Caveolae-mediated Endocytosis Research Articles

Alterations in EGF-Endocytosis, Lysosomal Enzyme Transport and Maturation of Cathepsins in Juvenile Neuronal Ceroid Lipofuscinosis Fibroblasts

Background: Juvenile neuronal ceroid lipofuscinosis (JNCL), one of the most frequent forms of the NCL storage diseases, is known to be caused by loss-of-function mutations in ceroid-lipofuscinosis neuronal protein 3 (CLN3), but its cell function has not been fully elucidated. We previously reported increased lysosomal pH in CLN3 deficient cells. In the present study, we analysed the consequences of this effect in the endo-lysosomal pathways in CLN3 cells. Methods: The present study investigated different endo-lysosomal pathways in control, CLN2, CLN3 human skin fibroblasts under high and low proteolysis conditions. Cell surface biotinylation after EGF (2 ng/mL) stimulation, EGF phosphorylation (Tyr-845), retromer and cation-independent mannose-6- phosphate receptor (CI-MPR) levels and stability, EGF degradation pathways and cathepsin L and D levels were analysed by western blots. Caveolae mediated endocytosis was analysed by flow cytometry. CIMPR subcellular localization was ascertained by immunocytochemistry, confocal microscopy and further image analysis. Results: Whereas caveolae-mediated endocytosis was not affected in CLN3 cells, clathrin-mediated epidermal growth factor (EGF) internalization was reduced, along with EGF receptor (EGFR) phosphorylation. In addition, cell surface EGFR levels and recycling to the cell membrane were increased. EGFR lysosomal degradation was impaired and our results suggest that the receptor was diverted to proteasomal degradation. We also analysed the machinery responsible for lysosomal hydrolase transport to the lysosome and found increased stability of CIMPR, a major receptor implicated in the transport of hydrolases. The subcellular distribution of the CI-MPR was also altered in CLN3 cells, since it accumulated within the Trans-Golgi network (TGN) and did not progress into the lysosomes. In addition, we found a reduced turnover of retromer subunits, a complex that retrieves the CI-MPR from endosomes to the TGN. Finally and as a possible consequence of these alterations in lysosomal enzyme transport, cathepsin L and D maturation were found suppressed in CLN3 cells. Conclusion: Altogether, these results point to increased lisosomal pH as a pivotal event causing various alterations in intracellular traffic associated to the development of JNCL disease.

Synaptotagmin-11 regulates the functions of caveolae and responds to mechanical stimuli in astrocytes.

Caveolae play crucial roles in intracellular membrane trafficking and mechanosensation. In this study, we report that synaptotagmin-11 (Syt11), a synaptotagmin isoform associated with Parkinson's disease and schizophrenia, regulates both caveolae-mediated endocytosis and the caveolar response to mechanical stimuli in astrocytes. Syt11-knockout (KO) accelerated caveolae-mediated endocytosis. Interestingly, the caveolar structures on the cell surface were markedly fewer in the absence of Syt11. Caveolar disassembly in response to hypoosmotic stimuli and astrocyte swelling were both impaired in Syt11-KO astrocytes. Live imaging revealed that Syt11 left caveolar structures before cavin1 during hypoosmotic stress and returned earlier than cavin1 after isoosmotic recovery. Chronic hypoosmotic stress led to proteasome-mediated Syt11 degradation. In addition, Syt11-KO increased the turnover of cavin1 and EH domain-containing protein 2 (EHD2), accompanied by compromised membrane integrity, suggesting a mechanoprotective role of Syt11. Direct interactions between Syt11 and cavin1 and EHD2, but not caveolin-1, are found. Altogether, we propose that Syt11 stabilizes caveolar structures on the cell surface of astrocytes and regulates caveolar functions under physiological and pathological conditions through cavin1 and EHD2.

A mechanistic explanation of the inhibitory role of the protein corona on liposomal gene expression

The past three decades have witnessed fast advances in the use of cationic liposome-DNA complexes (lipoplexes) for gene delivery applications. However, no lipoplex formulation has reached into the clinical practice so far. The primary drawback limiting clinical use of lipoplexes is the lack of mechanistic understanding of their low transfection efficiency (TE) in vivo. In physiological environments, lipoplexes are coated by a protein corona (PC) that mediates the interactions with the cell machinery. Here we show that the formation of PC can change the interactions of multicomponent (MC) lipoplexes with our cell model (i.e., HeLa). At the highest lipoplex concentration, the formation of PC can reduce the TE of MC lipoplexes from 60% to <5%. Combining dynamic light scattering and synchrotron small-angle X-ray scattering (SAXS), we clarify that the formation of PC modifies physical-chemical properties of MC lipoplexes so as to affect their TE. Moreover, we examined single transfection barriers by a combination of fluorescence-activated cell sorting, single-cell real-time fluorescence confocal microscopy, and synchrotron SAXS. We demonstrate that PC formation has the ability to modify the relative contribution of caveolae-mediated endocytosis and macropinocytosis in lipoplexes uptake, in favor of the latter, increasing accumulation of PC-decorated lipoplexes into degradative lysosomal compartments. Finally, we report evidences that PC reduces the structural stability of lipoplexes against solubilization by cellular lipids, likely favoring premature DNA release and cytosolic digestion by DNAase. These combined effects revealed here offer a comprehensive mechanistic explanation on the reason behind reduction in gene expression of MC lipoplexes.

Coomassie Bright Blue-Conjugated Human Serum Albumin Nanoparticles as a Tumor-Selective Weapon for Leukemia Therapy

Leukemia is a group of blood cancers which seriously endangers the health of many people, especially children and adolescents. Although some drugs have been developed to treat leukemia, these drugs always have harmful side effects, which seriously affect the quality of life of patients. Therefore, the development of a special drug which damages only leukemia cells and does not harm normal cells remains a challenge. In a previous study, we developed a novel nanomedicine, Cy5.5MSA-G250, which selectively kills fast-growing tumor cells without harming normal cells. Based on our previous work, we prepared Cy5.5-HSA-G250 nanoparticles (CHGNPs) by replacing mouse serum albumin (MSA) with low immunogenic human serum albumin (HSA), and then applied this nanomaterial in leukemia treatment. Under physiological conditions, the CHGNPs exhibited good dispersion and stability, and hemolytic experiments showed that this material has excellent blood compatibility. The cell endocytosis of CHGNPs was obviously inhibited after treatment with nystatin and chlorpromazine, implying that caveolae-mediated endocytosis and clathrin-mediated endocytosis are involved in the cellular uptake of CHGNPs. Cytotoxicity and apoptosis assays revealed that the CHGNPs severely inhibited leukemia cell proliferation and induced leukemia cell apoptosis but did not affect normal cells. The selective up-regulation of tumor suppressor protein P27 and inducement of G1 arrest in leukemia cells may be the main mechanisms for CHGNPs' selective cytotoxicity. Owing to these advantages of CHGNPs, we believe that CHGNPs can be used as a potential leukemia drug for clinical treatment.

Effect of hydrophobic tails of plier-like cationic lipids on nucleic acid delivery and intracellular trafficking.

In the optimization of transfection efficacy, one of the crucial barriers to effective gene delivery is in fact the intracellular trafficking of nucleic acids, besides the first and the last steps of gene transfer, i.e., delivery to the cell and transcription. Modifications of cationic lipid structure have been reported to have a significant effect on gene delivery. Therefore, the plier-like cationic lipids (PCLs) have been synthesized and the effect of the different types of hydrophobic tails (chain length and unsaturated hydrocarbon) on physicochemical properties, cellular uptake, trafficking process, transfection, and silencing efficiency has been investigated. In this study, the plier-like cationic niosomes (PCNs) containing PCL (A, B, and C) were evaluated their performance to deliver pDNA and siRNA to HeLa cells. Among the PCNs, PCN-B with saturated asymmetric hydrocarbon tails (C18 and C12) provided the highest efficiency for pDNA and siRNA delivery. Furthermore, the results revealed that the structure of the cationic lipids affected the internalization pathway and the intracellular trafficking. PCL-B and PCL-C with asymmetric tails preferred clathrin- and caveolae-mediated endocytosis as the predominant internalization pathways and were also involved in the polymerization process for transfection. However, PCL-A with symmetry hydrocarbon tails (C12) was predominantly taken up via macropinocytosis. All PCNs were able to escape from endosomal-lysosomal systems through facilitation of acidification. Results obtained from the cytotoxicity test revealed that the PCNs were safe in vitro. Therefore, PCNs provide a great prospect as an alternative effective gene delivery system.

Casting Light on Intracellular Tracking of a New Functional Graphene-Based MicroRNA Delivery System by FLIM and Raman Imaging.

The theranostic ability of a new fluorescently labeled cationic cyclodextrin-graphene nanoplatform (GCD@Ada-Rhod) was investigated by studying its intracellular trafficking and its ability to deliver plasmid DNA and microRNA. The nanoplatform was synthesized by both covalent and supramolecular approaches, and its chemical structure, morphology, and colloidal behavior were investigated by TGA, TEM, spectroscopic analysis such as UV-vis, fluorescence emission, DLS, and ζ-potential measurements. The cellular internalization of GCD@Ada-Rhod and its perinuclear localization were assessed by FLIM, Raman imaging, and fluorescence microscopy. Biological experiments with pCMS-EGFP and miRNA-15a evidenced the excellent capability of GCD@Ada-Rhod to deliver both pDNA and microRNA without significant cytotoxicity. The biological results evidenced an unforeseen caveolae-mediated endocytosis internalization pathway (generally expected for particles <200 nm), despite the fact that the GCD@Ada-Rhod size is about 400 nm (by DLS and TEM data). We supposed that the internalization pathway was driven by physical-chemical features of GCD@Ada-Rhod, and the caveolae-mediated uptake enhanced the transfection efficiency, avoiding the lysosomal acid degradation. The cellular effects of internalized miRNA-15a on the oncogene protein BCL-2 were investigated at two different concentrations (N/P = 10 and 5), and a reduction of the BCL-2 level was detected at a low concentration (i.e., N/P = 10). miRNA-15a is considered an ideal cancer therapy molecule due to its activity on multiple transcription factors, and the elucidation of the correlation between the concentration of delivered miRNA-15a and the down-/up-regulation of the BCL-2 level, documented for the first time in this work, could be an important contribution to guide its clinical application.

Optimum Preparation Method for Self-Assembled PEGylation Nano-Adjuvant Based on Rehmannia glutinosa Polysaccharide and Its Immunological Effect on Macrophages.

BackgroundRehmannia glutinosa polysaccharide is the main reason that contributes to the immunological function of R. glutinosa. Due to its disadvantages in clinical use, here we designed the PEGylation nano-RGP (pRL) to improve the drug-targeting effect and the immunological function. Our present work aims to establish the optimum condition of preparing the pRL and to investigate its immunological function on macrophages.MethodspRL was prepared by thin film hydration method combined with ultra-sonication technique. And its preparation conditions were optimized with response surface methodology. Also, the lyophilization method was optimized. The characteristics of the pRL were evaluated, including particle size, drug loading, encapsulation efficiency and morphology. The immunological function of pRL on macrophage was investigated through CCK-8 test, ELISA and flow cytometry.ResultsThe lipid-to-cholesterol molar ratio of 8:1, the addition of DSPE-PEG2000 of 9% and the lipid-to-drug ratio of 5.4:1 were the optimum preparation technology for pRL. The encapsulation efficiency (EE) of pRL under this preparation technology was 95.81±1.58%, with a particle size of 31.98 ± 2.6 nm. The lactose-to-lipid ratio (2:1) was the optimal lyophilization method. pRL promoted macrophage proliferation, which is significantly better than that of nano-RGP without PEGylation (RL). pRL-stimulated RAW264.7 cells showed a high secretion of pro-inflammatory cytokines, which is the characteristic indicator of M1 polarization. Enhanced cellular uptake through macropinocytosis-dependent and caveolae-mediated endocytosis was observed in pRL-treated RAW264.7 cells.ConclusionOur study concluded that PEGylation effectively overcame the poor targeting effect of Rehmannia glutinosa polysaccharide (RGP) and significantly improved the immunological profile of its nano-formulation, which suggested that pRL could serve as an immune adjuvant in clinical application.

Sertraline Reduces Albuminuria by Interfering with Caveolae-Mediated Endocytosis through Glomerular Endothelial and Epithelial Cells

Introduction: Previously, we reported the caveolae-mediated intracellular trafficking pathway of albumin through glomerular endothelial cells (GEnCs) as a new etiological hypothesis of urinary albumin excretion. The selective serotonin reuptake inhibitor, sertraline (Ser), inhibits dynamin, which plays a pivotal role in the fission of caveolae from the cell membrane during caveolae endocytosis. Objective: In this study, we evaluated whether Ser reduces albuminuria levels by interfering with albumin endocytosis through caveolae into GEnCs and podocytes as a novel treatment for glomerulonephritis. Methods: After treating the cells with Ser, albumin and caveolin-1 (Cav-1) expression levels were evaluated by immunofluorescence (IF) and western blot (WB) analyses. The albuminuria level was determined by histology in a puromycin aminonucleoside (PAN)-induced nephrotic syndrome mouse model (PAN mice) treated with or without Ser. Results: IF and WB analyses showed that the albumin expression level was significantly decreased by Ser treatment; however, Cav-1 expression was not decreased in GEnCs or podocytes based on the IF results. In PAN mice treated with or without Ser, Cav-1 expression increased, and the foot process effacement of podocytes and swelling of GEnCs were observed. However, proteinuria levels were not increased in PAN mice treated with Ser relative to that in normal control mice (p = 0.17), and a significant increase was observed in PAN mice without Ser treatment (p = 0.0027). Conclusions: Ser interfered with albumin internalization through the caveolae into GEnCs and podocytes and reduced albuminuria. Dynamin inhibitors may serve as a novel therapeutic option for reducing albuminuria in glomerulonephritis.

Abl family tyrosine kinases govern IgG extravasation in the skin in a murine pemphigus model

The pathway of homeostatic IgG extravasation is not fully understood, in spite of its importance for the maintenance of host immunity, the management of autoantibody-mediated disorders, and the use of antibody-based biologics. Here we show in a murine model of pemphigus, a prototypic cutaneous autoantibody-mediated disorder, that blood-circulating IgG extravasates into the skin in a time- and dose-dependent manner under homeostatic conditions. This IgG extravasation is unaffected by depletion of Fcγ receptors, but is largely attenuated by specific ablation of dynamin-dependent endocytic vesicle formation in blood endothelial cells (BECs). Among dynamin-dependent endocytic vesicles, IgG co-localizes well with caveolae in cultured BECs. An Abl family tyrosine kinase inhibitor imatinib, which reduces caveolae-mediated endocytosis, impairs IgG extravasation in the skin and attenuates the murine pemphigus manifestations. Our study highlights the kinetics of IgG extravasation in vivo, which might be a clue to understand the pathological mechanism of autoantibody-mediated autoimmune disorders.

Low density lipoprotein mimic nanoparticles composed of amphipathic hybrid peptides and lipids for tumor-targeted delivery of paclitaxel.

BackgroundLow density lipoprotein (LDL) has been regarded as a promising antitumor drug vehicle. However some problems, such as rare source, difficulty of large-scale production, and potential safety concerns, hinder its clinical application.PurposeThe objective of this study is to develop a biomimetic LDL nanocarrier by replacing the native apolipoprotein B-100 (apoB-100) with an artificial amphipathic peptide and demonstrate its antitumor efficacy.MethodsThe amphipathic hybrid peptide (termed as FPL) consisting of a lipid binding motif of apoB-100 (LBMapoB)-polyethylene glycol (PEG)-folic acid (FA) was synthesized and characterized by 1H NMR and circular dichroism. FPL decorated lipoprotein-mimic nanoparticles (termed as FPLM NPs) were prepared by a modified solvent emulsification method. Paclitaxel (PTX) was incorporated into NPs and its content was quantified by HPLC analysis. The morphology of NPs was observed by transmission electron microscopy (TEM), and the particle size and zeta potential of NPs were determined by dynamic light scattering (DLS). The colloidal stability of FPLM NPs was evaluated in PBS containing bovine serum albumin (BSA). In vitro release of PTX loaded FPLM NPs was evaluated using the dialysis method. Cellular uptake and cytotoxity assayswere evaluated on human cervical cancer cells (HeLa) and lung cancer cells (A549). Tumor inhibition in vivo was investigated in M109 tumor-bearing mice via tail vein injection of Taxol formulation and PTX loaded NPs.ResultsThe composition of FPLM NPs, including cholesteryl oleate, glyceryl trioleate, cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and FPL peptides, was optimized to be 5:1:1:3:10 (w/w). FPLM NPs had a spherical shape with a mean diameter of 83 nm and a negative charge (-12 mV). FPLM NPs with optimum formulation had good colloidal stability in BSA solution.The release of PTX from FPLM NPs was slow and sustained. The uptake of FPLM NPs was higher in folate receptor (FR) overexpressing tumor cells (HeLa cells) than in FR deficient tumor cells (A549 cells). The intracellular distribution indicated that FPLM NPs had the lysosome escape capacity. The internalization mechanism of FPLM NPs was involved with clathrin- and caveolae-mediated endocytosis and FR played a positive role in the internalization of FPLM NPs. The CCK-8 assay demonstrated that FPLM NPs exhibited notably better anti-tumor effect than Taxol formulation in vitro. Moreover, PTX loaded FPLM NPs produced very marked anti-tumor efficiency in M109 tumor-bearing mice in vivo.ConclusionFPLM NPs is a promising nanocarrier which can improve the therapeutic effect and reduce the side effects of antitumor drugs.

Enhanced Chemical Stability, Intestinal Absorption, and Intracellular Antioxidant Activity of Cyanidin-3-O-glucoside by Composite Nanogel Encapsulation.

A composite nanogel was developed for cyanidin-3-O-glucoside (C3G) delivery by combining Maillard reaction and heat gelation. The starting materials utilized were ovalbumin, dextran, and pectin. C3G-loaded nanogel was spherical with a diameter of ∼185 nm, which was maintained over a wide range of pH and NaCl concentrations. The composite nanogel enhanced the chemical stability of C3G under accelerated degradation models and a simulated gastrointestinal tract. Clathrin-mediated, caveolae-mediated, and macropinocytosis-related endocytosis contributed to the higher cellular uptake of nano-C3G than that of free-C3G. The apparent permeability coefficients of C3G increased 2.16 times after nanoencapsulation. The transcytosis of the C3G-bearing nanogel occurred primarily through the clathrin-related pathway and macropinocytosis and followed the "common recycling endosomes-endoplasmic reticulum-Golgi complex-basolateral plasma membrane" route. Moreover, nano-C3G was more efficient in restoring the viability of cells and activities of endogenous antioxidant enzymes than free-C3G in oxidative models, which may be attributed to the former's high cellular absorption.

Multi-Modal Visualization of Uptake and Distribution of Iron Oxide Nanoparticles in Macrophages, Cancer Cells, and Xenograft Models.

Iron oxide nanoparticles (IONPs) have shown great potential in various biomedical applications. However, information on the interaction between IONPs and biological systems, especially the uptake and distribution of IONPs in cells and tissues, as well as the mechanism of biological action, is relatively limited. In the present study, multi-modal visualization methods, including confocal fluorescence microscopy, transmission electron microscopy, magnetic resonance imaging, and fluorescence optical imaging, were utilized to unveil the uptake and distribution of IONPs in macrophages, cancer cells, and xenograft models. Our results demonstrated that uptake of IONPs in RAW264.7 macrophages and SKOV-3 cancer cells were dose- and cell type-dependent. Cellular uptake of IONPs was an energy-dependent process, and caveolae-mediated endocytosis was the main uptake pathway. All the IONPs were primarily present in endocytic compartments (e.g., endosomes, lysosomes) inside the cells. At 48 hours after intravenous injection of IONPs in SKOV-3 tumor bearing mice, most of the IONPs was distributed in the liver and spleen, with obvious uptake in the tumor, less but significant amount in the kidney and brain. Taken together, multi-modal visualization approaches in our study provide detailed information on the cellular uptake and tissue distribution of IONPs from multiple levels and perspectives.

GH625 Cell-Penetrating Peptide Promotes the Endosomal Escape of Nanovectorized siRNA in a Triple-Negative Breast Cancer Cell Line.

The use of small interfering RNA (siRNA) to regulate oncogenes appears as a promising strategy in the context of cancer therapy, especially if they are vectorized by a smart delivery system. In this study, we investigated the cellular trafficking of a siRNA nanovector (called CS-MSN) functionalized with the cell-penetrating peptide gH625 in a triple-negative breast cancer model. With complementary techniques, we showed that siRNA nanovectors were internalized by both clathrin- and caveolae-mediated endocytosis. The presence of gH625 at the surface of the siRNA nanovector did not modify the entry pathway of CS-MSN, but it increased the amount of siRNA found inside the cells. Results suggested an escape of siRNA from endosomes, which is enhanced by the presence of the peptide gH625, whereas nanoparticles continued their trafficking into lysosomes. The efficiency of CS-MSN to inhibit the GFP in MDA-MB-231 cells was 1.7-fold higher than that of the nanovectors without gH625.

Toxic effects and involved molecular pathways of nanoparticles on cells and subcellular organelles.

Owing to the increasing application of engineered nanoparticles (NPs), besides the workplace, human beings are also exposed to NPs from nanoproducts through the skin, respiratory tract, digestive tract and vein injection. This review states pathways of cellular uptake, subcellular distribution and excretion of NPs. The uptake pathways commonly include phagocytosis, micropinocytosis, clathrin- and caveolae-mediated endocytosis, scavenger receptor-related pathway, clathrin- or caveolae-independent pathway, and direct penetration or insertion. Then the ability of NPs to decrease cell viability and metabolic activity, change cell morphology, and destroy cell membrane, cytoskeleton and cell function was presented. In addition, the lowest dose decreasing cell metabolic viability compared with the control or IC50 of silver, titanium dioxide, zinc oxide, carbon black, carbon nanotubes, silica, silicon NPs and cadmium telluride quantum dots to some cell lines was gathered. Next, this review attempts to increase our understanding of NP-caused adverse effects on organelles, which have implications in mitochondrial dysfunction, endoplasmic reticulum stress and lysosomal rupture. In particular, the disturbance of mitochondrial biogenesis and mitochondrial dynamic fusion-fission, mitophagy and cytochrome c-dependent apoptosis are involved. In addition, prolonged endoplasmic reticulum stress will result in apoptosis. Rupture of the lysosomal membrane was associated with inflammation, and both induction of autophagy and blockade of autophagic flow can result in cytotoxicity. Finally, the network mechanism of the combined action of multiple organelle dysfunction, apoptosis, autophagy and oxidative stress was discussed.

Enzyme-activatable polymer-drug conjugate augments tumour penetration and treatment efficacy.

A tumour microenvironment imposes barriers to the passive diffusion of molecules, which renders tumour penetration an unresolved obstacle to an effective anticancer drug delivery. Here, we present a γ-glutamyl transpeptidase-responsive camptothecin-polymer conjugate that actively infiltrates throughout the tumour tissue through transcytosis. When the conjugate passes on the luminal endothelial cells of the tumour blood vessels or extravasates into the tumour interstitium, the overexpressed γ-glutamyl transpeptidase on the cell membrane cleaves the γ-glutamyl moieties of the conjugate to generate positively charged primary amines. The resulting cationic conjugate undergoes caveolae-mediated endocytosis and transcytosis, which enables transendothelial and transcellular transport and a relatively uniform distribution throughout the tumour. The conjugate showed a potent antitumour activity in mouse models that led to the eradication of small solid tumours (~100 mm3) and regression of large established tumours with clinically relevant sizes (~500 mm3), and significantly extended the survival of orthotopic pancreatic tumour-bearing mice compared to that with the first-line chemotherapeutic drug gemcitabine.

An endosomal LAPF is required for macrophage endocytosis and elimination of bacteria

Macrophages can internalize the invading pathogens by raft/caveolae and/or clathrin-dependent endocytosis and elicit an immune response against infection. However, the molecular mechanism for macrophage endocytosis remains elusive. Here we report that LAPF (lysosome-associated and apoptosis-inducing protein containing PH and FYVE domains) is required for caveolae-mediated endocytosis. Lapf-deficient macrophages have impaired capacity to endocytose and eliminate bacteria. Macrophage-specific Lapf-deficient mice are more susceptible to Escherichia coli (E. coli) infection with higher bacterial loads. Moreover, Lapf deficiency impairs TLR4 endocytosis, resulting in attenuated production of TLR-triggered proinflammatory cytokines. LAPF is localized to early endosomes and interacts with caveolin-1. Phosphorylation of LAPF by the tyrosine kinase Src is required for LAPF-Src-Caveolin complex formation and endocytosis and elimination of bacteria. Collectively, our work demonstrates that LAPF is critical for endocytosis of bacteria and induction of inflammatory responses, suggesting that LAPF and Src could be potential targets for the control of infectious diseases.

Promoting Early Diagnosis and Precise Therapy of Hepatocellular Carcinoma by Glypican-3-Targeted Synergistic Chemo-Photothermal Theranostics.

The specific-targeting approach could promote the specificity of diagnosis and the accuracy of cancer treatment. The choice of a specific-targeting receptor is the key step in this approach. Glypican-3 (GPC3) is an oncofetal proteoglycan anchored on the cell membrane. It is overexpressed even in the early stage of hepatocellular carcinoma (HCC), whereas it shows almost no expression in the healthy adult liver. Therefore, GPC3 may be applied as a specific-targeting receptor for HCC theranostics. In this study, a GPC3 specific-targeting theranostics nanodevice, GPC3 targeting peptide (named G12)-modified liposomes co-loaded with sorafenib (SF) and IR780 iodide (IR780), was developed (GSI-Lip), which aims to realize early diagnosis and precise chemo-photothermal therapy of HCC. SF was the first-line chemotherapy drug for the treatment of HCC. IR780 was used for photothermal therapy and near-infrared fluorescence imaging. The evaluation of early diagnosis verified that early-stage tumors (3.45 ± 0.98 mm3, 2 days after 5 × 105 H22 cells' inoculation in mice) could be clearly detected using GSI-Lip, which was significantly more sensitive than folic acid-modified liposomes ( p < 0.01, 32.90 ± 10.01 mm3, 4 days after 1 × 106 H22 cells' inoculation in mice). The study of the endocytic pathway indicated that specific G12/GPC3 recognition may induce caveolae-mediated endocytosis of GSI-Lip. Notably, the accumulation of GSI-Lip in tumors was significantly increased compared with that observed with folic acid-modified liposomes ( p < 0.01). Specific-targeting endowed the precise antitumor effect of GSI-Lip. GSI-Lip showed a higher antitumor efficacy in comparison with folic acid-modified liposomes (inhibition rate: 90.52% vs 84.22%, respectively; p < 0.01). During a period of 21 days, the synergistic chemo-photothermal therapy (GSI-Lip + laser) exhibited a better antitumor effect versus GSI-Lip without laser (inhibition rate: 94.93% vs 90.52%, respectively; p < 0.01). Overall, GPC3-targeted GSI-Lip promoted the sensitivity and specificity of HCC early diagnosis and achieved synergistic efficacy of chemo-photothermal theranostics, which has potential clinical applications. Furthermore, the present study revealed that a more specific-targeting ligand could further improve the efficacy of theranostics against HCC.

Fabrication of fluorescein labeled galactoxyloglucan polysaccharide for tumor and macrophage tagging

A simple and low-cost chemical modification process was employed for the synthesis of fluorescently labeled galactoxyloglucan (FITC-GXG). Reactive carbamoyl groups of fluorescent dye were covalently attached to activated hydroxyl groups of GXG through thiocarbamoyl linkage. Fourier transform infrared spectrometry, UV–visible absorbance spectrophotometry, thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) confirmed the successful introduction of the fluorescent groups. Compared with a mixture of uncoupled FITC and GXG, covalently linked FITC-GXG displayed a more obvious fluorescence intensity and higher photostability. Furthermore, FITC-GXG was biocompatible with cells and could be easily internalized. Mechanisms of internalization of FITC-GXG were studied using pharmacological inhibitors for clathrin-mediated and caveolae-mediated endocytosis in lung adenocarcinoma cell lines. In vitro and in vivo bio-distribution of these fluorescently labeled polysaccharides revealed the higher rate of accumulation in tumor tissues due to the tumor homing property of GXG.

Growth-inhibitory effects of anthracycline-loaded bacterial magnetosomes against hepatic cancer in vitro and in vivo.

Aim: This study aimed to develop anthracycline-loaded bacterial magnetosomes (BMs) with enhanced anticancer efficiency and elucidate their endocytosis mechanism. Methods: Drug-loaded BMs (DBMs) were successfully prepared and characterized. DBMs endocytosis was investigated within HepG2 cells. The anticancer effect of DBMs was studied both in vitro and in vivo. Results: Doxorubicin-BMs and daunorubicin-BMs showed enhanced growth inhibitory effect in vitro and in vivo with no notable toxicity to normal tissues. The DBMs were internalized into cells through caveolae-mediated endocytosis and macropinocytosis. The loaded drugs were released from DBMs in cytoplasm and entered the nucleus to exert their activity. Conclusion: Our findings offer promising candidates for improved cancer therapy with a clear mechanism of DBMs endocytosis and working principle.

Temporal Modulation of HER2 Membrane Availability Increases Pertuzumab Uptake and Pretargeted Molecular Imaging of Gastric Tumors.

Human epidermal growth factor receptor 2 (HER2) is used as a tumor biomarker and therapeutic target. Pertuzumab is an anti-HER2 antibody, and its binding to tumor cells requires HER2 to be present at the cell membrane. However, the cellular distribution of HER2 protein in gastric tumors is dynamic, and HER2 internalization decreases antibody binding to tumor cells. These features preclude the use of pretargeted strategies for molecular imaging and therapy. We explored the pharmacological modulation of HER2 endocytosis as a strategy to improve pertuzumab uptake in HER2-positive gastric tumors and allow the use of a pretargeted imaging approach. Methods: We conducted in vitro and in vivo studies with NCI-N87 gastric cancer cells to determine how HER2 endocytosis affects pertuzumab binding to tumor cells. Lovastatin, a clinically approved cholesterol-lowering drug, was used to modulate caveolae-mediated HER2 endocytosis. Results: Administration of lovastatin to NCI-N87 cancer cells resulted in significant accumulation of non-activated HER2 dimers at the cell surface. Pretreatment of NCI-N87 cells with lovastatin increased in vitro specific accumulation of membrane-bound 89Zr-labeled pertuzumab. Lovastatin-enhanced pertuzumab tumor uptake was also observed in NCI-N87 gastric cancer xenografts, allowing tumor detection as early as 4 h and high-contrast images at 48 h after tracer administration via PET. Temporal enhancement of HER2 membrane availability by lovastatin allowed imaging of cell surface HER2 with transcyclooctene-conjugated antibodies and 18F-labeled tetrazine. Conclusion: Temporal pharmacological modulation of membrane HER2 may be clinically relevant and exploitable for pretargeted molecular imaging and therapy in gastric tumors.