Search Results (9)

Background/Objectives: The development of polymer–lipid hybrid nanoparticles (PLNs) is a promising area of research, as it can help increase the stability of cationic lipid carriers. Hybrid PLNs are core–shell nanoparticle structures that combine the advantages of both polymer nanoparticles and liposomes, especially in terms of their physical stability and biocompatibility. Natural polymers such as polyhydroxyalkanoate (PHA) can be used as a matrix for the PLNs’ preparation. Methods: In this study, we first obtained stable cationic hybrid PLNs using a cationic liposome (CL) composed of a polycationic lipid 2X3 (1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride), helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and the hydrophobic polymer mcl-PHA, which was produced by the soil bacterium Pseudomonas helmantisensis P1. Results: The new polymer-lipid carriers effectively encapsulated and delivered model mRNA-eGFP (enhanced green fluorescent protein mRNA) to BHK-21 cells. We then evaluated the role of mcl-PHA in increasing the stability of cationic PLNs in ionic solutions using dynamic light scattering data, electrophoretic mobility, and transmission electron microscopy techniques. Conclusions: The results showed that increasing the concentration of PBS (phosphate buffered saline) led to a decrease in the stability of the CLs. At high concentrations of PBS, the CLs aggregate. In contrast, the presence of isotonic PBS did not result in the aggregation of PLNs, and the particles remained stable for 120 h when stored at +4 °C. The obtained results show that PLNs hold promise for further in vivo studies on nucleic acid delivery. Full article

Foot-and-mouth disease (FMD) is a highly contagious viral infection causing acute and severe vesicular lesions in cattle and pigs, which has prompted global vaccination policies. This study presents a technique for enhancing antigen yield in SAT1 BOT and SAT3 ZIM by treatment with calcium chloride (CaCl₂). We tested changes in cell viability in BHK-21 suspension cells treated with varying concentrations of CaCl₂. The optimal CaCl₂ concentration was determined based on antigen yield. The timing of CaCl₂ supplementation relative to FMD virus inoculation was tested. Finally, the optimal medium for antigen production was identified. We observed a concentration-dependent decrease in BHK-21 cell viability at >7.5 mM CaCl₂. A CaCl₂ concentration of 3 mM yielded the most antigens. CaCl₂ supplementation relative to FMD virus infection was optimal 2 h before or with viral inoculation. CD-BHK 21 medium supplemented with CaCl₂ was the most productive medium. Specifically, SAT1 BOT and SAT3 ZIM showed improved antigen production in CD-BHK 21 medium with 3 mM CaCl₂, while Provero-1 and Cellvento BHK-200 media showed no significant enhancement. Overall, CaCl₂ supplementation enhanced FMD antigen productivity. This study provides a useful framework for enhancing antigen production efficiently in the FMD vaccine industry. Full article

The complex [Ni₃L₆]•1.56CH₂Cl₂ (HL = (E)-2-(((3,4-dimethylphenyl)imino)methyl)phenol) was isolated in the monoclinic C 2/c space group. All nickel atoms are six-coordinate with a nickel atom bound to only O-phenol atoms, while the two terminal cations are surrounded by N₃O₃ atoms. This complex and its ligand (20 mg/mL in DMSO) were tested as antimicrobials. Against two fungi, the complex and amphotericin B caused 13 and 21 mm inhibition diameters, respectively, in Candida albicans plates. Against four bacteria, the ligand inhibited only Staphylococcus aureus with a 10 mm diameter, and the complex induced inhibitions with 10–13 mm (ampicillin afforded 21–26 mm inhibitions). Against cancer (MCF-7) and normal (BHK) cells, the ligand provided virtual inactivity, but great activities (IC₅₀ = 5.44 and 11.61 μM, respectively) were shown by the complex. Doxorubicin afforded activities with IC₅₀ = 9.66 and 36.42 μM in these cells, respectively. The ligand and its complex offered 100% germination of a drought-sensitive wheat cultivar (90% for control), but, under drought, the complex, ligand and control gave germination with 85, 75 and 95%, respectively. Under normal irrigation and drought, the control and complex afforded 100% germination, and the ligand afforded 95% germination for a drought-resistive wheat cultivar. Full article

Two antimony complexes {[Sb(L¹)Cl₂] C1 and [Sb(L²)Cl₂] C2} with the thiosemicarbazone ligands {HL¹ = 4-(2,4-dimethylphenyl)-1-((pyridin-2-yl)methylene)thiosemicarbazide and HL² = 4-(2,5-dimethoxyphenyl)-1-((pyridin-2-yl)methylene)thiosemicarbazide} were introduced. The structures were elucidated on the basis of a CHNS analysis, spectroscopic techniques (UV-Vis and FT-IR), and DMF solution electrical conductivities. Single crystal X-ray diffraction analysis of complex C1 assigned the complex pseudo-octahedral geometry and triclinic P-1 space group. Only the ligand HL¹ and its derived complex C1 displayed antifungal activities against Candida albicans and this activity was enhanced from 10 mm to 21 mm for the respective complex, which is the same activity given by the drug “Amphotericin B”. The ligands HL¹ and HL² gave inhibitions, respectively, of 14 and 10 mm against Staphylococcus aureus and 15 and 10 mm against Escherichia coli; however, complexes C1 and C2 increased these inhibitions to 36 and 32 mm against Staphylococcus aureus and 35 and 31 mm against Escherichia coli exceeding the activities given by the ampicillin standard (i.e., 21 mm against Staphylococcus aureus and 25 mm against Escherichia coli). Against MCF-7 human breast cancer cells, the IC₅₀ values of HL¹ (68.9 μM) and HL² (145.4 μM) were notably enhanced to the values of 34.7 and 37.4 μM for both complexes, respectively. Further, the complexes induced less toxicity in normal BHK cells (HL¹ (126.6 μM), HL² (110.6 μM), C1 (>210.1 μM), and C2 (160.6 μM)). As a comparison, doxorubicin gave an IC₅₀ value of 9.66 μM against MCF-7 cells and 36.42 μM against BHK cells. Full article

Oral vaccination of wildlife has shown to be a very effective management tool in rabies control. Evaluation of the genetic stability of vaccine viruses before distributing vaccine baits in the environment is essential because all available oral rabies vaccines, including the genetically engineered rabies virus vaccine strain SPBN GASGAS (Rabitec), are based on replication-competent viruses. To evaluate the genetic stability of this vaccine strain, five serial passages of the Master Seed Virus (MSV) in the production cell line BHK21 Cl13 were performed. Furthermore, to test possible reversion to virulence, a back-passage study in suckling mouse brain (SMB) was performed. Subsequently, the pooled 5th SMB passage was inoculated intracerebrally (i.c.) in adult and suckling mice. The full genome sequences of the isolated 5th passage, in vivo and in vitro, were compared at both the consensus and the quasispecies level with the MSV. Additionally, the full genome sequence of the 6th SMB passage from the individual animals was determined and compared. Full-length integration of the double glycoprotein and modified base substitutions at amino acid position 194 and 333 of the glycoprotein could be verified in all 5th and 6th passage samples. Overall, 11 single nucleotide polymorphisms (SNPs) were detected in the 5th pooled SMB passage, 4 with frequency between 10 and 20%, and 7 with between 2.5 and 10%. SNPs that resulted in amino acid exchange were found in genes: N (one SNP), G (four SNPs), and L (three SNPs). However, none of these SNPs were associated with reversion to virulence since all adult mice inoculated i.c. with this material survived. In the individual samples of the 6th SMB passage 24 additional SNPs (>2.5%) were found, of which only 1 SNP (L-gene, position 6969) had a prevalence of >50% in 3 of 17 samples. The obtained results confirmed the stable expression of genetic modifications and the genetic stability of the consensus strain after serial in vivo and in vitro passaging. Full article

Cisplatin and other metallodrugs have realised great success in clinical chemotherapeutic applications as anticancer drugs. However, severe toxicity to healthy cells and non-selectivity to cancer cells remains a challenge, warranting the further search for alternative agents. Herein, we report the anticancer potential of a series of complexes of the general formula [MCl(p-cym)(k²-N^N-L)]⁺ X⁻ and [MCl(Cp*)(k²-N^N-L)]⁺ X⁻, where M is the metal centre (Ru(II), Os(II), Rh(III) or Ir(III)), L = 1-benzyl-4-pyridinyl-1-H-1,2,3-triazole for L1 and 1-picolyl-4-pyridinyl-1-H-1,2,3-triazole for L2 and X⁻ = Cl⁻, BF₄⁻, BPh₄⁻. When evaluated for activity against some cancerous and non-cancerous cell lines (namely, HeLa, HEK293, A549 and MT4 cancer cells and the normal healthy kidney cells (BHK21)), most of the compounds displayed poor cytotoxicities against cancer cells except for complexes C2 ([RuCl(p-cym)(k²-N^N-L1)]⁺ BPh₄⁻, EC₅₀ = 9–16 µM and SI = 14), C7 ([RuCl(p-cym)(k²-N^N-L2)]⁺ BPh₄⁻, EC₅₀ = 17–53 µM and SI = 4) and C11 ([IrCl(Cp*)(k²-N^N-L2)]⁺ BF₄⁻, EC₅₀ < 5 µM and SI > 10). Selected complexes C1 ([RuCl(p-cym)(k²-N^N-L1)]⁺ BF₄⁻), C5 ([IrCl(Cp*)(k²-N^N-L1)]⁺ BF₄⁻) and C11 showed significant interactions with model biomolecules such as guanosine-5′-monophosphate (5′-GMP), bovine serum albumin (BSA) and amino acids under physiological conditions, possibly through carbenylation and N-coordination with 5′-GMP, N-coordination with L-Histidine and L-proline. While the compounds showed good activities in reducing pyruvate to lactate, there was no direct correlation between catalytic transfer hydrogenation of pyruvate and the observed cytotoxic activities. As observed in this work, the marked influence of single atom replacement in ligand may provide a pivotal approach to improving the cytotoxicity and fine-tuning the selectivity to cancer cells. Full article

Recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV)-1 and -2 initiate virus infection by binding of their spike glycoprotein with the cell-surface receptor angiotensin-converting enzyme 2 (ACE2) and enter into the host cells mainly via the clathrin-mediated endocytosis pathway. However, the internalization process post attachment with the receptor is not clear for both SARS-CoV-1 and -2. Understanding the cellular factor/s or pathways used by these CoVs for internalization might provide insights into viral pathogenesis, transmission, and development of novel therapeutics. Here, we demonstrated that the cytoplasmic tail of ACE2 is not essential for the entry of SARS-CoV-1 and -2 by using bioinformatics, mutational, confocal imaging, and pseudotyped SARS-CoVs infection studies. ACE2 cytoplasmic domain (cytACE2) contains a conserved internalization motif and eight putative phosphorylation sites. Complete cytoplasmic domain deleted ACE2 (∆cytACE2) was properly synthesized and presented on the surface of HEK293T and BHK21 cells like wtACE2. The SARS-CoVs S1 or RBD of spike protein binds and colocalizes with the receptors followed by internalization into the host cells. Moreover, pseudotyped SARS-CoVs entered into wtACE2- and ∆cytACE2-transfected cells but not into dipeptidyl peptidase 4 (DPP4)-expressing cells. Their entry was significantly inhibited by treatment with dynasore, a dynamin inhibitor, and NH₄Cl, an endosomal acidification inhibitor. Furthermore, SARS-CoV antibodies and the soluble form of ACE2-treated pseudotyped SARS-CoVs were unable to enter the wtACE2 and ∆cytACE2-expressing cells. Altogether, our data show that ACE2 cytoplasmic domain signaling is not essential for the entry of SARS-CoV-1 and -2 and that SARS-CoVs entry might be mediated via known/unknown host factor/s. Full article

Duck Tembusu virus (DTMUV), a pathogenic member of the Flavivirus family, was first discovered in the coastal provinces of South-Eastern China in 2010. Many previous reports have clearly shown that some Flaviviruses utilize several endocytic pathways to enter the host cells, however, the detailed mechanism of DTMUV entry into BHK-21 cells, which is usually employed to produce commercial veterinary vaccines for DTMUV, as well as of other Flaviviruses by serial passages, is still unknown. In this study, DTMUV entry into BHK-21 cells was found to be inhibited by noncytotoxic concentrations of the agents chloroquine, NH₄Cl, and Bafilomycin A1, which blocked the acidification of the endosomes. Inactivation of virions by acid pretreatment is a hallmark of viruses that utilize a low-pH-mediated entry pathway. Exposure of DTMUV virions to pH 5.0 in the absence of host cell membranes decreased entry into cells by 65%. Furthermore, DTMUV infection was significantly decreased by chlorpromazine treatment, or by knockdown of the clathrin heavy chain (CHC) through RNA interference, which suggested that DTMUV entry depends on clathrin. Taken together, these findings highlight that a low endosomal pH is an important route of entry for DTMUV. Full article

New amphiphilic 1,4-DHP derivative C12-Man-Q with remoted cationic moieties at positions 2 and 6 was synthesised to study DNA delivery activity. The results were compared with data obtained for cationic 1,4-DHP derivative D19, which is known to be the most efficient one among the previously tested 1,4-DHP amphiphiles. We analysed the effects of C12-Man-Q concentration, complexation media, and complex/cell contact time on the gene delivery effectiveness and cell viability. Transmission electron microscopy data confirms that lipoplexes formed by the compound C12-Man-Q were quite uniform, vesicular-like structures with sizes of about 50 nm, and lipoplexes produced by compound D19 were of irregular shapes, varied in size in the range of 25–80 nm. Additionally, confocal microscopy results revealed that both amphiphiles effectively delivered green fluorescent protein expression plasmid into BHK-21 cells and produced a fluorescent signal with satisfactory efficiency, although compound C12-Man-Q was more cytotoxic to the BHK-21 cells with an increase of concentration. It can be concluded that optimal conditions for C12-Man-Q lipoplexes delivery in BHK-21 cells were the serum free media without 0.15 M NaCl, at an N/P ratio of 0.9. Compound D19 showed higher transfection efficiency to transfect BHK-21 and Cos-7 cell lines, when transfecting active proliferating cells. Although D19 was not able to transfect all studied cell lines we propose that it could be cell type specific. The compound C12-Man-Q showed modest delivery activity in all used cell lines, and higher activity was obtained in the case of H2-35 and B16 cells. The transfection efficiency in cell lines MCF-7, HeLa, and Huh-7 appears to be comparable to the reference compound D19 and minimal in the HepG2 cell line. Full article