Self-Penetrating Oligonucleotide Derivatives: Features of Self-Assembly and Interactions with Serum and Intracellular Proteins.

Lipophilic oligonucleotide derivatives are a potent approach to the intracellular delivery of nucleic acids. The binding of these derivatives to serum albumin is a determinant of their fate in the body, as its structure contains several sites of high affinity for hydrophobic compounds. This study focuses on the features of self-association and non-covalent interactions with human serum albumin of novel self-penetrating oligonucleotide derivatives. The study revealed that the introduction of a triazinyl phosphoramidate modification bearing two dodecyl groups at the 3' end region of the oligonucleotide sequence has a negligible effect on its affinity for the complementary sequence. Dynamic light scattering verified that the amphiphilic oligonucleotides under study can self-assemble into micelle-like particles ranging from 8 to 15 nm in size. The oligonucleotides with dodecyl groups form stable complexes with human serum albumin with a dissociation constant of approximately 10-6 M. The oligonucleotide micelles are simultaneously destroyed upon binding to albumin. Using an electrophoretic mobility shift assay and affinity modification, we examined the ability of DNA duplexes containing triazinyl phosphoramidate oligonucleotides to interact with Ku antigen and PARP1, as well as the mutual influence of PARP1 and albumin or Ku antigen and albumin upon interaction with DNA duplexes. These findings, together with the capability of dodecyl-containing derivatives to effectively penetrate different cells, such as HEK293 and T98G, indicate that the oligonucleotides under study can be considered as a platform for the development of therapeutic preparations with a target effect.

Stability of ZIF-8 Nanoparticles in Most Common Cell Culture Media.

Zeolite imidazolate framework-8 (ZIF-8) is a promising platform for drug delivery, and information regarding the stability of ZIF-8 nanoparticles in cell culture media is essential for proper interpretation of in vitro experimental results. In this work, we report a quantitative investigation of the ZIF-8 nanoparticle's stability in most common cell culture media. To this purpose, ZIF-8 nanoparticles containing sterically shielded nitroxide probes with high resistance to reduction were synthesized and studied using electron paramagnetic resonance (EPR). The degradation of ZIF-8 in cell media was monitored by tracking the cargo leakage. It was shown that nanoparticles degrade at least partially in all studied media, although the degree of cargo leakage varies widely. We found a strong correlation between the amount of escaped cargo and total concentration of amino acids in the environment. We also established the role of individual amino acids in ZIF-8 degradation. Finally, 2-methylimidazole preliminary dissolved in cell culture media partially inhibits the degradation of ZIF-8 nanoparticles. The guidelines for choosing the proper cell culture medium for the in vitro study of ZIF-8 nanoparticles have been formulated.

Uptake of Cell-Penetrating Peptide RL2 by Human Lung Cancer Cells: Monitoring by Electron Paramagnetic Resonance and Confocal Laser Scanning Microscopy.

RL2 is a recombinant analogue of a human κ-casein fragment, capable of penetrating cells and inducing apoptosis of cancer cells with no toxicity to normal cells. The exact mechanism of RL2 penetration into cells remains unknown. In this study, we investigated the mechanism of RL2 penetration into human lung cancer A549 cells by a combination of electron paramagnetic resonance (EPR) spectroscopy and confocal laser scanning microscopy. EPR spectra of A549 cells incubated with RL2 (sRL2) spin-labeled by a highly stable 3-carboxy-2,2,5,5-tetraethylpyrrolidine-1-oxyl radical were found to contain three components, with their contributions changing with time. The combined EPR and confocal-microscopy data allowed us to assign these three forms of sRL2 to the spin-labeled protein sticking to the membrane of the cell and endosomes, to the spin-labeled protein in the cell interior, and to spin labeled short peptides formed in the cell because of protein digestion. EPR spectroscopy enabled us to follow the kinetics of transformations between different forms of the spin-labeled protein at a minimal spin concentration (3-16 µM) in the cell. The prospects of applications of spin-labeled cell-penetrating peptides to EPR imaging, DNP, and magnetic resonance imaging are discussed, as is possible research on an intrinsically disordered protein in the cell by pulsed dipolar EPR spectroscopy.

The Recombinant Fragment of Human κ-Casein Induces Cell Death by Targeting the Proteins of Mitochondrial Import in Breast Cancer Cells.

Breast cancer is still one of the most common cancers for women. Specified therapeutics are indispensable for optimal treatment. In previous studies, it has been shown that RL2, the recombinant fragment of human κ-Casein, induces cell death in breast cancer cells. However, the molecular mechanisms of RL2-induced cell death remain largely unknown. In this study, mechanisms of RL2-induced cell death in breast cancer cells were systematically investigated. In particular, we demonstrate that RL2 induces loss of mitochondrial membrane potential and cellular ATP loss followed by cell death in breast cancer cells. The mass spectrometry-based screen for RL2 interaction partners identified mitochondrial import protein TOM70 as a target of RL2, which was subsequently validated. Further to this, we show that RL2 is targeted to mitochondria after internalization into the cells, where it can also be found in the dimeric form. The importance of TOM70 and RL2 interaction in RL2-induced reduction in ATP levels was validated by siRNA-induced downregulation of TOM70, resulting in the partial rescue of ATP production. Taken together, this study demonstrates that RL2-TOM70 interaction plays a key role in RL2-mediated cell death and targeting this pathway may provide new therapeutic options for treating breast cancer.

Nucleic Acids Delivery Into the Cells Using Pro-Apoptotic Protein Lactaptin.

Cell penetrating peptides (CPP) are promising agents for transporting diverse cargo into the cells. The amino acid sequence and the mechanism of lactaptin entry into the cells allow it to be included into CPP group. Lactaptin, the fragment of human milk kappa-casein, and recombinant lactaptin (RL2) were initially discovered as molecules that induced apoptosis of cultured cancer cells and did not affect non-malignant cells. Here, we analyzed the recombinant lactaptin potency to form complexes with nucleic acids and to act as a gene delivery system. To study RL2-dependent delivery, three type of nucleic acid were used as a models: plasmid DNA (pDNA), siRNA, and non-coding RNA which allow to detect intracellular localization through their functional activity. We have demonstrated that RL2 formed positively charged noncovalent 110-nm-sized complexes with enhanced green fluorescent protein (EGFP)-expressing plasmid DNA. Ca2+ ions stabilized these complexes, whereas polyanion heparin displaced DNA from the complexes. The functional activity of delivered nucleic acids were assessed by fluorescent microscopy using A549 lung adenocarcinoma cells and A431 epidermoid carcinoma cells. We observed that RL2:pDNA complexes provided EGFP expression in the treated cells and that strongly confirmed the entering pDNA into the cells. The efficiency of cell transformation by these complexes increased when RL2:pDNA ratio increased. Pre-treatment of the cells with anti-RL2 antibodies partly inhibited the entry of pDNA into the cells. RL2-mediated delivery of siRNA against EGFP was analyzed when A549 cells were co-transfected with EGFP-pDNA and RL2:siRNA complexes. siRNA against EGFP efficiently inhibited the expression of EGFP being delivered as RL2:siRNA complexes. We have previously demonstrated that non-coding U25 small nucleolar RNA (snoRNA) can decrease cell viability. Cancer cell transfection with RL2-snoRNA U25 complexes lead to a substantial decrease of cell viability, confirming the efficiency of snoRNA U25 delivery. Collectively, these findings indicate that recombinant lactaptin is able to deliver noncovalently associated nucleic acids into cancer cells in vitro.

Structural and Aggregation Features of a Human κ-Casein Fragment with Antitumor and Cell-Penetrating Properties.

Intrinsically disordered proteins play a central role in dynamic regulatory and assembly processes in the cell. Recently, a human κ-casein proteolytic fragment called lactaptin (8.6 kDa) was found to induce apoptosis of human breast adenocarcinoma MCF-7 and MDA-MB-231 cells with no cytotoxic activity toward normal cells. Earlier, we had designed some recombinant analogs of lactaptin and compared their biological activity. Among these analogs, RL2 has the highest antitumor activity, but the amino acid residues and secondary structures that are responsible for RL2's activity remain unclear. To elucidate the structure-activity relations of RL2, we studied the structural and aggregation features of this fairly large intrinsically disordered fragment of human milk κ-casein by a combination of physicochemical methods: NMR, paramagnetic relaxation enhancement (PRE), Electron Paramagnetic Resonance (EPR), circular dichroism, dynamic light scattering, atomic force microscopy, and a cytotoxic activity assay. It was found that in solution, RL2 exists as stand-alone monomeric particles and large aggregates. Whereas the disulfide-bonded homodimer turned out to be more prone to assembly into large aggregates, the monomer predominantly forms single particles. NMR relaxation analysis of spin-labeled RL2 showed that the RL2 N-terminal region, which is essential not only for multimerization of the peptide but also for its proapoptotic action on cancer cells, is more ordered than its C-terminal counterpart and contains a site with a propensity for α-helical secondary structure.