Kim L. Wark

A general approach for DNA encapsulation in degradable polymer microcapsules.

We report a general and facile method for the encapsulation of DNA in nanoengineered, degradable polymer microcapsules. Single-stranded (ss), linear double-stranded (ds), and plasmid DNA were encapsulated into disulfide-cross-linked poly(methacrylic acid) (PMA) capsules. The encapsulation procedure involves four steps: adsorption of DNA onto amine-functionalized silica (SiO(2)(+)) particles; sequential deposition of thiolated PMA (PMA (SH)) and poly(vinylpyrrolidone) to form multilayers; cross-linking of the thiol groups of the PMA (SH) in the multilayers into disulfide linkages; and removal of the sacrificial SiO(2)(+) particles. Multilayer growth was dependent on the surface coverage of DNA on the SiO(2)(+) particles, with stable capsules formed from particles with up to 50% DNA surface coverage. The encapsulation strategy applies to nucleic acids with varied size and conformation and allows DNA to be concentrated over 100-fold from dilute solutions into monodisperse, uniformly loaded polymer capsules. The capsule loading can be controlled by the DNA:SiO(2)(+)particle ratio, and for 1 microm diameter capsules, loadings of approximately 1000 chains of 800 bp dsDNA and more than 10,000 chains of 20-mer ssDNA can be achieved. The encapsulated DNA was released and successfully used in polymerase chain reactions as both templates (linear dsDNA and plasmid DNA) and primer sequences (ssDNA), confirming the functionality and structural integrity of the encapsulated DNA. These DNA-loaded polymer microcapsules hold promise as delivery vehicles for gene therapy and diagnostic applications.

Engineered antibody intervention strategies for Alzheimer's disease and related dementias by targeting amyloid and toxic oligomers

Most neurodegenerative disorders, such as Alzheimers (AD), Parkinsons, Huntingtons and Creutzfeldt-Jakob disease, are characterised by the accumulation of insoluble filamentous aggregates known as amyloid. These pathologies share common pathways involving protein aggregation which can lead to fibril formation and amyloid plaques. The 4 kDa Abeta peptide (39-43 amino acids) derived from the proteolysis of the amyloid precursor protein is currently a validated target for therapy in AD. Both active and passive immunisation studies against Abeta are being trialled as potential AD therapeutic approaches. In this study, we have characterised engineered antibody fragments derived from the monoclonal antibody, WO-2 which recognises an epitope in the N-terminal region of Abeta (amino acids 2-8 of Abeta). A chimeric recombinant Fab (rFab) and single chain fragments (scFvs) of WO-2 were constructed and expressed in Escherichia coli. Rationally designed mutants to improve the stability of antibody fragments were also constructed. All antibody formats retained high affinity (K(D) approximately 8 x 10(-9) M) for the Abeta peptide, comparable with the intact parental IgG as measured by surface plasmon resonance. Likewise, all engineered fragments were able to: (i) prevent amyloid fibrillisation, (ii) disaggregate preformed Abeta(1-42) fibrils and (iii) inhibit Abeta(1-42) oligomer-mediated neurotoxicity in vitro as efficiently as the whole IgG molecule. These data indicate that the WO-2 antibody and its fragments have immunotherapeutic potential. The perceived advantages of using small Fab and scFv engineered antibody formats which lack the effector function include more efficient passage across the blood-brain barrier and minimising the risk of triggering inflammatory side reactions. Hence, these recombinant antibody fragments represent attractive candidates and safer formulations of passive immunotherapy for AD.

Metal-free and MRI visible theranostic lyotropic liquid crystal nitroxide-based nanoparticles.

The development of improved, low toxicity, clinically viable nanomaterials that provide MRI contrast have tremendous potential to form the basis of translatable theranostic agents. Herein we describe a class of MRI visible materials based on lyotropic liquid crystal nanoparticles loaded with a paramagnetic nitroxide lipid. These readily synthesized nanoparticles achieved enhanced proton-relaxivities on the order of clinically used gadolinium complexes such as Omniscan™ without the use of heavy metal coordination complexes. Their low toxicity, high water solubility and colloidal stability in buffer resulted in them being well tolerated in vitro and in vivo. The nanoparticles were initially screened in vitro for cytotoxicity and subsequently a defined concentration range was tested in rats to determine the maximum tolerated dose. Pharmacokinetic profiles of the candidate nanoparticles were established in vivo on IV administration to rats. The lyotropic liquid crystal nanoparticles were proven to be effective liver MRI contrast agents. We have demonstrated the effective in vivo performance of a T1 enhancing, biocompatible, colloidally stable, amphiphilic MRI contrast agent that does not contain a metal.

Noncovalent Liposome Linkage and Miniaturization of Capsosomes for Drug Delivery

We report the synthesis of poly(methacrylic acid)-co-(oleyl methacrylate) with three different amounts of oleyl methacrylate and compare the ability of these polymers with that of poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMA(c)) to noncovalently anchor liposomes to polymer layers. We subsequently assembled ∼1 μm diameter PMA(c)-based capsosomes, polymer hydrogel capsules that contain up to ∼2000 liposomal subcompartments, and investigate the potential of these carriers to deliver water-insoluble drugs by encapsulating two different antitumor compounds, thiocoraline or paclitaxel, into the liposomes. The viability of lung cancer cells is used to substantiate the cargo concentration-dependent activity of the capsosomes. These findings cover several crucial aspects for the application of capsosomes as potential drug delivery vehicles.

A Biomolecular “Ship-in-a-Bottle”: Continuous RNA Synthesis Within Hollow Polymer Hydrogel Assemblies

2010 WILEY-VCH Verlag Gm Synthetic counterparts to cellular compartments remain far from the complexity of living systems but hold tremendous promise for advancing studies into the synthesis, confinement, and delivery of biomolecules. Of all biomolecular candidates, RNA could benefit from encapsulation as it spans a range of cellular functions including information storage, regulation, and catalysis. RNA has proven to be a potent therapeutic for the modification of cellular function, yet unprotected, it remains highly susceptible to degradation. A practical and biomimetic approach to RNA encapsulation is the enzyme-catalyzed synthesis of RNA within the confines of a drug delivery vehicle, which has the potential to minimize the handling of RNA, bypassing isolation and purification steps. To date, the most successful examples of encapsulated transcription exploit liposomes and emulsions, yet controlled RNA loading and their subsequent use for the cellular internalization of the newly synthesized RNA have not been accomplished. Herein, we describe the use of micrometer-sized, monodisperse polymer hydrogel capsules (HCs) for the first successful example of encapsulated de novo RNA synthesis and subsequent cellular internalization of the RNA. The capsules act as both microreactors and drug carriers. Unlike other methods of encapsulated RNA transcription, polymer HCs also allow real-time monitoring of RNA synthesis and, therefore, precise control over the encapsulated RNA concentration via the reaction time. The recent development of polymer capsules produced via the layer-by-layer (LbL) deposition of polymers has provided a novel platform for encapsulated catalysis with a number of advantages, including precise control over the capsule size and permeability based on both the size and/or the charge of the solutes. Successful examples of DNA synthesis, hybridization, and degradation within the confines of polymer capsules highlight the potential of these assemblies as host compartments for biomolecular transformations. In addition, their shape, stability, and size endow them with characteristics suitable for the delivery of molecular therapeutics. The successful synthesis of RNA within the confines of a polymer HC represents a facile technique for the controlled capsule loading of RNA, en route to a drug-delivery platform for RNA therapeutics. The encapsulation of two macromolecules is essential for the synthesis of RNA within the interior of a polymer capsule: an RNA polymerase and a double-stranded DNA (dsDNA) template containing a specific promoter sequence required for enzyme binding and the initiation of RNA transcription. We have recently encapsulated DNA into poly(methacrylic acid) (PMA) HCs through LbL assembly. The PMA HCs entrap DNA through a combination of size exclusion and electrostatic repulsion. Herein, we exploit the encapsulated dsDNA to template the transcription of RNA by T7 RNA polymerase (T7Pol) (Scheme 1). T7Pol has sufficient diffusivity through the walls of PMA HCs, possibly via the ‘‘relay race’’ mechanism described for protein– hydrogel interactions. Once inside, the T7Pol binds the promoter sequence in the DNA template and is immobilized inside the capsule. Due to their small size, individual ribonucleotides freely diffuse into the PMA HCs, where T7Pol assemble them into single-stranded RNApolymers. Similar to the DNA template, the size, shape, and charge of the newly synthesized RNA polymers ensure they remained trapped within the capsules. The strategy outlined above (Scheme 1) was used to assemble PMA HCs (diameter of 4.35 0.25mm), each containing 9000 copies of a 777-base pair (bp) dsDNA polymerase chain reaction (PCR) product. The PCR products were designed to either include (þT7DNA) or exclude (–T7DNA) the T7 promoter sequence at the 50-end of the sense strand. Approximately 10 capsules were added to a 50mL transcription reaction supplemented with 0.1 nmol of fluorescently labeled uridine50-triphosphate (UTP, green) and the mixture was incubated at 37 8C. A concentration of 50 units of T7Pol in the 50mL reaction was sufficient to enable diffusion of enzyme into the core of the PMA HCs and initiate transcription. Confocal laser scanning microscopy (CLSM) was used to image fluorescently labeled HCs (red) following 3 h of incubation. Newly synthesized RNA was clearly visible in the interior of the PMA HCs containing DNA with the T7 promoter sequence (Fig. 1a), however, no fluorescencewas observed in the PMAHCs containing DNA without the T7 promoter sequence (Fig. 1b). An identical reaction was performed using 1.35 0.15mm PMA HCs, each containing 500 copies ofþT7DNA. Flow cytometry was used to measure the progress of the reaction within the PMA HCs (Fig. 1c), allowing real-time monitoring of RNA synthesis. Synthesis was slow in the first hour but then increased to a linear rate of synthesis between 1.5 h and 5 h, and finally leveled off over the next 24 h. The initial slow rate of synthesis possibly reflects the time required for the T7Pol to diffuse into the capsules and bind to the T7 promoter sequences.

Peptide-Functionalized, Low-Biofouling Click Multilayers for Promoting Cell Adhesion and Growth†

The nanoengineering of materials for biomedical applications LbL assembly has enabled the formation of layered, stable such as drug delivery, gene therapy and tissue engineering is an area of intense scientific research. To develop innovative materials with tailored properties, new synthetic methods that are highly specific and capable of delivering high yields of predesigned, functional building blocks, and complex molecular assemblies are required. Click chemistry meets such stringent requirements; it has provided a set of quantitative, highly selective covalent reactions that are simple and versatile, and can be performed undermild reaction conditions to produce high product yields. The most commonly used click reaction is the copper(I)-catalyzed variant of Huisgen’s 1,3-dipolar cycloaddition of alkynes and azides to produce 1,2,3-triazoles. The versatility and specificity of this click reaction has been demonstrated through the synthesis of a range of advanced materials, including hydrogels, crosslinkedmicelles, and dendritic copolymers as well as for the preparation of functionalized nanoparticles, nanotubes, and cotton and organic resin surfaces. This technique has also been popular in a number of bioapplications for the functionalization of DNA, site specific modification of proteins, and selective dye labeling within cells. Recently, we combined click chemistry with an inexpensive and simple assembly technique, known as layer-by-layer (LbL) assembly. The LbL technology is based on the serial adsorption of species with complementary interactions, and has been widely used to prepare a diverse range of layered materials. The recent combination of click chemistry and

Engineered antibody approaches for Alzheimer’s disease immunotherapy

The accumulation of amyloid-β-peptide (Aβ or A-beta) in the brain is considered to be a key event in the pathogenesis of Alzheimers disease (AD). Over the last decade, antibody strategies aimed at reducing high levels of Aβ in the brain and or neutralizing its toxic effects have emerged as one of the most promising treatments for AD. Early approaches using conventional antibody formats demonstrated the potential of immunotherapy, but also caused a range of undesirable side effects such meningoencephalitis, vasogenic edema or cerebral microhemorrhages in both murine and humans. This prompted the exploration of alternative approaches using engineered antibodies to avoid adverse immunological responses and provide a safer and more effective therapy. Encouraging results have been obtained using a range of recombinant antibody formats including, single chain antibodies, antibody domains, intrabodies, bispecific antibodies as well as Fc-engineered antibodies in transgenic AD mouse and primate models. This review will address recent progress using these recombinant antibodies against Aβ, highlighting their advantages over conventional monoclonal antibodies and delivery methods.

Cytotoxicity and internalization of polymer hydrogel capsules by mammalian cells.

Polymer hydrogel capsules comprised of poly(methacrylic acid) chains and cross-linked via disulfide linkages were investigated for their cytotoxicity and mechanism of internalization in a variety of mammalian cells. The capsules were internalized by all the tested cell lines which differed in their morphology and function and over short to medium term (24 h) revealed no reduction in viability and metabolic activity of cells. The mechanism of capsule uptake was analyzed using inhibitors of various cellular entry pathways. Of these, blocking the clathrin-mediated endocytotic pathway resulted in a statistically significant reduction in capsule uptake, suggesting this was the predominant pathway of capsule entry in these cell lines. The uptake of solid particles with similar surface chemistry was not significantly decreased by the inhibitor of the clathrin-mediated pathway, which suggested that softness and concomitant flexibility of the hydrogel capsules were factors governing the entry mechanism. This work represents the first systematic study of the interaction of polymer hydrogel capsules with mammalian cells and provides essential information for the application of these capsules in biomedicine.

Germline humanization of a murine Aβ antibody and crystal structure of the humanized recombinant Fab fragment

Alzheimers disease is the most common form of dementia, affecting 26 million people worldwide. The Aβ peptide (39–43 amino acids) derived from the proteolytic cleavage of the amyloid precursor protein is one of the main constituents of amyloid plaques associated with disease pathogenesis and therefore a validated target for therapy. Recently, we characterized antibody fragments (Fab and scFvs) derived from the murine monoclonal antibody WO‐2, which bind the immunodominant epitope (3EFRH6) in the Aβ peptide at the N‐terminus. In vitro, these fragments are able to inhibit fibril formation, disaggregate preformed amyloid fibrils, and protect neuroblastoma cells against oligomer‐mediated toxicity. In this study, we describe the humanization of WO‐2 using complementary determining region loop grafting onto the human germline gene and the determination of the three‐dimensional structure by X‐ray crystallography. This humanized version retains a high affinity for the Aβ peptide and therefore is a potential candidate for passive immunotherapy of Alzheimers disease.

Restricted V gene usage and VH/VL pairing of mouse humoral response against the N-terminal immunodominant epitope of the amyloid β peptide.

Over the last decade, the potential of antibodies as therapeutic strategies to treat Alzheimers disease (AD) has been growing, based on successful experimental and clinical trials in transgenic mice. Despite, undesirable side effects in humans using an active immunization approach, immunotherapy still remains one of the most promising treatments for AD. In this study, we analyzed the V genes of twelve independently isolated monoclonal antibodies raised against the N-terminal immunodominant epitope of the amyloid β peptide (Aβ or A beta). Surprisingly, we found a high and unusual level of restriction in the VH/VL pairing of these antibodies. Moreover, these antibodies mostly differ in their heavy chain complementary determining region 3 (HCDR3) and the residues in the antibodies which contact Aβ are already present in the germline V-genes. Based on these observations and or co-crystal structures of antibodies with Aβ, the aim of the current study was to better understand the role of antibody V-domains, HCDR3 regions, key contact residue (H58) and germline encoded residues in Aβ recognition. For that purpose, we designed and produced a range of recombinant Fab constructs. All the Fabs were tested and compared by surface plasmon resonance on Aβ(1-16), Aβ(1-42) high molecular weight and Aβ(1-42) low molecular weight soluble oligomers. Although all the Fabs recognized the Aβ(1-16) peptide and the Aβ(1-42) high molecular weight soluble oligomers, they did not bind the Aβ(1-42) low molecular weight soluble oligomers. Furthermore, we demonstrated that: (1) an aromatic residue at position H58 in the antibody is essential in the recognition of Aβ and (2) Fabs based on germline V-genes bind to Aβ monomers with a low affinity. These findings may have important implications in designing more effective therapeutic antibodies against Aβ.