Papers by Nikita Oskolkov
Acta neurobiologiae experimentalis
For centuries, inflammatory/foreign body reactions have plagued the attempts of clinicians to use... more For centuries, inflammatory/foreign body reactions have plagued the attempts of clinicians to use metals for tissue and bone reconstructions. Since corrosion contributes to the rejection of metal by the body, an extremely bioinert metal - tantalum - has been successfully used in medicine. The outstanding biocompatibility and flexibility of tantalum established the basis for a growing cadre of clinical applications. One important application which benefited from the introduction of powder (particle) metallurgy is use of tantalum as bone implants. Porous materials have re-shaped the landscape of bone implants, as they allow for bone ingrowth and biological fixation, and eliminate implant loosening and related treatment failures. The unique bone-mimicking properties of porous tantalum enabled the use of tantalum as a material for bulk implants, and not only for coatings, as is the case with other porous metals. Moreover, porous tantalum also facilitates the ingrowth of soft tissue, inc...
Journal of Controlled Release, 2009
Aberrations in splicing patterns play a significant role in several diseases, and splice correcti... more Aberrations in splicing patterns play a significant role in several diseases, and splice correction, together with other forms of gene regulation, is consequently an emerging therapeutic target. In order to achieve successful oligonucleotide transfection, efficient delivery vectors are generally necessary. In this study we present one such vector, the chemically modified cell-penetrating peptide (CPP) TP10, for efficient delivery of a
Acta neurobiologiae experimentalis, 2014
For centuries, inflammatory/foreign body reactions have plagued the attempts of clinicians to use... more For centuries, inflammatory/foreign body reactions have plagued the attempts of clinicians to use metals for tissue and bone reconstructions. Since corrosion contributes to the rejection of metal by the body, an extremely bioinert metal - tantalum - has been successfully used in medicine. The outstanding biocompatibility and flexibility of tantalum established the basis for a growing cadre of clinical applications. One important application which benefited from the introduction of powder (particle) metallurgy is use of tantalum as bone implants. Porous materials have re-shaped the landscape of bone implants, as they allow for bone ingrowth and biological fixation, and eliminate implant loosening and related treatment failures. The unique bone-mimicking properties of porous tantalum enabled the use of tantalum as a material for bulk implants, and not only for coatings, as is the case with other porous metals. Moreover, porous tantalum also facilitates the ingrowth of soft tissue, inc...
International Journal of Peptide Research and Therapeutics, 2011
Oligonucleotide-based gene regulation has a high potential in gene therapy, but the plasma membra... more Oligonucleotide-based gene regulation has a high potential in gene therapy, but the plasma membrane is impermeable for nucleic acid polymers and, consequently, an efficient and non-toxic transfection agent is needed for their delivery into the cell. In this study we present a novel series, NickFects, of chemically modified TP10 peptide-based delivery vectors used for the cellular delivery of single-stranded oligonucleotides.
ACS macro letters, Jan 20, 2015
The protamines are a low-molecular-weight, arginine-rich family of nuclear proteins that protect ... more The protamines are a low-molecular-weight, arginine-rich family of nuclear proteins that protect chromosomal DNA in germ cells by packing it densely using electrostatic interactions. Human protamine-1 (hPRM1) has been developed as a magnetic resonance imaging (MRI) chemical exchange saturation transfer (CEST) reporter gene, based on a sequence that is approximately 50% arginine, which has a side chain with rapidly exchanging protons. In this study, we have synthesized hPRM1 and determined how its CEST MRI contrast varies as a function of pH, phosphorylation state, and upon noncovalent interaction with nucleic acids and heparin (as antagonist). CEST contrast was found to be highly sensitive to phosphorylation on serine residues, intra- and intermolecular disulfide bridge formation, and the binding of negatively charged nucleotides and heparin. In addition, the nucleotide binding constants (K eq) for the protamines were determined through plotting the molar concentration of heparin ve...
PLoS ONE, 2013
Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to ... more Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cellpenetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells. Citation: Pärn K, Viru L, Lehto T, Oskolkov N, Langel Ü, et al. (2013) Transfection of Infectious RNA and DNA/RNA Layered Vectors of Semliki Forest Virus by the Cell-Penetrating Peptide Based Reagent PepFect6. PLoS ONE 8(7): e69659.
Nanomedicine: Nanotechnology, Biology and Medicine, 2015
Mucus barriers lining mucosal epithelia reduce the effectiveness of nanocarrier-based mucosal dru... more Mucus barriers lining mucosal epithelia reduce the effectiveness of nanocarrier-based mucosal drug delivery and imaging ("theranostics"). Here, we describe liposome-based mucus-penetrating particles (MPP) capable of loading hydrophilic agents, e.g., the diaCEST MRI contrast agent barbituric acid (BA). We observed that polyethylene glycol (PEG)-coated liposomes containing ≥7mol% PEG diffused only ~10-fold slower in human cervicovaginal mucus (CVM) compared to their theoretical speeds in water. 7mol%-PEG liposomes contained sufficient BA loading for diaCEST contrast, and provided improved vaginal distribution compared to 0 and 3mol%-PEG liposomes. However, increasing PEG content to ~12mol% compromised BA loading and vaginal distribution, suggesting that PEG content must be optimized to maintain drug loading and stability. Non-invasive diaCEST MRI illustrated uniform vaginal coverage and longer retention of BA-loaded 7mol%-PEG liposomes compared to unencapsulated BA. Liposomal MPP with optimized PEG content hold promise for drug delivery and imaging at mucosal surfaces. This team of authors characterized liposome-based mucus-penetrating particles (MPP) capable of loading hydrophilic agents, such as barbituric acid (a diaCEST MRI contrast agent) and concluded that liposomal MPP with optimized PEG coating enables drug delivery and imaging at mucosal surfaces.
Nucleic Acids Research, 2011
While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient a... more While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/ siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.
NeuroImage, 2013
Chemical exchange saturation transfer (CEST) is a magnetization transfer (MT) technique to indire... more Chemical exchange saturation transfer (CEST) is a magnetization transfer (MT) technique to indirectly detect pools of exchangeable protons through the water signal. CEST MRI has focused predominantly on signals from exchangeable protons downfield (higher frequency) from water in the CEST spectrum. Low power radiofrequency (RF) pulses can slowly saturate protons with minimal interference of conventional semi-solid based MT contrast (MTC). When doing so, saturation-transfer signals are revealed upfield from water, which is the frequency range of non-exchangeable aliphatic and olefinic protons. The visibility of such signals indicates the presence of a relayed transfer mechanism to the water signal, while their finite width reflects that these signals are likely due to mobile solutes. It is shown here in protein phantoms and the human brain that these signals build up slower than conventional CEST, at a rate typical for intramolecular nuclear Overhauser enhancement (NOE) effects in mobile macromolecules such as proteins/peptides and lipids. These NOE-based saturation transfer signals show a pH dependence, suggesting that this process is the inverse of the well-known exchange-relayed NOEs in high resolution NMR protein studies, thus a relayed-NOE CEST process. When studying 6 normal volunteers with a low-power pulsed CEST approach, the relayed-NOE CEST effect was about twice as large as the CEST effects downfield and larger in white matter than gray matter. This NOE contrast upfield from water provides a way to study mobile macromolecules in tissue. First data on a tumor patient show reduction in both relayed NOE and CEST amide proton signals leading to an increase in magnetization transfer ratio asymmetry, providing insight into previously reported amide proton transfer (APT) effects in tumors.
Journal of Controlled Release, 2009
Aberrations in splicing patterns play a significant role in several diseases, and splice correcti... more Aberrations in splicing patterns play a significant role in several diseases, and splice correction, together with other forms of gene regulation, is consequently an emerging therapeutic target. In order to achieve successful oligonucleotide transfection, efficient delivery vectors are generally necessary. In this study we present one such vector, the chemically modified cell-penetrating peptide (CPP) TP10, for efficient delivery of a splicecorrecting 2′-OMe RNA oligonucleotide. Utilizing a functional splice correction assay, we assessed the transfection efficiency of non-covalent complexes of oligonucleotides and stearylated or cysteamidated CPPs. Stearylation of the CPPs Arg9 and penetratin, as well as cysteamidation of MPG and TP10, did not improve transfection, whereas the presence of an N-terminal stearyl group on TP10 improved delivery efficiency remarkably compared to the unmodified peptide. The splice correction levels observed with stearyl-TP10 are in fact in parity with the effects seen with the commercially available transfection agent Lipofectamine ™ 2000. However, the inherent toxicity associated with cationic lipid-based transfections can be completely eliminated when using the stearylated TP10, making this vector highly promising for non-covalent delivery of negatively charged oligonucleotides.
In recent years, oligonucleotide-based molecules have been intensely used to modulate gene expres... more In recent years, oligonucleotide-based molecules have been intensely used to modulate gene expression. All these molecules share the common feature of being essentially impermeable over cellular membranes and they therefore require efficient delivery vectors. Cell-penetrating peptides are a group of delivery peptides that has been readily used for nucleic acid delivery. In particular, polyarginine and derivates thereof, i.e. the (RxR) 4 peptide, have been applied with success both in vitro and in vivo. A major problem, however, with these arginine-rich peptides is that they frequently remain trapped in endosomal compartments following internalization. The activity of polyarginine has previously been improved by conjugation to a stearyl moiety. Therefore, we sought to investigate what impact such modification would have on the pre-clinically used (RxR) 4 peptide for non-covalent delivery of plasmids and splice-correcting oligonucleotides (SCOs) and compare it with stearylated Arg9 and Lipofectamine™ 2000. We show that stearyl-(RxR) 4 mediates efficient plasmid transfections in several cell lines and the expression levels are significantly higher than when using unmodified (RxR) 4 or stearylated Arg9. Although the transfection efficiency is lower than with Lipofectamine™ 2000, we show that stearyl-(RxR) 4 is substantially less toxic. Furthermore, using a functional splice-correction assay, we show that stearyl-(RxR) 4 complexed with 2′-OMe SCOs promotes significant splice correction whereas stearyl-Arg9 fails to do so. Moreover, stearyl-(RxR) 4 promotes dose-dependent splice correction in parity with (RxR) 4 -PMO covalent conjugates, but at least 10-times lower concentration. These features make this stearic acid modified analog of (RxR) 4 an intriguing vector for future in vivo experiments.
Journal of Biomolecular Screening, 2005
A peptide library approach based on electrospray mass-spectrometric (ESI-MS) detection of phospho... more A peptide library approach based on electrospray mass-spectrometric (ESI-MS) detection of phosphopeptides was designed for rapid and quantitative characterization of protein kinase specificity. The k cat /K m values for the protein kinase Cβ (PKCβ) were determined for a systematically varied set of individual substrate peptides in library mixtures by the ESI-MS method. The analysis revealed a complex structural specificity profile in positions around the phosphorylated serine with hydrophobic and/or basic residues being mostly preferred. On the basis of the kinetic parameters, a highly efficient peptide substrate for PKCβ (K m value below 100 nM) FRRRRSFRRR and its alanine substituted pseudosubstrate-analog inhibitor (K i value of 76 nM) were designed. The quantitative specificity profiles obtained by the new approach contained more information about kinase specificity than the conventional substrate consensus motifs. The new method presents a promising basis for design of substrate-site directed peptide or peptidomimetic inhibitors of protein kinases. Second, highly specific substrates could be designed for novel applications such as high-throughput protein kinase activity screens on protein kinase chips. (Journal of Biomolecular Screening 2005:320-328) Key words: protein kinases, protein kinase inhibitors, protein kinase C, mass-spectrometry, peptide library 320 www.sbsonline.org
Journal of Biological Chemistry, 2012
Background: Uptake of various cell-penetrating peptides (CPPs) can be toxic to cells. Results: Am... more Background: Uptake of various cell-penetrating peptides (CPPs) can be toxic to cells. Results: Amphipathic CPPs disorder the plasma membrane inducing the influx of calcium ions that in turn can activate recovery mechanisms. Conclusion: Influx of calcium ions and subsequent toxicity induced by the uptake of CPPs can be averted efficiently. Significance: Membrane-active CPPs can be exploited as efficient transport vectors.
Drug Discovery Today, 2010
Drug Discovery Today, 2010
Bioconjugate Chemistry, 2013
Gliomas are therapeutically challenging cancers with poor patient prognosis. New drug delivery st... more Gliomas are therapeutically challenging cancers with poor patient prognosis. New drug delivery strategies are needed to achieve a more efficient chemotherapy-based approach against brain tumors. The current paper demonstrates development of a tumor-targeted delivery vector that is based on a cell-penetrating peptide pVEC and a novel glioma-targeting peptide sequence gHo. The unique tumor-homing peptide gHo was identified using in vitro phage display technology. The novel delivery vector, which we designated as gHoPe2, was constructed by a covalent conjugation of pVEC, gHo, and a cargo; the latter could be either a labeling moiety (such as a fluorescent marker) or a cytostatic entity. Using a fluorescent marker, we demonstrate efficient uptake of the vector in glioma cells and selective labeling of glioma xenograft tumors in a mouse model. This is the first time that we know where in vitro phage display has yielded an efficient, in vivo working vector. We also demonstrate antitumor efficacy of the delivery vector gHoPe2 using a well-characterized chemotherapeutic drug doxorubicin. Vectorized doxorubicin proved to be more efficient than the free drug in a mouse glioma xenograft model after systemic administration of the drugs. In conclusion, we have characterized a novel glioma-homing peptide gHo, demonstrated development of a new and potential glioma-targeted drug delivery vector gHoPe2, and demonstrated the general feasibility of the current approach for constructing cell-penetrating peptide-based targeted delivery systems.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2005
Mutants of L-type pyruvate kinase with modified peptide sequence around the Ser-12 phosphorylatio... more Mutants of L-type pyruvate kinase with modified peptide sequence around the Ser-12 phosphorylation site were prepared and kinetics of their phosphorylation by protein kinase A was studied. The profile of substrate specificity obtained for these proteins was compared with the kinetic data of phosphorylation of short peptide substrates. Alterations made in protein structure caused weaker effects than the corresponding alterations made in peptides, while the amino acid preferences and the overall specificity pattern remained similar in the both cases. Thus, similar consensus motif holds for both protein and peptide substrates, but is less critical for recognition of proteins if compared with short peptides.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2013
Harnessing of a branched structure is a novel approach in the design of cell-penetrating peptides... more Harnessing of a branched structure is a novel approach in the design of cell-penetrating peptides and it has provided highly efficient transfection reagents for intracellular delivery of nucleic acids. The new stearylated TP10 analogs, NickFects, condense plasmid DNA, splice correcting oligonucleotides and short interfering RNAs into stable nanoparticles with a size of 62-160nm. Such nanoparticles have a negative surface charge (-11 to -18mV) in serum containing medium and enable highly efficient gene expression, splice correction and gene silencing. One of the novel peptides, NickFect51 is capable of transfecting plasmid DNA into a large variety of cell lines, including refractory suspension and primary cells and in several cases exceeds the transfection level of commercially available reagent Lipofectamine™ 2000 without any cytotoxic side effects. Additionally we demonstrate the advantages of NickFect51 in a protein production system, QMCF technology, for expression and production of recombinant proteins in hardly transfectable suspension cells.
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Papers by Nikita Oskolkov