Conjugation of Triphenylphosphonium Cation to Hydrophobic Moieties to Prepare Mitochondria-Targeting Nanocarriers.

The contribution of mitochondrial dysfunctions to diseases such as cancer, diabetes, cardiovascular, and neurodegenerative diseases has made mitochondria an attractive pharmacological target. To deliver biologically active molecules to mitochondria, however, cellular and mitochondrial barriers must be first overcome. The mitochondrial transmembrane electric potential (negative inside) is among the most commonly used strategies to deliver molecules to mitochondria as it allows the accumulation of positively charged molecules. Thus, therapeutic molecules are either covalently conjugated to lipophilic cations like triphenylphosphonium (TPP) or loaded into nanocarriers conjugated to TPP.

PEGylated-nanoliposomal clusterin for amyloidogenic light chain-induced endothelial dysfunction.

Light chain (AL) amyloidosis is a disease associated with significant morbidity and mortality arising from multi-organ injury induced by amyloidogenic light chain proteins (LC). There is no available treatment to reverse the toxicity of LC. We previously showed that chaperone glycoprotein clusterin (CLU) and nanoliposomes (NL), separately, restore human microvascular endothelial function impaired by LC. In this work, we aim to prepare PEGylated-nanoliposomal clusterin (NL-CLU) formulations that could allow combined benefit against LC while potentially enabling efficient delivery to microvascular tissue, and test efficacy on human arteriole endothelial function. NL-CLU was prepared by a conjugation reaction between the carboxylated surface of NL and the primary amines of the CLU protein. NL were made of phosphatidylcholine (PC), cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG 2000 carboxylic acid) at 70:25:5 mol%. The protective effect of NL-CLU was tested by measuring the dilation response to acetylcholine and papaverine in human adipose arterioles exposed to LC. LC treatment significantly reduced the dilation response to acetylcholine and papaverine; co-treatment of LC with PEGylated-nanoliposomal CLU or free CLU restored the dilator response. NL-CLU is a feasible and promising approach to reverse LC-induced endothelial damage.

Mitochondria-Targeted Agents: Mitochondriotropics, Mitochondriotoxics, and Mitocans.

Mitochondria, the powerhouse of the cell, have been known for many years for their central role in the energy metabolism; however, extensive progress has been made and to date substantial evidence demonstrates that mitochondria play a critical role not only in the cell bioenergetics but also in the entire cell metabolome. Mitochondria are also involved in the intracellular redox poise, the regulation of calcium homeostasis, and the generation of reactive oxygen species (ROS), which are crucial for the control of a variety of signaling pathways. Additionally, they are essential for the mitochondrial-mediated apoptosis process. Thus, it is not surprising that disruptions of mitochondrial functions can lead or be associated with human pathologies. Because of diseases like diabetes, Alzheimer, Parkinson's, cancer, and ischemic disease are being increasingly linked to mitochondrial dysfunctions, the interest in mitochondria as a prime pharmacological target has dramatically risen over the last decades and as a consequence a large number of agents, which could potentially impact or modulate mitochondrial functions, are currently under investigation. Based on their site of action, these agents can be classified as mitochondria-targeted and non-mitochondria-targeted agents. As a result of the continuous search for new agents and the design of potential therapeutic agents to treat mitochondrial diseases, terms like mitochondriotropics, mitochondriotoxics, mitocancerotropics, and mitocans have emerged to describe those agents with high affinity to mitochondria that exert a therapeutic or deleterious effect on these organelles. In this chapter, mitochondria-targeted agents and some strategies to deliver agents to and/or into mitochondria will be reviewed.

Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile.

Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride ("DQAsomes") have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2'-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription-translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We describe the novel application of cationic bolasomes to deliver ASOs into bacteria. We also report the first successful in vitro antisense treatment to inhibit the growth of C. difficile.

Monosialoganglioside-Containing Nanoliposomes Restore Endothelial Function Impaired by AL Amyloidosis Light Chain Proteins.

BACKGROUND: Light chain amyloidosis (AL) is associated with high mortality, especially in patients with advanced cardiovascular involvement. It is caused by toxicity of misfolded light chain proteins (LC) in vascular, cardiac, and other tissues. There is no treatment to reverse LC tissue toxicity. We tested the hypothesis that nanoliposomes composed of monosialoganglioside, phosphatidylcholine, and cholesterol (GM1 ganglioside-containing nanoliposomes [NLGM1]) can protect against LC-induced human microvascular dysfunction and assess mechanisms behind the protective effect. METHODS AND RESULTS: The dilator responses of ex vivo abdominal adipose arterioles from human participants without AL to acetylcholine and papaverine were measured before and after exposure to LC (20 µg/mL) with or without NLGM1 (1:10 ratio for LC:NLGM1 mass). Human umbilical vein endothelial cells were exposed for 18 to 20 hours to vehicle, LC with or without NLGM1, or NLGM1 and compared for oxidative and nitrative stress response and cellular viability. LC impaired arteriole dilator response to acetylcholine, which was restored by co-treatment with NLGM1. LC decreased endothelial cell nitric oxide production and cell viability while increasing superoxide and peroxynitrite; these adverse effects were reversed by NLGM1. NLGM1 increased endothelial cell protein expression of antioxidant enzymes heme oxygenase 1 and NAD(P)H quinone dehydrogenase 1 and increased nuclear factor, erythroid 2 like 2 (Nrf-2) protein. Nrf-2 gene knockdown reduced antioxidant stress response and reversed the protective effects of NLGM1. CONCLUSIONS: NLGM1 protects against LC-induced human microvascular endothelial dysfunction through increased nitric oxide bioavailability and reduced oxidative and nitrative stress mediated by Nrf-2-dependent antioxidant stress response. These findings point to a potential novel therapeutic approach for light chain amyloidosis.

Nanoliposomes protect against human arteriole endothelial dysfunction induced by ß-amyloid peptide.

We tested whether nanoliposomes containing phosphatidylcholine, cholesterol and phosphatidic acid (NLPA) prevent ß-amyloid 1-42 (Aß42) fibrillation and Aß42-induced human arteriole endothelial dysfunction. NLPA abolished Aß42 fibril formation (thioflavin-T fluorescence/electron microscopy). In ex-vivo human adipose and leptomeningeal arterioles, Aß42 impaired dilator response to acetylcholine that was reversed by NLPA; this protection was abolished by L-NG-nitroarginine methyl ester. Aß42 reduced human umbilical vein endothelial cell NO production that was restored by NLPA. Nanoliposomes prevented Aß42 amyloid formation, reversed Aß42-induced human microvascular endothelial dysfunction and may be useful in Alzheimer's disease.

Nanocarrier-based antioxidant therapy: promise or delusion?

INTRODUCTION: Oxidative stress has generally been recognized as an important factor in the pathogenesis of human diseases, making antioxidant therapy a plausible strategy to either prevent or treat human disorders. Yet so far, numerous antioxidant-based clinical trials aimed at developing clinically approved protocols have been disappointing and many reasons for their failure are being discussed, including the limited bioavailability of most antioxidants. To overcome the hurdles associated with the direct administration of antioxidant molecules, a variety of nanotechnology-based drug delivery systems are being developed. All the strategies currently being explored, however, appear in our opinion to underappreciate the crucial role reactive oxygen and nitrogen species (RO/NS) play in the regulation of the metabolome, as revealed by recent progress made in redox biology. AREAS COVERED: We briefly review antioxidant-based clinical trials and discuss the functions of RO/NS as crucial intracellular messengers. We emphasize the probable existence of three distinct concentration levels of RO/NS: a physiological level reflecting their functions as messenger molecules, an elevated level crucial for activation of protective pathways and a toxic level causing oxidative damage to cellular components. EXPERT OPINION: Site-specific, multifunctional nanodrug delivery systems able to sense the actual intracellular concentrations of RO/NS and release antioxidants accordingly in order to only neutralize the pathologic excess of RO/NS need to be developed.

Nanopharmaceuticals (part 2): products in the pipeline.

In part I of this review we assessed nanoscience-related definitions as applied to pharmaceuticals and we discussed all 43 currently approved drug formulations, which are widely publicized as nanopharmaceuticals or nanomedicines. In continuation, here we review the currently ongoing clinical trials within the broad field of nanomedicine. Confining the definition of nanopharmaceuticals to therapeutic formulations, in which the unique physicochemical properties expressed in the nanosize range, when man-made, play the pivotal therapeutic role, we found an apparently low number of trials, which reflects neither the massive investments made in the field of nanomedicine nor the general hype associated with the term "nano." Moreover, after an extensive search for information through clinical trials, we found only two clinical trials with materials that show unique nano-based properties, ie, properties that are displayed neither on the atomic nor on the bulk material level.

Enhanced cellular uptake and gene silencing activity of siRNA molecules mediated by chitosan-derivative nanocomplexes.

The RNA interference (RNAi) constitutes a conservative mechanism in eukaryotic cells that induces silencing of target genes. In mammalians, the RNAi is triggered by siRNA (small interfering RNA) molecules. Due to its potential in silencing specific genes, the siRNA has been considered a potential alternative for the treatment of genetic and acquired diseases. However, the siRNA therapy has been limited by its low stability and rapid degradation in presence of nucleases, low cellular uptake, and immune response activation. In order to overcome these drawbacks, we propose the synthesis and characterization of non-viral delivery systems using chitosan derivatives to obtain siRNA complexes (polyplexes). The non-viral delivery systems synthesized included PEG-g-OCs (oligochitosan) and PEG-g-Cs (chitosan medium molecular weight). Both systems allowed the formation of siRNA polyplexes, increased the stability of siRNA in the presence of nucleases, enhanced cellular internalization, and showed low toxicity in the A549 cell line. Finally, the complexes obtained with the PEG-g-OCs system showed silencing activity in a GFP model in the cell line A549 in comparison with naked siRNA.

Design and in vitro evaluation of a new nano-microparticulate system for enhanced aqueous-phase solubility of curcumin.

Curcumin, a yellow polyphenol derived from the turmeric Curcuma longa, has been associated with a diverse therapeutic potential including anti-inflammatory, antioxidant, antiviral, and anticancer properties. However, the poor aqueous solubility and low bioavailability of curcumin have limited its potential when administrated orally. In this study, curcumin was encapsulated in a series of novel nano-microparticulate systems developed to improve its aqueous solubility and stability. The nano-microparticulate systems are based entirely on biocompatible, biodegradable, and edible polymers including chitosan, alginate, and carrageenan. The particles were synthesized via ionotropic gelation. Encapsulating the curcumin into the hydrogel nanoparticles yielded a homogenous curcumin dispersion in aqueous solution compared to the free form of curcumin. Also, the in vitro release profile showed up to 95% release of curcumin from the developed nano-microparticulate systems after 9 hours in PBS at pH 7.4 when freeze-dried particles were used.

Formulation approaches to short interfering RNA and MicroRNA: challenges and implications.

RNA interference has emerged as a potentially powerful tool in the treatment of genetic and acquired diseases by delivering short interfering RNA (siRNA) or microRNA (miRNA) to target genes, resulting in their silencing. However, many physicochemical and biological barriers have to be overcome to obtain efficient in vivo delivery of siRNA and miRNA molecules to the organ/tissue of interest, thereby enabling their effective clinical therapy. This review discusses the challenges associated with the use of siRNA and miRNA and describes the nonviral delivery strategies used in overcoming these barriers. More specifically, emphasis has been placed on those technologies that have progressed to clinical trials for both local and systemic siRNA and miRNA delivery.

A Novel Aerosol Method for the Production of Hydrogel Particles.

A novel method of generating hydrogel particles for various applications including drug delivery purposes was developed. This method is based on the production of hydrogel particles from sprayed polymeric nano/microdroplets obtained by a nebulization process that is immediately followed by gelation in a crosslinking fluid. In this study, particle synthesis parameters such as type of nebulizer, type of crosslinker, air pressure, and polymer concentration were investigated for their impact on the mean particle size, swelling behavior, and morphology of the developed particles. Spherical alginate-based hydrogel particles with a mean particle size in the range from 842 to 886 nm were obtained. Using statistical analysis of the factorial design of experiment it was found that the main factors influencing the size and swelling values of the particles are the alginate concentration and the air pressure. Thus, it was demonstrated that the method described in the current study is promising for the generation of hydrogel particles and it constitutes a relatively simple and low-cost system.