Inhibition of nonspecific polymerase activity using Poly(Aspartic) acid as a model anionic polyelectrolyte.

DNA polymerases with strand-displacement activity allow to amplify nucleic acids under isothermal conditions but often lead to undesirable by-products. Here, we report the increase of specificity of isothermal amplification in the presence of poly (aspartic) acids (pAsp). We hypothesized that side reactions occur due to the binding of the phosphate backbone of synthesized DNA strands with surface amino groups of the polymerase, and weakly acidic polyelectrolytes could shield polymerase molecules from DNA and thereby inhibit nonspecific amplification. Suppression of nonspecific polymerase activity by pAsp was studied on multimerization as a model side reaction. It was found that a low concentration of pAsp (0.01%) provides successful amplification of specific DNA targets. The inhibitory effect of pAsp is due to its polymeric structure since aspartic acid did affect neither specific nor nonspecific amplification. Strongly acidic polyelectrolyte heparin does not possess the same selectivity since it suppresses any DNA synthesis. The applicability of pAsp to prevent nonspecific reactions and reliable detection of the specific target has been demonstrated on the genetic material of SARS-CoV-2 coronavirus using Loop-mediated isothermal amplification.

Comparison of systemic and localized carrier-mediated delivery of methylprednisolone succinate for treatment of acute spinal cord injury.

Localized carrier-mediated administration of drugs is a promising approach to treatment of acute phase of spinal cord injury (SCI) as it allows enhanced and/or sustained drug delivery to damaged tissues along with minimization of systemic side effects. We studied the effect of locally applied self-assembling micellar formulation of methylprednisolone succinate (MPS) with trifunctional block copolymer of ethylene oxide and propylene oxide (TBC) on functional recovery and tissue drug content after SCI in rats in comparison with local and systemic administration of MPS alone. Variations in the amplitude of motor evoked responses in the hindlimb muscles induced by epidural stimulation during acute phase of SCI and restoration of movements during chronic period after local vs. systemic application of MPS were evaluated in this study. Results demonstrate that local delivery of MPS in combination with TBC facilitates spinal cord sensorimotor circuitry, increasing the excitability. In addition, this formulation was found to be more effective in improvement of locomotion after SCI compared to systemic administration. LC-MS/MS data shows that the use of TBC carrier increases the glucocorticoid content in treated spinal cord by more than four times over other modes of treatment. The results of this study demonstrate that the local treatment of acute SCI with MPS in the form of mixed micelles with TBC can provide improved therapeutic outcome by promoting drug accumulation and functional restoration of the spinal cord.

Oligo (Poly (Ethylene Glycol) Fumarate)-Based Multicomponent Cryogels for Neural Tissue Replacement.

Synthetic hydrogels provide a promising platform to produce neural tissue analogs with improved control over structural, physical, and chemical properties. In this study, oligo (poly (ethylene glycol) fumarate) (OPF)-based macroporous cryogels were developed as a potential next-generation alternative to a non-porous OPF hydrogel previously proposed as an advanced biodegradable scaffold for spinal cord repair. A series of OPF cryogel conduits in combination with PEG diacrylate and 2-(methacryloyloxy) ethyl-trimethylammonium chloride (MAETAC) cationic monomers were synthesized and characterized. The contribution of each component to viscoelastic and hydration behaviors and porous structure was identified, and concentration relationships for these properties were revealed. The rheological properties of the materials corresponded to those of neural tissues and scaffolds, according to the reviewed data. A comparative assessment of adhesion, migration, and proliferation of neuronal cells in multicomponent cryogels was carried out to optimize cell-supporting characteristics. The results show that OPF-based cryogels can be used as a tunable synthetic scaffold for neural tissue repair with advantages over their hydrogel counterparts.

A new triphenylphosphonium-conjugated amphipathic cationic peptide with improved cell-penetrating and ROS-targeting properties.

We study for the first time whether triphenylphosphonium (TPP) moiety can improve cellular delivery and redox properties of amphipathic cationic peptides based on YRFK/YrFK cell-penetrating and cytoprotective motif. TPP moiety was found to increase reducing activity of both stereoisomeric peptides in solution and on electrode surface in association with TPP-mediated intramolecular interactions. Among TPP-conjugated peptides, newly synthesized TPP3-YrFK featured both increased antioxidant efficacy and proteolytic resistance. TPP-conjugated peptides preferably mitigated endogenic ROS in mitochondria and cytoplasm of model glioblastoma cells with increased oxidative status. This anti-ROS effect was accompanied by mild reversible decrease of reduced glutathione level in the cells with relatively weak change in glutathione redox forms ratio. Such low interference with cell redox status is in accordance with non-cytotoxic nature of the compounds. Intracellular concentrations of label-free peptides were analyzed by LC-MS/MS, which showed substantial TPP-promoted penetration of YrFK motif across cell plasma membrane. However, according to ΔΨm analysis, TPP moiety did not profoundly enhance peptide interaction with mitochondrial inner membrane. Our study clarifies the role of TPP moiety in cellular delivery of amphipathic cationic oligopeptides. The results suggest TPP moiety as a multi-functional modifier for the oligopeptides which is capable of improving cellular pharmacokinetics and antioxidant activity as well as targeting increased ROS levels. The results encourage further investigation of TPP3-YrFK as a peptide antioxidant with multiple benefits.

Triphenylphosphonium Moiety Modulates Proteolytic Stability and Potentiates Neuroprotective Activity of Antioxidant Tetrapeptides in Vitro.

Although delocalized lipophilic cations have been identified as effective cellular and mitochondrial carriers for a range of natural and synthetic drug molecules, little is known about their effects on pharmacological properties of peptides. The effect of triphenylphosphonium (TPP) cation on bioactivity of antioxidant tetrapeptides based on the model opioid YRFK motif was studied. Two tetrapeptide variants with L-arginine (YRFK) and D-arginine (YrFK) were synthesized and coupled with carboxyethyl-TPP (TPP-3) and carboxypentyl-TPP (TPP-6) units. The TPP moiety noticeably promoted YRFK cleavage by trypsin, but effectively prevented digestion of more resistant YrFK attributed, respectively, to structure-organizing and shielding effects of the TPP cation on conformational variants of the tetrapeptide motif. The TPP moiety enhanced radical scavenging activity of the modified YRFK in a model Fenton-like reaction, whereas decreased reactivity was revealed for both YrFK and its TPP derivative. The starting motifs and modified oligopeptides, especially the TPP-6 derivatives, suppressed acute oxidative stress in neuronal PC-12 cells during a brief exposure similarly with glutathione. The effect of oligopeptides was compared upon culturing of PC-12 cells with CoCl2, L-glutamic acid, or menadione to mimic physiologically relevant oxidative states. The cytoprotective activity of oligopeptides significantly depended on the type of oxidative factor, order of treatment and peptide structure. Pronounced cell-protective effect was established for the TPP-modified oligopeptides, which surpassed that of the unmodified motifs. The protease-resistant TPP-modified YrFK showed the highest activity when administered 24 h prior to the cell damage. Our results suggest that the TPP cation can be used as a modifier for small therapeutic peptides to improve their pharmacokinetic and pharmacological properties.

Self-assembled nanoformulation of methylprednisolone succinate with carboxylated block copolymer for local glucocorticoid therapy.

A new self-assembled formulation of methylprednisolone succinate (MPS) based on a carboxylated trifunctional block copolymer of ethylene oxide and propylene oxide (TBC-COOH) was developed. TBC-COOH and MPS associated spontaneously at increased concentrations in aqueous solutions to form almost monodisperse mixed micelles (TBC-COOH/MPS) with a hydrodynamic diameter of 19.6 nm, zeta potential of -27.8 mV and optimal weight ratio ∼1:6.3. Conditions for the effective formation of TBC-COOH/MPS were elucidated by comparing copolymers and glucocorticoids with different structure. The micellar structure of TBC-COOH/MPS persisted upon dilution, temperature fluctuations and interaction with blood serum components. TBC-COOH increased antiradical activity of MPS and promoted its intrinsic cytotoxicity in vitro attributed to enhanced cellular availability of the mixed micelles. Intracellular transportation and hydrolysis of MPS were analyzed using optimized liquid chromatography tandem mass spectrometry with multiple reaction monitoring which showed increased level of both MPS and methylprednisolone in neuronal cells treated with the formulated glucocorticoid. Our results identify TBC-COOH/MPS as an advanced in situ prepared nanoformulation and encourage its further investigation for a potential local glucocorticoid therapy.

Non-invasive topical drug delivery to spinal cord with carboxyl-modified trifunctional copolymer of ethylene oxide and propylene oxide.

In this study the effect of oxidative modification on micellar and drug delivery properties of copolymers of ethylene oxide (EO) and propylene oxide (PO) was investigated. Carboxylated trifunctional copolymers were synthesized in the reaction with chromium(VI) oxide. We found that carboxylation significantly improved the uniformity and stability of polymeric micelles by inhibiting the microphase transition. The cytotoxicity of copolymers was studied in relation to their aggregative state on two cell types (cancer line vs. primary fibroblasts). The accumulation of rhodamine 123 in neuroblastoma SH-SY5Y cells was dramatically increased in the presence of the oxidized block copolymer with the number of PO and EO units of 83.5 and 24.2, respectively. The copolymer was also tested as an enhancer for topical drug delivery to the spinal cord when applied subdurally. The oxidized copolymer facilitated the penetration of rhodamine 123 across spinal cord tissues and increased its intraspinal accumulation. These results show the potential of using oxidized EO/PO based polymers for non-invasive delivery of protective drugs after spinal cord injury.