Fully collapsed (kippah) vesicles: preparation and characterization.

A study is presented of the formation of a kippah or hemispherical dome structure, a new morphology generated when a vesicle completely collapses to a hollow hemisphere. Justification for the new name is given in the Introduction. Relatively large vesicles of ca. approximately 500 nm in diameter were prepared from poly(acrylic acid)-block-polystyrene (PAA-b-PS) amphiphilic copolymer in the dioxane/water system. The vesicle specimens for transmission electron microscopy (TEM) were prepared using four different methods: drying under ambient conditions, freeze-drying, freeze-drying and subsequent resuspension in water, and drying under vacuum. The formation of the kippah was found to be strongly influenced by the method of preparation. When the vesicles were allowed to dry on the grid, either by drying under ambient conditions or by direct freeze-drying, "normal" vesicles (i.e., not kippah) with the classical indentation pattern were the only structures to be observed. Kippah vesicles, on the other hand, were obtained only by freeze-drying and subsequent rehydration in water or by direct drying under vacuum where no freezing is involved. The cause of the kippah vesicle formation is not yet completely understood for all methods of preparation; however, it was postulated to be strongly influenced by one or more of the following parameters: the relative flexibility of the vesicle wall, pressure gradient, and surface tension. Unlike "normal" vesicles, which exhibit, in TEM, a classical indentation pattern, kippah vesicles appear nearly round but with average wall thickness twice as large as in the "normal" vesicles. The study illustrates also the usefulness of specimen tilting in the analysis of the kippah. In addition, specimen tilting was found to allow the unambiguous determination of the orientation of the kippah on the surface (i.e., open-side-up or open-side-down).

"Breathing" vesicles.

A vesicle system is described that possesses a pH-induced "breathing" feature and consists of a three-layered wall structure. The "breathing" feature consists of a highly reversible vesicle volume change by a factor of ca. 7, accompanied by diffusion of species into and out of the vesicles with a relaxation time of ca. 1 min. The triblock copolymer poly(ethylene oxide)(45)-block-polystyrene(130)-block-poly(2-diethylaminoethyl methacrylate)(120) (PEO(45)-b-PS(130)-b-PDEA(120)) was synthesized via ATRP. Self-assembly into vesicles was carried out at a pH of ca.10.4. The vesicle wall was shown by cryo-TEM to consist of a sandwich of two external ca. 4 nm thick continuous PS layers and one ca. 17 nm thick PDEA layer in the middle. As the pH decreases, both the vesicle size and the thickness of all three layers increase. The increase of the thickness of the intermediate PDEA layer arises from the protonation and hydration, but the swelling is constrained by the PS layers. The increase of the thickness of the two PS layers is a result of an increasing incompatibility and an accompanying sharpening of the interface between the PS layers and the PDEA layer. Starting at a pH slightly below 6, progressive swelling of the PDEA layer with decreasing pH induces a cracking of the two PS layers and also a sharp increase of the vesicle size and the wall thickness. By pH 3.4, the vesicle size has increased by a factor of approximately 1.9 and the wall shows a cracked surface. These changes between pH 10.4 and 3.4 are highly reversible with the relaxation time of ca. 1 min and can be performed repeatedly. The change in the wall structure not only increases dramatically the wall permeability to water but also greatly expands the rate of proton diffusion from practically zero to extremely rapid.

Block-copolymer micelles as carriers of cell signaling modulators for the inhibition of JNK in human islets of Langerhans.

Here we investigate the potential of PCL-b-PEO micelles in preventing the cell death of isolated human islets of Langerhans. PCL-b-PEO micelles were loaded with c-Jun NH2-terminal kinases inhibitor SP600125 to rescue the isolated islets. Mechanistic studies of the uptake were conducted in PC12 cells. Incorporation of SP600125 afforded 8.2 fold greater solubility of SP600125 in micelle suspension. To investigate the effectiveness of micelle-incorporated SP600125 in preventing the islet cell death, we challenged the islets with TNF-alpha, IL-1, and IFN gamma. Micelle-incorporated SP600125 did not lose its inhibitory activity during incorporation into micelles, and it protected the islets against cytokine-induced loss of viability to the same extent as control SP600125. Moreover, the concentration of micelle-incorporated SP600125 used was 13-fold lower, demonstrating the greater efficacy of micelle delivered SP600125. Micelles maintained their cytoplasmic distribution without detectable nuclear localization in islets. The inhibition of JNK was confirmed by western blots. This study suggests that micelle-based intracellular delivery of potent, poorly water soluble, cell-death-pathway inhibitors may represent a valuable addition to established delivery of cytocidal block-copolymer micelle-incorporated bioactives.

Water-soluble surface-anchored gold and palladium nanoparticles stabilized by exchange of low molecular weight ligands with biamphiphilic triblock copolymers.

A study is presented of the stabilization of gold and palladium nanoparticles (NPs) via a place-exchange reaction. Au and Pd NPs of approximately 3.5 nm were prepared by a conventional method using tetraoctylammonium bromide (TOAB) as the stabilizing agent. The resulting nanoparticles, referred to as Au-TOAB or Pd-TOAB, were later used as templates for the replacement of TOAB ligand with poly(ethylene oxide)- b-polystyrene- b-poly(4-vinylpyridine) (PEO- b-PS- b-P4VP) triblock copolymer. This biamphiphilic triblock copolymer was synthesized by atom transfer radical polymerization (ATRP) with control over the molecular weight and polydispersity. The place-exchange reaction was mediated through strong coordination forces between the 4-vinylpyridine copolymer and the metal species located on the surface of the nanoparticles. In addition, the displacement of the outgoing low molecular weight TOAB ligands by high molecular weight polymers is an entropy-assisted process and is believed to contribute to stabilization. The prepared complex, polymer-NP, exhibits greatly improved stability over the metal-NP complex in common organic solvents for the triblock copolymer. Self-assembly in water after ligand exchange resulted in micellar structures of about approximately 20 nm (electron microscopy) with the metal NP found located on the surface of the micelles. The stability of the nanoparticles in water was shown to depend greatly on the grafting density of the copolymer, with high stability (more than 6 months) at high grafting density and low stability, accompanied with irreversible agglomeration, at relatively low grafting densities. The surprising location of the metal NP (for both Au and Pd) on the surface of the micelles in water is explained by the fact that, upon self-assembly in THF/water system, the most hydrophobic chains (i.e., PS) undergo self-assembly first at low water content forming the core, followed by the P4VP (whether or not associated with the metal) forming a shell, and finally the PEO forming the corona. In lower metal content assemblies, the P4VP chains located in the shell undergo swelling in an acidic medium causing a substantial increase in micellar corona size, as confirmed by dynamic light scattering measurements. The present study offers a simple approach for the stabilization of various metal nanoparticles of catalytic interest, using a unique polymeric support that can be dispersed in organic solvents as well as aqueous solutions.

Dendrimer influenced supramolecular structure formation of block copolymers: II. Dendrimer concentration dependence.

The dendrimer concentration dependence of the supramolecular structure formation of polystyrene-block-poly(acrylic acid) in dioxane/THF was investigated as a function of water content. The distribution as well as the localization of the dendrimer units inside the formed aggregates were determined by comparative studies of turbidity measurements and transmission electron microscopy. The strong and specific interactions present between the amine groups of the dendrimer (PAMAM) and the carboxylic acid residues of PAA in the copolymer have a strong influence on the structure formation. The PAMAM concentration as well as the character of the terminal groups of the dendrimer influence the strength of these interactions and consequently affect the structure formation process. As shown by fluorescence quenching experiments, on all supramolecular hierarchical structure levels, and specifically in vesicles, the dendrimer is coated by the PAA chains of the block copolymer due to the strong interactions; since the PAA blocks are connected to the PS blocks, which form the corona, the dendrimer is surrounded by PS chains and is thus encapsulated into the hydrophobic regions of the block copolymer aggregates. A high-resolution transmission electron microscopy image of a micelle is shown, in which the individual dendrimer cores are seen to be localized in the center of these aggregates, and thus, the structure proposed in the previous publication (Kroeger, A.; Li, X.; Eisenberg, A. Langmuir 2007, 23, 10732) is confirmed. Furthermore, the sizes of the resulting aggregates depend on the relative concentration of dendrimer, expressed as RAm/Ac (the ratio of amine to acid groups). With increasing RAm/Ac values, not only the sizes of the micelles but also the vesicle dimensions, especially vesicle wall thicknesses, increase, and this effect suggests the encapsulation of the dendrimer into the vesicle walls. Thus, the constitution of the vesicle structure is determined precisely. This feature allows the potential incorporation of a wide range of species into the vesicle walls or the center of the micelle cores.

Monolayer-protected gold nanoparticles by the self-assembly of micellar poly(ethylene oxide)-b-poly(epsilon-caprolactone) block copolymer.

A study is presented of the preparation of gold nanoparticles incorporated into biodegradable micelles. Poly(ethylene oxide)-b-poly(epsilon-caprolactone) (PEO-b-PCL) copolymer was synthesized by ring-opening polymerization, and the hydroxyl end group of the PCL block was modified with thioctic acid using dicyclohexyl carbodiimide as the coupling reagent. The PEO-b-PCL-thioctate ester (TE) thus obtained was used in a later step to form monolayer protected gold nanoparticles via the thioctate spacer. Gold nanoparticles stabilized with the PEO-b-PCL block (named Au/Block (x/y), where x/y is the mole feed ratio between HAuCl4 and PEO-b-PCL-TE) were prepared and analyzed. Au/Block (1/1), Au/Block (2/1), and Au/Block (3/1) nanoparticles were found to form stable dispersions in the organic solvents commonly used to dissolve the unlabeled block copolymer. The average diameter of the nanoparticles was determined by transmission electron microscopy (TEM) and found to be 6+/-2 nm. Au/Block (4/1) nanoparticle dispersions in organic solvents, on the other hand, were not stable and produced large gold clusters (50-100 nm). Cluster formation was attributed to the low grafting density of the block copolymer, which facilitates agglomeration. For Au/Block (12/1), along the same trend, only an insoluble product was isolated. Micelles in water were prepared by the slow addition of the dilute Au/Block solution in dimethylformamide into a large excess of water with vigorous stirring. Au/Block (1/1) and Au/Block (2/1) formed nanosized structures of 5-7 nm. TEM images of stained Au/Block (1/1) micelles, made in water, clearly showed the formation of core-shell structures. Au/Block (3/1) micelles, on the other hand, were not stable and large agglomerates a few microns in size were observed. The study focuses on the synthesis, characterization, and aggregation behavior of gold-loaded PEO-b-PCL block copolymer micelles, a potential system for drug delivery in conjunction with tissue and subcellular localization studies.

Biocompatible polymer vesicles from biamphiphilic triblock copolymers and their interaction with bovine serum albumin.

The self-assembly of the biamphiphilic triblock copolymer poly(ethylene oxide)-b-poly(caprolactone)-b-poly(acrylic acid) into polymer vesicles is studied. The vesicles provide both biocompatibility and biodegradability. Moreover, the biamphiphilic nature of the triblock copolymer provides different surface properties in the interior and in the outer interface of the vesicles. Preparation of the aggregates by direct dissolution of the copolymer in a solution of albumin does not alter the morphology of the aggregates, and thus, they have the potential to immobilize protein molecules. Since a part of the protein is encapsulated in the interior of the vesicles, they can be used as nanocontainers. A further fraction of the protein is bound to the outer interface, which is primarily composed of the poly(acrylic acid) tails. Immobilization of protein on the outer interface can stabilize the colloidal particles and also provide them with a biofunctional component.

Combination of 3D tissue engineered scaffold and non-viral gene carrier enhance in vitro DNA expression of mesenchymal stem cells.

The objective of this study is to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSC) by combination of 3-dimensional (3D) tissue engineered scaffolds and non-viral gene carrier. As a carrier of plasmid DNA, dextran-spermine cationic polysaccharide was prepared by means of reductive-amination between oxidized dextran and the natural oligoamine, spermine. As the MSC scaffold, collagen sponges reinforced by incorporation of poly(glycolic acid) (PGA) fibers were used. A complex of the cationized dextran and plasmid DNA of BMP-2 was impregnated into the scaffolds. MCS were seeded into each scaffold and cultured by a 3D culture method. When MSC were cultured in the PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by the cationized dextran-plasmid DNA complex impregnated into the scaffold than by the cationized dextran-plasmid DNA complex in 2-dimensional (2D) (tissue culture plate) culture method. The alkaline phosphatase activity and osteocalcin content of transfected MSC cultured in the PGA-reinforced sponge were significantly higher compared with 2D culture method. We conclude that combination of cationized dextran plasmid DNA complex and 3D tissue engineered scaffold was promising to promote the in vitro gene expression for MSC.

Assessment of the integrity of poly(caprolactone)-b-poly(ethylene oxide) micelles under biological conditions: a fluorogenic-based approach.

The integrity of block copolymer micelles is important for their effectiveness and successful delivery of the incorporated drugs. Here we evaluate the integrity of poly(caprolactone)-b-poly(ethylene oxide) micelles in media of varying chemical complexity and in cells by using fluorogenic micelles. Fluorogenic dye fluorescein-5-carbonyl azide diacetate was covalently attached to the micelle-core-forming part of the block copolymer, poly(caprolactone). The fluorescence was not detectable unless the poly(caprolactone)21-b-poly(ethylene oxide)45 micelles were destroyed and the fluorogenic dye was activated by deesterification. The fluorescence of the activated dye from destroyed micelles was easily detectable in various media and in cells. Micelles were stable in simple media such as phosphate-buffered saline but disassembled to varying extents with increasing chemical complexity of the media and addition of serum. The integrity of the internalized micelles within the cells showed a time-dependent decrease but remained largely preserved (80%) after 20 h of incubation with cells. A proof of principle was also demonstrated in vivo in mice. The fluorogenic approach to micelle integrity assessment presented herein should lend itself to other block copolymer micelles and assessments of their integrity in complex biological systems in vitro and in vivo.

Dextran-spermine-based polyplexes--evaluation of transgene expression and of local and systemic toxicity in mice.

Gene delivery using self-assembled polyplexes, formed between negatively charged nucleic acids and cationic polymers, have several drawbacks including low transgene expression and toxicity effects related to their positive charge. Recently, a novel cationic polymer based on dextran-spermine (D-SPM) was developed for gene delivery. This polymer showed systemic biodistribution upon local administration (intramuscular (i.m.) and intranasal (i.n.)) followed by transgene expression in organs remote from the site of injection (liver and lungs). Polyplexes based on D-SPM were administered both i.m. and i.n. to BALB/c female mice. LacZ expression in the liver, lungs, and muscles was assessed using whole-mount 5-bromo-4-chloro-3-indolyl beta-d-galactopyranoside (X-gal) staining and paraffin sectioning. The local toxicity in these organs was evaluated from hematoxylin and eosin stained sections. The systemic toxicity of the polymer and polyplexes was estimated by comparing the mice total weight, major organ weights, blood counts (primarily white blood cells (WBC) and platelets), and serum transaminases, to those of control animals (which received PBS). Transgene expression using D-SPM polyplexes was dependent upon the dosage and the polyplexes (+/-) charge ratio. Using the i.m. and i.n. routes of administration the transfection occurred primarily in the bronchial epithelial cells, pneumocytes, and bronchial alveoli of the lungs; in the muscle's fibrocytes; and in the liver's hepatocytes. Histopathological assays revealed mild toxicity in muscle and no abnormal findings in liver and lung. No systemic toxicity was obtained, as we did not find decrease in WBC count or platelet and no increase in serum transaminases. In addition, mice body weights and major organ weights were similar to the control group at both 2 and 28 days post-administration. This study demonstrates systemic transfection efficacy upon local administration of D-SPM complexes with good tolerability and low toxicity.

Relationships between chemical composition, physical properties and transfection efficiency of polysaccharide-spermine conjugates.

Biodegradable water-soluble polysaccharide-spermine (SPM) polycation conjugates for nucleic acid delivery were synthesized by oxidizing polysaccharides using potassium periodate, followed by SPM conjugation. The polycations differ in their polysaccharide type, arabinogalactan (AG) or dextran (D), and/or in the IO(4)- /saccharide mole ratio used for polysaccharide oxidation (1:1, 1:3, or 1:5), resulting in either D(1:1)-SPM, AG(1:1)-SPM, D(1:3)-SPM, AG(1:3)-SPM, or AG(1:5)-SPM. Chemical structure of the conjugates was characterized for total nitrogen and primary amino groups. Surface pH and electrical surface potential were determined by means of spectral changes of covalently attached 7-hydroxycoumarin (HC, a pH- and electrical surface potential-sensitive fluorophore). The binding and the electrostatic neutralization of the polycations by plasmid DNA, as well as the relationship between chemical structure, physical parameters, and transfection of NIH3T3 cells, were also studied. D(1:1)-SPM, the only polycation that showed efficient cell transfection in culture, was shown to have: (1) high SPM content (2000 nmol/mg); (2) high levels of cross-linked SPM (39-51%); (3) at DNA P-/NH3+ ratio of 2.0, a plateau in neutralization of cationic groups (+48 mV, as determined by HC-labeled D(1:1)-SPM titration with DNA), and a drop in zeta-potential from +42 mV for the polymer alone to 0 mV for the polyplex, suggesting that some of the charges are hidden from the DNA; (4) pH(surface) value of 9.2, suggesting that at physiological bulk pH the polymer is only partially ionized, and therefore can act as a "proton sponge" in the endosome; and (5) high sensitivity to serum-rich growth medium. An oleyl derivative, N-oleyl-dextran-spermine (ODS), was synthesized and demonstrated improved transfection efficiency in serum-rich medium.

Quaternary ammonium polysaccharides for gene delivery.

Cationic polysaccharides were synthesized by conjugation of various monoquaternary (MQ) ammonium oligoamines to oxidized dextran by reductive amination and tested for gene transfection. Polycations of dextran grafted with MQ ammonium oligoamines of two to four amino groups were investigated for their ability to cause pCMV-GFP encoding for green fluorescence protein and beta-Gal encoding for beta-galactosidase protein transfection on EPC and HEK-293 cell lines. These polycations were expected to strongly complex DNA due to increased surface cationic charge of the carrier, which may result in a higher transfection yield. However, the transfection yields were much lower compared to the parent vector, dextran-spermine conjugate, which was highly effective both in vitro and in vivo.

Cationic polysaccharides as antiprion agents.

Cationic polysaccharides were synthesized by conjugation of various oligoamines to oxidized polysaccharides by reductive amination and tested for antiprion activity. Polycations of dextran, pullulan and arabinogalactan grafted with oligoamines of 2 to 4 amino groups were investigated for their ability to eliminate PrP(Sc), the protease-resistant isoform of the prion protein, from chronically infected neuroblastoma cells, ScN2a-M. The proteinase K (PK)-resistant PrP elimination depends on both the concentration of the reagent and the duration of exposure. The most potent compound was found to be dextran-spermine that caused depletion of PrP(Sc) to undetectable levels at concentration of 31 ng/mL after 4 days of exposure. Activity analysis revealed that grafted oligoamine indentity of the polycation plays a significant role in elimination of PK-resistant PrP from chronically infected N2a-M cells, regardless of the polysaccharide used. Dextran-spermine conjugates were modified with oleic acid and with methoxypoly(ethylene glycol) (MPEG) at various degrees of substitution for further studies and their antiprion activity was examined. Substitution of dextran-spermine with MPEG or oleic acid slightly decreases its activity as a function of MPEG/oleic acid content. These findings confirm previous reports that polycations are effective in eliminating PrP(Sc) in vitro.

Hydrophobized dextran-spermine conjugate as potential vector for in vitro gene transfection.

Dextran polysaccharide was grafted by reductive-amination with mixtures of spermine and other natural/synthetic oligoamines of two to four amine groups. The transfection efficiencies of the polycations thus obtained were assessed in various cell lines, and found to depend on the spermine contents. Higher spermine ratios of grafted oligoamines resulted in high gene expression, whereas low to negligible expressions were obtained with lower spermine contents. The effect was explained by spermine residues which exhibit altered buffering capacity in comparison to other substituted oligoamines. Hydrophobization of dextran-spermine (D-SPM) was achieved by treating the polymer with N-hydroxysuccinimide derivatives of cholesterol and fatty acids in a mixture of water/THF. The degree of hydrophobization was in the range of 1-30% mol/mol (hydrophobic moieties/primary amine) and the coupling yields were >95% as determined by (1)H-NMR. The oleate-modified D-SPM remarkably enhanced the gene expression in serum rich media, in marked contrast to unmodified D-SPM which resulted with a drastic decrease in the transfection yields. Modified D-SPM derivatives of other fatty acids and cholesterol showed improved transfection yields in comparison to unmodified D-SPM, but to a lower extent when compared to oleate modification. The improvement in cell transfection was attributed to oleate residues which probably play a role in increasing stability and uptake of polycation-DNA complexes.

Current developments in gene transfection agents.

DNA can be delivered into the cell nucleus either using physical means or specific carriers that carry the genes into the cells for gene expression). Various carriers for delivering genes have been investigated which can be divided into two main groups: viral carriers where the DNA to be delivered is inserted into a virus, and cationic molecular carriers that form electrostatic interactions with DNA). Successful gene therapy depends on the efficient delivery of genetic materials into the cells nucleus and its effective expression within these cells). Although at present the in vivo expression levels of synthetic molecular gene vectors are lower than for viral vectors and gene expression is transient, these vehicles are likely to present several advantages including safety, low-immunogenicity, capacity to deliver large genes and large-scale production at low-cost). The two leading classes of synthetic gene delivery systems that have been mostly investigated are cationic lipids and cationic polymers). This review discusses recent developments in viral vectors, physical means and molecular gene carriers). The last part focuses on our recent studies in developing a new series of biodegradable polycations for in vitro and in vivo gene transfection).

Gene therapy for malignant brain tumors.

Malignant primary and metastatic brain tumors have remained fatal in spite of major advances in diagnostic tools and the improvement of conventional therapies. Recent discoveries in the molecular basis of the disease have allowed increased understanding of the events that lead to the development of brain tumors and have also brought a new spectrum of alternatives for treatment. By using gene therapy, brain tumors can be treated by targeting their fundamental molecular defects, delivering gene-drugs to the malignant cells. The possible targets for this type of treatment are progressively increasing but abundant clinical success has yet to be obtained, in part due to imperfect delivery systems. In this review, the genetic fundamentals of various cerebral neoplasms and neurogenetic syndromes, different strategies used for gene therapy, various available DNA delivery systems, status of ongoing clinical trials, and possible prospects for the future are discussed.

Polysaccharide-oligoamine based conjugates for gene delivery.

This work describes a versatile and universal polycation system based on oligoamines grafted on natural polysaccharides that is capable of complexing various plasmids and administering them into various cells in high yield to produce a desired protein. These polycations are expected to better meet the requirements for effective complexation and delivery of plasmid or an antisense and to biodegrade into nontoxic components at a controlled rate. The developed biodegradable polycations are based on spermine, a natural tetramine, conjugated to dextran or arabinogalactan. These polycations were prepared by reductive amination of oxidized polysaccharides with the desired oligoamines. The Schiff base conjugates thus obtained were reduced to the stable amine conjugates by sodium borohydride. Over 300 different polycations were prepared starting from various polysaccharides and oligoamines, mainly oligoamines of two to four amino groups. Although most of these conjugates formed stable complexes with various plasmids as determined by turbidity experiments, only a few polycations were found to be active in transfecting cells. This work indicates that the structure of the polycation plays a significant role in the transfection activity of polycations.