Caveolae-mediated Endocytosis Pathways Research Articles

PEGylation-Dependent Cell Uptake of Lipid Nanoparticles Revealed by Spatiotemporal Correlation Spectroscopy.

Polyethylene glycol (PEG) is a common surface modification for lipid nanoparticles (LNPs) to improve their stability and in vivo circulation time. However, the impact of PEGylation on LNP cellular uptake remains poorly understood. To tackle this issue, we systematically compared plain and PEGylated LNPs by combining dynamic light scattering, electrophoretic light scattering, and synchrotron small-angle X-ray scattering (SAXS) that unveils a striking similarity in size and core structure but a significant reduction in surface charge. Upon administration to human embryonic kidney (HEK 293) cells, plain and PEGylated LNPs were internalized through different endocytic routes, as revealed by spatiotemporal correlation spectroscopy. An imaging-derived mean square displacement (iMSD) analysis shows that PEGylated LNPs exhibit a significantly stronger preference for caveolae-mediated endocytosis (CAV) and clathrin-mediated endocytosis (CME) pathways compared to plain LNPs, with these latter being better tailored to MCR-dependent internalization and trafficking. This suggests that PEG plays a crucial role in directing LNPs toward specific cellular uptake routes. Further studies should explore how PEG-mediated endocytosis impacts intracellular trafficking and ultimately translates to therapeutic efficacy, guiding the design of next-generation LNP delivery systems.

Tris-assisted one-step fabrication of functional carbon dots for specific folate receptor positive-expressed cancer cell imaging

Specific staining of cancer cells is momentous for cancer research. Nanoprobe with multivalent recognition is emerging as powerful tools for bioimaging, but the nonspecific cell uptake and complex functional modification procedures are still obstacles for specific detection and convenient synthesis. Carbon dots (CDs) with an intrinsic targeting ability, excellent optical properties and biocompatibility acquired from an efficient one-step fabrication procedure were urgently desired in specific cancer cells visualization. Herein, inspired by the interrelationships between interface and biomolecular mechanisms, we suggested that it was possible to construct CDs with the desired characteristics for folate receptor (FR) positive-expressed cancer cell imaging via rich hydroxyl groups Tris-assisted one-step hydrothermal treatment of folate acid (FA) and l-Arginine (L-Arg) precursors. The prepared small-sized F-CDs were equipped with abundant hydroxyl, pterin and negative charge surface, and possessed environmental friendliness, outstanding photostability and biocompatibility. Moreover, F-CDs had an intrinsic FR positive-expressed cancer cell targeting ability without any post-modification of the ligands. Rich hydroxyl groups play a vital role in endowing the optical properties and biological effects of F-CDs. F-CDs could be used as a promising candidate for FR-expressed cancer cell labeling and tracking. In addition, the caveolae-mediated endocytosis pathway of F-CDs was ascertained. More importantly, experimental results confirmed that the combination of physicochemical properties may provide an efficient strategy to overcome non-specific cell uptake interactions for cell labeling. Our strategy put forward a promising alternative to design fluorescent CDs for extensive chemical and biomedical applications.

Sugar alcohol-modified polyester nanoparticles for gene delivery via selective caveolae-mediated endocytosis.

Nucleic acid-based drugs are changing the scope of emerging medicine in preventing and treating diseases. Nanoparticle systems based on lipids and polymers developed to navigate tissue-level and cellular-level barriers are now emerging as vector systems that can be translated to clinical settings. A class of polymers, poly(β-amino esters) (PBAEs) known for their chemical flexibility and biodegradability, has been explored for gene delivery. These polymers are sensitive to changes in the monomer composition affecting transfection efficiency. Hence to add functionality to these polymers, we partially substituted ligands to an identified effective polymer chemistry. We report here a new series of statistical copolymers based on PBAEs where the backbone is modified with sugar alcohols to selectively facilitate the caveolae-mediated endocytosis pathway of cellular transport. These ligands are grafted at the polymer's backbone, thereby establishing a new strategy of modification in PBAEs. We demonstrate that these polymers form nanoparticles with DNA, show effective complexation and cargo release, enter the cell via selective caveolae-mediated endocytosis, exhibit low cytotoxicity, and increase transfection in neuronal cells.

Caveolae-mediated endocytosis pathway regulates endothelial fenestra homeostasis in the rat pituitary

Endothelial fenestrae are transcellular pores separated by diaphragms formed by plasmalemma vesicle-associated proteins (PLVAP) and function as channels for peptide hormones and other substances. Caveola, a key regulator of clathrin-independent endocytosis, may be involved in the invagination and fusion of plasma membranes, which are essential for fenestra formation. In this study, we first found that caveolin-1 and -2, the major components of caveolae, was localized in fenestrated endothelial cells in the anterior lobe of the rat pituitary by immunohistochemistry. As we also observed caveolae in the endothelial cells of the anterior lobe of the rat pituitary by transmission electron microscopy, we studied the relationship between the caveolae-mediated endocytosis pathway and fenestrae structure in cultured endothelial cells isolated from the anterior lobe of the rat pituitary (CECAL) by immunofluorescence staining and scanning electron microscopy. The inhibition of caveolae-mediated endocytosis by genistein enlarged the PLVAP-positive oval-shaped structure that represented the sieve plate and induced the formation of a doughnut-shaped bulge around the fenestra in CECAL. In contrast, the acceleration of caveolae-mediated endocytosis by okadaic acid induced the diffusion of PLVAP-positive signals in the cytoplasm and reduced the number of fenestrae in CECAL. These results indicate that the caveolae-mediated endocytosis pathway is involved in the fenestra homeostasis in the fenestrated endothelial cells of the rat pituitary.

Carotenoid containing cationic nanoparticles for effective therapy for suppressing oxidative stress: An intranasal approach

Alzheimer's disease is the most common type of dementia. Oxidative stress is involved in the progression of aging and Alzheimer's disease. It is known that lutein is a carotenoid having antioxidant properties. The present research explores Lutein loaded chitosan nanoparticles for Suppressing Oxidative Stress in the treatment of Alzheimer's. The developed nanoparticles are administered through the nose to target brain via nose-to-brain pathway. For optimization of formulation a systematic QbD approach was used. The developed nanoparticles further characterized for Physicochemical parameters, Morphology, In-vitro drug releases, Ex-vivo diffusion, In-vitro Cell viability, cellular uptake, In-vitro BBB Permeation, antioxidant properties, In-vivo biodistribution, and stability study. The developed nanoparticles' surface morphology suggested homogeneously dispersed spherical nanoparticles having < 200 nm size. The drug release study demonstrate the controlled release of lutein for more than 96 h while less than 50% lutein was released after 24 h in an ex-vivo diffusion study. The cell cytotoxicity assay confirms the nontoxicity of l-CNPs. The cellular uptake study shows enhanced internalization of l-CNPs through the caveolae-mediated endocytosis pathway. The ROS generation confirmed the absence of any significant ROS generation nanoparticles. The antioxidant assay shows significant ROS scavenging activity of l-CNPs. In-vitro BBB permeation demonstrates the efficient passage of l-CNPs through BBB compared to pure lutein. This was further supported by bio-distribution demonstrating the deposition of nanoparticles in the brain through nasal administration. The acquired outcomes prove the possible activities of lutein-loaded l-CNPs for reducing oxidative stress in the brain for Alzheimer's treatment.

Folic Acid–Chitosan Oligosaccharide Conjugates Decorated Nanodiamond as Potential Carriers for the Oral Delivery of Doxorubicin

Oral administration of doxorubicin (DOX) is preferred but challenged owing to poor permeability in the gastrointestinal tract (GIT), efflux of P-glycoprotein, short residence time in the intestine, and rapid hydrolysis. Herein, folic acid-chitosan oligosaccharide conjugate (FA-COS)-modified hydroxylated nanodiamond (ND-OH) was designed to enhance the oral bioavailability of DOX. The carboxyl surface of ND was modified into hydroxyl terminal group to increase the colloidal stability of the system under different pH conditions in GIT. FA-COS modification could prolong retention time, endow the drug with sustained release properties, and actively target intestinal FA receptors. In contrast to DOX/ND-OH, the particle size of DOX/ND-OH/FA-COS increased from 189.5 ± 2.8 to 224.5 ± 1.4nm, and the zeta potential reversed from - 9.1 ± 0.2 to 14.8 ± 0.4mV. At 48h, DOX/ND-OH and DOX/ND-OH/FA-COS released 69.07 ± 5.70% and 35.87 ± 5.64%, respectively. FA-COS modification effectively enhanced the cytotoxicity and intracellular uptake of ND-OH/DOX by Caco-2 cells and prolonged intestinal retention in rats. The internalization of DOX/ND-OH and DOX/ND-OH/FA-COS was mainly mediated by energy-dependent clathrin- and caveolae-mediated endocytosis pathways. Pharmacokinetic study demonstrated that the AUC0-t of DOX/ND-OH and DOX/ND-OH/FA-COS was enhanced by 3.94- and 6.08-fold compared to DOX solution, respectively. These results illustrated that DOX/ND-OH/FA-COS could be an effective strategy to enhance the oral bioavailability of DOX.

Enzymatic Synthesis of Peptide Nanofibers for Self-Delivery of Indomethacin and Tyroservatide in Cancer Therapy.

Non-steroidal anti-inflammatory drugs (NSAIDs) have drawn considerable attention in the field of cancer treatment, yet these drugs display limited potency and selectivity against cancer cells. To address these problems, we designed a peptide-based self-delivery system [Indomethacin-Phe-Phe-Tyr (H2PO3)-Ser-Val, IDM-FFpYSV] that combines an NSAID molecule (indomethacin, or IDM) and a segment of anticancer tripeptide (tyroservatide, or YSV). IDM-FFpYSV is capable of self-assembling in an aqueous solution to afford nanofibrillar hydrogels under the catalysis of alkaline phosphatases (ALPs), which are overexpressed on the plasma membrane of cancer cells. The IDM-FFpYSV + ALP hydrogel displays a continuous release profile of peptide drugs, whereas a solution mixture of pure drugs (IDM-OH + pYSV + ALP) shows burst release of drug moieties. The treatment of IDM-FFpYSV selectively inhibits the proliferation of HeLa cells in vitro, with precise regulations of intracellular targeting proteins (COX-2 and AC-H3). The enhanced potency and selectivity of IDM-FFpYSV are found to be attributed to enhanced cellular uptake of peptide drugs, which involves a caveolae-mediated endocytosis pathway. Furthermore, intravenous administration of the IDM-FFpYSV formulation significantly inhibits the tumor growth in a HeLa-xenografted mouse model, whereas treatment of solution mixtures of pure drugs (IDM-OH + pYSV) fails to do so. Taken together, the study provides a viable strategy to augment anticancer efficacies of self-delivery system through molecular integration of multiple anticancer elements with an enzyme-instructed self-assembly process.

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.

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.

Engineering Extracellular Vesicles Restore the Impaired Cellular Uptake and Attenuate Intervertebral Disc Degeneration.

Extracellular vesicles (EVs) are potential alternatives for mesenchymal stem cells (MSCs) in the treatment of musculoskeletal degenerative diseases, including intervertebral disc degeneration (IDD). Usually, EVs are internalized and then deliver bioactive molecules that impart phenotypic changes in recipient cells. For effective utilization of EVs in the IDD therapy, understanding the mechanism of EV uptake is of vital importance. In this study, we found that EVs delivered antioxidant proteins to protect against pyroptosis of nucleus pulposus cells (NPCs). In particular, the therapeutic effect of EVs decreased in TNF-α-treated NPCs due to the impaired caveolae-mediated endocytosis pathway. Transcriptome sequencing and functional verification revealed that caveolae associated protein 2 (Cavin-2) played an important role in the uptake process of EVs. We then constructed the Cavin-2-modified engineering EVs via the gene-editing of parental MSCs. These kinds of modified EVs presented an improved uptake rate in TNF-α-treated NPCs, which effectively ameliorated the cell death of NPCs in a three-dimensional hydrogel culture model and retarded the progression of IDD in the ex vivo organ culture model. Collectively, these findings illustrate the mechanism of EV uptake in NPCs and explore the application of engineering EVs in the treatment of IDD.

Chiral mesoporous silica nano-screws as an efficient biomimetic oral drug delivery platform through multiple topological mechanisms

In the microscale, bacteria with helical body shapes have been reported to yield advantages in many bio-processes. In the human society, there are also wisdoms in knowing how to recognize and make use of helical shapes with multi-functionality. Herein, we designed atypical chiral mesoporous silica nano-screws (CMSWs) with ideal topological structures (e.g., small section area, relative rough surface, screw-like body with three-dimension chirality) and demonstrated that CMSWs displayed enhanced bio-adhesion, mucus-penetration and cellular uptake (contributed by the macropinocytosis and caveolae-mediated endocytosis pathways) abilities compared to the chiral mesoporous silica nanospheres (CMSSs) and chiral mesoporous silica nanorods (CMSRs), achieving extended retention duration in the gastrointestinal (GI) tract and superior adsorption in the blood circulation (up to 2.61- and 5.65-times in AUC). After doxorubicin (DOX) loading into CMSs, DOX@CMSWs exhibited controlled drug release manners with pH responsiveness in vitro. Orally administered DOX@CMSWs could efficiently overcome the intestinal epithelium barrier (IEB), and resulted in satisfactory oral bioavailability of DOX (up to 348%). CMSWs were also proved to exhibit good biocompatibility and unique biodegradability. These findings displayed superior ability of CMSWs in crossing IEB through multiple topological mechanisms and would provide useful information on the rational design of nano-drug delivery systems.

Construction of Oral Insulin-Loaded Solid Lipid Nanoparticles and Their Intestinal Epithelial Cell Transcytosis Study

To construct solid lipid nanoparticle (SNPs) drug delivery system loaded with peptide and protein drugs by using mixedsolvents, to study the transcytosis mechanisms of SNPs across intestinal epithelial cells, and to improve the endocytosis and transcytosis efficiency of peptide and protein drugs. The formulation of insulin-loaded water-in-oil-in-water solid lipid nanoparticles (INS-SNPs) was prepared by using a methanol-chloroform mixed solvent. The formulation was optimized with the single factor screening method. The optimized INS-SNPs were then characterized in terms of their morphology, stability and drug release properties. The cytotoxicity, cellular uptake and endocytosis mechanisms of INS-SNPs were then assessed on Caco-2 cells. The transcytosis efficiency of INS-SNPs was finally evaluated by using cellular monolayer in Transwell ® insert. The size, zeta potentials and drug loading efficiency of the optimized INS-SNPs were observed to be (145.4±0.5) nm, (-12.9±0.2) mV and (7.93±0.02)%, respectively. INS-SNPs were then shown to maintain desirable colloidal stability and sustained release of insulin in the simulated intestinal fluid. It was revealed that the cellular uptake of INS-SNPs reached its maximum after cellular incubation for 2 hours and was 1.53-fold higher than that of free insulin. Investigation of the endocytic mechanism revealed that INS-SNPs enter intestinal epithelial cells mainly through the clathrin-mediated and caveolae-mediated endocytosis pathways. Further investigation revealed that the amount of INS-SNPs permeating the cell monolayers was 1.54-fold higher than that of free insulin, which was comparable to the increase in their cellular uptake efficiency, indicating that INS-SNPs displayed enhanced absorption across the intestinal epithelium. The INS-SNPs prepared with mixed solvents in this study could significantly enhance the transcytosis efficiency of peptide and protein drugs, displaying great potentials in the application of oral drug delivery. This study may provide information and reference for the designing of efficient oral nano-drug delivery system in the future.

Overexpression of Toll-like receptor 4 contributes to the internalization and elimination of Escherichia coli in sheep by enhancing caveolae-dependent endocytosis

BackgroundGram-negative bacterial infections have a major economic impact on both the livestock industry and public health. Toll-like receptor 4 (TLR4) plays a crucial role in host defence against Gram-negative bacteria. Exploring the defence mechanism regulated by TLR4 may provide new targets for treatment of inflammation and control of bacterial infections. In a previous study, we generated transgenic sheep overexpressing TLR4 by microinjection to improve disease resistance. The defence mechanism through which TLR4 overexpression protected these sheep against pathogens is still not fully understood.ResultsIn the present study, we used Escherichia coli to infect monocytes isolated from peripheral blood of the animal model. The overexpression of TLR4 strongly enhanced the percentage of endocytosis and capacity of elimination in monocytes during the early stages of infection. This phenomenon was mainly due to overexpression of TLR4 promoting caveolae-mediated endocytosis. Pretreatment of the transgenic sheep monocytes with inhibitors of TLR4, Src signalling, or the caveolae-mediated endocytosis pathway reduced the internalization of bacteria, weakened the ability of the monocytes to eliminate the bacteria, and increased the pH of the endosomes.ConclusionTogether, our results reveal the effects of TLR4 on the control of E. coli infection in the innate immunity of sheep and provide crucial evidence of the caveolae-mediated endocytosis pathway required for host resistance to invading bacteria in a large animal model, providing theoretical support for breeding disease resistance in the future. Furthermore, Src and caveolin 1 (CAV1) could be potentially valuable targets for the control of infectious diseases.

Endocytosis and Organelle Targeting of Nanomedicines in Cancer Therapy.

Nanomedicines (NMs) have played an increasing role in cancer therapy as carriers to efficiently deliver therapeutics into tumor cells. For this application, the uptake of NMs by tumor cells is usually a prerequisite to deliver the cargo to intracellular locations, which mainly relies on endocytosis. NMs can enter cells through a variety of endocytosis pathways. Different endocytosis pathways exhibit different intracellular trafficking routes and diverse subcellular localizations. Therefore, a comprehensive understanding of endocytosis mechanisms is necessary for increasing cellular entry efficiency and to trace the fate of NMs after internalization. This review focuses on endocytosis pathways of NMs in tumor cells, mainly including clathrin- and caveolae-mediated endocytosis pathways, involving effector molecules, expression difference of those molecules between normal and tumor cells, as well as the intracellular trafficking route of corresponding endocytosis vesicles. Then, the latest strategies for NMs to actively employ endocytosis are described, including improving tumor cellular uptake of NMs by receptor-mediated endocytosis, transporter-mediated endocytosis and enabling drug activity by changing intracellular routes. Finally, active targeting strategies towards intracellular organelles are also mentioned. This review will be helpful not only in explicating endocytosis and the trafficking process of NMs and elucidating anti-tumor mechanisms inside the cell but also in rendering new ideas for the design of highly efficacious and cancer-targeted NMs.

Tumor cell-derived autophagosomes (DRibbles)-activated B cells induce specific naïve CD8+ T cell response and exhibit antitumor effect.

Dendritic cell (DC) vaccine has been proved to be an effective way in cancer immunotherapy in both preclinical and clinical studies. However, limitations in DC isolation and culture have hampered its practice and promoted the development of other antigen-presenting cells (APCs) sources to fulfill that role. Our previous studies have shown that B cells loaded by tumor cell-derived autophagosomes, which we named as DRibbles (defective ribosomal products-containing blebs), could reactivate DC-induced effector T cell response. In this study, the roles of DRibble-loaded B cells in priming naïve CD8+ T cell responses and controlling tumors were investigated. We found that high-mobility group box 1 protein (HMGB1) on DRibbles was involved in DRibble-induced B cell activation, and the DRibble-triggered B cell phagocytosis via the caveolae-mediated endocytosis pathway. By using OT-I mouse-derived T cells, we demonstrated that DRibble-loaded B cells could activate specific naïve CD8+ T cells in vitro and ex vivo. In a tumor-bearing mouse model, DRibble-loaded B cells elicited systemic antitumor immunity and significantly suppressed the tumor growth. Moreover, the antitumor efficacy of DRibble-loaded B cells was enhanced when they were combined with CpG and anti-CD40 stimulation. These results suggest that DRibble-loaded B cells represent a viable and practical therapeutic vaccination strategy that might have important clinical implications for tumor immunotherapy.

Paclitaxel/IR1061-Co-Loaded Protein Nanoparticle for Tumor-Targeted and pH/NIR-II-Triggered Synergistic Photothermal-Chemotherapy.

PurposeThe aim of this study was to develop an “all-in-one” nanoplatform that integrates at the second near-infrared (NIR-II) region dye IR1061 and anticancer drug paclitaxel (PTX) into an apoferritin (AFN) nanocage (IR-AFN@PTX). Simultaneously, folic acid (FA), tumor target molecule, was conjugated onto IR-AFN@PTX to be IR-AFN@PTX-FA for tumor-targeted and pH/NIR-II-triggered synergistic photothermal-chemotherapy.MethodsIR1061 was firstly reacted with PEG and then conjugated with AFN to be IR-AFN. Then, FA was conjugated onto the surface of IR-AFN to be IR-AFN-FA. At last, PTX was incorporated into IR-AFN-FA to fabricate a nanoplatform IR-AFN@PTX-FA. The NIR-II photothermal properties and pH/NIR-II triggered drug release were evaluated. The ability of IR-AFN@PTX-FA to target tumors was estimated using optical bioluminescence. In vitro and in vivo synergistic therapeutic effects of pH/NIR-II-triggered and tumor-targeted photothermal-chemotherapy were investigated in 4T1 tumor model.ResultsIR-AFN@PTX-FA showed excellent water solubility and physiological stability, which significantly enhanced the solubility of both IR1061 and PTX. After 5 min of laser irradiation at 1064 nm, IR-AFN@PTX-FA exhibited an effective photothermal effect compared with laser irradiation at 808 nm, even when blocked with 0.6 cm thick chicken breast. Cellular uptake experiments showed IR-AFN@PTX-FA utilized clathrin-mediated and caveolae-mediated endocytosis pathways to enter 4T1 cells, and was then delivered by the endosome to the lysosome. NIR-II laser irradiation and pH could synergistically trigger PTX release, inducing significant tumor inhibition in vitro and in vivo.ConclusionAs a novel “all-in-one” nanoplatform, IR-AFN@PTX-FA was found to selectively target tumors and showed very efficient NIR-II photothermal effects and pH/NIR-II triggered drug release effects, showing a remarkable, synergistic photothermal-chemotherapy effect.

Amino Acid-Linked Low Molecular Weight Polyethylenimine for Improved Gene Delivery and Biocompatibility.

The construction of efficient and low toxic non-viral gene delivery vectors is of great significance for gene therapy. Herein, two novel polycations were constructed via Michael addition from low molecular weight polyethylenimine (PEI) 600 Da and amino acid-containing linkages. Lysine and histidine were introduced for the purpose of improved DNA binding and pH buffering capacity, respectively. The ester bonds afforded the polymer biodegradability, which was confirmed by the gel permeation chromatography (GPC) measurement. The polymers could well condense DNA into nanoparticles and protect DNA from degradation by nuclease. Compared with PEI 25 kDa, these polymers showed higher transfection efficiency, lower toxicity, and better serum tolerance. Study of this mechanism revealed that the polyplexes enter the cells mainly through caveolae-mediated endocytosis pathway; this, together with their biodegradability, facilitates the internalization of polyplexes and the release of DNA. The results reveal that the amino acid-linked low molecular weight PEI polymers could serve as promising candidates for non-viral gene delivery.

Mediated pathways of exosomes uptake by stem cells of apical papilla

To evaluate the uptake of exosomes by stem cells from apical papilla (SCAP), thus to provide experimental basis for mechanism of the exosomes endocytosis by SCAP. (1) Exosomes of dental pulp stem cells (DPSCs) were isolated by hypercentrifugation combined with ultrafiltration method. The exosomes were identified by transmission electron microscopy, nanoparticle tracking analysis and western blot. (2) PKH-26 membrane labeling technology was used to mark the DPSCs derived exosomes. The labeled exosomes were co-cultured with SCAP at 37 °C as positive control group, and co-cultured with SCAP at 4 °C as the low-temperature treatment group, while the negative control group was set up. (3) Using clathrin-mediated endocytosis inhibitor chlorpromazine (CPZ, 10 μmol /L) as CPZ group, caveolae-mediated endocytosis Genistein (200 μmol/L) as Genistein group, and macropinocytosis inhibitor LY294002 (50 μmol/L) as LY294002 group to treat the SCAP respectively. Solvent control group (DMSO group) was set. Immunofluorescence staining was used to detect the red fluorescence SCAP and flow cytometry was used to analyze the proportion of SCAP labeled with red fluorescence. (1) The bilayer membrane and cup-shaped appearance of representative exosomes were observed. The peak of the size of DPSCs-derived exosomes was at 144 nm. The exosomes expressed exosomal marker proteins TSG101 and CD63, but not GAPDH which was the cellular internal control protein. (2) Immunofluorescence staining showed that after being co-cultured at 37 °C for 6 hours, red fluorescence could be detected in SCAP but it could not be detected after being co-cultured at 4 °C for 6 hours. After endocytosis inhibition, the red fluorescence in SCAP was reduced. Flow cytometry showed that the proportion of SCAP labeled with red fluorescence in positive group was 35.0%, in negative control group was 0.5%, and in solvent control group was 29.7%, in CPZ group, Genistein group and Genistein group were reduced to 13.7%, 16.6%, and 20.9%, respectively. SCAP could uptake the DPSCs derived exosomes, and low temperature could inhibit this process. The exosomes uptake of SCAP was mediated by the clathrin endocytosis pathway, caveolae-mediated endocytosis and macropinocytosis pathway.

PEDV enters cells through clathrin-, caveolae-, and lipid raft-mediated endocytosis and traffics via the endo-/lysosome pathway

With the emergence of highly pathogenic variant strains, porcine epidemic diarrhea virus (PEDV) has led to significant economic loss in the global swine industry. Many studies have described how coronaviruses enter cells, but information on PEDV invasion strategies remains insufficient. Given that the differences in gene sequences and pathogenicity between classical and mutant strains of PEDV may lead to diverse invasion mechanisms, this study focused on the cellular entry pathways and cellular transport of the PEDV GI and GII subtype strains in Vero cells and IPEC-J2 cells. We first characterized the kinetics of PEDV entry into cells and found that the highest invasion rate of PEDV was approximately 33% in the IPEC-J2 cells and approximately 100% in the Vero cells. To clarify the specific endocytic pathways, systematic research methods were used and showed that PEDV enters cells via the clathrin- and caveolae-mediated endocytosis pathways, in which dynamin II, clathrin heavy chain, Eps15, cholesterol, and caveolin-1 were indispensably involved. In addition, lipid raft extraction assay showed that PEDV can also enter cells through lipid raft-mediated endocytosis. To investigate the trafficking of internalized PEDV, we found that PEDV entry into cells relied on low pH and internalized virions reached lysosomes through the early endosome–late endosome–lysosome pathway. The results concretely revealed the entry mechanisms of PEDV and provided an insightful theoretical basis for the further understanding of PEDV pathogenesis and guidance for new targets of antiviral drugs.

Mechanical cues modulate cellular uptake of nanoparticles in cancer via clathrin-mediated and caveolae-mediated endocytosis pathways.

To investigate the influence of tissue mechanics on the cellular uptake efficiency of nanoparticles (NPs) in cancer. Collagen-coated polyacrylamide gels were prepared as model substrates. Coumarin 6-loaded poly(lactic-co-glycolic) acid micelles (C6-NPs) were prepared to investigate the cellular uptake of NPs. We demonstrated that substrate stiffness modulated the cellular uptake of NPs of cancer. Mechanistically, mechanical cues exerted influence on the clathrin-mediated endocytosis and caveolae-mediated endocytosis pathways, which mediated stiffness-regulated cellular uptake of NPs. Our findings shed light on the regulatory role of the mechanical cues on the cellular uptake of NPs and will facilitate the selection of clinical patients who might benefit from a given nanotherapy.