Sarah F. Hamm-Alvarez

Intracellular trafficking of nonviral vectors

Nonviral vectors continue to be attractive alternatives to viruses due to their low toxicity and immunogenicity, lack of pathogenicity, and ease of pharmacologic production. However, nonviral vectors also continue to suffer from relatively low levels of gene transfer compared to viruses, thus the drive to improve these vectors continues. Many studies on vector–cell interactions have reported that nonviral vectors bind and enter cells efficiently, but yield low gene expression, thus directing our attention to the intracellular trafficking of these vectors to understand where the obstacles occur. Here, we will review nonviral vector trafficking pathways, which will be considered here as the steps from cell binding to nuclear delivery. Studies on the intracellular trafficking of nonviral vectors has given us valuable insights into the barriers these vectors must overcome to mediate efficient gene transfer. Importantly, we will highlight the different approaches used by researchers to overcome certain trafficking barriers to gene transfer, many of which incorporate components from biological systems that have naturally evolved the capacity to overcome such obstacles. The tools used to study trafficking pathways will also be discussed.

Targeting receptor-mediated endocytotic pathways with nanoparticles: rationale and advances

Targeting of drugs and their carrier systems by using receptor-mediated endocytotic pathways was in its nascent stages 25 years ago. In the intervening years, an explosion of knowledge focused on design and synthesis of nanoparticulate delivery systems as well as elucidation of the cellular complexity of what was previously-termed receptor-mediated endocytosis has now created a situation when it has become possible to design and test the feasibility of delivery of highly specific nanoparticle drug carriers to specific cells and tissue. This review outlines the mechanisms governing the major modes of receptor-mediated endocytosis used in drug delivery and highlights recent approaches using these as targets for in vivo drug delivery of nanoparticles. The review also discusses some of the inherent complexity associated with the simple shift from a ligand-drug conjugate versus a ligand-nanoparticle conjugate, in terms of ligand valency and its relationship to the mode of receptor-mediated internalization.

Site-Specific Labeling of Enveloped Viruses with Quantum Dots for Single Virus Tracking

This study reports a general method of labeling enveloped viruses with semiconductor quantum dots (QDs) for use in single virus trafficking studies. Retroviruses, including human immunodeficiency virus (HIV), could be successfully tagged with QDs through the membrane incorporation of a short acceptor peptide (AP) that is susceptible to site-specific biotinylation and attachment of streptavidin-conjugated QDs. It was found that this AP tag-based QD labeling had little effect on the viral infectivity and allowed for the study of the kinetics of the internalization of the recombinant lentivirus enveloped with vesicular stomatitis virus glycoprotein (VSVG) into the early endosomes. It also allows for the live cell imaging of the trafficking of labeled virus to the Rab5(+) endosomal compartments. This study further demonstrated by direct visualization of QD-labeled virus that VSVG-pseudotyped lentivirus enters cells independent of clatherin- and caveolin-pathways, while the entry of VSVG-pseudotyped retrovirus occurs via the clathrin pathway. The studies monitoring HIV particles using QD-labeling showed that we could detect single virions on the surface of target cells expressing either CD4/CCR5 or DC-SIGN. Further internalization studies of QD-HIV evidently showed that the clathrin pathway is the major route for DC-SIGN-mediated uptake of viruses. Taken together, our data demonstrate the potential of this QD-labeling for visualizing the dynamic interactions between viruses and target cell structures.

Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells

The acinar epithelial cells of the lacrimal gland exocytose the contents of mature secretory vesicles containing tear proteins at their apical membranes in response to secretagogues. Here we use time-lapse confocal fluorescence microscopy and fluorescence recovery after photobleaching to investigate the changes in actin filaments located beneath the apical membrane during exocytosis evoked by the muscarinic agonist, carbachol (100 μM). Time-lapse confocal fluorescence microscopy of apical actin filaments in reconstituted rabbit lacrimal acini transduced with replication-deficient adenovirus containing GFP-actin revealed a relatively quiescent apical actin array in resting acini. Carbachol markedly increased apical actin filament turnover and also promoted transient actin assembly around apparent fusion intermediates. Fluorescence recovery after photobleaching measurements revealed significant (P≤0.05) increases and decreases, respectively, in mobile fraction (Mf) and turnover times (t½) for apical actin filaments in carbachol-stimulated acini relative to untreated acini. The myosin inhibitors, 2,3-butanedione monoxime (BDM, 10 mM, 15 minutes) and ML-7 (40 μM, 15 minutes), significantly decreased carbachol-stimulated secretion of bulk protein and the exogenous secretory vesicle marker, syncollin-GFP; these agents also promoted accumulation of actin-coated structures which were enriched, in transduced acini, in syncollin-GFP, confirming their identity as fusion intermediates. Actin-coated fusion intermediates were sized consistent with incorporation of multiple rather than single secretory vesicles; moreover, BDM and ML-7 caused a shift towards formation of multiple secretory vesicle aggregates while significantly increasing the diameter of actin-coated fusion intermediates. Our findings suggest that the increased turnover of apical actin filaments and the interaction of actin with non-muscle myosin II assembled around aggregates of secretory vesicles facilitate exocytosis in lacrimal acinar epithelial cells.

Mechanisms of Alveolar Epithelial Translocation of a Defined Population of Nanoparticles

To explore mechanisms of nanoparticle interactions with and trafficking across lung alveolar epithelium, we utilized primary rat alveolar epithelial cell monolayers (RAECMs) and an artificial lipid bilayer on filter model (ALBF). Trafficking rates of fluorescently labeled polystyrene nanoparticles (PNPs; 20 and 100 nm, carboxylate (negatively charged) or amidine (positively charged)-modified) in the apical-to-basolateral direction under various experimental conditions were measured. Using confocal laser scanning microscopy, we investigated PNP colocalization with early endosome antigen-1, caveolin-1, clathrin heavy chain, cholera toxin B, and wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from RAECMs, and trafficking of (22)Na and (14)C-mannitol across ALBF, were measured in the presence and absence of PNPs. Results showed that trafficking of positively charged PNPs was 20-40 times that of negatively charged PNPs across both RAECMs and ALBF, whereas translocation of PNPs across RAECMs was 2-3 times faster than that across ALBF. Trafficking rates of PNPs across RAECMs did not change in the presence of EGTA (which decreased transepithelial electrical resistance to zero) or inhibitors of endocytosis. Confocal laser scanning microscopy revealed no intracellular colocalization of PNPs with early endosome antigen-1, caveolin-1, clathrin heavy chain, cholera toxin B, or wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from alveolar epithelial cells, and sodium ion and mannitol flux across ALBF, were not different in the presence or absence of PNPs. These data indicate that PNPs translocate primarily transcellularly across RAECMs, but not via known major endocytic pathways, and suggest that such translocation may take place by diffusion of PNPs through the lipid bilayer of cell plasma membranes.

Cellular uptake of cyclotide MCoTI-I follows multiple endocytic pathways

Cyclotides are plant-derived proteins that naturally exhibit various biological activities and whose unique cyclic structure makes them remarkably stable and resistant to denaturation or degradation. These attributes, among others, make them ideally suited for use as drug development tools. This study investigated the cellular uptake of cyclotide, MCoTI-I in live HeLa cells. Using real time confocal fluorescence microscopy imaging, we show that MCoTI-I is readily internalized in live HeLa cells and that its endocytosis is temperature-dependent. Endocytosis of MCoTI-I in HeLa cells is achieved primarily through fluid-phase endocytosis, as evidenced by its significant colocalization with 10K-dextran, but also through other pathways as well, as evidenced by its colocalization with markers for cholesterol-dependent and clathrin-mediated endocytosis, cholera toxin B and EGF respectively. Uptake does not appear to occur only via macropinocytosis as inhibition of this pathway by Latrunculin B-induced disassembly of actin filaments did not affect MCoTI-I uptake and treatment with EIPA which also seemed to inhibit other pathways collectively inhibited approximately 80% of cellular uptake. As well, a significant amount of MCoTI-I accumulates in late endosomal and lysosomal compartments and MCoTI-I-containing vesicles continue to exhibit directed movements. These findings demonstrate internalization of MCoTI-I through multiple endocytic pathways that are dominant in the cell type investigated, suggesting that this cyclotide has ready access to general endosomal/lysosomal pathways but could readily be re-targeted to specific receptors through addition of targeting ligands.

Polystyrene nanoparticle trafficking across alveolar epithelium

We investigated trafficking of polystyrene nanoparticles (PNP; 20 and 100 nm; carboxylate, sulfate, or aldehyde-sulfate modified [negatively charged] and amidine-modified [positively charged]) across rat alveolar epithelial cell monolayers (RAECM). Apical-to-basolateral fluxes of nanoparticles were estimated as functions of apical PNP concentration ([PNP]) and temperature. Uptake of nanoparticles into RAECM was determined using confocal microscopy. Fluxes increased as charge density became less negative/more positive, with positively charged PNPs trafficking 20-40 times faster than highly negatively charged PNP of comparable size. Trafficking rates decreased with increasing PNP diameter. PNP fluxes tended to level off at high apical [PNP]. Fluxes at 4 degrees C were significantly lower than those at 37 degrees C. Confocal microscopy revealed nanoparticles localized to cell cytoplasm, whereas cell junctions and nuclei appeared free of PNP. These data indicate that (1) trafficking of PNP across RAECM is strongly influenced by charge density, size, and temperature, (2) PNP translocate primarily transcellularly, and (3) PNP translocation requires cellular energy.

Alveolar Epithelial Cell Injury Due to Zinc Oxide Nanoparticle Exposure

RATIONALE Although inhalation of zinc oxide (ZnO) nanoparticles (NPs) is known to cause systemic disease (i.e., metal fume fever), little is known about mechanisms underlying injury to alveolar epithelium. OBJECTIVES Investigate ZnO NP-induced injury to alveolar epithelium by exposing primary cultured rat alveolar epithelial cell monolayers (RAECMs) to ZnO NPs. METHODS RAECMs were exposed apically to ZnO NPs or, in some experiments, to culture fluid containing ZnCl₂ or free Zn released from ZnO NPs. Transepithelial electrical resistance (R(T)) and equivalent short-circuit current (I(EQ)) were assessed as functions of concentration and time. Morphologic changes, lactate dehydrogenase release, cell membrane integrity, intracellular reactive oxygen species (ROS), and mitochondrial activity were measured. MEASUREMENTS AND MAIN RESULTS Apical exposure to 176 μg/ml ZnO NPs decreased R(T) and I(EQ) of RAECMs by 100% over 24 hours, whereas exposure to 11 μg/ml ZnO NPs had little effect. Changes in R(T) and I(EQ) caused by 176 μg/ml ZnO NPs were irreversible. ZnO NP effects on R(T) yielded half-maximal concentrations of approximately 20 μg/ml. Apical exposure for 24 hours to 176 μg/ml ZnO NPs induced decreases in mitochondrial activity and increases in lactate dehydrogenase release, permeability to fluorescein sulfonic acid, increased intracellular ROS, and translocation of ZnO NPs from apical to basolateral fluid (most likely across injured cells and/or damaged paracellular pathways). CONCLUSIONS ZnO NPs cause severe injury to RAECMs in a dose- and time-dependent manner, mediated, at least in part, by free Zn released from ZnO NPs, mitochondrial dysfunction, and increased intracellular ROS.

Cytoplasmic dynein participates in apically targeted stimulated secretory traffic in primary rabbit lacrimal acinar epithelial cells

A major function of the acinar cells of the lacrimal gland is the production and stimulated release of tear proteins into ocular surface fluid. We investigate the participation of cytoplasmic dynein in carbachol-stimulated traffic to the apical plasma membrane in primary rabbit lacrimal acinar epithelial cells. Confocal fluorescence microscopy revealed a major carbachol-induced, microtubule-dependent recruitment of cytoplasmic dynein and the dynactin complex into the subapical region. Colocalization studies, sorbitol density gradient/phase partitioning analysis and microtubule-affinity purification of membranes showed that some dynein and dynactin complex were associated with VAMP2-enriched membranes. Adenovirus-mediated overexpression of p50/dynamitin inhibited the recruitment and colocalization of dynein, the dynactin complex and VAMP2 in the subapical region. Nocodazole treatment and p50/dynamitin overexpression also depleted subapical stores of rab3D in resting acini, suggesting that dynein activity was also involved in maintenance of rab3D-enriched secretory vesicles. These data implicate cytoplasmic dynein in stimulated traffic to the apical plasma membrane in these secretory epithelial cells.

A rapamycin-binding protein polymer nanoparticle shows potent therapeutic activity in suppressing autoimmune dacryoadenitis in a mouse model of Sjögren's syndrome

Sjögrens syndrome (SjS) is a chronic autoimmune disease characterized initially by lymphocytic infiltration and destruction of exocrine glands, followed by systemic organ damage and B-cell lymphoma. Conventional treatment is based on management of symptoms and there is a shortage of therapies that address the underlying causes of inflammation at source exocrine tissue. The aim of this study was to test a novel protein polymer-based platform consisting of diblock copolymers composed from Elastin-like Polypeptides (ELPs) fused with FKBP12, to deliver a potent immunosuppressant with dose-limiting toxicity, rapamycin (Rapa) also known as Sirolimus, and evaluate its effects on the inflamed lacrimal gland (LG) of non-obese diabetic mouse (NOD), a classic mouse model of SjS. Both soluble and diblock copolymer ELPs were fused to FKBP12 and characterized with respect to purity, hydrodynamic radii, drug entrapment and release. Both formulations showed successful association with Rapa; however, the nanoparticle formulation, FSI, released drug with nearly a 5 fold longer terminal half-life of 62.5h. The strong interaction of FSI nanoparticles with Rapa was confirmed in vivo by a shift in the monoexponential pharmacokinetic profile for free drug to a biexponential profile for the nanoparticle formulation. When acutely administered by injection into NOD mice via the tail vein, this FSI formulation significantly suppressed lymphocytic infiltration in the LG relative to the control group while reducing toxicity. There was also a significant effect on inflammatory and mammalian target of Rapamycin (mTOR) pathway genes in the LG and surprisingly, our nanoparticle formulation was significantly better at decreasing a proposed tear biomarker of SjS, cathepsin S (CATS) compared to free drug. These findings suggest that FSI is a promising tool for delivering Rapa for treatment of SjS in a murine model and may be further explored to meet the unmet medical challenge of SjS.