No abstract

The design of nanoparticle delivery materials possessing biological activities is an attractive strategy for the development of various therapies. In this study, 11 cationic amphiphilic 4-(N-alkylpyridinium)-1,4-dihydropyridine (1,4-DHP) derivatives differing in alkyl chain length and propargyl moiety/ties number and position were selected for the study of their self-assembling properties, evaluation of their cytotoxicity in vitro and toxicity on microorganisms, and the characterisation of their interaction with phospholipids. These lipid-like 1,4-DHPs have been earlier proposed as promising nanocarriers for DNA delivery. We have revealed that the mean diameter of freshly prepared nanoparticles varied from 58 to 513 nm, depending upon the 4-(N-alkylpyridinium)-1,4-DHP structure. Additionally, we have confirmed that only nanoparticles formed by 4-(N-dodecylpyridinium)-1,4-DHP derivatives 3 and 6, and by 4-(N-hexadecylpyridinium)-1,4-DHP derivatives 10 and 11 were stable after two weeks of storage. The nanoparticles of these compounds were found to be homogenous in size distribution, ranging from 124 to 221 nm. The polydispersity index (PDI) values of 1,4-DHPs samples 3, 6, 10, and 11 were in the range of 0.10 to 0.37. We also demonstrated that the nanoparticles formed by 4-(N-dodecylpyridinium)-1,4-DHP derivatives 3, 6, and 9, and 4-(N-hexadecylpyridinium)-1,4-DHP derivatives 10 and 11 had zeta-potentials from +26.07 mV (compound 6) to +62.80 mV (compound 11), indicating a strongly positive surface charge and confirming the relative electrostatic stability of these nanoparticle solutions. Transmission electron microscopy (TEM) images of nanoaggregates formed by 1,4-DHPs 3 and 11 confirmed liposome-like structures with diameters around 70 to 170 nm. The critical aggregation concentration (CAC) value interval for 4-(N-alkylpyridinium)-1,4-DHP was from 7.6 µM (compound 11) to 43.3 µM (compound 6). The tested 4-(N-alkylpyridinium)-1,4-DHP derivatives were able to quench the fluorescence of the binary 1,6-diphenyl-1,3,5-hexatriene (DPH)—1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) system, demonstrating hydrophobic interactions of 1,4-DHPs with phospholipids. Thus, 4-(N-dodecylpyridinium)-1,4-DHP derivative 3 quenched the fluorescence of the DPH–DPPC system more efficiently than the other 4-(N-alkylpyridinium)-1,4-DHP derivatives. Likewise the compound 3, also 4-(N-dodecylpyridinium)-1,4-DHP derivative 9 interacted with the phospholipids. Moreover, we have established that increasing the length of the alkyl chain at the quaternised nitrogen of the 4-(N-alkylpyridinium)-1,4-DHP molecule or the introduction of propargyl moieties in the 1,4-DHP molecule significantly influences the cytotoxicity on HT-1080 (human fibrosarcoma) and MH-22A (mouse hepatocarcinoma) cell lines, as well as the estimated basal cytotoxicity. Additionally, it was demonstrated that the toxicity of the 4-(N-alkylpyridinium)-1,4-DHP derivatives on the Gram-positive and Gram-negative bacteria species and eukaryotic microorganism depended on the presence of the alkyl chain length at the N-alkyl pyridinium moiety, as well as the number of propargyl groups. These lipid-like compounds may be proposed for the further development of drug formulations to be used in cancer treatment.

The icosahedral capsid of duck hepatitis B virus (DHBV) is formed by a single core protein species (DHBc).DHBc is much larger than HBc from human HBV, and no high-resolution structure is available. In an accompanying study (M. Nassal, I. Leifer, I. Wingert, K. Dallmeier, S. Prinz, and J. Vorreiter, J. Virol. 81:13218-13229, 2007), we used extensive mutagenesis to derive a structural model for DHBc. For independent validation, we here mapped the epitopes of seven anti-DHBc monoclonal antibodies. Using numerous recombinant DHBc proteins and authentic nucleocapsids from different avihepadnaviruses as test antigens, plus a panel of complementary assays, particle-specific and exposed plus buried linear epitopes were revealed. These data fully support key features of the model. Duck hepatitis B virus (DHBV)is an important model hepadnavirus (4, 14); however, structural knowledge of DHBV is much less advanced than for human HBV (17). With 262 amino acids (aa) in length, the DHBV core protein (DHBc) constituting the nucleocapsid shell is much larger than its ϳ180-aa HBV counterpart HBc. In the accompanying study (10a) we used a large panel of recombinant DHBc variants to derive a structural model for DHBc (shown there in Fig. 8C). The model predicts, inter alia, that the N-proximal ϳ185 aa of DHBc, forming the assembly domain, adopt an architecture similar to that of the first 140 aa of HBc and yet with an ϳ45-aa central insertion (residues 75 to 120). This insertion would be largely exposed on the DHBc spikes seen in low resolution cryo-electron microscopic reconstructions (7).As yet, no direct proof for the spatial disposition of specific DHBc protein segments is available, except that peptides from six antigenic regions (AR1 to AR6), recognized by sera from DHBV-infected and/or DHBc-immunized ducks, have been suggested as candidates for surface exposure (18). However, their availability on intact capsids could not experimentally be tested. Furthermore, peptides usually mimic only linear but not conformational epitopes which abound on HBV capsids (2, 6).To independently test the mutagenesis-derived DHBc model, we generated anti-DHBc monoclonal antibodies (MAbs) and characterized their epitopes. Three MAbs (all immunoglobulin G3 [IgG3]; their full names are 20-2-17C, 21-5-10C, and 27-15-12D [here abbreviated as MAbs n1, n2, and n3]) were obtained by immunization of mice with nearnative DHBc particles that precipitated in highly pure form from concentrated recombinant preparations upon storage (10a); four additional MAbs were obtained by immunization (Abnova, Taiwan) with a Sarkosyl-solubilized, partly denatured formulation of the same antigen (all IgG1; full/abbreviated names are as follows: 2B9-4F8/d1, 2B9-4E12/d2, 1A6-3E9/d3, and 2E9-4D10/d4). Finally, freshly gradient-purified, nonprecipitated particles were used to generate polyclonal rabbit (termed 12/99) and chicken (ch anti-DHBc) antisera (at Eurogentec, Belgium; Biogenes, Germany).To address both linear and conformational epitopes, we used as test antigens nume...

The virus-neutralising domain III (DIII) of the West Nile virus glycoprotein E was exposed on the surface of RNA phage AP205 virus-like particles (VLPs) in mosaic form. For this purpose, a 111 amino acid sequence of DIII was added via amber or opal termination codons to the C-terminus of the AP205 coat protein, and mosaic AP205-DIII VLPs were generated by cultivation in amber- or opal-suppressing Escherichia coli strains. After extensive purification to 95 % homogeneity, mosaic AP205-DIII VLPs retained up to 11-16 % monomers carrying DIII domains. The DIII domains appeared on the VLP surface because they were fully accessible to anti-DIII antibodies. Immunisation of BALB/c mice with AP205-DIII VLPs resulted in the induction of specific anti-DIII antibodies, of which the level was comparable to that of the anti-AP205 antibodies generated against the VLP carrier. The AP205-DIII-induced anti-DIII response was represented by a significant fraction of IgG2 isotype antibodies, in contrast to parallel immunisation with the DIII oligopeptide, which failed to induce IgG2 isotype antibodies. Formulation of AP-205-DIII VLPs in alum adjuvant stimulated the level of the anti-DIII response, but did not alter the fraction of IgG2 isotype antibodies. Mosaic AP205-DIII VLPs could be regarded as a promising prototype of a putative West Nile vaccine.

Our previous research showed that the best yield of virus-like particles (VLPs) formed by RNA bacteriophage GA coat protein was obtained by expression in yeast Pichia pastoris, while other used expression systems in Saccharomyces cerevisiae gave much lower amounts of capsids. The main reasons to attempt further studies in Saccharomyces cerevisiae were to improve the yield of GA-based VLPs using constructs with optimised nucleotide triplets in coding sequences, and to exploit the possibilities of the two-promoter Gal1/Gal10 system of expression vector pESC-URA for production of the desired mosaic VLPs and for packaging of mRNAs into VLPs in vivo