Pathways of cellular internalisation of liposomes delivered siRNA and effects on siRNA engagement with target mRNA and silencing in cancer cells

Design of an efficient delivery system is a generally recognised bottleneck in translation of siRNA technology into clinic. Despite research efforts, cellular processes that determine efficiency of siRNA silencing achieved by different delivery formulations remain unclear. Here, we investigated the mechanism(s) of cellular internalisation of a model siRNA-loaded liposome system in a correlation to the engagement of delivered siRNA with its target and consequent silencing by adopting siRNA molecular beacon technology. Probing of cellular internalisation pathways by a panel of pharmacological inhibitors indicated that clathrin-mediated (dynamin-dependent) endocytosis, macropinocytosis (dynamine independent), and cell membrane cholesterol dependent process(es) (clathrin and caveolea-independent) all play a role in the siRNA-liposomes internalization. The inhibition of either of these entry routes was, in general, mirrored by a reduction in the level of siRNA engagement with its target mRNA, as well as in a reduction of the target gene silencing. A dramatic increase in siRNA engagement with its target RNA was observed on disruption of endosomal membrane (by chloroquine), accompanied with an increased silencing. The work thus illustrates that employing molecular beacon siRNA technology one can start to assess the target RNA engagement – a stage between initial cellular internalization and final gene silencing of siRNA delivery systems

A Lipid Based Antigen Delivery System Efficiently Facilitates MHC Class-I Antigen Presentation in Dendritic Cells to Stimulate CD8+ T Cells

The most effective strategy for protection against intracellular infections such as Leishmania is vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8+cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4+ and CD8+ T cell responses for longterm protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to efficient presentation of antigen to specific CD4+ and CD8+ T cells. Furthermore, lymphoid CD8+ T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8+ T cell responses when compared to free and liposomal ntigen. These liposomal formulations presented to CD8+ T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine

Cellular attachment and internalization of cationic liposomes containing mycobacterial cell wall

[[abstract]]The cell wall derived from Mycobacterium bovis bacillus Calmette-Gue?rin (BCG) is apotent immunopotentiator and has recently been suggested as an alternative treatment for in situ bladder carcinoma. In contrast to the live BCG, the loss of infectivity and the negatively charged nature of BCG cell wall physically inhibit its attachment and subsequent internalization to the urothelial bladder cells. As part of our research involving the delivery of macromolecules to target cells, we developed cationic liposomes that anchor arginine octamers on the liposome surface. In this study, we used cationic liposomes as a delivery tool to facilitate the attachment and internalization of the BCG cell wall. Using confocal scanning microscopy, we verified that cationic liposomes incorporated with BCG cell wall could attach to the cellular membrane of murine bladder tumour (MBT-2) cells and become internalized. Cationic liposomes containing BCG cell wall were taken up by MBT-2 cells mainly via clathrin-mediated endocytosis. These results would be useful to understand the mechanism of action of BCG cell wall against bladder tumour cells as well as to develop an immunotherapeutic agent for clinical use

Maturation of dendritic cells induced by nano-liposomes containing imiquimod

[[abstract]]Background: Imiquimod (IMQ), a small-molecule immune response modifier of the imidazoquinoline family, has been shown to activate functional maturation of dendritic cells (DCs) through the Toll-like receptor (TLR)7-signaling pathway. IMQ has the potential to develop into a novel vaccine adjuvant. Currently, a topical cream of IMQ has been approved for the treatment of genital warts, superficial basal cell carcinoma and actinic keratosis. Objective: To develop nano-liposomes with encapsulated IMQ as a new vaccine adjuvant. Methods: Two types of nano-lipid particles containing IMQ which differed in their surface charge (i.e. positively and negatively charged liposomes) were prepared and characterized. Murine bone marrow derived DCs were cultured and treated with liposomal formulations. The surface marker expression of DCs was then determined by flow cytometry. Results: Both liposomes with IMQ encapsulated enhanced DCs maturation as determined by the surface marker expression of DCs (CD80, CD86, and MHC class II). Cationic liposomes induced high levels of surface marker expression of DCs in similar levels as those induced by lipopolysaccharides (LPS). Conclusion: The novel IMQ formulation, nano-liposome, has the benefits of simplicity and it enhances the adjuvant effect of IMQ

Virosome and ISCOM vaccines against Newcastle disease: preparation, characterization and immunogenicity

The purpose of this study was to prepare and characterize virosomes and ISCOMs containing envelope proteins of Newcastle disease virus (NDV) and to evaluate their immunogenicity in target animals (chickens). Virosomes were prepared by solubilization of virus with either Triton X-100 or octyl glucoside (OG) followed by detergent removal. Biochemical analysis revealed that these virosomes contained both the haemagglutinin-neuraminidase protein (HN) and the fusion protein (F), with preserved biological activity. Acidic environment triggered the fusion between virosomes and chicken erythrocyte ghosts. Formation of ISCOMs was achieved by solubilizing phospholipids, cholesterol, envelope protein antigen and Quil A in Triton X-100. The ISCOM particles were formed by removal of the detergent. In each formulation the relative HN content correlated with the capability to agglutinate red blood cells. The immunogenicity of these lipid-based subunit vaccines was determined in chickens after subcutaneous immunization. The relative HN content of the subunit vaccines correlated with the haemagglutination-inhibition (HI) antibody titres. Virosomes prepared with Triton X-100 and ISCOMs offered high clinical protection (> 80%) upon challenge with virulent NDV. Virosomes prepared with OG yielded lower clinical protection despite high HI antibody titres. Virosomes with reduced antigen density showed poor immunogenicity and protection. In conclusion, ND virosomes and ISCOMs were found to be immunogenic and provided good protection