Photo-control of DNA oligonucleotides with cage compounds

Many biochemical processes in which DNA and other nucleic acids participate are central to functions in both living cells and in molecular biology assays. While many compounds have been used to regulate the activity of DNA, these strategies are limited to the aqueous-based diffusion of the activator to the target DNA molecule. An improvement to the induction of DNA bioactivity is to move to a light-based modulation. This research demonstrates a light-based technique using a photo-cleavable cage compound to transiently inactivate DNA hybridization. Function can be restored with exposure to near-UV light, allowing for temporal control of DNA oligonucleotide (ODN) activity. This method has demonstrated the control of hybridization in molecular biology assays, and provides the framework for in vivo experimentation. A similar light-activated strategy has been shown useful in controlling expression of plasmid transgenes (Monroe 1999). By adapting this method to DNA oligonucleotides (ODNs), we have partially blocked hybridization with the cage compound (1-(4,5-dimethoxy-2-nitrophenyl)ethyl ester (DMNPE) for both phosphodiester and phosphorothioate DNA ODNs. The production and purification of DMNPE-caged DNA ODNs yields products with similar spectrophotometric properties to caged plasmids. In hybridization studies, 20-mer (20 base long) caged DNA ODNs were hybridized with complementary 30-mer molecular beacon probes, and fluorescence measurements were used to assess hybridization of native (non-caged), caged, and caged-light-exposed ODNs. Developments of the molecular beacon assays were studied to improve sensitivity of the assay to caged and caged-flashed ODN hybridization control. Results demonstrated that hybridization can be blocked and subsequently restored by light through the attachment of the DMNPE cage compound, and were further characterized with gel electrophoresis assays. ODN hybridization was restricted to as little as 2% when compared to native (non-caged) ODNs and restored to up to nearly 80% of the native (non-caged) ODN hybridization activity levels. Additional studies on adduction, purification, and characterization of the DMNPE-caged ODNs were performed to optimize their production and efficacy in controlling hybridization. These results suggest that this light-based technology can be used as a tool for the spatial and temporal regulation of hybridization-based DNA bioactivity, including applications with antisense ODNs as a form of controlled gene therapy

Durability Evaluation of Hemp Fibers and Recycled Aggregates Concrete

Hemp and Recycled Aggregates Concrete (HRAC) is a sustainable concrete where coarse aggregates are partially replaced with industrial hemp fibers and recycled concrete aggregates (RCA). This replacement has two main benefits: it saves on natural resources and it recycles and reuses waste material. Previous studies showed that the mechanical performance of HRAC is satisfactory. On the other hand, concrete’s durability is also an important criterion to evaluate concrete’s performance and HRAC’s durability can be affected by the presence of both RCA and hemp fibers in the concrete’s alkaline environment. This paper aims at investigating the durability of HRAC concrete mixes by comparing the mechanical performance of HRAC specimens at the ages of 2 and 28 days. Furthermore, the performance of HRAC under freeze/thaw cycles is evaluated and compared to that of regular concrete. Results showed that the mechanical performance of HRAC improves at older age and the resistance of HRAC mixes to freeze-thaw cycles is similar to that of control mixes

Development and evaluation of an imidazole-modified chitosan for nucleic acid and contrast agent delivery

textOver the past several decades, gene therapy technologies have been developed for a diverse number of applications ranging from DNA-based vaccines to gene silencing with RNAi. While all are powerful tools, a common limitation for these technologies is the need for effective and safe delivery to target sites within the body. Such delivery vectors are necessary for retention of bioactivity and stability, while also providing a method of cellular and tissue uptake and distribution, which may require endosomal escape. Although, viral and lipid-based technologies have shown promise as nucleic acid delivery vectors, both have inherent issues such as cytoxicity, oncogenicity, and immunogenicity. Thus, the development of polymer-based non-viral vectors has been an area of great focus over the past decade. While many polymeric vectors have been developed for plasmid DNA (pDNA) delivery, very few have shown effective delivery of short interfering RNA (siRNA), a powerful tool for gene silencing via the RNA interference mechanism. Furthermore, very few prospective delivery vectors have shown versatility for the administration of siRNA through multiple routes of administration. The overall goal of this research was to develop a biocompatible non-viral delivery system for the delivery of plasmid DNA, siRNA, and contrast agents through the modification of the natural biopolymer chitosan. We have synthesized an imidazole modified chitosan (chitosan-IAA) by conjugation of imidazole acetic acid to chitosan. Extensive evaluation and characterization of the modified polymer demonstrates enhanced solubility and buffering capacity within the physiological and endosomal pHs, thus providing enhanced endosomal escape by exploiting the "proton sponge" effect. We have demonstrated effective in vitro gene expression and gene silencing with chitosan-IAA mediated delivery of pDNA and siRNA, respectively. Furthermore, we have demonstrated in vivo gene silencing by delivery of siRNA through both intranasal and intravenous routes of delivery with chitosan-IAA/siRNA nanocomplexes. We have also demonstrated delivery of contrast agents up to 45 nm in size through mucosal tissue following treatment with chitosan and no contrast agent modification in both human and animal tissue. In conclusion, we have successfully developed a versatile and highly effective delivery vector for both nucleic acids and contrast agents.Biomedical Engineerin

Control of DNA hybridization with photocleavable adducts

Previous reports have shown that 1-(4,5-dimethoxy-2-nitrophenyl)ethyl ester (DMNPE) adducts coupled to DNA plasmids block transcription in vitro and in vivo until removed with light. In this report, we explore the use of DMNPE to control DNA hybridization. We found that DMNPE-caged oligonucleotides have changed spectrophotometric and electrophoretic properties that can be restored with light exposure. Caged oligonucleotides have slower electrophoretic mobility than noncaged oligonucleotides and caged oligonucleotides exposed to light. Effects of caging on hybridization were assessed in a fluorescence-based assay using a 20mer caged DNA oligonucleotide complementary to a 30mer molecular beacon. Fluorescence results indicate that hybridization is reduced and subsequently restored by light. Subsequent gel shift assays confirmed these results. Hybridization activity of caged oligonucleotides with an average of 14-16 DMNPE adducts per oligonucleotide was 14% of noncaged control oligonucleotides and after 365 nm photolysis, increased to nearly 80% of controls. Spectrophotometric characterization of caged oligonucleotides exposed to light and then filtered to remove the released DMNPE adducts indicates two to four attached cage groups remaining following photoactivation. These results suggest that this light-based technology can be used as a tool for the spatial and temporal regulation of hybridization-based DNA bioactivity

Synthesis and Characterization of Transferrin-Targeted Chemotherapeutic Delivery Systems Prepared Via RAFT Copolymerization of High Molecular Weight PEG Macromonomers

Reversible addition-fragmentation chain transfer (RAFT) polymerization was employed to prepare a nanoparticulate drug delivery system for chemotherapeutics. The nanoparticles contain a PEG stealth corona as well as a reactive anhydride functionality designed for conjugating targeting proteins. The multifunctional carrier functionality was achieved by controlling the copolymerization of the hydrophobic monomer lauryl methacrylate (LMA), with a reactive anhydride functional methacrylate (TMA), and a large polyethylene glycol methacrylate monomer (Mn ~ 950 Da) (O950). RAFT polymerization kinetics of O950 were evaluated as a function of target degrees of polymerization (DPs), initial chain transfer agent to initiator ratio ([CTA]o/[I]o), and solvent concentration. Excellent control over the polymerization was observed for target DPs of 25 and 50 at a [CTA]o/[I]o ratio of 10 as evidenced by narrow and symmetric molecular weight distributions and the ability to prepare block copolymers. The TMA-functional copolymers were conjugated to the tumor targeting protein transferrin (Tf). The targeted copolymer was shown to encapsulate docetaxel at concentrations comparable to the commercial single vial formulation of docetaxel (Taxotere). In Vitro cytotoxicity studies conducted in HeLa cells show that the Tf targeting enhances the cancer killing properties relative to the polymer encapsulated docetaxel formulation

Efficient mucosal delivery of optical contrast agents using imidazole-modified chitosan

The clinical applicability of antibodies and plasmonic nanosensors as topically applied, molecule-specific optical diagnostic agents for noninvasive early detection of cancer and precancer is severely limited by our inability to efficiently deliver macromolecules and nanoparticles through mucosal tissues. We have developed an imidazole-functionalized conjugate of the polysaccharide chitosan (chitosan-IAA) to enhance topical delivery of contrast agents, ranging from small molecules and antibodies to gold nanoparticles up to 44 nm in average diameter. Contrast agent uptake and localization in freshly resected mucosal tissues was monitored using confocal microscopy. Chitosan-IAA was found to reversibly enhance mucosal permeability in a rapid, reproducible manner, facilitating transepithelial delivery of optical contrast agents. Permeation enhancement occurred through an active process, resulting in the delivery of contrast agents via a paracellular or a combined paracellular∕transcellular route depending on size. Coadministration of epidermal growth factor receptor–targeted antibodies with chitosan-IAA facilitated specific labeling and discrimination between paired normal and malignant human oral biopsies. Together, these data suggest that chitosan-IAA is a promising topical permeation enhancer for mucosal delivery of optical contrast agents

In Vivo Targeting of Alveolar Macrophages Via RAFT-Based Glycopolymers

Targeting cell populations via endogenous carbohydrate receptors is an appealing approach for drug delivery. However, to be effective, this strategy requires the production of high affinity carbohydrate ligands capable of engaging with specific cell-surface lectins. To develop materials that exhibit high affinity towards these receptors, we synthesized glycopolymers displaying pendent carbohydrate moieties from carbohydrate-functionalized monomer precursors via reversible addition-fragmentation chain transfer (RAFT) polymerization. These glycopolymers were fluorescently labeled and used to determine macrophage-specific targeting both in vitro and in vivo. Mannose- and N-acetylglucosamine-containing glycopolymers were shown to specifically target mouse bone marrow-derived macrophages (BMDMs) in vitro in a dose-dependent manner as compared to a galactose-containing glycopolymer (30- and 19-fold higher uptake, respectively). In addition, upon macrophage differentiation, the mannose glycopolymer exhibited enhanced uptake in M2-polarized macrophages, an anti-inflammatory macrophage phenotype prevalent in injured tissue. This carbohydrate-specific uptake was retained in vivo, as alveolar macrophages demonstrated 6-fold higher internalization of mannose glycopolymer, as compared to galactose, following intratracheal administration in mice. We have shown the successful synthesis of a class of functional RAFT glycopolymers capable of macrophage-type specific uptake both in vitro and in vivo , with significant implications for the design of future targeted drug delivery systems