Biodegradable polymer nanoparticles (NPs) are a promising approach for intracellular delivery of drugs, proteins, and nucleic acids, but little is known about their intracellular fate, particularly in epithelial cells, which represent a major target. Rhodamine-loaded PLGA (polylactic co-glycolic acid) NPs were used to explore particle uptake and intracellular fate in three different epithelial cell lines modeling the respiratory airway (HBE), gut (Caco-2), and renal proximal tubule (OK). To track intracellular fate, immunofluorescence techniques and confocal microscopy were used to demonstrate colocalization of NPs with specific organelles: early endosomes, late endosomes, lysosomes, endoplasmic reticulum (ER), and Golgi apparatus. Confocal analysis demonstrated that NPs are capable of entering cells of all three types of epithelium. NPs appear to colocalize with the early endosomes at short times after exposure (~2 hr), but are also found in other compartments within the cytoplasm, notably Golgi and, possibly, ER, as time progressed over the period of 4 to 24 hrs. The rate and extent of uptake differed among these cell lines: at a fixed particle/cell ratio, cellular uptake was most abundant in OK cells and least abundant in Caco-2 cells. We present a model for the intracellular fate of particles that is consistent with our experimental data.

The host factors required for in vivo infection have not been investigated for any papillomavirus. Using a recently developed murine cervicovaginal challenge model, we evaluated the importance of heparan sulfate proteoglycans (HSPGs) in human papillomavirus (HPV) infection of the murine female genital tract. We examined HPV type 16 (HPV16) as well as HPV31 and HPV5, for which some evidence suggests that they may differ from HPV16 in their utilization of HSPGs as their primary attachment factor in vitro. Luciferaseexpressing pseudovirus of all three types infected the mouse genital tract, although HPV5, which normally infects nongenital epidermis, was less efficient. Heparinase III treatment of the genital tract significantly inhibited infection of all three types by greater than 90% and clearly inhibited virion attachment to the basement membrane and cell surfaces, establishing that HSPGs are the primary attachment factors for these three viruses in vivo. However, the pseudoviruses differed in their responses to treatment with various forms of heparin, a soluble analog of heparan sulfate. HPV16 and HPV31 infections were effectively inhibited by a highly sulfated form of heparin, but HPV5 was not, although it bound the compound. In contrast, a Ndesulfated and N-acylated variant preferentially inhibited HPV5. Inhibition of infection paralleled the relative ability of the variants to inhibit basement membrane and cell surface binding. We speculate that cutaneous HPVs, such as HPV5, and genital mucosal HPVs, such as HPV16 and -31, may have evolved to recognize different forms of HSPGs to enable them to preferentially infect keratinocytes at different anatomical sites.Papillomaviruses (PVs) are icosahedral DNA viruses that have evolved into numerous genotypes that productively infect diverse vertebrates in a species-specific manner. These viruses also display strict tissue specificity, productively infecting only epithelial cells in the skin and mucosa. These features have inhibited viral propagation in vitro and retarded the development of in vivo models for infection. The human PVs (HPVs) belonging to the alpha genus preferentially infect the genital mucosa, and a subset of this genus include the types (e.g., HPV16, that are the causative agents of cervical carcinoma. HPV types belonging to the beta genus generally cause asymptomatic skin infections, but certain beta types (e.g., HPV5 and -8) are associated with cutaneous squamous cell carcinomas in individuals with the rare genetic disorder epidermodysplasia verruciformis.As with other viruses, virion attachment to the host cell is required for successful PV infection. In vitro studies have implicated cell surface heparan sulfate (HS) proteoglycans (HSPGs) as the primary attachment factors for most HPV types (13, 15). HSPGs are composed of a core protein with covalently attached repeating disaccharide units known as glycosaminoglycans. Posttranslational modification of the glycosaminoglycans by acetylation and sulfation leads to substantial heterogeneity that vari...

Hepatocytes metabolize energy-rich cytoplasmic lipid droplets (LDs) in the lysosome-directed process of autophagy. An organelle-selective form of this process (macrolipophagy) results in the engulfment of LDs within double-membrane delimited structures (autophagosomes) before lysosomal fusion. Whether this is an exclusive autophagic mechanism used by hepatocytes to catabolize LDs is unclear. It is also unknown whether lysosomes alone might be sufficient to mediate LD turnover in the absence of an autophagosomal intermediate. We performed live-cell microscopy of hepatocytes to monitor the dynamic interactions between lysosomes and LDs in real-time. We additionally used a fluorescent variant of the LD-specific protein (PLIN2) that exhibits altered fluorescence in response to LD interactions with the lysosome. We find that mammalian lysosomes and LDs undergo interactions during which proteins and lipids can be transferred from LDs directly into lysosomes. Electron microscopy (EM) of primary hepatocytes or hepatocyte-derived cell lines supports the existence of these interactions. It reveals a dramatic process whereby the lipid contents of the LD can be “extruded” directly into the lysosomal lumen under nutrient-limited conditions. Significantly, these interactions are not affected by perturbations to crucial components of the canonical macroautophagy machinery and can occur in the absence of double-membrane lipoautophagosomes. These findings implicate the existence of an autophagic mechanism used by mammalian cells for the direct transfer of LD components into the lysosome for breakdown. This process further emphasizes the critical role of lysosomes in hepatic LD catabolism and provides insights into the mechanisms underlying lipid homeostasis in the liver.