BODIPY-based fluorescent liposomes with sesquiterpene lactone trilobolide

Like thapsigargin, which is undergoing clinical trials, trilobolide is a natural product with promising anticancer and anti-inflammatory properties. Similar to thapsigargin, it has limited aqueous solubility that strongly reduces its potential medicinal applications. The targeted delivery of hydrophobic drugs can be achieved using liposome-based carriers. Therefore, we designed a traceable liposomal drug delivery system for trilobolide. The fluorescent green-emitting dye BODIPY, cholesterol and trilobolide were used to create construct 6. The liposomes were composed of dipalmitoyl-3-trimethylammoniumpropane and phosphatidylethanolamine. The whole system was characterized by atomic force microscopy, the average size of the liposomes was 150 nm in width and 30 nm in height. We evaluated the biological activity of construct 6 and its liposomal formulation, both of which showed immunomodulatory properties in primary rat macrophages. The uptake and intracellular distribution of construct 6 and its liposomal formulation was monitored by means of live-cell fluorescence microscopy in two cancer cell lines. The encapsulation of construct 6 into the liposomes improved the drug distribution in cancer cells and was followed by cell death. This new liposomal trilobolide derivative not only retains the biological properties of pure trilobolide, but also enhances the bioavailability, and thus has potential for the use in theranostic applications

The Position of His-Tag in Recombinant OspC and Application of Various Adjuvants Affects the Intensity and Quality of Specific Antibody Response after Immunization of Experimental Mice.

Lyme disease, Borrelia burgdorferi-caused infection, if not recognized and appropriately treated by antibiotics, may lead to chronic complications, thus stressing the need for protective vaccine development. The immune protection is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies, associated with the Th1 immune response. Surface antigen OspC is involved in Borrelia spreading through the host body. Previously we reported that recombinant histidine tagged (His-tag) OspC (rOspC) could be attached onto liposome surfaces by metallochelation. Here we report that levels of OspC-specific antibodies vary substantially depending upon whether rOspC possesses an N' or C' terminal His-tag. This is the case in mice immunized: (a) with rOspC proteoliposomes containing adjuvants MPLA or non-pyrogenic MDP analogue MT06; (b) with free rOspC and Montanide PET GEL A; (c) with free rOspC and alum; or (d) with adjuvant-free rOspC. Stronger responses are noted with all N'-terminal His-tag rOspC formulations. OspC-specific Th1-type antibodies predominate post-immunization with rOspC proteoliposomes formulated with MPLA or MT06 adjuvants. Further analyses confirmed that the structural features of soluble N' and C' terminal His-tag rOspC and respective rOspC proteoliposomes are similar including their thermal stabilities at physiological temperatures. On the other hand, a change in the position of the rOspC His-tag from N' to C' terminal appears to affect substantially the immunogenicity of rOspC arguably due to steric hindrance of OspC epitopes by the C' terminal His-tag itself and not due to differences in overall conformations induced by changes in the His-tag position in rOspC variants

CD spectra of rOspC proteins.

<p>Secondary structure of N' and C' terminal His-tag rOspC proteins determined by CD and FTIR. <b>A</b>) CD and FTIR spectra were obtained at room temperature (22°C) using a Chirascan CD spectrometer and Tensor 27 FTIR, respectively. <b>B)</b> Comparison of secondary structures of rOspC proteins obtained by calculations based on FTIR and CD measurements.</p

SDS-PAGE and MS analyses of recombinant N' and C' terminal His-tag rOspC.

<p><b>A)</b> Recombinant N' and C' terminal His-tag rOspC proteins were separated using SDS-PAGE and stained by CBB G-250. The differences in mobility of both rOspC variants are caused by the length of labeling tags and spacers and correspond to theoretical prediction based on cDNA sequences: 23.96 kDa for C' and 27.12 for N' terminal His-tag rOspC. <b>B, C</b>) MALDI-TOF peptide mass fingerprinting of rOspC proteins. All spectra were acquired using CHCA matrix on Microflex LRF20 MALDI-TOF mass spectrometer. Panel <b>B</b>) refers to C' terminal His-tag rOspC and panel <b>C</b>) N' terminal His-tag rOspC.</p

Doses and vaccine formulations.

<p>Doses and vaccine formulations.</p

Synthetic molecular adjuvants MT06 and MPLA.

<p>Left panel shows MT06, a non-pyrogenic lipophilic derivative of muramyl dipeptide (MDP) analogue—Nor-AbuMDP. The right panel shows monophosphoryl lipid A, a derivative of lipopolysaccharide from <i>S</i>. <i>minnesota</i>.</p

Characterization of metallochelating N' and C´ terminal His-tag rOspC proteoliposomes by immuno EM and DLS.

<p>The EM picture of <b>A)</b> plain nanoliposome, <b>B</b>) metallochelation nanoliposome with surface bound N´ terminal His-tag rOspC proteins. Metallochelation liposomes were incubated with N' and C' terminal His-tag rOspC proteins followed by incubation with pooled sera from rOspC-immunized mice (1:50 dilution) followed by addition of protein A-labeled 10-nm colloidal gold particles. After 12-h, the proteoliposomes were negatively stained by ammonium molybdate and observed using Philips Morgagni transmission EM. Black dots represent immunogold particles on rOspC proteins bound to the liposome surface (black arrows). rOspC protein molecules (white dots) forms chains on proteoliposome surfaces (white arrows). <b>C)</b> The increase of hydrodynamic radius after binding of rOspC proteins was measured by DLS. <b>D)</b> Tabular data characterizing the size of plane and rOspC proteoliposomes in detail.</p

Determination of thermal stability of His-tag rOspC proteins by DLS, DSC and circular dichroism.

<p><b>A, B</b>) rOspC ellipticity induced by temperature changes monitored by circular dichroism. CD spectra were obtained at room temperature (22°C) using a Chirascan CD spectrometer. Data were collected from 185 to 260 nm at 100 nm/min, 1 s response time, and 2 nm bandwidth using a 0.1 cm quartz cuvette. Thermal unfolding of rOspC proteins was followed by monitoring the ellipticity at 195 and 222 nm over the temperature range of 20 to 80°C, with a resolution of 0.1°C, at a heating rate of 1°C/min. <b>C</b>) Determination of thermal stability by measurement transition midpoints of rOspC proteins using DSC. Scans ran from 10 to 85°C at the scan rate of 1°C per minute. <b>D</b>) Thermal stability of rOspC determined by DLS. The hydrodynamic diameter of the proteins was monitored over the temperature range of 25–55°C. The lines are created from hydrodynamic radius measurements at the temperature gradient of 1°C/ min. <b>E</b>) Transition temperatures (°C) of of N' and C' terminal His-tag rOspC calculated from DLS, DSC, and FTIR measurements.</p

Hyaluronic Acid Surface Modified Liposomes Prepared via Orthogonal Aminoxy Coupling: Synthesis of Nontoxic Aminoxylipids Based on Symmetrically α‑Branched Fatty Acids, Preparation of Liposomes by Microfluidic Mixing, and Targeting to Cancer Cells Expressing CD44

New synthetic aminoxy lipids are designed and synthesized as building blocks for the formulation of functionalized nanoliposomes by microfluidization using a NanoAssemblr. Orthogonal binding of hyaluronic acid onto the outer surface of functionalized nanoliposomes via aminoxy coupling (<i>N</i>-oxy ligation) is achieved at hemiacetal function of hyaluronic acid and the structure of hyaluronic acid-liposomes is visualized by transmission electron microscopy and cryotransmission electron microscopy. Observed structures are in a good correlation with data obtained by dynamic light scattering (size and ζ-potential). In vitro experiments on cell lines expressing CD44 receptors demonstrate selective internalization of fluorochrome-labeled hyaluronic acid-liposomes, while cells with down regulated CD44 receptor levels exhibit very low internalization of hyaluronic acid-liposomes. A method based on microfluidization mixing was developed for preparation of monodispersive unilamellar liposomes containing aminoxy lipids and orthogonal binding of hyaluronic acid onto the liposomal surface was demonstrated. These hyaluronic acid-liposomes represent a potentially new drug delivery platform for CD44-targeted anticancer drugs as well as for immunotherapeutics and vaccines

Nonpyrogenic Molecular Adjuvants Based on norAbu-Muramyldipeptide and norAbu-Glucosaminyl Muramyldipeptide: Synthesis, Molecular Mechanisms of Action, and Biological Activities in Vitro and in Vivo

Fatty acyl analogues of muramyldipeptide (MDP) (abbreviated <i>N</i>-L18 norAbuGMDP, <i>N</i>-B30 norAbuGMDP, norAbuMDP-Lys­(L18), norAbuMDP-Lys­(B30), norAbuGMDP-Lys­(L18), norAbuGMDP-Lys­(B30), B30 norAbuMDP, L18 norAbuMDP) are designed and synthesized comprising the normuramyl-l-α-aminobutanoyl (norAbu) structural moiety. All new analogues show depressed pyrogenicity in both free (micellar) state and in liposomal formulations when tested in rabbits in vivo (sc and iv application). New analogues are also shown to be selective activators of NOD2 and NLRP3 (inflammasome) in vitro but not NOD1. Potencies of NOD2 and NLRP3 stimulation are found comparable with free MDP and other positive controls. Analogues are also demonstrated to be effective in stimulating cellular proliferation when the sera from mice are injected sc with individual liposome-loaded analogues, causing proliferation of bone marrow-derived GM-progenitors cells. Importantly, vaccination nanoparticles prepared from metallochelation liposomes, His-tagged antigen rOspA from Borrelia burgdorferi, and lipophilic analogue norAbuMDP-Lys­(B30) as adjuvant, are shown to provoke OspA-specific antibody responses with a strong Th1-bias (dominance of IgG2a response). In contrast, the adjuvant effects of Alum or parent MDP show a strong Th2-bias (dominance of IgG1 response)