaging flow cytometry further demonstrated that 100% fucose-coated AuNPs had enhanced co-localisation with lysosomes, while 100% mannose-coated AuNPs exhibited higher co-localisation with endosomes.Furthermore, our data showed that the uptake of carbohydrate-coated AuNPs predominantly occurred through receptor-mediated endocytosis, as evidenced by a marked reduction of uptake upon treatment of DCs with methyl-β-cyclodextrins, known to disrupt receptor-mediated endocytosis. These findings highlight the utility of carbohydrate coatings to enable more targeted delivery of nanoparticles and their payload to distinct intracellular compartments in immune cells with potential applications in drug delivery and immunotherapy.

XAF1 is secreted from stressed tumor cells to activate T cell-mediated tumor surveillance via Lck-ERK signaling

X-linked inhibitor of apoptosis-associated factor 1 (XAF1) is a stress-inducible tumor suppressor that is commonly inactivated in multiple types of human malignancies. Nevertheless, the molecular basis for the XAF1-mediated tumor suppression remains largely undefined. Here, we report that XAF1 is secreted from cells under various cytotoxic stress conditions and activates T cell-mediated tumor surveillance. In cancer cells exposed to interferon −γ, tumor necrosis factor −α, and etoposide, XAF1 is elevated and actively secreted through the unconventional endo-lysosomal trafficking pathway and the zinc finger 4 domain of XAF1 plays an essential for this secretion. Secreted XAF1 is internalized into nearby T cells through clathrin-mediated endocytosis and stimulates proliferation, migration, and tumor infiltration of T cells. Internalized XAF1 activates RAF-MEK-ERK signaling through the direct interaction with and phosphorylation of lymphocyte-specific protein tyrosine kinase. In response to interferon −γ injection, Xaf1+/+ tumors display significantly higher regression rate and T cell infiltration compared to Xaf1−/− tumors while Xaf1−/− tumors are markedly reduced by injection of recombinant Xaf1. XAF1 expression is associated with overall survival in T cell-enriched cancer patients and also correlates with prognosis in T cell-based immunotherapies. Together, our study identifies XAF1 as a novel secretory immune-modulatory tumor suppressor, illuminating the mechanistic consequence of its inactivation in tumorigenesis.

The ultrastructural and proteomic analysis of mitochondria-associated endoplasmic reticulum membrane in the midbrain of a Parkinson's disease mouse model.

Recent studies indicated that the dysregulation of mitochondria-associated endoplasmic reticulum membrane (MAM) could be a significant hub in the pathogenesis of Parkinson's disease (PD). However, little has been known about how MAM altered in PD. This study was aimed to observe morphological changes and analyze proteomic profiles of MAM in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse models. In MPTP-treated mice, transmission electron microscopy was applied for MAM ultrastructural visualization. Nano ultra-high performance liquid chromatography-tandem mass spectrum and bioinformatic analysis were adopted to obtain underlying molecular data of MAM fractions. The loosened, shortened and reduced MAM tethering was found in substantia nigral neurons from MPTP-treated mice. In midbrain MAM proteomics, 158 differentially expressed proteins (DEPs) were identified between two groups. Specific DEPs were validated by western blot and exhibited significantly statistical changes, aligning with proteomic results. Bioinformatic analysis indicated that membrane, cytoplasm and cell projection were three major localizations for DEPs. Biological processes including metabolism, lipid transport, and immunological and apoptotic signaling pathways were greatly affected. For consensus MAM proteins, the enriched pathway analysis revealed the potential relationship between neurodegenerative diseases and MAM. Several biological processes such as peroxisome function and clathrin-mediated endocytosis, were clustered, which provided additional insights into the fundamental molecular pathways associated with MAM. In our study, we demonstrated disrupted ER-mitochondria contacts in an MPTP-induced PD mouse model. The underlying signatures of MAM were revealed by proteomics and bioinformatic analysis, providing valuable insights into its potential role in PD pathogenesis.

Impact of pH-Shifting and Autoclaving on the Allergenic Potential of Red Kidney Bean (Phaseolus vulgaris L.) Lectins.

The ingestion of red kidney bean products is hindered by the persistent allergenicity of lectins, even after autoclaving. This study examined the modification of lectin allergenicity in red kidney beans by pH-shifting and autoclaving treatments, utilizing BALB/c mouse sensitization, in situ recirculating perfusion, and a bone marrow-derived dendritic cell (BMDC) model for allergenicity evaluation. Compared to autoclaving alone, combined pH-shifting and autoclaving reduced allergic symptoms in BALB/c mice, as evidenced by lower serum IgE, mMCPT-1, GM-CSF, HIS, IL-2, IL-4, IL-9, IL-13, and IL-17 levels and higher IgG1, IgG2a, IL-10, IFN-γ, and IFN-α cytokine release. Moreover, lectin continued to affect intestinal permeability and damaged the barrier despite undergoing pH-shifting and autoclaving treatments. Additionally, the uptake of lectin by BMDCs through mannose receptor-mediated endocytosis was diminished, with an increased susceptibility to endolysosomal degradation. The T-cell polarization was consistent with the mouse experiments, where the balance of Th1 and Th2 cells remained in lectin with pH-shifting and autoclaving treatments though the decreased abundance ratios of peptide YKYDSNAHT and increased abundance ratios of peptide ITKGNVETN in endolysosomal degradation. Therefore, the immunogenicity of lectins could be decreased by pH-shifting and autoclaving treatments, offering insights into the development of hypoallergenic legume products.

High-affinity ELR+ chemokine ligands show G protein bias over β-arrestin recruitment and receptor internalization in CXCR1 signalling

The human CXC chemokine receptor 1 (CXCR1), a G protein-coupled receptor (GPCR), plays significant roles in inflammatory diseases and cancer. While CXCL8 is a well-established high-affinity ligand for CXCR1, there is no consensus regarding the binding ability of the other ELR+ chemokines (CXCL1-3 and CXCL5-8). Since research has predominantly focused on CXCL8-mediated CXCR1 signalling, insight into potential signalling bias induced by different CXCR1 ligands is lacking. Therefore, in this study we first compared and clarified the binding ability of all ELR+ chemokines using a competition binding assay. In this assay CXCL1-3 and CXCL5 behaved as low-affinity ligands while CXCL6-8 were high affinity ligands. We further investigated potential ligand bias within the CXCR1 signalling system. Using NanoBRET-based assays heterotrimeric G protein dissociation, β-arrestin recruitment and receptor internalisation induced by chemokine binding to CXCR1 were investigated. A quantitative and qualitative investigation of ligand bias showed that the high-affinity ELR+ chemokines were biased towards G protein activation over β-arrestin recruitment and receptor internalisation, when CXCL8 was used as a reference ligand.

Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells

Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells

Long-Circulating and Targeted Liposomes Co-loading Cisplatin and Mifamurtide: Formulation and Delivery in Osteosarcoma Cells.

Osteosarcoma (OS) is one of the most common primary bone sarcoma with high malignant degree and poor prognosis, for which there is an urgent need to develop novel therapeutic approaches. Recent research has revealed that mifamurtide significantly improved the outcome of OS patients when combined with adjuvant chemotherapy drugs including cisplatin (DDP). The present study aimed to construct a drug delivery system co-loading DDP and mifamurtide. Long-circulating targeted liposomes co-loading DDP and mifamurtide were constructed with Soy lecithin (SPC), cholesterol (Chol) and 1,2-distearoylglycero-3-phosphoethanolamine-n-[poly(ethyleneglycol)] (DSPE-PEG), modified with MMP14 targeting peptide BCY-B in the surface of liposomes. In addition to characterization, the cellular uptake, endocytosis pathway and inhibition on cell viability, migration, invasion and cell apoptosis of MG-63 cells were explored. The constructed liposomal delivery possessed the basic characteristics of liposomes and showed high affinity to MG-63 cells, resulting in high uptake efficiency in MG-63 cells. The endocytosis might be involved in multiple pathways including caveolae-mediated endocytosis, clathrin-mediated endocytosis and macropinocytosis, dependently on energy. The constructed long-circulating targeted liposomes co-loading DDP and mifamurtide significantly inhibited the cell viability, migration, invasion and cell apoptosis of MG-63 cells, improving the antitumor effect of DDP and mifamurtide in vitro. The constructed liposomal delivery system is suitable for co-loading DDP and mifamurtide to achieve active tumor targeting, supplying a new strategy for the treatment of OS.

Surface-modified dendrimer nanoconjugate for targeting delivery of rifampicin

ObjectiveThe study aimed to formulate and evaluate the efficiency of surface-modified Polyamidoamine dendrimer (PAMAM) as a targeted nanocarrier for the antimycobacterial agent rifampicin. MethodsThe periphery of dendrimer was modified with mannose using α-D-mannopyranosylphenyl isothiocyanate and characterized using analytical techniques i.e., infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR). Then, rifampicin was encapsulated into mannosylated dendrimers and evaluated for size, shape, encapsulation efficiency (EE%), drug-dendrimer interaction, and drug release. The toxicity of the nanoconjugates were assessed towards macrophages using the MTT technique. The study investigated the internalization of dendrimer nanoparticles into macrophages to assess the impact of mannose conjugation. Dendrimers were fluorescently labelled and the internalization was quantified using a fluorescence spectroscopy and flow cytometer methods. ResultsMannosylated dendrimers with different mannose densities were successfully developed. The nanoparticles were within the nanoscopic scale. The dendrimers appeared spheroidal under scanning electron microscopy (SEM), and thermal analysis confirmed the conjugation of rifampicin into the dendrimers. The nanoparticles exhibited a good EE% for rifampicin at 10.34 % w/w. After surface mannosylation, the EE% further increased, ranging from 43.43 % to 57.91 % w/w. Mannose-functionalized dendrimers prolonged rifampicin release in physiological pH, while a rapid release in pH 4.5 medium was noticed. The cytotoxicity of the dendrimers in RAW macrophages was considerably reduced after mannose functionalization. In vitro studies indicated that mannose significantly increased the macrophage uptake of nanoconjugates, likely via lectin receptor-mediated endocytosis. ConclusionOverall results suggested mannosylated dendrimers as an effective targeted pulmonary delivery system for rifampicin with negligible cytotoxicity.

PD-L1 antibody conjugated dihydrotanshinone I-loaded polymeric nanoparticle for targeted cancer immunotherapy combining PD-L1 blockade with immunogenic cell death

PurposeCombination immune checkpoint inhibitors (ICI) with chemotherapeutic agents has proven to be highly promising in cancer therapy. However, low response rate, immune-related adverse events, and lack of effectively targeted co-delivery strategy are still major hurdles to overcome for this combination therapeutic regimen. Herein, programmed death-L1 (PD-L1) antibody modified and dihydrotanshinone I (DHT) loaded nanoparticle was prepared for tumor targeting drug delivery, thus achieving immune checkpoint blockade (ICB) and immunogenic cell death (ICD) synergistic anti-tumor effects. MethodsThe DHT-loaded nanoparticle (DHT NP) was prepared by the emulsion solvent diffusion method. Atezolizumab (ATEZO) was thiolated with 2-iminothiolane and conjugated to the surface of DHT NP to prepare the ATEZO DHT NP. The drug encapsulation efficiency, drug loading, particle size and drug release were determined. The in vitro cellular uptake, cell proliferation inhibition and apoptosis were evaluated on the HGC-27 tumor cell. The in vivo tumor targeting, anti-tumor efficiency and immune regulation were assessed on tumor bearing mice. ResultsThe optimized ATEZO DHT NP was a spherical nanoparticle of about 250 nm with a continuous drug release profile. It was selectively taken up by the tumor cells through PD-L1 receptor-mediated endocytosis, which resulted in enhanced cytotoxicity and cell apoptosis. In vivo imaging further demonstrated its superior tumor tissue targeting ability. When tumor bearing mice were treated with the ATEZO DHT NP, its synergistic anti-tumor effect was much stronger than that of a single drug. Moreover, the tumor targeting delivery of DHT caused tumor necrosis and initiated ICD with release of tumor-associated antigens, which efficiently up-regulated the population of CD4+ and CD8+ T cells. Notably, there were no obvious system toxicity or tissue damage occur during the whole treatment period. ConclusionThe ATEZO DHT NP could specifically target to tumor and enhance treatment efficiency through combination of PD-L1 blockade with ICD effect.

PIEZO1 attenuates Marfan syndrome aneurysm development through TGF-β signaling pathway inhibition via TGFBR2.

Marfan syndrome (MFS) is a hereditary disorder primarily caused by mutations in the FBN1 gene. Its critical cardiovascular manifestation is thoracic aortic aneurysm (TAA), which poses life-threatening risks. Owing to the lack of effective pharmacological therapies, surgical intervention continues to be the current definitive treatment. In this study, the role of Piezo-type mechanosensitive ion channel component 1 (Piezo1) in MFS was investigated and the activation of PIEZO1 was identified as a potential treatment for MFS. PIEZO1 expression was detected in MFS mice (Fbn1C1041G/+) and patients. Piezo1 conditional knockout mice in vascular smooth muscle cells of MFS mice (MFS × CKO) was generated, and bioinformatics analysis and experiments in vitro and in vivo were performed to investigate the role of Piezo1 in MFS. PIEZO1 expression decreased in the aortas of MFS mice; MFS × CKO mice showed aggravated TAA, inflammation, extracellular matrix remodelling, and TGF-β pathway activation compared to MFS mice. Mechanistically, PIEZO1 knockout exacerbated the activation of the TGF-β signalling pathway by inhibiting the endocytosis and autophagy of TGF-β receptor 2 mediated by Rab GTPase 3C. Additionally, the pharmacological activation PIEZO1 through Yoda1 prevented TGF-β signalling pathway activation and reversed TAA in MFS mice. Piezo1 deficiency aggravates MFS aneurysms by promoting TGF-β signalling pathway activation via TGF-β receptor 2 endocytosis and a decrease in autophagy. These data suggest that PIEZO1 may be a potential therapeutic target for MFS treatment.

Tuned Manganese-Impregnated Mesoporous Silica Nanoparticles as a pH-Responsive Dual Imaging Probe.

The limitations of individual imaging modalities have led to significant interest in hybrid imaging methods that combine the advantages of multiple techniques. The development of diverse dual imaging agents, which offer the exceptional sensitivity of single-photon emission computed tomography (SPECT) and the high spatial resolution of magnetic resonance imaging (MRI), has been addressing the demand for more advanced diagnostic pharmaceuticals. In this study, 99mTc-labeled manganese oxide-loaded mesoporous silica nanoparticles (MSNs), conjugated with folic acid as the targeting moiety and the chelating agent H2pentapa-en-NH2 (99mTc-MnOx-MSN-FA-pa), were developed for targeted SPECT-MRI dual imaging. The toxicity of the nanoparticles was confirmed through an MTT assay, showing >90% viability in HEK-293 and MDA-MB-231 cells at concentrations up to 200 μg/mL, indicating nonsignificant toxicity. Cellular uptake studies showed that folic acid functionalization effectively accentuated tumor-specific intracellular uptake of nanoparticles in MDA-MB-231 cells through folate receptor-mediated endocytosis. Additionally, the radiolabeling yield of 99mTc-MnOx-MSN-FA-pa was found to be 99.6 ± 0.8% (n = 3), and the pH-responsive release of paramagnetic manganese ions increased the r1 relaxivity of the nanoprobe to 11.37 mM-1 s-1. In vivo SPECT imaging demonstrated rapid tracer accumulation in MDA-MB-231 xenografts, with a tumor-to-muscle ratio of 6.01 ± 0.51 at 2 h, and minimal uptake in nontargeted organs. In vivo MRI studies indicated the strongest tumor contrast at 2 h postinjection. Given its desirable contrast enhancement in T1 MRI and SPECT imaging, along with low toxicity, MnOx-MSN-FA-pa shows potential as an effective multifunctional nanoprobe for precise tumor imaging.

Self-assembled Peptide-derived Proteolysis-targeting Chimera (PROTAC) Nanoparticles for Tumor-targeted and Durable PD-L1 Degradation in Cancer Immunotherapy.

Proteolysis-targeting chimeras (PROTACs) are a promising technique for the specific and durable degradation of cancer-related proteins via the ubiquitin-proteasome system in cancer treatment. However, the therapeutic efficacy of PROTACs is restricted due to their hydrophobicity, poor cell permeability and insufficient tumor-targeting ability. Herein, we develop the self-assembled peptide-derived PROTAC nanoparticles (PT-NPs) for precise and durable programmed death-ligand 1 (PD-L1) degradation in targeted tumors. The PT-NPs with an average size of 211.8 nm are formed through the self-assembly of amphiphilic peptide-derived PROTAC (CLQKTPKQC-FF-ALAPYIP), comprising a PD-L1-targeting 'CLQKTPKQC', self-assembling linker 'FF' and E3 ligase recruiting 'ALAPYIP'. Particularly, PT-NPs strongly bind to tumor cell surface PD-L1 to form PD-L1/PT-NPs complex, then internalized through receptor-mediated endocytosis and degraded in lysosomes. Second, free PROTACs released from PT-NPs to the cytoplasm further induce the durable proteolysis of cytoplasmic PD-L1 via the ubiquitin-proteasome system. In colon tumor models, intravenously injected PT-NPs accumulate significantly at targeted tumor tissues through nanoparticle-derived passive and active targeting. At the targeted tumor tissues, PT-NPs promote durable PD-L1 degradation and ultimately trigger a substantial antitumor immune response. Collectively, this study provides valuable insights into the rational design of self-assembled peptide-derived PROTAC NPs to ensure noticeable accuracy and enhanced efficacy in cancer treatment.