Discovery of neuroprotective Agents: Potent, brain Penetrating, lipoic acid derivatives for the potential treatment of ischemic stroke by regulating oxidative stress and inflammation - a Preliminary study.

Stroke poses a serious risk to the physical and mental health of patients. Endogenous compounds are widely used to treat ischemic stroke. Lipoic acid, a naturally occurring (R)-5-(1,2-dithiolan-3-yl)pentanoic acid, has therapeutic potential for the treatment of ischemic stroke. However, the direct application of lipoic acid is limited by its relatively low efficacy and instability. Therefore, there is a need to modify the structure of lipoic acid to improve its pharmaceutical capabilities. Currently, 37 lipoic acid derivatives have been synthesized, and compound AA-9 demonstrated optimal therapeutic potential in an in vitro model of induced oxidative damage using tert-butyl hydroperoxide (t-BHP). In addition, in vitro experiments have shown that compound AA-9 has an excellent safety profile. Subsequently, the therapeutic effect of AA-9 was significant in the rat MCAO ischemic stroke model, which may be attributed to the antioxidant and anti-inflammatory effects of compound AA-9 by activating PGC-1α and inhibiting NLRP3. Notably, compound AA-9 exhibited higher stability and better bioavailability properties than ALA in plasma stability and pharmacokinetic properties. In conclusion, AA-9 may be a promising neuroprotective agent for the treatment of ischemic stroke and warrants further investigation.

Lipoic Acid-Modified Oligoethyleneimine-Mediated miR-34a Delivery to Achieve the Anti-Tumor Efficacy.

MiR-34a, an important tumor suppressor, has been demonstrated to possess great potential in tumor gene therapy. To achieve the upregulation of miR-34a expression level, an oligoethyleneimine (OEI) derivative was constructed and employed as the carrier through the modification with lipoic acid (LA), namely LA-OEI. In contrast to OEI, the derivative LA-OEI exhibited superior transfection efficiency measured by confocal laser scanning microscopy and flow cytometry, owing to rapid cargo release in the disulfide bond-based reduction sensitive pattern. The anti-proliferation and anti-migration effects were tested after the miR-34a transfection to evaluate the anti-tumor response, using human cervical carcinoma cell line HeLa as a model. The delivery of LA-OEI/miR-34a nanoparticles could achieve obvious anti-proliferative effect caused by the induction of cell apoptosis and cell cycle arrest at G1 phase. In addition, it could inhibit the migration of tumor cells via the downregulation of MMP-9 and Notch-1 level. Overall, the LA-OEI-mediated miR-34a delivery was potential to be used as an effective way in the tumor gene therapy.

Novel anti-Alzheimer phenol-lipoyl hybrids: Synthesis, physico-chemical characterization, and biological evaluation.

To date, drugs that hit a single target are inadequate for the treatment of neurodegenerative diseases, such as Alzheimer's or Parkinson's diseases. The development of multitarget ligands, able to interact with the different pathways involved in the progession of these disorders, represents a great challenge for medicinal chemists. In this context, we report here the synthesis and biological evaluation of phenol-lipoyl hybrids (SV1-13), obtained via a linking strategy, to take advantage of the synergistic effect due to the antioxidant portions and anti-amyloid properties of the single constituents present in the hybrid molecule. Biological results showed that SV5 and SV10 possessed the best protective activity against Aß1-42 induced neurotoxicity in differentiated SH-SY5Y cells. SV9 and SV10 showed remarkable antioxidant properties due to their ability to counteract the damage caused by H2O2 in SHSY-5Y-treated cells. Hovewer, SV5, showing moderate antioxidant and good neuroprotective activities, resulted the best candidate for further experiments since it also resulted stable both simulated and plasma fluids.

Single-Molecule Kinetics of Growth and Degradation of Cell-Penetrating Poly(disulfide)s.

The delivery of therapeutic agents into target cells is a challenging task. Cell penetration and intracellular targeting were recently addressed with biodegradable cell-penetrating poly(disulfide)s (CPDs). Cellular localization is determined by the length of these polymers, emphasizing the significance of initial chain length and the kinetics of intracellular depolymerization for targeted delivery. In the present study, the kinetics of CPD polymer growth and degradation were monitored in a single-molecule nanoreactor. The chain lengths achievable under synthetic conditions with high concentrations of dithiolanes were then predicted by using the rate constants. For example, CPDs comprising 40 units are generated in 1 s at pH 7.4 and 0.3 s at pH 8.4 at dithiolane concentrations of 200 mM. The rate constants for degradation suggest that the main depolymerization pathway in the cell is by monomer removal by self-cyclization, rather than by intrachain cleavage by endogenous thiols.

Functionalization of Elongated Tetrahexahedral Au Nanoparticles and Their Antimicrobial Activity Assay.

Gold nanoparticles are inert for the human body, and therefore, they have been functionalized to provide them with antibacterial properties. Here, elongated tetrahexahedral (ETHH) Au nanoparticles were synthesized, characterized, and functionalized with lipoic acid (LA), a natural antioxidant with a terminal carboxylic acid and a dithiolane ring, to generate ETHH-LA Au nanoparticles. The antioxidant activity of Au nanoparticles was investigated in vitro, showing that LA enhances the 2,2-diphenyl-1-picrylhydrazyl free-radical scavenging and Fe3+ ion reducing activity of ETHH-LA at higher amounts. The antimicrobial propensities of the nanoparticles were investigated against Gram-positive ( Bacillus subtilis) and Gram-negative ( Escherichia coli) bacteria through propidium iodide assay as well as disk diffusion assay. ETHH-LA Au nanoparticles showed significantly higher antimicrobial activity against B. subtilis compared with E. coli. Furthermore, ETHH-LA Au nanoparticles also showed significantly better antimicrobial activity against both bacterial strains when compared with ETHH. ETHH Au nanoparticles also bring about the oxidation of bacterial cell membrane fatty acids and produce lipid peroxides. ETHH-LA showed higher lipid peroxidation potential than that of ETHH against both bacteria tested. The hemolytic potential of Au nanoparticles was investigated using human red blood cells and ETHH-LA showed reduced hemolytic activity than that of ETHH. The cytotoxicity of Au nanoparticles was investigated using human cervical cancer cells, HeLa, and ETHH-LA Au nanoparticles showed reduced cytotoxicity than that of ETHH. Taken together, LA enhances the antimicrobial activity of ETHH Au nanoparticles and Au nanoparticles interact with the bacteria through electrostatic interactions as well as hydrophobic interactions and damage the bacterial cell wall followed by oxidation of cell membrane fatty acids.

Set of Highly Stable Amine- and Carboxylate-Terminated Dendronized Au Nanoparticles with Dense Coating and Nontoxic Mixed-Dendronized Form.

The synthesis of a novel poly(propyleneimine) (PPI) dendron in gram scale as well as its use in the formation of a highly stable, dendronized gold nanoparticle (AuNP)-based drug delivery platform is described herein. The AuNP-based platform is composed of three complementary parts: (i) a 15 nm AuNP core, (ii) a heterofunctional thioctic acid-terminated tetraethylene glycol spacer, and (iii) a third-generation PPI dendron with a unique protonation profile and diverse end-group functionalization that allows for further derivatization. The prepared dendronized AuNPs are able to withstand several rounds of lyophilization cycles with no sign of aggregation, are stable in phosphate-buffered saline and Hanks' buffer as well as in serum, and are resistant to degradation by glutathione exchange reactions. This nanocarrier platform displays a dense coating, with >1400 dendrons/AuNPs, which will enable very high payload. Furthermore, while amine-terminated AuNPs expectedly showed cytotoxicity against the MCF-7 breast cancer cell line from a NP concentration of 1 nM, the mixed monolayer AuNPs (coated with 40/60 amine/carboxylate dendrons) interestingly did not exhibit any sign of toxicity at concentrations as high as 15 nM, similar to the carboxylate-terminated AuNPs. The described dendronized AuNPs address the current practical need for a stable NP-based drug delivery platform which is scalable and easily conjugable, has long-term stability in solution, and can be conveniently formulated as a powder and redispersed in desired buffer or serum.

Synthesis, antioxidant and Aß anti-aggregation properties of new ferulic, caffeic and lipoic acid derivatives obtained by the Ugi four-component reaction.

We report herein the synthesis antioxidant and Aß anti-aggregation capacity of (E)-N-benzyl-N-[2-(benzylamino)-2-oxoethyl]-3-(aryl)acrylamides and related (R)-N-benzyl-N-(2-(benzylamino)-2-oxoethyl)-5-(1,2-dithiolan-3-yl)pentanamides 1-12. These compounds have been obtained, via Ugi four-component reaction, from modest to good yields. Their antioxidant analysis, using the DPPH and ORAC assays, allowed us to identify compounds 8 and 9, as potent antioxidant agents, showing also strong Aß1-40 self-aggregation inhibition, two biological properties of interest in pathologies linked to the oxidative stress, such as Alzheimer's disease.

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides.

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the Aß peptide associated with Alzheimer's disease, the peptide is shown to self-assemble into cross-ß amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

Design, synthesis and evaluation of novel multi-target-directed ligands for treatment of Alzheimer's disease based on coumarin and lipoic acid scaffolds.

A novel series of coumarin-lipoic acid conjugates were synthesized via cycloaddition click reaction to find out new multi-target-directed ligands (MTDLs) for treatment of Alzheimer's disease (AD). All of synthesized compounds were screened for neuroprotective and anti-cholinesterase activities. Based on primary screening, two compounds (5 and 11) were subjected to further biological evaluations. In particular, compound 11 which was the most potent AChE inhibitor showed good inhibitory effect on Aß-aggregation and intracellular ROS (reactive oxygen species) formation, as well as the ability of selective bio-metal chelation and neuroprotection against H2O2- and Aß1-42-induced cytotoxicity. In the light of these results, the applied hybridization approach introduced new promising lead compound with desired multifunctional properties, being useful in the treatment of Alzheimer's disease.

Novel 3-phenylcoumarin-lipoic acid conjugates as multi-functional agents for potential treatment of Alzheimer's disease.

New series of triazole-containing 3-phenylcoumarin-lipoic acid conjugates were designed as multi-functional agents for treatment of Alzheimer's disease. The target compounds 4a-o were synthesized via the azide-alkyne cycloaddition reaction and their biological activities were primarily evaluated in terms of neuroprotection against H2O2-induced cell death in PC12 cells and AChE/BuChE inhibition. The promising compounds 4j and 4i containing four carbons spacer were selected for further biological evaluations. Based on the obtained results, the benzocoumarin derivative 4j with IC50 value of 7.3 µM was the most potent AChE inhibitor and displayed good inhibition toward intracellular reactive oxygen species (ROS). This compound with antioxidant and metal chelating ability showed also protective effect on cell injury induced by Aß1-42 in SH-SY5Y cells. Although the 8-methoxycoumarin analog 4i was slightly less active than 4j against AChE, but displayed higher protection ability against H2O2-induced cell death in PC12 and could significantly block Aß-aggregation. The results suggested that the prototype compounds 4i and 4j might be promising multi-functional agents for the further development of the disease-modifying treatments of Alzheimer's disease.

Lipoyl Ester Terminated Star PLGA as a Simple and Smart Material for Controlled Drug Delivery Application.

PLGA, a copolymer of lactide and glycolide, is one of the most used biodegradable polymers that find a wide range of biomedical applications including drug delivery and tissue engineering. However, in spite of remarkable advancement, nanotherapeutics based on PLGA might have drawbacks of inadequate stability, drug leakage, and slow drug release at the tumor site, which reduces its targeting ability and therapeutic efficacy. Here, we report that direct modification of star PLGA ends with lipoic acid, a natural antioxidant present in our human body, affords a smart material (sPLGA-LA) that forms reversibly crosslinked and bioresponsive multifunctional nanoparticles (sPLGA XNPs). Interestingly, sPLGA XNPs obtained in the presence of 23.0 wt % PEG-PDLLA displayed a small hydrodynamic size of 73 ± 1.2 nm, high stability against dilution and 10% serum, while fast destabilization under a reductive environment. Moreover, sPLGA XNPs achieved efficient loading of lipophilic anticancer drug model, doxorubicin (DOX), at a theoretical drug loading content of 13.3 wt %, giving DOX-loaded sPLGA XNPs with reduced drug leakage under physiological conditions as well as significantly accelerated drug release under 10 mM glutathione condition compared with both linear and star PLGA controls (denoted as lPLGA NPs and sPLGA NPs, respectively). Confocal microscopy and flow cytometry displayed obviously stronger DOX fluorescence in B16F10 melanoma cells treated with DOX-loaded sPLGA XNPs than with lPLGA and sPLGA counterparts. MTT assays revealed that DOX-sPLGA XNPs caused 2.4- and 4.2-fold higher antitumor activity toward B16F10 cells than DOX-sPLGA NPs and DOX-lPLGA NPs, respectively. Notably, in vivo pharmacokinetics studies showed prolonged circulation time and significantly improved AUC for DOX-sPLGA XNPs over lPLGA NPs control. Hence, lipoyl ester terminated star PLGA emerges as a simple and smart material for better-controlled anticancer drug delivery.

A Multifunctional Polypeptide via Ugi Reaction for Compact and Biocompatible Quantum Dots with Efficient Bioconjugation.

The growing application of quantum dots (QDs) in biomedical research necessitates, in turn, continuous development of surface functionalizing ligands to optimize their performance for ever more challenging and diverse biological applications. Here, we demonstrate the novel multifunctional polypeptide ligands for compact and biocompatible QDs. The target ligand preparation exploits the efficient, activating agent-free Ugi reaction of four functional components to incorporate lipoic acid, pyridine, zwitterion motifs, and reactive functionalities in a one-pot procedure under mild conditions. Cap exchange with these multifunctional polypeptide ligands generates hydrophilic QD dispersions, which are colloidally stable for prolonged periods of time. The zwitterionic ligation delivers compact and small QDs, and the existence of reactive functionalities enables coupling of the QDs to biologics through bio-orthogonal coupling chemistry, such as ligation of azide-modified QDs to DNA. Therefore, this QD functionalization strategy via Ugi reaction is believed to be a viable approach for compact and biocompatible QDs with efficient bioconjugation.

Lipoic acid based core cross-linked micelles for multivalent platforms: design, synthesis and application in bio-imaging and drug delivery.

Natural lipoic acid derived small-molecule amphiphiles self-assemble into micelles in water. The presence of numerous disulfides accumulated in the core makes the micelles readily cross-linked to achieve the establishment of core cross-linked micelles (CCMs). Thanks to the inherent biocompatibility, the resulting lipoic acid based CCMs (LA-CCMs) are good multivalent platforms for biomedical applications.

Robust, active tumor-targeting and fast bioresponsive anticancer nanotherapeutics based on natural endogenous materials.

The clinical success of cancer nanomedicines critically depends on availability of simple, safe and highly efficient nanocarriers. Here, we report that robust and multifunctional nanoparticles self-assembled from hyaluronic acid-g-poly(γ-benzyl-l-glutamate)-lipoic acid conjugates achieve a remarkably high loading (up to 25.8wt.%) and active targeted delivery of doxorubicin (DOX) to human breast tumor xenograft in vivo. DOX-loaded nanoparticles following auto-crosslinking (DOX-CLNPs) are highly stable with little drug leakage under physiological conditions while quickly release ca. 92% DOX in 30h under a cytoplasmic-mimicking reductive environment. The in vitro assays reveal that DOX-CLNPs possess a superior selectivity and antitumor activity to clinically used pegylated liposomal doxorubicin hydrochloride (DOX-LPs) in CD44 receptor overexpressing MCF-7 human breast cancer cells. Strikingly, DOX-CLNPs exhibit a superb tolerated dose of over 100mg DOX equiv./kg, which is more than 5 times higher than DOX-LPs, and an extraordinary breast tumor accumulation of 8.6%ID/g in mice. The in vivo therapeutic studies in MCF-7 human breast tumor-bearing nude mice show that DOX-CLNPs effectively inhibit tumor growth, improve survival rate, and significantly decrease adverse effects as compared to DOX-LPs. DOX-CLNPs based on natural endogenous materials with high drug loading, great stability and CD44-targetability are highly promising for precision cancer chemotherapy. STATEMENT OF SIGNIFICANCE: We demonstrate that with rational design, simple and multifunctional anticancer nanotherapeutics can be developed to achieve highly efficient and targeted cancer chemotherapy. Doxorubicin-loaded multifunctional nanoparticles based on hyaluronic acid-g-poly(γ-benzyl-l-glutamate)-lipoic acid conjugates exhibit a high drug loading, superior stability, fast bioresponsivity, high tolerability, and obvious selectivity toward CD44-overexpressing tumors in vivo. These nanotherapeutics achieve effective tumor suppression, drastically improved survival rate and reduced side effects as compared to clinically used pegylated liposomal doxorubicin in MCF-7 human breast tumor-bearing nude mice. Unlike previously reported multifunctional nanomedicines, the present nanotherapeutics primarily based on natural endogenous materials are simple and straightforward to fabricate, which makes them potentially interesting for clinical translation.

Novel Synthetic PEGylated Conjugate of α-Lipoic Acid and Tempol Reduces Cell Death in a Neuronal PC12 Clonal Line Subjected to Ischemia.

α-Lipoic acid (α-LA), a natural thiol antioxidant, and Tempol, a synthetic free radical scavenger, are known to confer neuroprotection following ischemic insults in both in vivo and in vitro models. The aim of this study was to synthesize and characterize a conjugate of α-LA and Tempol linked by polyethylene glycol (PEG) in order to generate a more efficacious neuroprotectant molecule. AD3 (α-Tempol ester-ω-lipo ester PEG) was synthesized, purified, and characterized by flash chromatography and reverse phase high pressure liquid chromatography and by 1H nuclear magnetic resonance, infrared spectroscopy, and mass spectrometry. AD3 conferred neuroprotection in a PC12 pheochromocytoma cell line of dopaminergic origin, exposed to oxygen and glucose deprivation (OGD) insult measured by LDH release. AD3 exhibited EC50 at 10 µM and showed a 2-3-fold higher efficacy compared to the precursor moieties, indicating an intrinsic potent neuroprotective activity. AD3 attenuated by 25% the intracellular redox potential, by 54% lipid peroxidation and prevented phosphorylation of ERK, JNK, and p38 by 57%, 22%, and 21%, respectively. Cumulatively, these findings indicate that AD3 is a novel conjugate that confers neuroprotection by attenuation of MAPK phosphorylation and by modulation of the redox potential of the cells.

The Protective Effects of 5-Methoxytryptamine-α-lipoic Acid on Ionizing Radiation-Induced Hematopoietic Injury.

Antioxidants are prospective radioprotectors because of their ability to scavenge radiation-induced reactive oxygen species (ROS). The hematopoietic system is widely studied in radiation research because of its high radiosensitivity. In the present study, we describe the beneficial effects of 5-methoxytryptamine-α-lipoic acid (MLA), which was synthesized from melatonin and α-lipoic acid, against radiation-induced hematopoietic injury. MLA administration significantly enhanced the survival rate of mice after 7.2 Gy total body irradiation. The results showed that MLA not only markedly increased the numbers and clonogenic potential of hematopoietic cells but also decreased DNA damage, as determined by flow cytometric analysis of histone H2AX phosphorylation. In addition, MLA decreased the levels of ROS in hematopoietic cells by inhibiting NOX4 expression. These data demonstrate that MLA prevents radiation-induced hematopoietic syndrome by increasing the number and function of and by inhibiting DNA damage and ROS production in hematopoietic cells. These data suggest MLA is beneficial for the protection of radiation injuries.

A silver complex of hyaluronan-lipoate (SHLS12): Synthesis, characterization and biological properties.

In this study we present a novel silver complex of hyaluronan-lipoate (SHLS12) in a gel-state form. NMR analysis, conductometry and elemental analysis demonstrated stable non-covalent interactions between silver ions and the polysaccharide-lipoate backbone, whereas rheological investigations confirmed its gel-like physical-chemical behavior. Biological studies showed the ability of SHLS12 to exert a straightforward activity against different bacterial strains grown in sessile/planktonic state. The biocompatibility was also proved toward two eukaryotic cell lines. By considering both its ability to preserve antibacterial properties when exposed to the serum protein BSA and its low susceptibility to be degraded by hyaluronidase enzyme, this novel complex may be considered as a promising biomaterial for future in vivo applications.

Gas-saturated solution process to obtain microcomposite particles of alpha lipoic acid/hydrogenated colza oil in supercritical carbon dioxide.

Alpha lipoic acid (ALA), an active substance in anti-aging products and dietary supplements, need to be masked with an edible polymer to obscure its unpleasant taste. However, the high viscosity of the ALA molecules prevents them from forming microcomposites with masking materials even in supercritical carbon dioxide (scCO2). Therefore, the purpose of this study was to investigate and develop a novel production method for microcomposite particles for ALA in hydrogenated colza oil (HCO). Microcomposite particles of ALA/HCO were prepared by using a novel gas-saturated solution (PGSS) process in which the solid-dispersion method is used along with stepwise temperature control (PGSS-STC). Its high viscosity prevents the formation of microcomposites in the conventional PGSS process even under strong agitation. Here, we disperse the solid particles of ALA and HCO in scCO2 at low temperatures and change the temperature stepwise in order to mix the melted ALA and HCO in scCO2. As a result, a homogeneous dispersion of the droplets of ALA in melted HCO saturated with CO2 is obtained at high temperatures. After the rapid expansion of the saturated solution through a nozzle, microcomposite particles of ALA/HCO several micrometers in diameter are obtained.

Studies on the sulfation of cellulose α-lipoate and ability of the sulfated product to stabilize colloidal suspensions of gold nanoparticles.

A versatile method for the synthesis of cellulose α-lipoate with a low degree of substitution (DS) has been developed using N,N-dimethylacetamide (DMA)/LiCl as a solvent and N,N'-carbonyldiimidazole (CDI) as an esterification reagent. The cellulose α-lipoate with DS of α-lipoate groups of 0.26 was converted with sulfur trioxide-pyridine complex in dimethyl sulfoxide (DMSO) as solvent. The sulfation is accompanied by an unexpected partial oxidation of the disulfide moiety leading to the formation of the corresponding stereoisomers of S-oxides. The resulting mixture of water-soluble cellulose α- and ß-lipoate sulfate possesses a DS of sulfuric acid half ester groups of 1.78. This cellulose-α/ß-lipoate sulfate derivative can be used as an effective stabilizer and solubilizer for the formation of colloidal suspensions of gold nanoparticles formed in situ in aqueous solution.

Formulation of highly functionalizable DNA nanoparticles based on 1,2-dithiolane derivatives.

We describe the formulation of synthetic virus models based on ionic compounds bearing the polymerizable 1,2-dithiolane moiety. First, cationic amphiphiles containing the polymeric inducer were prepared and used to efficiently condense a DNA plasmid (pDNA) into a highly monodisperse population of small polymeric cationic DNA nanoparticles (NPs; Dh ∼100 nm). These nonspecific cationic particles were then functionalized with anionic PEGylated conjugates, also based on the 1,2-dithiolane motifs, in order to produce stable and fully dispersible stealth DNA nanoparticles. Our results show that both ionic interactions and polymerization based on the 1,2-dithiolane pattern occur and that they produce highly functionalizable nonviral DNA NPs.