Caveolae-mediated Endocytosis Research Articles

LPS transport during fat absorption is inhibited in caveolin‐1 knockout murine jejunum

Lipopolysaccharides (LPS) entry from the intestinal lumen to the circulation is associated with systemic inflammation. We have found that LPS is transcellularly transported to portal vein during long‐chain fatty acid (LCFA) exposure via CD36‐ and lipid raft‐mediated pathways in rats, suggesting the involvement of caveolae‐mediated endocytosis. We thus examined LPS transport in caveolin‐1 (Cav1) knockout (KO) murine jejunum.FITC‐LPS was applied to the mucosal bath of Ussing chambered muscle‐stripped jejunal mucosa‐submucosa preparations of Cav1 KO and wild type (WT) mice. Serosal appearance of FITC‐LPS was measured with or without luminal application of oleic acid (OA, 30 mM) with taurocholic acid (TCA, 0.1 mM), or medium‐chain fatty acid sodium caprate (C10, 10 mM).Luminal application of OA/TCA increased FITC‐LPS m‐to‐s transport in WT jejunum, inhibited by the CD36 inhibitor sulfosuccinimidyl oleate (SSO, 0.1 mM) or the lipid raft inhibitor methyl‐β‐cyclodextrin (MβCD, 0.1 mM), not by the clathrin inhibitor chlorpromazine (0.1 mM) or Pitstop2 (30 µM), suggesting that LCFA‐induced LPS transport is mediated by caveolae‐mediated endocytosis, not by clathrin‐mediated endocytosis. In contrast, OA/TCA‐induced FITC‐LPS transport was abolished in Cav1 KO jejunum. Nevertheless, luminal application of C10 increased FITC‐LPS transport both in WT and Cav1 KO jejuna without transepithelial electrical resistance changes, suggesting that C10 enhances transcellular LPS transport via caveolin‐independent endocytosis in the jejunum.These results suggest that LPS transport during LCFA exposure is mediated by Cav1‐mediated endocytosis, whereas MCFA‐induced LPS transport is likely via clathrin‐mediated endocytosis. Modulation of epithelial endocytosis may be a new therapeutic target for LPS‐associated diseases mediated by dietary fatty acids, including metabolic syndrome.

Cellular Uptake Pathways of Nanoparticles: Process of Endocytosis and Factors Affecting their Fate.

Efficient and controlled internalization of NPs into the cells depends on their physicochemical properties and dynamics of the plasma membrane. NPs-cell interaction is a complex process that decides the fate of NPs internalization through different endocytosis pathways. The aim of this review is to highlight the physicochemical properties of synthesized nanoparticles (NPs) and their interaction with the cellular-dynamics and pathways like phagocytosis, pinocytosis, macropinocytosis, clathrin, and caveolae-mediated endocytosis, and the involvement of effector proteins domain such as clathrin, AP2, caveolin, Arf6, Cdc42, dynamin and cell surface receptors in the endocytosis process of NPs. An electronic search was performed to explore the focused reviews and research articles on types of endocytosis and physicochemical properties of nanoparticles and their impact on cellular internalizations. The search was limited to peer-reviewed journals in the PubMed database. This article discusses in detail, how different types of NPs and their physicochemical properties such as size, shape, aspect ratio, surface charge, hydrophobicity, elasticity, stiffness, corona formation, and surface functionalization change the pattern of endocytosis in the presence of different pharmacological blockers. Some external forces like a magnetic field, electric field, and ultrasound exploit the cell membrane dynamics to permeabilize them for efficient internalization with respect to fundamental principles of membrane bending and pore formation. This review will be useful to attract and guide the audience to understand the endocytosis mechanism and its pattern with respect to physicochemical properties of NPs to improve their efficacy and targeting to achieve the impactful outcome in drug-delivery and theranostic applications.

Porcine Deltacoronavirus (PDCoV) Entry into PK-15 Cells by Caveolae-Mediated Endocytosis.

(1) Background: Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus affecting pig breeding industries worldwide, and its pathogenic mechanism remains unclear. (2) Methods: In this study, we preliminarily identified the endocytic pathway of PDCoV in PK-15 cells, using six chemical inhibitors (targeting clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis pathway and endosomal acidification), overexpression of dominant-negative (DN) mutants to treat PK-15 cells and proteins knockdown. (3) Results: The results revealed that PDCoV entry was not affected after treatment with chlorpromazine (CPZ), 5-(N-ethyl-N-isopropyl) amiloride (EIPA)or ammonium chloride (NH4Cl), indicating that the entry of PDCoV into PK-15 cells were clathrin-, micropinocytosis-, PH-independent endocytosis. Conversely, PDCoV infection was sensitive to nystatin, dynasore and methyl-β-cyclodextrin (MβCD) with reduced PDCoV internalization, indicating that entry of PDCoV into PK-15 cells was caveolae-mediated endocytosis that required dynamin and cholesterol; indirect immunofluorescence and shRNA interference further validated these results. (4) Conclusions: In conclusion, PDCoV entry into PK-15 cells depends on caveolae-mediated endocytosis, which requires cholesterol and dynamin. Our finding is the first initial identification of the endocytic pathway of PDCoV in PK-15 cells, providing a theoretical basis for an in-depth understanding of the pathogenic mechanism of PDCoV and the design of new antiviral targets.

Tumor-Selective Cascade-Amplified Dual-Prodrugs Activation for Synergistic Oxidation-Chemotherapy

Tumor-Selective Cascade-Amplified Dual-Prodrugs Activation for Synergistic Oxidation-Chemotherapy

Self-Assembled Inhalable Immunomodulatory Silk Fibroin Nanocarriers for Enhanced Drug Loading and Intracellular Antibacterial Activity.

In this study, a pH-induced self-assembly-based method has been developed to form silk fibroin nanoparticles (SFN-2) with a higher drug loading capacity (21.0 ± 2.1%) and cellular uptake than that of silk fibroin particles produced by a conventional desolvation method (SFN-1). Using the self-assembly method, rifampicin-encapsulated silk fibroin nanoparticles (R-SFN-2) were prepared with a size of 165 ± 38 nm at an optimum pH of 3.8. In silico analysis reveals that at acidic pH, the amino acid side chain charge neutralization of acidic residues, especially GLU64, promotes the formation of additional favorable interactions between the silk fibroin and the drug. The SFN-2 also possess a good aerosol property with a mass median aerodynamic diameter of 3.82 ± 0.71 μm and fine particle fraction of 64.0 ± 1.4%. These SFN-2 particles were selectively endocytosed by macrophages through clathrin- and caveolae-mediated endocytosis with a higher uptake efficiency (66.2 ± 2.1%) and were found to exhibit a sustained drug release in the presence of macrophage intracellular lysates. The cytokine and biomarker expression analyses revealed that SFN-2 could exhibit an immunomodulatory effect by polarizing the macrophages to an initial M1 phase and later M2 phase. Further, R-SFN-2 also exhibited an enhanced and sustained intracellular antibacterial activity against Mycobacterium smegmatis-infected macrophages than free rifampicin. Thus, the self-assembled silk fibroin particles with immunomodulatory action combined with a good aerosol and intracellular drug release property can be an attractive choice as a carrier for developing pulmonary drug delivery systems.

Histologic and proteomic remodeling of the pulmonary veins and arteries in a porcine model of chronic pulmonary venous hypertension.

In heart failure (HF), pulmonary venous hypertension (PVH) produces pulmonary hypertension (PH) with remodeling of pulmonary veins (PV) and arteries (PA). In a porcine PVH model, we performed proteomic-based bioinformatics to investigate unique pathophysiologic mechanisms mediating PA and PV remodeling. Large PV were banded (PVH, n = 10) or not (Sham, n = 9) in piglets. At sacrifice, PV and PA were perfusion labelled for vessel-specific histology and proteomics. The PA and PV were separately sampled with laser-capture micro-dissection for mass spectrometry. Pulmonary vascular resistance [Wood Units; 8.6 (95% confidence interval: 6.3, 12.3) vs. 2.0 (1.7, 2.3)] and PA [19.9 (standard error of mean, 1.1) vs. 10.3 (1.1)] and PV [14.2 (1.2) vs. 7.6 (1.1)] wall thickness/external diameter (%) were increased in PVH (P < 0.05 for all). Similar numbers of proteins were identified in PA (2093) and PV (2085) with 94% overlap, but biological processes differed. There were more differentially expressed proteins (287 vs. 161), altered canonical pathways (17 vs. 3), and predicted upstream regulators (PUSR; 22 vs. 6) in PV than PA. In PA and PV, bioinformatics indicated activation of the integrated stress response and mammalian target of rapamycin signalling with dysregulated growth. In PV, there was also activation of Rho/Rho-kinase signalling with decreased actin cytoskeletal signalling and altered tight and adherens junctions, ephrin B, and caveolae-mediated endocytosis signalling; all indicating disrupted endothelial barrier function. Indeed, protein biomarkers and the top PUSR in PV (transforming growth factor-beta) suggested endothelial to mesenchymal transition in PV. Findings were similar in human autopsy specimens. These findings provide new therapeutic targets to oppose pulmonary vascular remodeling in HF-related PH.

Multivalent Display of Lipophilic DNA Binders for Dual-Selective Anti-Mycobacterium Peptidomimetics with Binary Mechanism of Action

Multivalent Display of Lipophilic DNA Binders for Dual-Selective Anti-Mycobacterium Peptidomimetics with Binary Mechanism of Action

A cationic cyclodextrin derivative-lipid hybrid nanoparticles for gene delivery effectively promotes stability and transfection efficiency

Genetic medicines hold great promise for treatment of a number of diseases, however, the development of effective gene delivery carrier is still a challenge. The commonly used gene carrier liposomes and cationic polymers have limited their clinical application due to their respective disadvantages. Lipid-polymer hybrid nanoparticles (LHNPs) are novel drug delivery system which exhibit complementary characteristics of both polymeric nanoparticles and liposomes. In this account, we developed the α-cyclodextrin conjugated generation-2 polyamidoamine dendrimers-lipids hybrid nanoparticles (CDG2-LHNPs) for gene delivery. The pDNA/CDG2-LHNPs was stable during 15 days storage period both at 4 °C, 25 °C and 37 °C, whereas the particle size of pDNA/CDG2 and pDNA/liposomes dramatically increased after storage at 4 °C for 8 h. CDG2-LHNPs showed significantly superior transfection efficiencies compared to either CDG2 or liposomes. The mechanism of high transfection efficiency of pDNA/CDG2-LHNPs was further explored using pharmacological inhibitors chlorpromazine, filipin and cytochalasion D. The result demonstrated that cell uptake of pDNA/CDG2-LHNPs was mediated by clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME) and macropinocytosis together. pDNA/CDG2-LHNPs were more likely be taken up by cells through CVME, which avoided lysosomal degradation to a large extent. Moreover, the liposome component of pDNA/CDG2-LHNPs increased its cell uptake efficiency, and the CDG2 polymer component increased its proton buffer capacity, so the hybrid nanoparticles taken up by CME could also successfully escape from the lysosome. This CDG2-LHNPs with stability and high transfection efficiency overcome the shortcomings of liposomes and polymers applied separately, and has great potential for gene drug delivery.

A new approach based on CXCR4-targeted combination liposomes for the treatment of liver fibrosis.

Liver fibrosis results from excessive extracellular matrix accumulation due to injury and leads to cirrhosis, cancer, and death. Herein, we propose a chemokine receptor 4 (CXCR4)-targeted combination (CTC) liposomal therapy to treat carbon tetrachloride (CCl4)-induced liver fibrosis in a mouse model. This study aims to combine small molecules such as pirfenidone and AMD3100 in a single nanoplatform to investigate their synergistic antifibrotic effects in a setting of CCl4-induced liver fibrosis. CTC liposomes (CTC lipo) were prepared using the thin-film hydration method. CTC lipo exhibited a spherical shape, and the particle size was recorded at the nanoscale which confirms its appropriateness for in vitro and in vivo applications. CTC lipo had good storage and serum stability. The entrapped drugs in CTC lipo showed reduced toxicity at higher concentrations. CTC lipo displayed CXCR4 mediated cell uptake and were internalized by caveolae-mediated endocytosis. CTC lipo showed CXCR4 targeting and stromal cell-derived factor 1α (SDF1-α)/CXCR4 axis blocking activity. CTC lipo reduced the elevated serum aspartate aminotransferase (AST), alanine transaminase (ALT), and hydroxyproline (HYP) levels. The histological studies showed improved liver architecture and reduced collagen deposition after treatment. Transforming growth factor β (TGFβ), alpha-smooth muscle actin (α-SMA), and collagen I were elevated by CCl4 in comparison with the Sham. Upon CTC liposomal treatment, the quantitative score for the elevated fibrotic proteins such as TGFβ, α-SMA, and collagen I was normalized. CTC lipo displayed significant downregulation of the upregulated TGFβ, α-SMA, collagen I, and P-p38 expressions at the molecular level. The CXCR4 targeted liposomes showed prolonged biodistribution at 24 h. Our findings indicated that CTC lipo might be an alternative antifibrotic therapy that may offer new access to research and development. In a nutshell, the present study suggests that systemic administration of CTC lipo has efficient antifibrotic potential and deserves to be investigated for further clinical applications.

Highly Luminescent, Stable, and Red-Emitting CsMgxPb1-xI3 Quantum Dots for Dual-Modal Imaging-Guided Photodynamic Therapy and Photocatalytic Activity.

In this study, for the first time, red-emitting CsMgxPb1-xI3 quantum dots (QDs) are prepared by doping with magnesium (Mg) ions via the one-pot microwave pyrolysis technique. The X-ray diffraction and X-ray photoelectron spectroscopy results have confirmed partial substitution of Pb2+ by Mg2+ inside the CsPbI3 framework. The as-synthesized CsMgxPb1-xI3 QDs have exhibited excellent morphology, higher quantum yield (upto ∼89%), better photostability and storage stability than undoped CsPbI3. Next, the bioavailability of as-synthesized hydrophobic CsMgxPb1-xI3 QDs is improved by encapsulating them into gadolinium-conjugated pluronic 127 (PF127-Gd) micelles through hydrophobic interactions (PQD@Gd). The optical properties of perovskite quantum dots (PQDs) and the presence of Gd could endow the PQD@Gd with fluorescence imaging, magnetic resonance imaging (MRI), and phototherapeutic properties. Accordingly, the MRI contrasting effects of PQD@Gd nanoagents are demonstrated by employing T1 and T2 studies, which validated that PQD@Gd nanoagents had superior MR contrasting effect with a r2/r1 ratio of 1.38. In vitro MRI and fluorescence imaging analyses have shown that the PQD@Gd nanoagents are internalized into the cancer cells via a caveolae-mediated endocytosis pathway. The PQD@Gd nanoagents have exhibited excellent biocompatibility even at concentrations as high as 450 ppm. Interestingly, the as-prepared PQD@Gd nanoagents have efficiently produced cytotoxic reactive oxygen species in the cancer cells under 671 nm laser illumination and thereby induced cell death. Moreover, the PQD@Gd nanoagent also demonstrated excellent photocatalytic activity toward organic pollutants under visible light irradiation. The organic pollutants rhodamine b, methyl orange, and methylene blue were degraded by 92.11, 89.21, and 76.21%, respectively, under 60, 80, and 100 min, respectively, irradiation time. The plausible mechanism for the photocatalytic activity is also elucidated. Overall, this work proposes a novel strategy to enhance the optical properties, stability, and bioapplicability of PQDs. The multifunctional PQD@Gd nanoagents developed in this study could be the potential choice of components not only for cancer therapy due to dual-modal imaging and photodynamic therapeutic properties but also for organic pollutant or bacterial removal due to excellent photocatalytic properties.

Glycyrrhiza acid-Licochalcone A complexes for enhanced bioavailability and anti-melanogenic effect of Licochalcone A: cellular uptake and in vitro experiments

Licochalcone A (LicA) is a natural chalcones, well-known for its anti-melanogenic activity, however, its poor solubility leads to a limited topical bioavailability and clinical outcomes. Therefore, LicA-encapsulated glycyrrhiza acid (GA) (GA-LicA) micelles were formulated for higher cellular uptake and improved cutaneous applications. The physicochemical properties (morphology, size and charge), topical penetration, endocytic pathways, cellular uptake amount (CUA) and intracellular distribution of the GA-LicA micelles were studied. Moreover, the anti-melanogenic mechanisms including antioxidant properties, melanogenesis-mediated enzymes and melanin generation as well as cell apoptosis in A375 cells were demonstrated. We found that significant increase amount of LicA was obtained into the epidermis after encapsulated in GA micelles, and the hair follicles were the preferential route for LicA permeation. Moreover, HaCaT and melanocytes possessed a significantly higher effective uptake and CUA for GA-LicA micelles than that of the crude LicA. Caveolae-mediated and actin filaments-mediated endocytosis, caveolae-mediated endocytosis were respectively involved in cellular uptake of GA-LicA micelles for HaCaT cells and melanocytes. Furthermore, GA-LicA micelles possessed a synergistic anti-melanogenic effect by scavenging the free radicals, reducing the activity of α-glucosidase and tyrosinase, and inducing the cell apoptosis. Taken together, GA micelles can enhance the bioavailability and anti-melanogenic effect of LicA, and the article provided principles for rational design of formulations in whitening cosmetics.

Fabrication of chitosan-coated epigallocatechin-3-gallate (EGCG)-hordein nanoparticles and their transcellular permeability in Caco-2/HT29 cocultures

Epigallocatechin-3-gallate (EGCG) has gained appreciable attention because of its health benefits. However, the poor permeability across the intestine limits its use. In this study, we have fabricated chitosan-coated EGCG-hordein nanoparticles (Cs-EHNs), with the aim to enhance the intestinal permeability of EGCG. Cs-EHNs were fabricated by layer-by-layer electrostatic stacking method, and its uptake and transcellular permeability were studied in the Caco-2/HT29 co-culture model. The constructed Cs-EHNs had the average diameter of 296 nm, polymer dispersity index (PDI) of 0.30, zeta potential of 59.6 mV, and showed a spherical morphology. Encapsulation efficiency of EGCG was 87.3%. The transcellular permeability experiments indicated that the apparent permeability coefficient (Papp) of Cs-EHNs was higher than that of free EGCG. Furthermore, the cellular uptake of Cs-EHNs was studied by specific endocytosis inhibitors, and results showed that the uptake mechanisms of Cs-EHNs were through caveolae-mediated endocytosis and macropinocytosis. This study demonstrated that encapsulation of EGCG using chitosan-coated hordein nanoparticles could be a promising approach to improve the absorption of EGCG.

Protein Delivery to Cytosol by Cell-Penetrating Peptide Bearing Tandem Repeat Penetration-Accelerating Sequence.

Pas2r12 is comprised of a repeat of the penetration-accelerating sequence (Pas) (Pas2: FFLIG-FFLIG) and D-form dodeca-arginine (r12), a cell-penetrating peptide. Pas2r12 significantly enhances cytosolic delivery of cargo proteins, including enhanced green fluorescent protein and immunoglobulin G. Simply incubating Pas2r12 with cargo leads to their cytosolic tranlsocation. Cytosolic delivery of cargo by Pas2r12 involves caveolae-mediated endocytosis. In this chapter, we describe methods of cytosolic delivery of cargo using Pas2r12 and provide methods for investigating the cellular uptake pathway of cargo by Pas2r12.

Persistence and Recovery of ZIF-8 and ZIF-67 Phytotoxicity.

Zeolitic imidazolate frameworks (ZIFs) have been developed quickly and have attracted considerable attention for use in the detection and removal of various pollutants. Understanding the environmental risks of ZIFs is a prerequisite to their safe application by industry and new chemical registration by governments; however, the persistence and recovery of toxicity induced by ZIFs remain largely unclear. This study finds that typical ZIFs (e.g., ZIF-8 and ZIF-67) at a concentration of 0.01-1 mg/L induce significant algal growth inhibition, plasmolysis, membrane permeability, chloroplast damage, and chlorophyll biosynthesis, and the above alterations are recoverable. Unexpectedly, a persistent decrease in reactive oxygen species (ROS) is observed due to the quenching of hydroxyl free radicals. The adverse effects of ZIF-8 are weak and easily alleviated compared with those of ZIF-67. ZIF-8 is internalized mainly by caveolae-mediated endocytosis, while ZIF-67 is internalized mainly by clathrin-mediated endocytosis. Omics studies reveal that the downregulation of mRNA associated with oxidative phosphorylation and the inhibition of chlorophyll and adenosine triphosphate (ATP) synthesis in mitochondria are related to the persistence of phytotoxicity. These findings highlight the phenomena and mechanisms of the persistence and recovery of phytotoxicity, indicating the need to reconsider the environmental risk assessments of ZIFs.

Hypoxia-responsive pullulan-based nanoparticles as erlotinib carriers

A hypoxia-responsive pullulan-based co-polymer was developed to assess its efficacy to deliver erlotinib (ERL) to the cervical cancer cells. Upon exposure to hypoxic condition, the synthesized and structurally characterized co-polymer i.e. succinyl pullulan-g-6-(2-nitroimidazole) hexylamine (Pull-SA-HA-NI) exhibited a hypochromic shift in the UV spectra and alteration in its self-assembled structures as compared to the control co-polymer, succinyl pullulan-g-hexylamine (Pull-SA-HA). Its corresponding ERL-loaded nanoparticles (NPs) displayed an attenuated crystallinity of pure ERL with excellent drug-trapping capacity (DEE, 94.23 ± 1.36%) and acceptable zeta potential (+39.21 ± 1.09 mV) and diameter (84.10 ± 2.10 nm) values. These also evidenced a faster drug release profile under hypoxic condition relative to the normoxic condition. The cellular internalization of the NPs was mediated through the energy-dependent endocytic process, which could utilize its multiple pathways (i.e., macropinocytosis, clathrin- and caveolae-mediated endocytosis). The ERL-loaded NPs suppressed HeLa cell proliferation and induced apoptosis more efficiently than the pristine drug.

Fate of CdSe/ZnS quantum dots in cells: Endocytosis, translocation and exocytosis

Semiconductor quantum dots (QDs) have been extensively explored for extensive bioapplications, yet their cellular fate, especially exocytosis, has not been thoroughly investigated. Herein, we systematically investigated the whole cellular process from the endocytosis, intercellular trafficking, to the exocytosis of a typical QD, core/shell CdSe/ZnS QD. Using confocal laser scanning microscopy and flow cytometry, and after carefully eliminating the effect of cell division, we found that the QDs were internalized by HeLa cells with a time-, dose-, and serum-dependent manner. The cellular uptake was inhibited by serum, but eventually peaked after 4–6 h incubation with or without serum. The primary endocytosis pathway was clathrin-mediated, and actin- and microtubule-dependent in the medium with serum, while the caveolae-mediated endocytosis and macropinocytosis were more important for the QDs in the serum-free medium. Inside cells, most QDs distributed in lysosomes, and some entered mitochondria, endoplasmic reticulum, and Golgi apparatus. The translocation of the QDs from other organelles to Golgi apparatus was observed. The exocytosis of QDs was faster than the endocytosis, reaching the maximum in about one hour after cultured in fresh culture medium, with around 60% of the internalized QDs remained undischarged. The exocytosis process was energy- and actin-dependent, and the lysosome exocytosis and endoplasmic reticulum/Golgi pathway were the main routes. This study provides a full picture of behavior and fate of QDs in cells, which may facilitate the design of ideal QDs applied in biomedical and other fields.

Albumin-EDTA-Vanadium Is a Powerful Anti-Proliferative Agent, Following Entrance into Glioma Cells via Caveolae-Mediated Endocytosis.

Human serum albumin (HSA) is efficiently taken up by cancer cells as a source of carbon and energy. In this study, we prepared a monomodified derivative of HSA covalently linked to an EDTA derivative and investigated its efficacy to shuttle weakly anti-proliferative EDTA associating ligands such as vanadium, into a cancer cell line. HSA-S-MAL-(CH2)2-NH-CO-EDTA was found to associate both with the vanadium anion (+5) and the vanadium cation (+4) with more than thrice the associating affinity of those ligands toward EDTA. Both conjugates internalized into glioma tumor cell line via caveolae-mediated endocytosis pathway and showed potent anti-proliferative capacities. IC50 values were in the range of 0.2 to 0.3 µM, potentiating the anti-proliferative efficacies of vanadium (+4) and vanadium (+5) twenty to thirty fold, respectively. HSA-EDTA-VO++ in particular is a cancer permeable prodrug conjugate. The associated vanadium (+4) is not released, nor is it active anti-proliferatively prior to its engagement with the cancerous cells. The bound vanadium (+4) dissociates from the conjugate under acidic conditions with half maximal value at pH 5.8. In conclusion, the anti-proliferative activity feature of vanadium can be amplified and directed toward a cancer cell line. This is accomplished using a specially designed HSA-EDTA-shuttling vehicle, enabling vanadium to be anti-proliferatively active at the low micromolar range of concentration.

The protective effect of harpagoside on angiotensin II (Ang II)-induced blood-brain barrier leakage in vitro.

Hypertension and its associated dysfunction of the blood-brain barrier (BBB) contribute to cerebral small vessel disease (cSVD). Angiotensin II (Ang II), a vasoactive peptide of the renin-angiotensin system (RAS), is not only a pivotal molecular signal in hypertension but also causes BBB leakage, cSVD, and cognitive impair. Harpagoside, the major bioactive constituent of Scrophulariae Radix, has been commonly used for the treatment of multiple diseases including hypertension in China. The effect of harpagoside on Ang II-induced BBB damage is unclear. We employed an immortalized endothelial cell line (bEnd.3) to mimic a BBB monolayer model in vitro and investigated the effect of harpagoside on BBB and found that harpagoside alleviated Ang II-induced BBB destruction, inhibited Ang II-associated cytotoxicity in a concentration-dependent manner and attenuated Ang II-induced reactive oxygen species (ROS) impair by downregulation of Nox2, Nox4, and COX-2. Harpagoside prevented Ang II-induced apoptosis via keeping Bax/Bcl-2 balance, decreasing cytochrome c release, and inactivation of caspase-8, caspase-9, and caspase-3 (the mitochondria-dependent and death receptor-mediated apoptosis pathways). Moreover, harpagoside can alleviate Ang II-induced BBB damage through upregulation of tight junction proteins and decrease of caveolae-mediated endocytosis. Thus, harpagoside might be a potential drug to treat Ang II-induced cSVD.

Mucoadhesive versus mucopenetrating nanoparticles for oral delivery of insulin

Mucoadhesive and mucopenetrating nanoparticles are commonly designed to improve mucosal drug delivery efficiency. Herein, in order to better understand the contribution of mucoadhesion and mucopenetration in oral delivery of biomacromolecules, insulin-loaded poly (n-butylcyanoacrylate) nanoparticles (Ins/PBCA NPs) with different coating layers, chitosan (CS) or alginate (Alg), were designed and their different absorption enhancing mechanisms were explored. It was demonstrated that both the mucoadhesive (Ins/PBCA/CS) and the mucopenetrating (Ins/PBCA/CS/Alg) nanoparticles showed good stability and similar release profiles in the gastrointestinal fluid, the mucoadhesive nanoparticles presented an enrichment in mucus (70%, 10 min) while most of the mucopenetrating nanoparticles penetrated through the mucus (80%, 10 min). Uptake mechanism studies revealed clathrin- and caveolae-mediated endocytosis were mainly involved in the intestinal transport of mucoadhesive nanoparticles while caveolae-mediated endocytosis and macropinocytosis contributed to the absorption of mucopenetrating nanoparticles, and especially, M cells favored the absorption of mucoadhesive nanoparticles. In vivo studies revealed that the mucopenetrating nanoparticles had a fast onset of action while the mucoadhesive nanoparticles presented a sustained hypoglycemic effect in diabetic rats, and overall no significant difference in pharmacological availability was found between the mucopenetrating (8.80%) and mucoadhesive nanoparticles (8.44%). To sum up, due to the varied absorption mechanism in intestine, the mucoadhesive nanoparticles designed herein had a comparable effect in enhancing oral insulin absorption compared with the mucopenetrating nanoparticles. Statement of significanceIn order to improve oral delivery efficiency of insulin, insulin-loaded nanoparticles with opposite properties namely mucoadhesion and mucopenetration have been widely developed to either prolong their residence at the absorption site or improve their penetration across mucus. However, their individual contribution in oral insulin absorption is still unclear. In this paper, insulin-loaded poly (n-butylcyanoacrylate) nanoparticles with both properties were designed via different surface coating and their absorption enhancing mechanisms were explored. It was demonstrated that the mucoadhesive and mucopenetrating nanoparticles showed varied retention and mucus-penetration ability in mucus, with different absorption mechanism in intestine, but no statistical difference in pharmacological availability was found between them. Overall, the present work provides us a guidance for the design of oral nano-delivery system.

Nanoparticle-mediated specific elimination of soft cancer stem cells by targeting low cell stiffness

As the driving force of tumor progression, cancer stem cells (CSCs) hold much lower cellular stiffness than bulk tumor cells across many cancer types. However, it remains unclear whether low cell stiffness can be harnessed in nanoparticle-based therapeutics for CSC targeting. We report that breast CSCs exhibit much lower stiffness but considerably higher uptake of nitrogen-doped graphene quantum dots (N-GQDs) than bulk tumor cells. Softening/stiffening cells enhances/suppresses nanoparticle uptake through activating/inhibiting clathrin- and caveolae-mediated endocytosis, suggesting that low cell stiffness mediates the elevated uptake in soft CSCs that may lead to the specific elimination. Further, soft CSCs enhance drug release, cellular retention, and nuclear accumulation of drug-loaded N-GQDs by reducing intracellular pH and exocytosis. Remarkably, drug-loaded N-GQDs specifically eliminate soft CSCs both in vitro and in vivo, inhibit tumor but not animal growth, and reduce the tumorigenicity of xenograft cells. Our findings unveil a new mechanism by which low cellular stiffness can be harnessed in nanoparticle-based strategies for specific CSC elimination, opening a new paradigm of cancer mechanomedicine. Statement of significanceLow cell stiffness is associated with high malignancy of tumor cells and thus serves as a mechanical hallmark of CSCs. However, it remains unclear whether cellular stiffness can be exploited for specific targeting of soft CSCs. This work reports that soft CSCs exhibit high N-GQD uptake compared to stiff tumor cells, which is regulated by cellular stiffness. Further, soft CSCs have enhanced drug release, cellular retention, and nuclear accumulation of drug-loaded N-GQDs, which enable the specific elimination of malignant CSCs both in vitro and in vivo with minimal side effect. In summary, our study demonstrates that CSC's low stiffness can be harnessed as a mechanical target for specific eradication, which provides a new paradigm of cancer mechanomedicine.