Background & Aims It is a challenge to deliver nucleic acids to gastrointestinal (GI) tissues due to their size and need for intracellular delivery. They are also extremely susceptible to degradation by nucleases, which are ubiquitous in the GI tract. We investigated whether ultrasound, which can permeabilize tissue through a phenomenon known as transient cavitation, can be used to deliver RNA to the colonic mucosa of living mice. Methods We investigated delivery of fluorescently labeled permeants to colon tissues of Yorkshire pigs ex vivo and mice in vivo. Colon tissues were collected and fluorescence was measured by confocal microscopy. We then evaluated whether ultrasound is effective in delivering small interfering (si)RNA to C57/Bl6 mice with dextran sodium sulfate-induced colitis. Some mice were given siRNAs against tumor necrosis factor (Tnf) mRNA for 6 days; colon tissues were collected and analyzed histologically and TNF protein levels measured by ELISA. Feces were collected and assessed for consistency and occult bleeding. We delivered mRNA encoding firefly luciferase to colons of healthy C57/Bl6 mice. Results Exposure of ex vivo pig colon tissues to 20 kHz ultrasound for 1 min increased the level of delivery of 3 kDa dextran 7-fold, compared with passive diffusion (P=.037); 40 kHz ultrasound application for 0.5 sec increased the delivery 3.3-fold in living mice (P = .041). Confocal microscopy analyses of colon tissues from pigs revealed regions of punctuated fluorescent dextran signal, indicating intracellular delivery of macromolecules. In mice with colitis, ultrasound delivery of unencapsulated siRNA against Tnf mRNA reduced protein levels of TNF in colon tissues, compared to mice with colitis given siRNA against Tnf mRNA without ultrasound (P≤.014), and reduced features of inflammation (P≤4.1×10–5). Colons of mice given ultrasound delivery of an mRNA encoding firefly luciferase and the D-luciferin substrate had levels of bioluminescence 11-fold greater than colons of mice given the mRNA alone (P=.0025). Ultrasound exposures of 40 kHz ultrasound for 0.5 seconds were well tolerated, even in mice with acute colitis. Conclusions Ultrasound can be used to deliver mRNAs and siRNAs to the colonic mucosa of mice and knock down expression of target mRNAs.
Background: Hyaluronic acid (HA) is a major component of the extracellular matrix (ECM) in the central nervous system and the only purely supramolecular glycosaminoglycan. Much focus has been given to using this high molecular weight polysaccharide for tissue engineering applications. In most studies, HA is covalently functionalized with moieties that can facilitate network formation through physical self-assembly, or covalent crosslinking (e.g. photo-catalyzed) as the polysaccharide does not gel on its own. However, these crosslinks are not the driving force of HA self-assembly in biological tissues. Methods: Oscillatory rheology, dynamic light scattering, and scanning electron microscopy were used to study albumin/HA structures. Dynamic light scattering and transmission electron microscopy were used to study albumin/chondroitin sulfate (CS) structures. UV-vis spectroscopy was used to demonstrate the potential for using protein-crosslinked polymers as an ECM-mimetic model to study transport of hydrophilic small molecules. Results: We examine the intermolecular interactions of two major glycosaminoglycans found in the human brain, HA and the lower molecular weight CS, with the model protein albumin. We report the properties of the resulting micro- and nano-materials. Albumin/HA mixtures formed supramolecular gels, and albumin/CS mixtures formed micro- and nanoparticles. These systems are formed from unfunctionalized polysaccharides, which is an attractive and simpler method of forming HA hydrogels and CS nanoparticles than functional chemistry-based approaches such as chemically modifying the polymer backbones. We also summarize the concentrations of HA and CS found in various mammalian brains, which could potentially be useful for biomimetic scaffold development in tissue engineering. Conclusions: Simple preparation and combination of commercially available charged biomacromolecules rapidly result in interesting self-assembled materials with structures at the micron and nanometer length-scales. Such materials may have utility in serving as cost-effective and simple models of nervous system electrostatic interactions and as in vitro drug release and model system for ECM transport studies.
Background: Hyaluronic acid (HA) is a major component of the extracellular matrix (ECM) in the central nervous system and the only purely supramolecular glycosaminoglycan. Much focus has been given to using this high molecular weight polysaccharide for tissue engineering applications. In most studies, the backbone of HA is functionalized with moieties that can facilitate network formation through physical self-assembly, or covalent crosslinking (e.g. photo-catalyzed) at concentrations where the polysaccharide does not gel on its own. However, these crosslinks often utilize functional groups not found in biological tissues. Methods: Oscillatory rheology, dynamic light scattering, and scanning electron microscopy were used to study albumin/HA structures. Dynamic light scattering and transmission electron microscopy were used to study albumin/chondroitin sulfate (CS) structures. UV-vis spectroscopy was used to demonstrate the potential for using protein-polymer blends as an ECM-mimetic model to study transport of small molecules. Results: We examine the intermolecular interactions of two major glycosaminoglycans found in the human brain, HA and the lower molecular weight CS, with the model protein albumin. We report the properties of the resulting micro- and nano materials. Our albumin/HA systems formed gels, and albumin/CS systems formed micro- and nanoparticles. These systems are formed from unfunctionalized polysaccharides, which is an attractive and simple method of forming HA hydrogels and CS nanoparticles. We also summarize the concentrations of HA and CS found in various mammalian brains, which could potentially be useful for biomimetic scaffold development. Conclusions: Simple preparation of commercially available charged biomacromolecules results in interesting materials with structures at the micron and nanometer length-scales. Such materials may have utility in serving as cost-effective models of nervous system electrostatic interactions and as in vitro drug release and model system for ECM transport studies.
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