Johannes F.J. Engbersen

Molecular recognition by self-assembled monolayers of cavitand receptors.

It is shown by angle-resolved x-ray photoelectron spectroscopy that cavitands derived from resorcin[4]arenes provided with four dialkylsulfide chains form stable monolayers on gold surfaces that are well organized by self-assembly. The cavitand headgroups at the surface of the resorcin[4]arene monolayer act as molecular recognition sites for small organic molecules with remarkable selectivity for perchloroethylene (C2Cl4). Comparative thermal desorption experiments indicate binding sites with high interaction energies of C2Cl4 at the surface of the resorcin[4]arene monolayers. Fast and reversible host-guest interactions were found by the monitoring of extremely small mass changes (in the nanogram range) with a quartz microbalance oscillator provided with gold electrodes coated by resorcin[4]arene monolayers.

Effects of charge density and hydrophobicity of poly(amido amine)s for non-viral gene delivery.

High cationic charge densities in polymeric vectors result in tight DNA condensation, leading to small highly positively charged polyplexes which show generally high cellular uptake in vitro. However, high cationic charge densities also introduce membrane-disruptive properties to the polymers, thereby frequently causing high cytotoxities. We previously developed poly(amido amine)s with repetitive disulfide linkages in the main chain (SS-PAAs) that are significantly less toxic than PEI, due to fast intracellular degradation of these polymers by bioreductive cleavage of the disulfide bonds. In this study we have investigated the effects of variation in charge density and hydrophobicity on the gene delivery properties of these polymers by varying the degree of acetylation and benzoylation in SS-PAAs with aminobutyl side chains. It was found that introduction of hydrophobic benzoyl groups results in higher transfection efficiencies, both in the absence and presence of serum.

Role of boronic acid moieties in poly(amido amine)s for gene delivery

The effects of the presence of two different types of phenylboronic acids as side groups in disulfide-containing poly(amido amine)s (SS-PAA) were investigated in the application of these polymers as gene delivery vectors. To this purpose, a para-carboxyphenylboronic acid was grafted on a SS-PAA with pending aminobutyl side chains, resulting in p(DAB-4CPBA) and an ortho-aminomethylphenylboronic acid was incorporated through copolymerization, resulting in p(DAB-2AMPBA). Both polymers have 30% of phenylboronic acid side chains and 70% of residual aminobutyl side chains and were compared with the non-boronated benzoylated analogue p(DAB-Bz) of similar M(w). It was found that the presence of phenylboronic acid moieties improved polyplex formation with plasmid DNA since smaller and more monodisperse polyplexes were formed as compared to their non-boronated counterparts. The transfection efficiency of polyplexes of p(DAB-4CPBA) was approximately similar to that of p(DAB-Bz) and commercial PEI (Exgen), both in the absence and the presence of serum, indicating that p(DAB-4CPBA) and p(DAB-Bz) are potent gene delivery vectors. However, the polymers with phenylboronic acid functionalities showed increased cytotoxicity, which is stronger for the ortho-aminophenylboronic acid containing polyplexes of p(DAB-2AMPBA) than for the p(DAB-4CPBA) analog. The cytotoxic effect may be caused by increased membrane disruptive interaction as was indicated by the increased hemolytic activity observed for these polymers.

Functionalized linear poly(amidoamine)s are efficient vectors for intracellular protein delivery

An effective intracellular protein delivery system was developed based on functionalized linear poly(amidoamine)s (PAAs) that form self-assembled cationic nanocomplexes with oppositely charged proteins. Three differently functionalized PAAs were synthesized, two of these having repetitive disulfide bonds in the main chain, by Michael-type polyaddition of 4-amino-1-butanol (ABOL) to cystamine bisacrylamide (CBA), histamine (HIS) to CBA, and ABOL to bis(acryloyl)piperazine (BAP). These water-soluble PAAs efficiently condense β-galactosidase by self-assembly into nanoscaled and positively-charged complexes. Stable under neutral extracellular conditions, the disulfide-containing nanocomplexes rapidly destabilized in a reductive intracellular environment. Cell-internalization and cytotoxicity experiments showed that the PAA-based nanocomplexes were essentially non-toxic. β-Galactosidase was successfully internalized into cells, with up to 94% of the cells showing β-galactosidase activity, whereas the enzyme alone was not taken up by the cells. The results indicate that these poly(amidoamine)s have excellent properties as highly potent and non-toxic intracellular protein carriers, which should create opportunities for novel applications in protein delivery.

Synthesis and binding properties of novel cyclodextrin dimers

The synthesis of three cyclodextrin dimers from the novel building block 3-amino-3-deoxy-heptakis(6-O-tert-butyldimethylsilyl)-s-cyclodextrin is reported. The cyclodextrins are linked with an amide bond on their secondary sides to an ethylene, octamethylene, or a metal binding bipyridyl group. Complexation studies with the substrate 6-(p-toluidino)-2-naphthalenesulfonic acid are presented and discussed.

Influence of plasticizer on the selectivity of nitrate-sensitive CHEMFETs

By chemical modification of an ion-sensitive field-effect transistor, a sensor (CHEMFET) has been developed whose sensitivity for nitrate has been tested. A plasticized poly(vinyl chloride) (PVC) membrane, which contains lipophilic tetra-n-octyl-ammonium ions, is responsible for the nitrate sensitivity of the device. This membrane has been attached physically on top of a pH-buffered hydrogel, which itself has been attached covalently to the gate oxide of the field-effect transistor. The influence of the plasticizer in the PVC membrane on the selectivity pattern of the CHEMFET for the detection of nitrate has been investigated.

Enhanced performance of potassium CHEMFETs by optimization of a polysiloxane membrane

In a way to enhance performance of CHEMFETs based on chemically modified polysiloxane membrane matrix, the effect of the polarity of polysiloxane material on the membrane homogeneity in relation to the covalent binding of the ionophore was investigated. Potassium sensors with the membrane based on the new polysiloxane showed a response with a Nernstian slope in a whole investigated range of cation activity. A new polysiloxane as the membrane matrix improved its homogeneity and increased the yield of functioning CHEMFETs.

Carbohydrate-interactive pDNA and siRNA gene vectors based on boronic acid functionalized poly(amido amine)s.

In order to evaluate the influence of incorporation of boronic acid groups on the properties of poly(amido amine)s as gene vectors, a novel poly(amido amine) copolymer p(CBA-ABOL/2AMPBA) containing ortho-aminomethylphenylboronic acid (2AMPBA) moieties was prepared by Michael-type polyaddition of a mixture of 1,4-aminobutanol (ABOL) and 2-((4-aminobutylamino)methyl)phenyl boronic acid to N,N-cystamine bisacrylamide (CBA). It appeared that the presence of the boronic acid moieties as side groups along the polymer chain strongly enhances the stability of the self-assembled nanoparticles and nanosized polyplexes formed from this polymer; no aggregation was observed after storage for 6days at 37°C. This strong stabilization can be attributed to intermolecular Lewis acid-base interactions between the 2AMPBA groups and the alcohol and amine groups present in the polymer, leading to dynamical (reversible) crosslinking in the nanoparticles. Moreover, since the boronic acids can reversibly form boronic esters with vicinal diol groups, the presence of the 2AMPBA groups add carbohydrate-interactive properties to these polymers that strongly influence their behavior as gene delivery vectors. DNA transfection with p(CBA-ABOL/2AMPBA) polyplexes gave transfection efficiencies that were approximately similar to commercial PEI in different cell lines (COS-7, HUH-6 and H1299-Fluc), but lower than those obtained with reference polyplexes from p(CBA-ABOL). It is hypothesized that the uptake of the boronated polyplexes is suppressed by binding to the glycocalyx of the cells. This is supported by the observation that addition of sorbitol or dextran to the transfection medium significantly enhances the transfection efficiency, which can be attributed to increased cellular uptake of the polyplexes due to boronic ester formation with these agents. AFM, SEM and confocal microscopy showed that polyplexes of p(CBA-ABOL/2AMPBA) become decorated with a dextran layer in the presence of 0.9% (w/v) dextran in the polyplex medium. In siRNA-mediated gene silencing experiments the use of p(CBA-ABOL/2AMPBA) as polymeric vector gave a knockdown of luciferase expression in H1229-Fluc cells of 35%. Also in this case addition of 0.9% (w/v) sorbitol or dextran to the transfection medium strongly increased the knockdown efficiency to 59% and 76%, respectively.

Novel calixarenes in thin films for efficient second harmonic generation

Calix[4]arenes are a novel type of molecules for nonlinear optics. In a single molecule four pi-conjugated systems are combined. Corona poled guest-host polymer systems [calix[4]arenes-poly(methyl-methacrylate)] with transparency into the UV and loading up to 100% show a high degree of orientation. The maximum resonant d33 nonlinear coefficient measured at a fundamental wavelength of 590 nm is 51 pm/V. The d33 values of the thin films relax after poling to 65% of their maximum value. Results indicate self-organization of the molecules upon poling.

Polymeric carrier systems for siRNA delivery.

RNA interference is a technique to induce sequence-specific gene silencing, but is hampered by inefficient delivery of its mediator, short interfering RNA, into target cells. This review describes recent advances in siRNA delivery using polymeric carrier systems. Structural variations that have been applied to these polymers for optimizing their intracellular trafficking are discussed, as well as strategies for stabilization and targeting to diseased tissues in vivo. Recent findings have highlighted safety issues that need to be taken into account in the design of nanoparticles for clinical application.