New Electroactive Hybridization Indicators 2‐Phthalimido‐N‐Substitutedphenylethanesulfonamide Derivatives for Biosensor Applications: Ring Substituent Effect on Interaction between Compound and DNA

In this work, an electrochemical DNA‐based sensor was developed for the detection of the interaction between the anticonvulsant compounds 2‐phthalimido‐N‐substituted phenylethanesulfonamides (PMPES‐derivatives) and 24‐mer short DNA sequences by using differential pulse voltammetry (DPV) based on both compound and guanine oxidation signals at the renewable carbon graphite electrode (CGE) surface. The influence of compounds on DNA showed differences depending on the nature and position of the substituent on the N‐phenyl ring. Compounds bearing 3‐methoxy, 4‐chloro and 2,6‐dimethyl substituents bind to single stranded probe DNA more strongly than the other derivatives of PMPES. Thus, these compounds were evaluated for use as an electrochemical hybridization label (indicator).     

Design of a Vitamin B1‐Selective Electrode Based on an Ion‐Pair and Its Application to Pharmaceutical Analysis

Vitamin B₁‐selective electrodes with PVC membrane were developed that contain ion associates of vitamin B₁ with an inorganic anion, BiI₄^?, and an organic anion, brilliant yellow, as electrode‐active substances. The linearity ranges of the electrode function are 1.0×10^?5–1.0×10^?2 and 1.0×10^?4–1.0×10^?2 M, the electrode function slopes are 33.0±1.0 and 33.1±1.1 mV decade^?1, the detection limits are 5.5×10^?6 and 8.3×10^?5 M for BiI₄^? and brilliant yellow respectively. The working range of pH is 5–12. The efficiency of the use of electrodes for the vitamin B₁ content control in multivitamin pharmaceutical preparations was shown by direct potentiometry and potentiometric titration methods.     

Nanogel engineering for new nanobiomaterials: from chaperoning engineering to biomedical applications

Nanosize hydrogels (nanogels) are polymer nanoparticles with three‐dimensional networks, formed by chemical and/or physical cross‐linking of polymer chains. Recently, various nanogels have been designed, with a particular focus on biomedical applications. In this review, we describe recent progress in the synthesis of nanogels and nanogel‐integrated hydrogels (nanogel cross‐linked gels) for drug‐delivery systems (DDS), regenerative medicine, and bioimaging. We also discuss chaperone‐like functions of physical cross‐linking nanogel (chaperoning engineering) and organic‐inorganic hybrid nanogels. © 2010 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Published online in Wiley InterScience ( www.interscience.wiley.com ) DOI 10.1002/tcr.201000008     

Micellar Nanocarriers Assembled from Doxorubicin‐Conjugated Amphiphilic Macromolecules (DOX–AM)

Amphiphilic macromolecules (AMs) have unique branched hydrophobic domains attached to linear PEG chains. AMs self‐assemble in aqueous solution to form micelles that are hydrolytically stable in physiological conditions (37 °C, pH 7.4) over 4 weeks. Evidence of AM biodegradability was demonstrated by complete AM degradation after 6 d in the presence of lipase. Doxorubicin (DOX) was chemically conjugated to AMs via a hydrazone linker to form DOX–AM conjugates that self‐assembled into micelles in aqueous solution. The conjugates were compared with DOX‐loaded AM micelles (i.e., physically loaded DOX) on DOX content, micellar sizes and in vitro cytotoxicity. Physically encapsulated DOX loading was higher (12 wt.‐%) than chemically bound DOX (6 wt.‐%), and micellar sizes of DOX‐loaded AMs (≈16 nm) were smaller than DOX–AMs (≈30 nm). In vitro DOX release from DOX–AM conjugates was faster at pH 5.0 (100%) compared to pH 7.4 (78%) after 48 h, 37 °C. Compared to free DOX and physically encapsulated DOX, chemically bound DOX had significantly higher cytotoxicity at 10^?7 M DOX dose against human hepatocellular carcinoma cells after 72 h. Overall, DOX–AM micelles showed promising characteristics as stable, biodegradable DOX nanocarriers.

     

Solid peptide nanoparticles--structural characterization and quantification of cargo encapsulation

CD3ac, an uncharged and strongly hydrophobic 10 amino acid peptide (Ac-LK(Ac)-LK(Ac)-LK(Ac)-LW-DL-LW-DL-LW-DL-LW-NH2) was synthesized and purified. The peptide readily dissolves in ethanol and--upon solvent exchange to water--assembles into solid spherical particles with diameters of around 500 nm and low size-polydispersity. CD3ac self-assembles in a convenient one-step-process in the absence of a templating two-phase solvent system or any other templating agents. Circular dichroism reveals a gramicidin-like secondary structure, which can be attributed to the presence of D-leucine, whereas LCD3ac, a peptide of identical constitution yet composed entirely of L-amino acids precipitates amorphously. The unacetylated derivative of LCD3ac (LCD3) displays α-helical character in circular dichroism. During the process of bead formation, CD3ac can take up and enrich water-soluble and--insoluble cargo compounds, which is exemplified by the encapsulation of rose bengal (RB) and 5-carboxy-fluorescein (CF), two xanthene derivatives. We confirmed their presence in CD3ac beads by confocal fluorescence microscopy and quantified the encapsulation efficiency by absorption measurements of dissolved RB-containing peptide bead suspensions. Loaded CD3ac beads consist of up to 40 mol-% RB, which corresponds to a logarithmic partition coefficient of 2.95. To the best of our knowledge CD3ac is the first peptide synthesized by Fmoc chemistry which forms solid particles in the nano- and micrometer size range and holds promise for drug delivery applications.     

Minocycline Block Copolymer Micelles and their Anti‐Inflammatory Effects on Microglia

MH, a semisynthetic tetracycline antibiotic with promising neuroprotective properties, was encapsulated into PIC micelles of CMD‐PEG as a potential new formulation of MH for the treatment of neuroinflammatory diseases. PIC micelles were prepared by mixing solutions of a Ca²⁺/MH chelate and CMD‐PEG copolymer in a Tris‐HCl buffer. Light scattering and ¹H NMR studies confirmed that Ca²⁺/MH/CMD‐PEG core‐corona micelles form at charge neutrality having a hydrodynamic radius ≈100 nm and incorporating ≈ 50 wt.‐% MH. MH entrapment in the micelles core sustained its release for up to 24 h under physiological conditions. The micelles protected the drug against degradation in aqueous solutions at room temperature and at 37 °C in the presence of FBS. The micelles were stable in aqueous solution for up to one month, after freeze drying and in the presence of FBS and BSA. CMD‐PEG copolymers did not induce cytotoxicity in human hepatocytes and murine microglia (N9) in concentrations as high as 15 mg·mL^?1 after incubation for 24 h. MH micelles were able to reduce the inflammation in murine microglia (N9) activated by LPS. These results strongly suggest that MH PIC micelles can be useful in the treatment of neuroinflammatory disorders.

     

Poly(ethylene glycol)‐block‐Poly(glycidyl methacrylate) with Oligoamine Side Chains as Efficient Gene Vectors

Well‐defined diblock copolymers, poly(ethylene glycol)‐block‐poly(glycidyl methacrylate)s (PEG‐b‐PGMAs), with different poly(glycidyl methacrylate) (PGMA) chains, were prepared via atom transfer radical polymerization (ATRP) from the same macromolecular initiator 2‐bromoisobutyryl‐terminated poly(ethylene glycol) (PEG). Ethyldiamine (EDA), diethylenetriamine (DETA), triethylenetetramine (TETA), and polyethyleneimine (PEI) with an of 400 (PEI₄₀₀) were used to decorate PEG‐b‐PGMAs to get the cationic polymers PEG‐b‐PGMA‐ oligoamines. These cationic polymers possessed high buffer capability and could condense plasmid DNA (pDNA) into nanoscaled complexes of 125–530 nm. These complexes showed the positive zeta potential of 20–35 mV at N/P ratios of 10–50. Most of them exhibited very low cytotoxicity and good transfection efficiency in 293T cells. The presence of the serum medium did not decrease the transfection efficiency due to the steric stabilization of the PEG chains.

     

Liquid,Injectable, Hydrophobic and Biodegradable Polymers as Drug Delivery Vehicles

New delivery approaches to achieve minimally invasive, sustained and local release of drugs are needed for more effective treatment of conditions such as cancer and ischemia. Hydrophobic, biodegradable, liquid injectable polymers possess a number of potential advantages for this purpose. This review examines various approaches that have been explored for the preparation of these types of polymers, their ability to control the release of various drugs ranging from low‐molecular‐weight hydrophobic compounds to protein therapeutics, and finally their degradation rates and the tissue response to them upon implantation.

     

An in vitro feasibility study of controlled drug release from encapsulated nanometer liposomes using high intensity focused ultrasound

A liposome with a diameter ranging from 150 to 200 nm has been considered to be one of the optimal vehicles for targeted drug delivery in vivo since it is able to encapsulate drug and also circulate in the blood stream stably. Its small size, however, makes controlled release of its encapsulated content difficult. A feasibility study for applications of high intensity focused ultrasound (HIFU) of the mega-hertz frequency to induce controlled release of its content was carried out. This study, using the dynamic light scattering and transmission electron microscopic observation, demonstrated 21.2% of encapsulated fluorescent materials (FITC) could be released from liposomes with an average diameter of 210 nm when exposed to continuous (cw) ultrasound at 1.1 MHz (I_SPTA = 900 W/cm²) for 10 s and the percentage release efficiency can reach to 70% after 60 s irradiation. This result also reveals that rupture of relatively large liposomes (>100 nm) and generation of pore-like defects in the membrane of small liposomes (<100 nm) due to HIFU excitation might be the main causes of the release; the inertial cavitation took place during the irradiation. The controlled drug release from liposomes by HIFU may be proven to be a potential useful modality for clinical applications.     

Microbubble mediated sonoporation of cells in suspension: Clonogenic viability and influence of molecular size on uptake

This work investigates whether the application of sonoporation is limited by the size of a macromolecule being delivered and by the ability of cells to proliferate following uptake. KHT-C cells in suspension were exposed to variations in ultrasound pressure (0-570 kPa) and microbubble shell-type (lipid and protein) at fixed settings of 500 kHz centre frequency, 32 μs pulse duration, 3 kHz pulse repetition frequency and 2 min insonation. Reversible permeability (P_R), defined as the number of cells stained with FITC-dextran and unstained with propidium iodide (i.e., PI-viable), was measured with flow cytometry for marker molecules ranging from 10 kDa to 2 MDa in size. Viable permeability (P_V) defined as the number of permeabilised cells that maintained their ability to proliferate, was measured by clonogenic assay. Comparable intracellular delivery of all sizes of molecules was achieved, indicating that intracellular delivery of common therapeutic drugs may not be limited by molecular size. Maximum P_R’s of 80% (at 10 kDa) and 55% (at 10 kDa) were achieved with lipid coated bubbles at 3.3% v/v and protein coated bubbles at 6.7% v/v concentrations. The PI-viability was approximately 80% at 570 kPa in both cases. The maximum P_V achieved with both agents was 22%, while inducing a lower overall clonogenic viability with the lipid (39%) compared to the protein (56%) shelled bubbles. This study demonstrates that large macromolecules, up to 2 MDa in size, can be delivered with high efficiency to cells which undergo reversible permeabilisation, maintaining long-term viability in approximately half of the cells.