Endocytic Uptake Research Articles

Genetic and Cell Biology Methods to Study ESCRTs in Drosophila melanogaster.

Mosaic analysisin Drosophila representsa convenient entry point for studying the role of ESCRT (Endosomal Sorting Complex Required for Transport) genes in multiple cell processes crucial for organ development and homeostasis. Here, we describe the procedure to generate populations of ESCRT-mutant cells within Drosophila larval epithelial organs and to study them in whole-mount preparations using confocal microscopy. The use of antibodies directed to endocytic cargoes, vesicular trafficking, cell proliferation, death, and polarity markers allows one to investigate the consequences of loss of ESCRT activity at the subcellular and tissue level. The protocols described here can be used in fixed tissue as well as in unfixed tissue using endocytic uptake assays.

Ovarian cancer cell-secreted exosomal miR-205 promotes metastasis by inducing angiogenesis.

Background: By providing oxygen, nutrients and metastatic conduits, tumour angiogenesis is essential for cancer metastasis. Cancer cell-secreted microRNAs can be packaged into exosomes and are implicated in different aspects of tumour angiogenesis. However, the underlying mechanisms are incompletely understood.Methods: The GEPIA database and in situ hybridization assay were used to analyse expression of miR-205 in ovarian tissues. Immunohistochemistry was performed to examine the relationship between miR-205 and microvessel density. Expression of circulating miR-205 was evaluated by RT-PCR and GEO database analysis. Co-culture and exosome labelling experiments were performed to assess exosomal miR-205 transfer from ovarian cancer (OC) cells to endothelial cells ECs. Exosome uptake assays were employed to define the cellular pathways associated with the endocytic uptake of exosomal miR-205. The role of exosomal miR-205 in angiogenesis was further investigated in vivo and in vitro. Western blotting and rescue experiments were applied to detect regulation of the PTEN-AKT pathway by exosomal miR-205 in ECs.Results: miR-205 was up-regulated in OC tissues, and high expression of miR-205 was associated with metastatic progression in OC patients. Moreover, miR-205 was highly enriched in cancer-adjacent ECs, and up-regulation of miR-205 correlated positively with high microvessel density in OC patients. Importantly, miR-205 was markedly enriched in the serum of OC patients, and a high level of miR-205 in circulating exosomes was associated with OC metastasis. In addition, OC-derived miR-205 was secreted into the extracellular space and efficiently transferred to adjacent ECs in an exosome-dependent manner, and the lipid raft-associated pathway plays an important role in regulating uptake of exosomal miR-205. Exosomal miR-205 from OC cells significantly promoted in vitro angiogenesis and accelerated angiogenesis and tumour growth in a mouse model. Furthermore, we found that exosomal miR-205 induces angiogenesis via the PTEN-AKT pathway.Conclusion: These findings demonstrate an exosome-dependent mechanism by which miR-205 derived from cancer cells regulates tumour angiogenesis and implicate exosomal miR-205 as a potential therapeutic target for OC.

Host Cell Rab GTPases in Hepatitis B Virus Infection.

Hepatitis B virus (HBV) is a leading cause of liver disease and is presently estimated to infect more than 250 million humans. The extremely successful spread of this virus among the human population is explained by its effective transmission strategies and its manifold particle types, including virions, empty envelopes and naked capsids. Due to its tiny genome, HBV depends on cellular machineries to thrive in infected hepatocytes. To enter, traverse and exit the cell, HBV exploits host membrane trafficking pathways, including intracellular highways directed by Rab GTPases. Here, we review recent discoveries focused on how HBV co-opts and perturbs host Rab GTPase functions with an emphasis on Rab7A- and Rab33B-mediated trafficking pathways. Rab7A plays bidirectional roles in the viral life cycle, as it promotes the endocytic uptake of HBV in early stages, but restricts exocytic virion release in late stages. In intermediate stages of HBV propagation, Rab33B is needed to guide the assembly of replicative progeny nucleocapsids. Rab33B acts together with its Atg5-12/16L1 effector, a protein complex required for autophagosome formation, suggesting the concept that HBV exploits this Rab/effector complex as an assembly scaffold and machine. We also discuss whether Rab-directed trafficking pathways engaged by HBV may be applicable to other virus families. Identification of overlapping Rab functions may offer new chances to develop broad-spectrum host-targeted antiviral strategies.

Enhanced anticancer activity and intracellular uptake of paclitaxel-containing solid lipid nanoparticles in multidrug-resistant breast cancer cells.

PurposeThe aim of this study was to show enhanced anticancer activity of paclitaxel (Ptx) incorporated into solid lipid nanoparticles (SLNs) and reveal reversal of multidrug resistance (MDR) by SLNs mediated by increased uptake through different entry mechanisms from that in drug-sensitive cells.MethodsAnticancer activity of Ptx incorporated in SLNs (Ptx-SLNs) was measured in the drug-sensitive human breast cancer cell line MCF7 and its MDR variant MCF7/ADR. Cellular uptake of cargo molecules in SLNs was compared using Ptx-SLNs and rhodamine 123-loaded SLNs (Rho-SLNs) in both cell lines. In addition, endocytic uptake was evaluated using genistein (Gen) and chlorpromazine (Cpz) as inhibitors of clathrin- and caveola-mediated endocytosis, respectively.ResultsPtx-SLNs showed remarkably enhanced anticancer activity in MCF7/ADR compared to Ptx delivered in dimethyl sulfoxide (DMSO) and Cremophor EL-based vehicles. SLNs significantly increased intracellular uptake of Ptx and Rho in MCF7/ADR. Western blotting demonstrated that clathrin was expressed in both cell lines, while caveolin 1 was expressed only in MCF7/ADR. In MCF7/ADR, uptake of Ptx-SLNs and Rho-SLNs was reduced by Gen, while there was no change by Cpz, suggesting the involvement of caveola-mediated endocytosis. Size reduction of Rho-SLNs through high-pressure homogenization (Rho-SLNs) appeared to cause a shift of the endocytosis mechanism from a clathrin-independent pathway to a clathrin-dependent one. In contrast to MCF7/ADR, the uptake of SLNs into MCF7 was not changed by Gen or Cpz, suggesting involvement of clathrin- and caveola-independent mechanism for the entry of SLNs.ConclusionMDR was reversed by incorporating drug into SLNs, and the reversal was mediated by increased uptake of SLNs evading efflux pumps in MDR cells. The enhanced uptake could also be due to the use of different endocytosis pathways by SLNs in MDR cells from drug-sensitive cancer cells.

Single-Stranded Nucleic Acids Regulate TLR3/4/7 Activation through Interference with Clathrin-Mediated Endocytosis

Recognition of nucleic acids by endosomal Toll-like receptors (TLR) is essential to combat pathogens, but requires strict control to limit inflammatory responses. The mechanisms governing this tight regulation are unclear. We found that single-stranded oligonucleotides (ssON) inhibit endocytic pathways used by cargo destined for TLR3/4/7 signaling endosomes. Both ssDNA and ssRNA conferred the endocytic inhibition, it was concentration dependent, and required a certain ssON length. The ssON-mediated inhibition modulated signaling downstream of TLRs that localized within the affected endosomal pathway. We further show that injection of ssON dampens dsRNA-mediated inflammatory responses in the skin of non-human primates. These studies reveal a regulatory role for extracellular ssON in the endocytic uptake of TLR ligands and provide a mechanistic explanation of their immunomodulation. The identified ssON-mediated interference of endocytosis (SOMIE) is a regulatory process that temporarily dampens TLR3/4/7 signaling, thereby averting excessive immune responses.

SERS-Active 3D Interconnected Nanocarbon Web toward Nonplasmonic in Vitro Sensing of HeLa Cells and Fibroblasts.

A noninvasive intracellular component analysis technique is important in cancer treatment and the initial identification of cancer. Carbon nanomaterials/nanostructures, such as carbon nanotubes and graphene, have little to no surface enhanced Raman scattering (SERS) ability. Because of these structures' low Raman responses, they are conjugated with gold or silver to attain the SERS-active ability to detect normal fibroblasts and HeLa cancer cells. To the best of our knowledge, the effectiveness of the individual use of carbon nanomaterials as a nonplasmonic SERS-active platform for in vitro cancer/normal cell detection has not been investigated to date. Here, for the first time, we introduce a unique nonplasmonic SERS-based biosensing platform that uses a biocompatible self-assembled three-dimensional interconnected nanocarbon web (INW) for in vitro detection and differentiation of HeLa cells and fibroblasts. The sub-10-nm morphology of the INW facilitates the endocytic uptake of INW clusters to the cells, and its SERS functionality introduces live cell Raman sensing. The INW platform has achieved an enhancement factor (EF) of 3.66 × 104 and 9.10 × 103 with crystal violet and Rhodamine 6G dyes, respectively, significant in comparison to the EF of graphene surfaces (2-17). The results of the time-based Raman spectroscopy of live HeLa cells and fibroblasts revealed chemical fingerprints of intracellular components, such as DNA/RNA, proteins, and lipids. The components' spectroscopic differences facilitate and elucidate the specification of each cell. The highest Raman enhancement achieved was fourfold for fibroblasts (protein) and sixfold for HeLa cells (DNA). Furthermore, the SERS spectra along with scanning electron microscopy and fluorescence microscopy analysis of the immobilized cells after 24 and 48 h shed light on the health of fibroblasts and HeLa cells. A photon energy-induced ionization achieved with a femtosecond laser fabricated a biocompatible INW platform with the designated unique attributes. This simple, label-free, in vitro diagnosis approach for HeLa cells and fibroblasts has strong potential for cancer research.

Bovine Herpesvirus 1 Entry by a Low-pH Endosomal Pathway.

Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that poses a significant challenge to health and welfare in the cattle industry. We investigated the cellular entry route utilized by BoHV-1. We report that BoHV-1 enters Madin Darby bovine kidney (MDBK) cells, bovine turbinate cells, and African green monkey kidney (Vero) cells via a low-pH-mediated endocytosis pathway. Treatment of MDBK cells with hypertonic medium, which inhibits receptor-mediated endocytosis, prevented infection as measured by a beta-galactosidase reporter assay. Treatment of cells with noncytotoxic concentrations of the lysosomotropic agents ammonium chloride and monensin, which block the acidification of endosomes, inhibited BoHV-1 entry in a concentration-dependent fashion. The kinetics of endocytic uptake of BoHV-1 from the cell surface was rapid (50% uptake by ∼5 min). Time-of-addition experiments indicated that the lysosomotropic agents acted at early times postinfection, consistent with entry. Inactivation of virions by pretreatment with mildly acidic pH is a hallmark characteristic of viruses that utilize a low-pH-activated entry pathway. When BoHV-1 particles were exposed to pH 5.0 in the absence of target membrane, infectivity was markedly reduced. Lastly, treatment of cells with the proteasome inhibitor MG132 inhibited BoHV-1 entry in a concentration-dependent manner. Together, these results support a model of BoHV-1 infection in which low endosomal pH is a critical host trigger for fusion of the viral envelope with an endocytic membrane and necessary for successful infection of the target cell.IMPORTANCE BoHV-1 is a ubiquitous pathogen affecting cattle populations worldwide. Infection can result in complicated, polymicrobial infections due to the immunosuppressive properties of the virus. While there are vaccines on the market, they only limit disease severity and spread but do not prevent infection. The financial and animal welfare ramifications of this virus are significant, and in order to develop more effective prevention and treatment regimens, a more complete understanding of the initial steps in viral infection is necessary. This research establishes the initial entry pathway of BoHV-1, which provides a foundation for future development of effective treatments and preventative vaccines. Additionally, it allows comparisons to the entry pathways of other alphaherpesviruses, such as HSV-1.

Cholesterol and phosphatidylserine are engaged in adenoviral dodecahedron endocytosis

Adenoviral dodecahedron is a virus-like particle composed of twelve penton base proteins, derived from the capsid of human adenovirus type 3. Due to the high cell penetration capacity, it was used as a vector for protein, peptide and drug delivery. Two receptors are known to be involved in the endocytic dodecahedron uptake, namely αv integrins and heparan sulfate proteoglycans. Since it has been observed, that dodecahedron efficiently penetrates a wide range of cancer cells, it suggests that other cellular compounds may play a role in the particle endocytosis. To shed some light onto the interactions with membrane lipids and their potential role in dodecahedron entry, we performed a series of experiments including biochemical assays, fluorescence confocal imaging of giant unilamellar vesicles and surface plasmon resonance, which indicated specific preference of the particle to anionic phosphatidylserine. Experiments performed on cholesterol-depleted epithelial cells showed that cholesterol is essential in the endocytic uptake, however a direct interaction was not observed. We believe that the results will allow to better understand the role of lipids in dodecahedron entry and to design more specific dodecahedron-based vectors for drug delivery to cancer cells.

Impact of the hypoxic phenotype on the uptake and efflux of nanoparticles by human breast cancer cells

Breast cancer cells adapt to the hypoxic tumoral environment by undergoing changes in metabolism, cell signalling, endo-lysosomal receptor uptake and recycling. The resulting hypoxic cell phenotype has the potential to undermine the therapeutic efficacy of nanomedicines designed for endocytic uptake and specific intracellular trafficking. The aim of this study was to examine the impact of hypoxia and simulated reperfusion on the in vitro uptake and release of nanomedicines by human breast cancer cells. Cells were exposed to a hypoxic preconditioning treatment in 1% oxygen for 6 and 24 hours to induce temporal changes in the hypoxic circuit (e.g. HIF-1α expression). The preconditioned cells were then dosed with nanoparticles for 45 or 180 minutes emulating nanomedicine access following tumor reperfusion. Hypoxic preconditioning significantly increased nanoparticle retention by up to 10% when compared to normoxic cultures, with the greatest relative difference between normoxic and hypoxic cultures occurring with a 45 minute dosing interval. Exocytosis studies indicated that the preconditioned cells had a significantly increased nanoparticle efflux (up to 9%) when compared to normoxic cells. Overall, we were able to show that hypoxic preconditioning regulates both the endocytosis and exocytosis of nanomedicines in human breast cancer cells.

Concerted ESCRT and clathrin recruitment waves define the timing and morphology of intraluminal vesicle formation

The endosomal sorting complex required for transport (ESCRT) machinery mediates cargo sorting, membrane deformation and membrane scission on the surface of endosomes, generating intraluminal vesicles (ILVs) to degrade signaling receptors. By live-cell imaging of individual endosomes in human cells, we find that ESCRT proteins are recruited in a repetitive pattern: ESCRT-0 and -I show a gradual and linear recruitment and dissociation, whereas ESCRT-III and its regulatory ATPase VPS4 display fast and transient dynamics. Electron microscopy shows that ILVs are formed consecutively, starting immediately after endocytic uptake of cargo proteins and correlating with the repeated ESCRT recruitment waves, unraveling the timing of ILV formation. Clathrin, recruited by ESCRT-0, is required for timely ESCRT-0 dissociation, efficient ILV formation, correct ILV size and cargo degradation. Thus, cargo sorting and ILV formation occur by concerted, coordinated and repetitive recruitment waves of individual ESCRT subcomplexes and are controlled by clathrin.

A fast and straightforward procedure for vault nanoparticle purification and the characterization of its endocytic uptake

BackgroundVaults are eukaryotic ribonucleoprotein particles composed of up 78 copies of the 97 kDa major vault protein that assembles into a barrel-like, “nanocapsule” enclosing poly(ADP-ribose) polymerase, telomerase-associated protein-1 and small untranslated RNAs. Overall, the molecular mass of vault particles amounts to about 13 MDa. Although it has been implicated in several cellular functions, its physiological roles remain poorly understood. Also, the possibility to exploit it as a nanovector for drug delivery is currently being explored in several laboratories. MethodsUsing the baculovirus expression system, vaults were expressed and purified by a dialysis step using a 1 MDa molecular weight cutoff membrane and a subsequent size exclusion chromatography. Purity was assessed by SDS-PAGE, transmission electron microscopy and dynamic light scattering. Particle's endocytic uptake was monitored by flow cytometry and confocal microscopy. ResultsThe purification protocol here reported is far simpler and faster than those currently available and lead to the production of authentic vault. We then demonstrated its clathrin-mediated endocytic uptake by normal fibroblast and glioblastoma, but not carcinoma cell lines. In contrast, no significant caveolin-mediated endocytosis was detected. ConclusionsThese results provide the first evidence for an intrinsic propensity of the vault complex to undergo endocytic uptake cultured eukaryotic cells. General significanceThe newly developed purification procedure will greatly facilitate any investigation based on the use of the vault particle as a natural nanocarrier. Its clathrin-mediated endocytic uptake observed in normal and in some tumor cell lines sheds light on its physiological role.

Targeted Perturbation of Nuclear Envelope Integrity with Vapor Nanobubble-Mediated Photoporation.

The nuclear envelope (NE) has long been considered to dismantle only during mitosis. However, recent observations in cancer cells and laminopathy patient cells have revealed that the NE can also transiently rupture during interphase, thereby perturbing cellular homeostasis. Although NE ruptures are promoted by mechanical force and the loss of lamins, their stochastic nature and variable frequency preclude the study of their direct downstream consequences. We have developed a method based on vapor nanobubble-mediated photoporation that allows for deliberately inducing NE ruptures in a spatiotemporally controlled manner. Our method relies on wide-field laser illumination of perinuclear gold nanoparticles, resulting in the formation of short-lived vapor nanobubbles that inflict minute mechanical damage to the NE, thus creating small pores. We demonstrate that perinuclear localization of gold nanoparticles can be achieved after endocytic uptake or electroporation-facilitated delivery and that both strategies result in NE rupture upon laser irradiation. Furthermore, we prove that photoporation-induced nuclear ruptures are transient and recapitulate hallmarks of spontaneous NE ruptures that occur in A-type lamin-depleted cells. Finally, we show that the same approach can be used to promote influx of macromolecules that are too large to passively migrate through the NE. Thus, by providing unprecedented control over nuclear compartmentalization, nuclear photoporation offers a powerful tool for both fundamental cell biology research and drug delivery applications.

Efficient Delivery of Macromolecules into Human Cells by Improving the Endosomal Escape Activity of Cell-Penetrating Peptides: Lessons Learned from dfTAT and its Analogs

Cell-penetrating peptides (CPPs) are typically prone to endocytic uptake into human cells. However, they are often inefficient at escaping from endosomes, which limits their ability to deliver cargos into cells. This review highlights the efforts that our laboratory has devoted toward developing CPPs that can mediate the leakage of endosomal membranes, and consequently gain better access to the intracellular milieu. In particular, we have identified a CPP named dimeric fluorescent TAT (dfTAT) with high endosomolytic activity. We describe how we have used this reagent and its analogs to develop efficient cytosolic delivery protocols and learn about molecular and cellular parameters that control the cell permeation process. Specifically, we discuss how late endosomes represent exploitable gateways for intracellular entry. We also describe how certain features in CPPs, including guanidinium content, charge density, multimerization, chirality, and susceptibility to degradation modulate the activity that these peptidic agents take toward endosomal membranes and cytosolic egress.

Down-regulation of acid sphingomyelinase and neutral sphingomyelinase-2 inversely determines the cellular resistance to plasmalemmal injury by pore-forming toxins.

Bacterial pore-forming toxins compromise plasmalemmal integrity, leading to Ca2+ influx, leakage of the cytoplasm, and cell death. Such lesions can be repaired by microvesicular shedding or by the endocytic uptake of the injured membrane sites. Cells have at their disposal an entire toolbox of repair proteins for the identification and elimination of membrane lesions. Sphingomyelinases catalyze the breakdown of sphingomyelin into ceramide and phosphocholine. Sphingomyelin is predominantly localized in the outer leaflet, where it is hydrolyzed by acid sphingomyelinase (ASM) after lysosomal fusion with the plasma membrane. The magnesium-dependent neutral sphingomyelinase (NSM)-2 is found at the inner leaflet of the plasmalemma. Because either sphingomyelinase has been ascribed a role in the cellular stress response, we investigated their role in plasma membrane repair and cellular survival after treatment with the pore-forming toxins listeriolysin O (LLO) or pneumolysin (PLY). Jurkat T cells, in which ASM or NSM-2 was down-regulated [ASM knockdown (KD) or NSM-2 KD cells], showed inverse reactions to toxin-induced membrane damage: ASM KD cells displayed reduced toxin resistance, decreased viability, and defects in membrane repair. In contrast, the down-regulation of NSM-2 led to an increase in viability and enhanced plasmalemmal repair. Yet, in addition to the increased plasmalemmal repair, the enhanced toxin resistance of NSM-2 KD cells also appeared to be dependent on the activation of p38/MAPK, which was constitutively activated, whereas in ASM KD cells, the p38/MAPK activation was constitutively blunted.-Schoenauer, R., Larpin, Y., Babiychuk, E. B., Drücker, P., Babiychuk, V. S., Avota, E., Schneider-Schaulies, S., Schumacher, F., Kleuser, B., Köffel, R., Draeger, A. Down-regulation of acid sphingomyelinase and neutral sphingomyelinase-2 inversely determines the cellular resistance to plasmalemmal injury by pore-forming toxins.

The gene transfection and endocytic uptake pathways mediated by PEGylated PEI-entrapped gold nanoparticles

The development of gene vectors is the vital step in gene therapy. The cationic polymer polyethylenimine (PEI) is widely applied as an efficient and low cost nonviral gene vehicle. However, its further practical applications in gene therapy are limited due to its high cytotoxicity. To obtain safe and efficient gene vectors, hydrophilic polyethylene glycol (PEG) has been conjugated onto its surface due to its good biocompatibility, and the PEGylated PEI was used as a template to entrap gold nanoparticles (Au NPs) with different Au atom/PEI molar ratios (25:1, 50:1, 100:1, and 200:1, respectively). The formed PEGylated PEI-entrapped Au NPs (PEG-Au PENPs) and their cytotoxic effects as well as ability to transfect plasmid DNA (pDNAs) to HeLa cells were analyzed using Cell Counting Kit-8 (CCK-8) assay, flow cytometry and confocal microscopic imaging. To further understand cell internalization pathway of PEG-Au PENPs, several pharmacologic inhibitions of endocytic pathways were conducted. Our results revealed that the PEG-Au PENPs were not only able to transfect pDNAs into cells with decreased cytotoxicity, but also showed high transfection efficiency compared with PEI alone. The cellular uptake data indicated that the clathrin-mediated endocytosis is the main pathway in the internalization of the formed polyplexes. These findings suggested that the developed PEG-Au PENPs may serve as a safe gene carrier with non-compromised DNA transfection efficiency and promote the further development of efficient and safe gene delivery strategies based on nanoparticles.

Generation of Potent Anti-HER1/2 Immunotoxins by Protein Ligation Using Split Inteins.

Cell targeting protein toxins have gained increasing interest for cancer therapy aimed at increasing the therapeutic window and reducing systemic toxicity. Because recombinant expression of immunotoxins consisting of a receptor-binding and a cell-killing moiety is hampered by their high toxicity in a eukaryotic production host, most applications rely on recombinant production of fusion proteins consisting of an antibody fragment and a protein toxin in bacterial hosts such as Escherichia coli ( E. coli). These fusions often lack beneficial properties of whole antibodies like extended serum half-life or efficient endocytic uptake via receptor clustering. Here, we describe the production of full-length antibody immunotoxins using self-splicing split inteins. To this end, the short (11 amino acids) N-terminal intein part of the artificially designed split intein M86, a derivative of the Ssp DnaB intein, was recombinantly fused to the heavy chain of trastuzumab, a human epidermal growth factor receptor 2 (HER2) receptor targeting antibody and to a nanobody-Fc fusion targeting the HER1 receptor, respectively. Both antibodies were produced in Expi293F cells. The longer C-terminal counterpart of the intein was genetically fused to the protein toxins gelonin or Pseudomonas Exotoxin A, respectively, and expressed in E. coli via fusion to maltose binding protein. Using optimized in vitro splicing conditions, we were able to generate a set of specific and potent immunotoxins with IC50 values in the mid- to subpicomolar range.

Enhanced nanoparticle uptake into virus infected cells: Could nanoparticles be useful in antiviral therapy?

Virus infections cause diseases of different severity ranged from mild infection e.g. common cold into life threatening diseases e.g. Human Immunodeficiency virus (HIV), Hepatitis B. Virus infections represent 44% of newly emerging infections. Although there are many efficient antiviral agents, they still have drawbacks due to accumulation at off target organs and developing of virus resistance due to virus mutation. Therefore, developing a delivery system that can selectively target drug into affected organs and avoid off target accumulation would be a highly advantageous strategy to improve antiviral therapy. Nanoparticles (NP) can be effectively targeted to the liver, and therefore it could be used for improving therapy of hepatic virus infections including hepatitis B virus and hepatitis C virus (HCV). Many studies were performed to encapsulate antiviral agents into nano-delivery system to improve their pharmacokinetics parameters to have a better therapeutic efficacy with lower side effects. However, the effect of virus infection on the uptake of NP has not yet been studied in detail. The latter is a crucial area as modulation of endocytic uptake of nanoparticles could impact on reduce potential therapeutic usefulness of antiviral agents loaded into nano-delivery system. In this study, a fluorescently-labelled polymeric nanoparticle was prepared and used to track NP uptake into Huh7.5, human hepatoma cells transfected with replicating HCV genomes, compared with non-transfected cells as a model representing hepatocyte uptake. Confocal microscopy and flow cytometry of virus transfected Huh7.5 cells unexpectedly demonstrated two-fold increase in uptake of NP compared to non-transfected cells. Therefore, virus transfection enhanced NP uptake into Huh7.5 cells and NP could be considered as a promising delivery system for targeted treatment of hepatitis viruses.

Polymeric nanoparticles as a platform for permeability enhancement of class III drug amikacin.

Amikacin (A), a water soluble aminoglycoside antibiotic is commercially available for intravenous administration only. Present investigation is aimed at the development of poly-lactic-co-glycolic acid (PLGA) nanoparticles (A-NPs).1 for oral permeability enhancement of amikacin. The pharmaceutical attributes of the A-NPs revealed particle size, 260.3 ± 2.05 nm, zeta potential, -12.9 ± 1.12 mV and drug content, 40.10 ± 1.87 μg/mg with spherical shape and smooth surface. In vitro antibacterial studies showed that the A-NPs were active against P. aeruginosa, K. pneumoniae and E. coli. The permeation study across rat ileum showed 2.6-fold improvement in Papp for A-NPs than A-S2 This increase in permeability is due to the uptake of nanoparticles by Peyer's patches of intestinal epithelium and endocytic uptake via enterocytes. Flow cytometric analysis demonstrated 2.2-fold higher uptake of Rh B-NPs3 than Rh B-S4 and elucidated the dominance of enterocytes mediated endocytosis of nanoparticles. Furthermore, stability data collected as per ICH guidelines for three months under accelerated conditions had shown that the A-NPs were stable. The purported drug delivery system hence, seems a promising tool to replace successfully the current intravenous therapy and is used to support relevant patient compliance thereby, adding value to the "patient care at home".

Extracellular Conformational Changes in the Capsid of Human Papillomaviruses Contribute to Asynchronous Uptake into Host Cells.

Human papillomavirus 16 (HPV16) is the leading cause of cervical cancer. For initial infection, HPV16 utilizes a novel endocytic pathway for host cell entry. Unique among viruses, uptake occurs asynchronously over a protracted period of time, with half-times between 9 and 12 h. To trigger endocytic uptake, the virus particles need to undergo a series of structural modifications after initial binding to heparan sulfate proteoglycans (HSPGs). These changes involve proteolytic cleavage of the major capsid protein L1 by kallikrein-8 (KLK8), exposure of the N terminus of the minor capsid protein L2 by cyclophilins, and cleavage of this N terminus by furin. Overall, the structural changes are thought to facilitate the engagement of an elusive secondary receptor for internalization. Here, we addressed whether structural changes are the rate-limiting steps during infectious internalization of HPV16 by using structurally primed HPV16 particles. Our findings indicate that the structural modifications mediated by cyclophilins and furin, which lead to exposure and cleavage, respectively, of the L2 N terminus contribute to the slow and asynchronous internalization kinetics, whereas conformational changes elicited by HSPG binding and KLK8 cleavage did not. However, these structural modifications accounted for only 30 to 50% of the delay in internalization. Therefore, we propose that limited internalization receptor availability for engagement of HPV16 causes slow and asynchronous internalization in addition to rate-limiting structural changes in the viral capsid.IMPORTANCE HPVs are the main cause of anogenital cancers. Their unique biology is linked to the differentiation program of skin or mucosa. Here, we analyzed another unique aspect of HPV infections using the prototype HPV16. After initial cell binding, HPVs display an unusually protracted residence time on the plasma membrane prior to asynchronous uptake. As viruses typically do not expose themselves to host immune sensing, we analyzed the underlying reasons for this unusual behavior. This study provides evidence that both extracellular structural modifications and possibly a limited availability of the internalization receptor contribute to the slow internalization process of the virus. These findings indicate that perhaps a unique niche for initial infection that could allow for rapid infection exists. In addition, our results may help to develop novel, preventive antiviral measures.

Clathrin-mediated endocytic uptake of PUFA enriched self-nanoemulsifying lipidic systems (SNELS) of an anticancer drug against triple negative cancer and DMBA induced preclinical tumor model

The current studies envisage unravelling the underlying cellular internalisation mechanism of the systematically developed docetaxel (DTH) polyunsaturated fatty acid (PUFA) enriched self-nanoemulsifying lipidic micellar systems (SNELS). The concentration-, time- and cytotoxicity-related effects of DTH-SNELS on triple negative breast cancer (TNBC) MDA-MB-231 and non-TNBC MCF-7 cell lines were assessed through Presto-blue assay. Subsequently, rhodamine-123 (Rh-123) loaded SNELS were employed for evaluating their internalisation through flow cytometry and fluorescence microscopy, establishing it to be “clathrin-mediated” endocytic pathway. Apoptosis assay (65% cell death) and cell cycle distribution (47% inhibition at G2/M phase) further corroborated the cytotoxicity of DTH-SNELS towards cancerous cells. Biodistribution, histopathology and haematology studies indicated insignificant toxicity of the optimized formulation on vital organs. Preclinical anticancer efficacy studies using 7,12-dimethylbenzantracene (DMBA)-induced model construed significant reduction in breast tumor-volume. Overall, extensive in vitro and in vivo studies indicated the intracellular localization and cytotoxicity, suggesting DTH-SNELS as promising delivery systems for breast tumor therapeutics including TNBC.