Immobilization of histidine-tagged proteins on monodisperse metallochelation liposomes: Preparation and study of their structure.

Liposomes represent a biocompatible platform for the construction of self-assembling proteoliposomes using nickel or zinc metallochelation. Potential applications of such structures consist in the development of new biocompatible vaccination nanoparticles and drug delivery nanoparticle systems. Here, we describe the design and construction of a flow-through ultrafiltration cell suitable for the preparation of monodisperse liposomes enabled for metallochelation and, hence, the formation of proteoliposomes. The linkage of the cell with a fast protein liquid chromatography system facilitates automation of the procedure, which fits the criteria for upscaling. Proof-of-concept experiments are performed using a mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS-NTA-Ni (1,2-dioleoyl-sn-glycero-3-{[N(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl}(nickel salt)) to formulate proteoliposomes with proteins attached by metallochelation, including histidine (His)-tagged recombinant green fluorescent protein and rgp120 (derived from HIV-1 Env). These model proteoliposomes are characterized by gel permeation chromatography and by dynamic light scattering. Transmission electron microscopy and immunogold staining are used to characterize surface-bound proteins, revealing the tendency of rgp120 to form microdomains on liposome surfaces. These microdomains possess a two-dimensional crystal-like structure that is seen more precisely by atomic force microscopy.

Enhancement of immune response towards non-lipidized Borrelia burgdorferi recombinant OspC antigen by binding onto the surface of metallochelating nanoliposomes with entrapped lipophilic derivatives of norAbuMDP.

Lyme disease caused by spirochete Borrelia burgdorferi sensu lato, is a tick-born illness. If the infection is not eliminated by the host immune system and/or antibiotics, it may further disseminate and cause severe chronic complications. The immune response to Borrelia is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies associated with Th1 cell activation. A new experimental vaccine was constructed using non-lipidized form of recombinant B. burgdorferi s.s. OspC protein was anchored by metallochelating bond onto the surface of nanoliposomes containing novel nonpyrogenic lipophilized norAbuMDP analogues denoted MT05 and MT06. After i.d. immunization, the experimental vaccines surpassed Alum with respect to OspC-specific titers of IgG2a, IgG2b isotypes when MT06 was used and IgG3, IgM isotypes when MT05 was used. Both adjuvants exerted a high adjuvant effect comparable or better than MDP and proved themselves as nonpyrogenic.

Antiviral effect of HPMPC (Cidofovir®), entrapped in cationic liposomes: in vitro study on MDBK cell and BHV-1 virus.

We designed and synthesised a series of new cationic lipids based on spermine linked to various hydrophobic anchors. These lipids could be potentially useful for the preparation of stable cationic liposomes intended for the construction of drug targeting systems applicable in the field of anticancer/antiviral therapy, vaccine carriers, and vectors for the gene therapy. Low in vitro toxicity was found for these compounds, especially for LD1, in several cell lines. The delivery of both a fluorescence marker (calcein) and antiviral drugs into cells has been achieved owing to a large extent of internalization of cationic liposomes (labelled by Lyssamine-Rhodamine PE or fluorescein-PE) as demonstrated by fluorescent microscopy and quantified by flow cytometry. The bovine herpes virus type 1 (BHV-1) virus infection in vitro model using MDBK cells was employed to study the effect of the established antiviral drug HPMPC (Cidofovir®) developed by Prof. A. Holý. Inhibition of BHV-1 virus replication was studied by quantitative RT-PCR and confirmed by both Hoffman modulation contrast microscopy and transmission electron microscopy. We found that in vitro antiviral activity of HPMPC was significantly improved by formulation in cationic liposomes, which decreased the viral replication by about 2 orders of magnitude.

Metallochelating liposomes with associated lipophilised norAbuMDP as biocompatible platform for construction of vaccines with recombinant His-tagged antigens: preparation, structural study and immune response towards rHsp90.

Hsp90-CA is present in cell wall of Candida pseudohyphae or hyphae-typical pathogenic morphotype for both systemic and mucosal Candida infections. Heat shock protein from Candida albicans (hsp90-CA) is an important target for protective antibodies during disseminated candidiasis of experimental mice and human. His-tagged protein rHsp90 was prepared and used as the antigen for preparation of experimental recombinant liposomal vaccine. Nickel-chelating liposomes (the size around 100nm, PDI≤0.1) were prepared from the mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS-NTA-Ni (molar ratio 95:5%) by hydration of lipid film and extrusion methods. New non-pyrogenic hydrophobised derivative of MDP (C18-O-6-norAbuMDP) was incorporated into liposomes as adjuvans. rHsp90 was attached onto the surface of metallochelating liposomes by metallochelating bond and the structure of these proteoliposomes was studied by dynamic light scattering, AF microscopy, TEM and GPC. The liposomes with surface-exposed C18-O-6-norAbuMDP were well recognised and phagocyted by human dendritic cells in vitro. In vivo the immune response towards this experimental vaccine applied in mice (i.d.) demonstrated both TH1 and TH2 response comparable to FCA, but without any side effects. Metallochelating liposomes with lipophilic derivatives of muramyl dipeptide represent a new biocompatible platform for construction of experimental recombinant vaccines and drug-targeting systems.

Liposomes with high encapsulation capacity for paclitaxel: Preparation, characterisation and in vivo anticancer effect.

Paclitaxel (PTX) is approved for the treatment of ovarian and breast cancer. The commercially available preparation of PTX, Cremophor EL(R) is associated with hypersensitivity reactions in spite of a suitable premedication. In general, the developed liposomal PTX formulations are troubled with low PTX encapsulation capacity (maximal content, 3 mol%) and accompanied by PTX crystallisation. The application of "pocket-forming" lipids significantly increased the encapsulation capacity of PTX in the liposomes up to 10 mol%. Stable lyophilised preparation of PTX (7 mol%) encapsulated in the liposomes composed of SOPC/POPG/MOPC (molar ratio, 60:20:20) doped with 5 mol% vitamin E had the size distribution of 180-190 nm (PDI, 0.1) with zeta-potential of -31 mV. Sucrose was found to be a suitable cryoprotectant at the lipid:sugar molar ratios of 1:5-1:10. This liposomal formulation did not show any evidence of toxicity in C57BL/6 mice treated with the highest doses of PTX (100 mg/kg administered as a single dose and 150 mg/kg as a cumulative dose applied in three equivalent doses in 48-h intervals). A dose-dependent anticancer effect was found in both hollow fibre implants and syngenic B16F10 melanoma mouse tumour models.