Targeted delivery to neuroblastoma of novel siRNA-anti-GD2-liposomes prepared by dual asymmetric centrifugation and sterol-based post-insertion method.

PURPOSE: To optimise and simplify preparation of targeted liposomes for efficient siRNA delivery to neuroblastoma, the most common solid tumour in early childhood. METHODS: Liposomes containing siRNA were prepared by combining the novel dual asymmetric centrifugation (DAC) method and the recently optimised sterol-based post-insertion technique (SPIT) to couple anti-GD2 antibody for selective interaction with neuroblastoma cells. Cultured human neuroblastoma cell lines were used to evaluate the efficiency of siRNA delivery. RESULTS: The size of liposomes prepared by DAC ranged from 190 to 240 nm; siRNA encapsulation efficiency was up to 50%. An average of 70 and 100 molecules of anti-GD2 antibody per particle were coupled. A significant association of liposomes with neuroblastoma cells as well as effective siRNA delivery was observed only when anti-GD2 antibody was coupled. Preliminary data suggest delivery of siRNA using anti-GD2-liposomes occurs via GD2-mediated endocytosis. Vascular endothelial growth factor A (VEGF-A) was down-regulated using siRNA delivered by anti-GD2-liposomes. CONCLUSIONS: DAC and SPIT allow for the straightforward preparation of liposomes for the targeted delivery of siRNA. Anti-GD2-liposomes thus produced can serve as versatile carriers of siRNA to neuroblastoma cells.

Targeted SAINT-O-Somes for improved intracellular delivery of siRNA and cytotoxic drugs into endothelial cells.

In non-phagocytic cells such as endothelial cells, processing of liposomes and subsequent release of drug content is often inefficient due to the absence of professional processing machinery, which limits pharmacological efficacy. We therefore developed a liposome based drug delivery system with superior intracellular release characteristics. The design was based on long circulating conventional liposomes that were formulated with a cationic amphiphile, 1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chlorid (SAINT-C18). These so-called SAINT-O-Somes had a diameter of 100 nm, were as stable as conventionally formulated liposomes, and showed superior release of their content at pH conditions that liposomes encounter when they are endocytosed by cells. Attachment of anti-E-selectin specific antibodies to the distal end of surface grafted poly(ethylene glycol) resulted in immuno-SAINT-O-Somes that were as efficiently taken up by inflammation activated endothelial cells as conventional anti-E-selectin specific immunoliposomes. More importantly, intracellular release of calcein encapsulated in these targeted SAINT-O-Somes was 10 fold higher as compared to the release of calcein from conventional liposomes. For intracellular delivery siRNA into activated endothelial cells, formulation with SAINT-C18 was a necessity to induce a specific down-regulation of gene expression of VE-cadherin. Additionally, targeted doxorubicin loaded SAINT-O-Somes decreased endothelial cell viability significantly more than targeted conventional doxorubicin liposomes. SAINT-O-Somes therefore represent a new class of lipid based particles with superior drug release characteristics that can be applied for the efficacious intracellular delivery of hydrophilic drugs including siRNA.

Receptor-specific targeting with liposomes in vitro based on sterol-PEG(1300) anchors.

PURPOSE: The challenge in developing liposomes to be used in active drug targeting is to design a method that can be used for modifying liposomal membranes that is applicable for a number of different specific ligands. In this study, the post insertion technique was used with activated sterol-PEG(1300) anchors and was evaluated with regard to its effectiveness in active targeting in vitro. The key advantage of these anchors is that the insertion step into the liposomal membrane takes place at room temperature and is very fast. MATERIALS AND METHODS: For in vitro experiments, neuroblastoma cell lines overexpressing GD2 antigen on their surface as a target structure were chosen. This allowed the use of anti-GD2 antibodies coupled to the liposomal surface for testing of specific binding. These modified liposomes were labelled with rhodamine-PE and their cellular association was analyzed by flow cytometry. RESULTS: It was shown that the activated sterol-PEG(1300) anchors allow specific and significant interactions of the modified liposomes with GD2 positive cells. CONCLUSION: Coupling using sterol-PEG(1300) anchors is both simple and rapid. It is reproducible and applicable for all ligands bearing amino groups. This method demonstrates the advantage of a ready-to-use system for the modification of pre-formed liposomes with different ligands.

Addressing liver fibrosis with liposomes targeted to hepatic stellate cells.

Liver fibrosis is a chronic disease that results from hepatitis B and C infections, alcohol abuse or metabolic and genetic disorders. Ultimately, progression of fibrosis leads to cirrhosis, a stage of the disease characterized by failure of the normal liver functions. Currently, the treatment of liver fibrosis is mainly based on the removal of the underlying cause of the disease and liver transplantation, which is the only treatment for patients with advanced fibrosis. Hepatic stellate cells (HSC) are considered to be key players in the development of liver fibrosis. Chronically activated HSC produces large amounts of extracellular matrix and enhance fibrosis by secreting a broad spectrum of cytokines that exert pro-fibrotic actions in other cells, and in an autocrine manner perpetuate their own activation. Therefore, therapeutic interventions that inhibit activation of HSC and its pro-fibrotic activities are currently under investigation worldwide. In the present study we applied targeted liposomes as drug carriers to HSC in the fibrotic liver and explored the potential of these liposomes in antifibrotic therapies. Moreover, we investigated effects of bioactive compounds delivered by these liposomes on the progression of liver fibrosis. To our knowledge, this is the first study demonstrating that lipid-based drug carriers can be selectively delivered to HSC in the fibrotic liver. By incorporating the bioactive lipid DLPC, these liposomes can modulate different processes such as inflammation and fibrogenesis in the fibrotic liver. This dual functionality of liposomes as a drug carrier system with intrinsic biological effects may be exploited in new approaches to treat liver fibrosis.

A novel lipid-based drug carrier targeted to the non-parenchymal cells, including hepatic stellate cells, in the fibrotic livers of bile duct ligated rats.

In fibrotic livers, collagen producing hepatic stellate cells (HSC) represent a major target for antifibrotic therapies. We designed liposomes with surface-coupled mannose 6-phosphate (M6P) modified human serum albumin (HSA) to target HSC via the M6P receptor. In this study we determined the pharmacokinetics and target specificity of M6P-HSA-liposomes in a rat model of liver fibrosis. Ten minutes after injection of [(3)H]-M6P-HSA-liposomes 90% of the dose has cleared the circulation. The blood elimination of these liposomes was counteracted by free M6P-HSA and polyinosinic acid, a competitive inhibitor of scavenger receptors. The M6P-HSA-liposomes accumulated in HSC. However, also Kupffer cells and endothelial cells contributed to the uptake of M6P-HSA-liposomes in the fibrotic livers. Polyinosinic acid inhibited the accumulation of the liposomes in Kupffer cells and liver endothelial cells, but not in HSC. PCR analysis revealed that cultured HSC express scavenger receptors. This was confirmed by Western blotting, although activation of HSC diminishes scavenger receptor protein expression. In conclusion, in a rat model for liver fibrosis M6P-HSA-liposomes can be efficiently targeted to non-parenchymal cells, including HSC. M6P receptors and scavenger receptors are involved in the cellular recognition of these liposomes, allowing multiple pharmacological interference in different pathways involved in the fibrosis.

Delivery of viral vectors to hepatic stellate cells in fibrotic livers using HVJ envelopes fused with targeted liposomes.

Hepatic stellate cells (HSC) are a major target for antifibrotic therapies in the liver and in particular gene delivery to these cells would be relevant. Previously, we demonstrated that mannose 6-phosphate human serum albumin (M6P-HSA) coupled liposomes accumulate in HSC in fibrotic livers. Here we prepared a M6P-HSA modified viral vector that allows the targeted delivery of plasmid DNA to HSC. Therefore, UV inactivated hemagglutinating virus of Japan (HVJ) containing plasmid DNA was fused with M6P-HSA liposomes to yield HVJ liposomes targeted to HSC. These new particles had a diameter of approximately 200 nm, as determined by electron microscopy. In a carbon tetrachloride mouse model of liver fibrosis, M6P-HSA-HVJ-liposomes associated with HSC. In conclusion, our results demonstrate that fusion of M6P-HSA liposomes with HVJ envelopes results in HVJ particles that accumulate in HSC, allowing for new possibilities to interfere with fibrosis in the liver.

Effects of a new bioactive lipid-based drug carrier on cultured hepatic stellate cells and liver fibrosis in bile duct-ligated rats.

In the fibrotic liver, hepatic stellate cells (HSC) produce large amounts of collagen and secrete variety of mediators that promote development of fibrosis in this organ. Therefore, these cells are considered an attractive target for antifibrotic therapies. We incorporated the bioactive lipid dilinoleoylphosphatidylcholine (DLPC) into the membrane of liposomes, and then we evaluated its effect on hepatic stellate cell activation and liver fibrosis. To target DLPC-liposomes to HSC, human serum albumin modified with mannose 6-phosphate (M6P-HSA) was coupled to the surface of these liposomes. In vitro, the effects of the carrier were determined in primary cultures of HSC, Kupffer cells, and liver endothelial cells using real-time reverse transcription-polymerase chain reaction. In vivo DLPC-liposomes were tested in bile duct-ligated rats. Targeted M6P-HSA-DLPC-liposomes and DLPC-liposomes significantly reduced gene expression levels for collagen 1alpha1, alpha-smooth muscle actin (alpha-SMA), and transforming growth factor-beta (TGF-beta) in cultured HSC. In fibrotic livers, DLPC-liposomes decreased gene expression for TGF-beta and collagen 1alpha1 as well as alpha-SMA and collagen protein expression. In contrast, M6P-HSA-DLPC-liposomes enhanced expression of profibrotic and proinflammatory genes in vivo. In cultured Kupffer and endothelial cells M6P-HSA liposomes influenced the expression of proinflammatory genes. Both types of liposomes increased hepatocyte glycogen content in fibrotic livers, indicating improved functionality of the hepatocytes. We conclude that DLPC-containing liposomes attenuate activation of cultured HSC. In fibrotic livers, M6P-HSA-mediated activation of Kupffer and endothelial cells probably counteracts this beneficial effect of DLPC-liposomes. Therefore, these bioactive drug carriers modulate the activity of all liver cells during liver fibrosis.

Interaction of targeted liposomes with primary cultured hepatic stellate cells: Involvement of multiple receptor systems.

BACKGROUND/AIMS: In designing a versatile liposomal drug carrier to hepatic stellate cells (HSC), the interaction of mannose 6-phosphate human serum albumin (M6P-HSA) liposomes with cultured cells was studied. METHODS: M6P-HSA was covalently coupled to the liposomal surface and the uptake and binding of 3H-labelled M6P-HSA liposomes by primary rat HSC and liver endothelial cells was determined. The targeting ability of M6P-HSA liposomes to HSC was tested in bile duct ligated rats using immunohistochemical methods. RESULTS: The association of M6P-HSA liposomes with HSC was 4-fold higher than of control liposomes. An excess of M6P-HSA inhibited this association by 58%, indicating M6P receptor specificity. The scavenger receptor competitor polyinosinic acid abolished association of M6P-HSA liposomes with HSC. M6P-HSA liposomes also amply associated with endothelial cells, which abundantly express scavenger receptors. Endocytosis of M6P-HSA liposomes by HSC was temperature dependent and could be inhibited by monensin. In the fibrotic liver M6P-HSA liposomes co-localised with HSC. CONCLUSIONS: Coupling of M6P-HSA to liposomes strongly increases the in vitro uptake of these liposomes by HSC and endothelial cells. Both the mannose 6-phosphate receptor and the scavenger receptors are involved in the uptake process. M6P-HSA liposomes are potential drug carriers to HSC in the fibrotic liver.