Number Concentration Measurements of Polystyrene Submicrometer Particles.

The number of techniques to measure number concentrations and size distributions of submicrometer particles has recently increased. Submicrometer particle standards are needed to improve the accuracy and reproducibility of these techniques. The number concentrations of fluorescently labeled polystyrene submicrometer sphere suspensions with nominal 100 nm, 200 nm and 500 nm diameters were measured using seven different techniques. Diameter values were also measured where possible. The diameter values were found to agree within 20%, but the number concentration values differed by as much as a factor of two. Accuracy and reproducibility related with the different techniques are discussed with the goal of using number concentration standards for instrument calibration. Three of the techniques were used to determine SI-traceable number concentration values, and the three independent values were averaged to give consensus values. This consensus approach is proposed as a protocol for certifying SI-traceable number concentration standards.

Nanotherapeutics for inhibition of atherogenesis and modulation of inflammation in atherosclerotic plaques.

AIMS: Atherosclerotic development is exacerbated by two coupled pathophysiological phenomena in plaque-resident cells: modified lipid trafficking and inflammation. To address this therapeutic challenge, we designed and investigated the efficacy in vitro and ex vivo of a novel 'composite' nanotherapeutic formulation with dual activity, wherein the nanoparticle core comprises the antioxidant α-tocopherol and the shell is based on sugar-derived amphiphilic polymers that exhibit scavenger receptor binding and counteract atherogenesis. METHODS AND RESULTS: Amphiphilic macromolecules were kinetically fabricated into serum-stable nanoparticles (NPs) using a core/shell configuration. The core of the NPs comprised either of a hydrophobe derived from mucic acid, M12, or the antioxidant α-tocopherol (α-T), while an amphiphile based on PEG-terminated M12 served as the shell. These composite NPs were then tested and validated for inhibition of oxidized lipid accumulation and inflammatory signalling in cultures of primary human macrophages, smooth muscle cells, and endothelial cells. Next, the NPs were evaluated for their athero-inflammatory effects in a novel ex vivo carotid plaque model and showed similar effects within human tissue. Incorporation of α-T into the hydrophobic core of the NPs caused a pronounced reduction in the inflammatory response, while maintaining high levels of anti-atherogenic efficacy. CONCLUSIONS: Sugar-based amphiphilic macromolecules can be complexed with α-T to establish new anti-athero-inflammatory nanotherapeutics. These dual efficacy NPs effectively inhibited key features of atherosclerosis (modified lipid uptake and the formation of foam cells) while demonstrating reduction in inflammatory markers based on a disease-mimetic model of human atherosclerotic plaques.

Sugar-based amphiphilic nanoparticles arrest atherosclerosis in vivo.

Atherosclerosis, the build-up of occlusive, lipid-rich plaques in arterial walls, is a focal trigger of chronic coronary, intracranial, and peripheral arterial diseases, which together account for the leading causes of death worldwide. Although the directed treatment of atherosclerotic plaques remains elusive, macrophages are a natural target for new interventions because they are recruited to lipid-rich lesions, actively internalize modified lipids, and convert to foam cells with diseased phenotypes. In this work, we present a nanomedicine platform to counteract plaque development based on two building blocks: first, at the single macrophage level, sugar-based amphiphilic macromolecules (AMs) were designed to competitively block oxidized lipid uptake via scavenger receptors on macrophages; second, for sustained lesion-level intervention, AMs were fabricated into serum-stable core/shell nanoparticles (NPs) to rapidly associate with plaques and inhibit disease progression in vivo. An AM library was designed and fabricated into NP compositions that showed high binding and down-regulation of both MSR1 and CD36 scavenger receptors, yielding minimal accumulation of oxidized lipids. When intravenously administered to a mouse model of cardiovascular disease, these AM NPs showed a pronounced increase in lesion association compared with the control nanoparticles, causing a significant reduction in neointimal hyperplasia, lipid burden, cholesterol clefts, and overall plaque occlusion. Thus, synthetic macromolecules configured as NPs are not only effectively mobilized to lipid-rich lesions but can also be deployed to counteract atheroinflammatory vascular diseases, highlighting the promise of nanomedicines for hyperlipidemic and metabolic syndromes.

Amphiphilic nanoparticles repress macrophage atherogenesis: novel core/shell designs for scavenger receptor targeting and down-regulation.

Atherosclerosis, an inflammatory lipid-rich plaque disease is perpetuated by the unregulated scavenger-receptor-mediated uptake of oxidized lipoproteins (oxLDL) in macrophages. Current treatments lack the ability to directly inhibit oxLDL accumulation and foam cell conversion within diseased arteries. In this work, we harness nanotechnology to design and fabricate a new class of nanoparticles (NPs) based on hydrophobic mucic acid cores and amphiphilic shells with the ability to inhibit the uncontrolled uptake of modified lipids in human macrophages. Our results indicate that tailored NP core and shell formulations repress oxLDL internalization via dual complementary mechanisms. Specifically, the most atheroprotective molecules in the NP cores competitively reduced NP-mediated uptake to scavenger receptor A (SRA) and also down-regulated the surface expression of SRA and CD36. Thus, nanoparticles can be designed to switch activated, lipid-scavenging macrophages to antiatherogenic phenotypes, which could be the basis for future antiatherosclerotic therapeutics.

Guanidine-Containing Methacrylamide (Co)polymers via aRAFT: Toward a Cell Penetrating Peptide Mimic().

We report the synthesis and controlled radical homo- and block copolymerization of 3-guanidinopropyl methacrylamide (GPMA) utilizing aqueous reversible addition-fragmentation chain transfer (aRAFT) polymerization. The resulting homopolymer and block copolymer with N-(2-hydroxypropyl) methacrylamide (HPMA) were prepared to mimic the behavior of cell penetrating peptides (CPPs) and poly(arginine) (> 6 units) which have been shown to cross cell membranes. The homopolymerization mediated by 4-cyano-4-(ethylsulfanylthiocarbonylsulfanyl)pentanoic acid (CEP) in aqueous buffer exhibited pseudo-first-order kinetics and linear growth of molecular weight with conversion. Retention of the "living" thiocarbonylthio ω-end-group was demonstrated through successful chain extension of the GPMA macroCTA yielding GPMA(37)-b-GPMA(61) (M(w)/M(n) =1.05). Block copolymers of GPMA with the non-immunogenic, biocompatible HPMA were synthesized yielding HPMA(271)-b-GPMA(13) (M(w)/M(n) = 1.15). Notably, intracellular uptake was confirmed by fluorescence microscopy, confocal laser scanning microscopy, and flow cytometry experiments after 2.5 h incubation with KB cells at 4 °C and at 37 °C utilizing FITC-labeled, GPMA-containing copolymers. The observed facility of cellular uptake and the structural control afforded by aRAFT polymerization suggest significant potential for these synthetic (co)polymers as drug delivery vehicles in targeted therapies.

Polymer-based therapeutics: nanoassemblies and nanoparticles for management of atherosclerosis.

Coronary arterial disease, one of the leading causes of adult mortality, is triggered by atherosclerosis. A disease with complex etiology, atherosclerosis results from the progressive long-term combination of atherogenesis, the accumulation of modified lipoproteins within blood vessel walls, along with vascular and systemic inflammatory processes. The management of atherosclerosis is challenged by the localized flare-up of several multipronged signaling interactions between activated monocytes, atherogenic macrophages and inflamed or dysfunctional endothelial cells. A new generation of approaches is now emerging founded on multifocal, targeted therapies that seek to reverse or ameliorate the atheroinflammatory cascade within the vascular intima. This article reviews the various classes and primary examples of bioactive configurations of nanoscale assemblies. Of specific interest are polymer-based or polymer-lipid micellar assemblies designed as multimodal receptor-targeted blockers or drug carriers whose activity can be tuned by variations in polymer hydrophobicity, charge, and architecture. Also reviewed are emerging reports on multifunctional nanoassemblies and nanoparticles for improved circulation and enhanced targeting to atheroinflammatory lesions and atherosclerotic plaques.

Tailored design of Au nanoparticle-siRNA carriers utilizing reversible addition-fragmentation chain transfer polymers.

The facile synthesis of polymer-stabilized Au nanoparticles (AuNPs) capable of forming neutral, sterically stable complexes with small interfering RNA (siRNA) is reported. The amine-containing cationic block of poly(N-2-hydroxypropyl methacrylamide(70)-block-N-[3-(dimethylamino)propyl] methacrylamide(24)) [P(HPMA(70)-b-DMAPMA(24))] was utilized to promote the in situ reduction of Au(3+) to AuNPs and subsequently bind small interfering RNA, while the nonimmunogenic, hydrophilic block provided steric stabilization. The ratio of [DMAPMA](0)/[Au(3+)](0) utilized in the reduction reaction was found to be critical to the production of polymer-stabilized AuNPs capable of complexing siRNA. Significant protection ( approximately 100 times) against nucleases was demonstrated by enzymatic tests, while gene down-regulation experiments indicated successful delivery of siRNA to cancerous cells.

Rational design of targeted cancer therapeutics through the multiconjugation of folate and cleavable siRNA to RAFT-synthesized (HPMA-s-APMA) copolymers.

A well-defined N-(2-hydroxypropyl)methacrylamide-s-N-(3-aminopropyl)methacrylamide (HPMA-s-APMA) copolymer, synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, was utilized for the rational design of multiconjugates containing both a gene therapeutic, small interfering RNA (siRNA), and a cancer cell targeting moiety, folate. The copolymer contains a biocompatible poly(HPMA) portion (91 mol %) and a primary amine, APMA, portion (9 mol %). A fraction (20 mol %) of the APMA repeats were converted to activated thiols utilizing the amine- and sulfhydryl-reactive molecule N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP). 5'-Thiolated sense strand RNAs were then coupled to the polymer through a disulfide exchange with pendant pyridyldithio moieties, giving an 89 +/- 4% degree of conjugation. The unmodified APMA units (80 mol %) were subsequently coupled to amine reactive folates with 81 +/- 1% efficiency. This yielded a multiconjugate copolymer with 91 mol % HPMA, 2 mol % RNA, and 6 mol % folate. siRNA formation was achieved by annealing antisense strands to the conjugated RNA sense strands. Subsequent siRNA cleavage under intracellular conditions demonstrated the potential utility of this carrier in gene delivery. The multiconjugate copolymer and siRNA release were characterized by UV-vis spectroscopy and polyacrylamide gel electrophoresis.

Facile synthesis of multivalent folate-block copolymer conjugates via aqueous RAFT polymerization: targeted delivery of siRNA and subsequent gene suppression.

Cell specific delivery of small interfering ribonucleic acid (siRNA) using well-defined multivalent folate-conjugated block copolymers is reported. Primary amine functional, biocompatible, hydrophilic-block-cationic copolymers were synthesized via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization. N-(2-hydroxypropyl)methacrylamide) (HPMA), a permanently hydrophilic monomer, was copolymerized with a primary amine containing monomer, N-(3-aminopropyl)methacrylamide (APMA). Poly(HPMA) confers biocompatibility, while APMA provides amine functionality, allowing conjugation of folate derivatives. HPMA-stat-APMA was chain extended with a cationic block, poly(N-[3-(dimethylamino)propyl]methacrylamide), to promote electrostatic complexation between the copolymer and the negatively charged phosphate backbone of siRNA. Notably, poly(HPMA) stabilizes the neutral complexes in aqueous solution, while APMA allows the conjugation of a targeting moiety, thus, dually circumventing problems associated with the delivery of genes via cationically charged complexes (universal transfection). Fluorescence microscopy and gene down-regulation studies indicate that these neutral complexes can be specifically delivered to cancer cells that overexpress folate receptors.

Advances in the synthesis of amphiphilic block copolymers via RAFT polymerization: stimuli-responsive drug and gene delivery.

Controlled/'living' radical polymerization methods, including the versatile reversible addition-fragmentation chain transfer (RAFT) polymerization process, are rapidly moving to the forefront in construction of drug and gene delivery vehicles. The RAFT technique allows an unprecedented latitude in the synthesis of water soluble or amphiphilic architectures with precise dimensions and appropriate functionality for attachment and targeted delivery of diagnostic and therapeutic agents. This review focuses on the chemistry of the RAFT process and its potential for preparing well-defined block copolymers and conjugates capable of stimuli-responsive assembly and release of bioactive agents in the physiological environment. Recent examples of block copolymers with designed structures and segmental compositions responsive to changes in pH or temperature are reviewed and hurdles facing further development of these novel systems are discussed.

Facile synthetic procedure for omega, primary amine functionalization directly in water for subsequent fluorescent labeling and potential bioconjugation of RAFT-synthesized (co)polymers.

We describe a facile method to amine functionalize and subsequently fluorescently label polymethacrylamides synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. RAFT-generated poly(N-(2-hydroxypropyl) methacrylamide-b-N-[3-(dimethylamino)propyl] methacrylamide) (poly(HPMA-b-DMAPMA)), a water soluble biocompatible polymer, is first converted to a polymeric thiol and functionalized with a primary amine through a disulfide exchange reaction with cystamine and subsequently reacted with the amine-functionalized fluorescent dye, 6-(fluorescein-5-carboxamido)hexanoic acid, succinimidyl ester (5-SFX). Poly(HPMA258-b-DMAPMA13) (Mn = 39 700 g/mol, Mw/Mn = 1.06), previously synthesized by RAFT polymerization, was used to demonstrate this facile labeling method. The problem with labeling the omega-terminal chain end of a RAFT-synthesized polymethacrylamide is that the reduced end yields a tertiary thiol with low reactivity. The key to labeling poly(HPMA-b-DMAPMA) is to first reduce the dithioester chain end with a strong reducing agent such as NaBH4, and then functionalize the tertiary polymeric thiol with a primary amine through a disulfide exchange reaction with dihydrochloride cystamine. We show that the disulfide exchange reaction is efficient and that the amine-functionalized poly(HPMA-b-DMAPMA) can be easily labeled with the fluorescent dye, 5-SFX. This concept is proven by using a ninhydrin assay to detect primary amines and UV-vis spectroscopy to measure the degree of conjugation.