Polyunsaturated liposomes are antiviral against hepatitis B and C viruses and HIV by decreasing cholesterol levels in infected cells.

The pressing need for broad-spectrum antivirals could be met by targeting host rather than viral processes. Cholesterol biosynthesis within the infected cell is one promising target for a large number of viral systems, including hepatitis C virus (HCV), hepatitis B virus (HBV) and HIV. Liposomes developed for intracellular, endoplasmic reticulum (ER)-targeted in vivo drug delivery have been modified to include polyunsaturated fatty acids that exert an independent antiviral activity through the reduction of cellular cholesterol. These polyunsaturated ER liposomes (PERLs) have greater activity than lovastatin (Mevacor, Altoprev), which is clinically approved for lowering cholesterol and preventing cardiovascular disease. Treatment of HCV, HBV, and HIV infections with PERLs significantly decreased viral secretion and infectivity, and pretreatment of naïve cells reduced the ability of both HCV and HIV to establish infections because of the decreased levels of plasma membrane cholesterol. Direct competition for cellular receptors was an added effect of PERLs against HCV infections. The greatest antiviral activity in all three systems was the inhibition of viral infectivity through the reduction of virus-associated cholesterol. Our study demonstrates that PERLs are a broadly effective antiviral therapy and should be developed further in combination with encapsulated drug mixtures for enhanced in vivo efficacy.

Uptake and trafficking of liposomes to the endoplasmic reticulum.

Liposomes are vesicular structures consisting of an aqueous core surrounded by a lipid bilayer. Apart from the cytosol and lysosomes, no other intracellular compartment has been successfully targeted using liposomal delivery. Here, we report the development of liposomes capable of specific targeting to the endoplasmic reticulum (ER) and associated membranes. Using competition and inhibitor assays along with confocal microscopy, we have determined that ER liposomes utilize scavenger and low-density lipoprotein receptors for endocytosis and enter cells through a caveolin- and microtubule-dependent mechanism. They traffic intact to the ER, where fusion with the ER membrane occurs after 22-25 min, which was confirmed by fluorescence-dequenching assays. Once inside the ER, tagged lipids intercalate with the ER membrane and are subsequently incorporated into ER-assembling entities, such as the ER-budding viruses hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV), lipid droplets, and secreted lipoproteins. ER liposomes are superior to cytosolic liposome formulations for the intracellular delivery of aqueous cargo, such as HIV-1 antivirals, and are especially suited for the prolonged delivery of lipids and lipophilic drugs into human cells.

N-Butyldeoxynojirimycin is a broadly effective anti-HIV therapy significantly enhanced by targeted liposome delivery.

OBJECTIVE: N-Butyldeoxynojirimycin (NB-DNJ), an inhibitor of HIV gp120 folding, was assessed as a broadly active therapy for the treatment of HIV/AIDS. Furthermore, to reduce the effective dose necessary for antiviral activity, NB-DNJ was encapsulated inside liposomes and targeted to HIV-infected cells. METHODS: Thirty-one primary isolates of HIV (including drug-resistant isolates) were cultured in peripheral blood mononuclear cells to quantify the effect of NB-DNJ on viral infectivity. pH-sensitive liposomes capable of mediating the intracellular delivery of NB-DNJ inside peripheral blood mononuclear cells were used to increase drug efficacy. RESULTS: NB-DNJ decreased viral infectivity with a single round of treatment by an average of 80% in HIV-1-infected and 95% in HIV-2-infected cultures. Two rounds of treatment reduced viral infectivity to below detectable levels for all isolates tested, with a calculated IC50 of 282 and 211 micromol/l for HIV-1 and HIV-2, respectively. When encapsulated inside liposomes, NB-DNJ inhibited HIV-1 with final concentrations in the nmol/l range (IC50 = 4 nmol/l), a 100 000-fold enhancement in IC50 relative to free NB-DNJ. Targeting liposomes to the gp120/gp41 complex with a CD4 molecule conjugated to the outer bilayer increased drug/liposome uptake five-fold in HIV-infected cells compared with uninfected cells. NB-DNJ CD4 liposomes demonstrated additional antiviral effects, reducing viral secretion by 81% and effectively neutralizing free viral particles to prevent further infections. CONCLUSION: The use of targeted liposomes encapsulating NB-DNJ provides an attractive therapeutic option against all clades of HIV, including drug-resistant isolates, in an attempt to prevent disease progression to AIDS.

pH-sensitive liposomes are efficient carriers for endoplasmic reticulum-targeted drugs in mouse melanoma cells.

Tyrosinase, the key enzyme of melanin biosynthesis, is inactivated in melanoma cells following the incubation with the imino-sugar N-butyldeoxynojirimycin, an inhibitor of the endoplasmic reticulum N-glycosylation processing. We have previously shown that tyrosinase inhibition requires high NB-DNJ concentrations, suggesting an inefficient cellular uptake of the drug. Here we show that the use of pH-sensitive liposomes composed of dioleoylphosphatidylethanolamine and cholesteryl hemisuccinate for the delivery of NB-DNJ reduced the required dose for tyrosinase inhibition by a factor of 1000. The results indicate that these pH-sensitive liposomes are efficient carriers for imino-sugars delivery in the endoplasmic reticulum of mammalian cells.

Accumulation of glycosphingolipids in Niemann-Pick C disease disrupts endosomal transport.

Glycosphingolipids are endocytosed and targeted to the Golgi apparatus but are mistargeted to lysosomes in sphingolipid storage disorders. Substrate reduction therapy utilizes imino sugars to inhibit glucosylceramide synthase and potentially abrogate the effects of storage. Niemann-Pick type C (NPC) disease is a disorder of intracellular transport where glycosphingolipids (GSLs) and cholesterol accumulate in endosomal compartments. The mechanisms of altered intracellular trafficking are not known but may involve the mistargeting and disrupted function of proteins associated with GSL membrane microdomains. Membrane microdomains were isolated by Triton X-100 and sucrose density gradient ultracentrifugation. High pressure liquid chromatography and mass spectrometric analysis of NPC1(-/-) mouse brain revealed large increases in GSL. Sphingosine was also found to be a component of membrane microdomains, and in NPC liver and spleen, large increases in cholesterol and sphingosine were found. GSL and cholesterol levels were increased in mutant NPC1-null Chinese hamster ovary cells as well as U18666A and progesterone induced NPC cell culture models. However, inhibition of GSL synthesis in NPC cells with N-butyldeoxygalactonojirimycin led to marked decreases in GSL but only small decreases in cholesterol levels. Both annexin 2 and 6, membrane-associated proteins that are important in endocytic trafficking, show distorted distributions in NPC cells. Altered BODIPY lactosylceramide targeting, decreased endocytic uptake of a fluid phase marker, and mistargeting of annexin 2 (phenotypes associated with NPC) are reversed by inhibition of GSL synthesis. It is suggested that accumulating GSL is part of a mislocalized membrane microdomain and is responsible for the deficit in endocytic trafficking found in NPC disease.