The strawberry-derived permeation enhancer pelargonidin enables oral protein delivery.

Although patients generally prefer oral drug delivery to injections, low permeability of the gastrointestinal tract makes this method impossible for most biomacromolecules. One potential solution is codelivery of macromolecules, including therapeutic proteins or nucleic acids, with intestinal permeation enhancers; however, enhancer use has been limited clinically by modest efficacy and toxicity concerns surrounding long-term administration. Here, we hypothesized that plant-based foods, which are well tolerated by the gastrointestinal tract, may contain compounds that enable oral macromolecular absorption without causing adverse effects. Upon testing more than 100 fruits, vegetables, and herbs, we identified strawberry and its red pigment, pelargonidin, as potent, well-tolerated enhancers of intestinal permeability. In mice, an oral capsule formulation comprising pelargonidin and a 1 U/kg dose of insulin reduced blood glucose levels for over 4 h, with bioactivity exceeding 100% relative to subcutaneous injection. Effects were reversible within 2 h and associated with actin and tight junction rearrangement. Furthermore, daily dosing of mice with pelargonidin for 1 mo resulted in no detectable side effects, including weight loss, tissue damage, or inflammatory responses. These data suggest that pelargonidin is an exceptionally effective enhancer of oral protein uptake that may be safe for routine pharmaceutical use.

A Potent Branched-Tail Lipid Nanoparticle Enables Multiplexed mRNA Delivery and Gene Editing In Vivo.

The clinical translation of messengerRNA (mRNA) drugs has been slowed by a shortage of delivery vehicles that potently and safely shuttle mRNA into target cells. Here, we describe the properties of a particularly potent branched-tail lipid nanoparticle that delivers mRNA to >80% of three major liver cell types. We characterize mRNA delivery spatially, temporally, and as a function of injection type. Following intravenous delivery, our lipid nanoparticle induced greater protein expression than two benchmark lipids, C12-200 and DLin-MC3-DMA, at an mRNA dose of 0.5 mg/kg. Lipid nanoparticles were sufficiently potent to codeliver three distinct mRNAs (firefly luciferase, mCherry, and erythropoietin) and, separately, Cas9 mRNA and single guide RNA (sgRNA) for proof-of-concept nonviral gene editing in mice. Furthermore, our branched-tail lipid nanoparticle was neither immunogenic nor toxic to the liver. Together, these results demonstrate the unique potential of this lipid material to improve the management of diseases rooted in liver dysfunction.

Engineering Aligned Skeletal Muscle Tissue Using Decellularized Plant-Derived Scaffolds.

To achieve organization and function, engineered tissues require a scaffold that supports cell adhesion, alignment, growth, and differentiation. For skeletal muscle tissue engineering, decellularization has been an approach for fabricating 3D scaffolds that retain biological architecture. While many decellularization approaches are focused on utilizing animal muscle as the starting material, decellularized plants are a potential source of highly structured cellulose-rich scaffolds. Here, we assessed the potential for a variety of decellularized plant scaffolds to promote mouse and human muscle cell alignment and differentiation. After decellularizing a range of fruits and vegetables, we identified the green-onion scaffold to have appropriate surface topography for generating highly confluent and aligned C2C12 and human skeletal muscle cells (HSMCs). The topography of the green-onion cellulose scaffold contained a repeating pattern of grooves that are approximately 20 µm wide by 10 µm deep. The outer white section of the green onion had a microstructure that guided C2C12 cell differentiation into aligned myotubes. Quantitative analysis of C2C12 and HSMC alignment revealed an almost complete anisotropic organization compared to 2D isotropic controls. Our results demonstrate that the decellularized green onion cellulose scaffolds, particularly from the outer white bulb segment, provide a simple and low-cost substrate to engineer aligned human skeletal muscle.

Thrifty, Rapid Intestinal Monolayers (TRIM) Using Caco-2 Epithelial Cells for Oral Drug Delivery Experiments.

PURPOSE: Caco-2 monolayers are the most common model of the intestinal epithelium and are critical to the development of oral drug delivery strategies and gastrointestinal disease treatments. However, current monolayer systems are cost- and/or time-intensive, hampering progress. This study evaluates two separate methods to reduce resource input: FB Essence as a fetal bovine serum (FBS) alternative and a new, 3-day Caco-2 system deemed "thrifty, rapid intestinal monolayers" (TRIM). METHODS: Caco-2 cells were cultured with FB Essence and compared to cells in 10% FBS for proliferation and monolayer formation. TRIM were compared to commonly-used 21-day and Corning® HTS monolayer systems, as well as mouse intestines, for permeability behavior, epithelial gene expression, and tight junction arrangement. RESULTS: No amount of FB Essence maintained Caco-2 cells beyond 10 passages. In contrast, TRIM compared favorably in permeability and gene expression to intestinal tissues. Furthermore, TRIM cost $109 and required 1.3 h of time per 24-well plate, compared to $164 and 3.7 h for 21-day monolayers, and $340 plus 1.0 h for the HTS system. CONCLUSIONS: TRIM offer a new approach to generating Caco-2 monolayers that resemble the intestinal epithelium. They are anticipated to accelerate the pace of in vitro intestinal experiments while easing financial burden.

Branched-Tail Lipid Nanoparticles Potently Deliver mRNA In Vivo due to Enhanced Ionization at Endosomal pH.

The potential of mRNA therapeutics will be realized only once safe and effective delivery systems are established. Unfortunately, delivery vehicle development is stymied by an inadequate understanding of how the molecular properties of a vehicle confer efficacy. Here, a small library of lipidoid materials is used to elucidate structure-function relationships and identify a previously unappreciated parameter-lipid nanoparticle surface ionization-that correlates with mRNA delivery efficacy. The two most potent materials of the library, 306O10 and 306Oi10 , induce substantial luciferase expression in mice following a single 0.75 mg kg-1 mRNA dose. These lipidoids, which have ten-carbon tails and identical molecular weights, vary only in that the 306O10 tail is straight and the 306Oi10 tail has a one-carbon branch. Remarkably, this small difference in structure conferred a tenfold improvement in 306Oi10 efficacy. The enhanced potency of this branched-tail lipidoid is attributed to its strong surface ionization at the late endosomal pH of 5.0. A secondary lipidoid library confirms that Oi10 materials ionize more strongly and deliver mRNA more potently than lipidoids containing linear tails. Together, these data highlight the exquisite control that lipid chemistry exerts on the mRNA delivery process and show that branched-tail lipids facilitate protein expression in animals.

Lipid Nanoparticle Formulations for Enhanced Co-delivery of siRNA and mRNA.

Although mRNA and siRNA have significant therapeutic potential, their simultaneous delivery has not been previously explored. To facilitate the treatment of diseases associated with aberrant gene upregulation and downregulation, we sought to co-formulate siRNA and mRNA in a single lipidoid nanoparticle (LNP) formulation. We accommodated the distinct molecular characteristics of mRNA and siRNA in a formulation consisting of an ionizable and biodegradable amine-containing lipidoid, cholesterol, DSPC, DOPE, and PEG-lipid. Surprisingly, the co-formulation of siRNA and mRNA in the same LNP enhanced the efficacy of both drugs in vitro and in vivo. Compared to LNPs encapsulating siRNA only, co-formulated LNPs improved Factor VII gene silencing in mice from 44 to 87% at an siRNA dose of 0.03 mg/kg. Co-formulation also improved mRNA delivery, as a 0.5 mg/kg dose of mRNA co-formulated with siRNA induced three times the luciferase protein expression compared to when siRNA was not included. As not all gene therapy applications require both RNA drugs, we sought to extend the benefit of co-formulated LNPs to formulations encapsulating only a single type of RNA. We accomplished this by substituting the "helper" RNA with a negatively charged polymer, polystyrenesulfonate (PSS). LNPs containing PSS mediated the same level of protein silencing or expression as standard LNPs using 2-3-fold less RNA. For example, LNPs formulated with and without PSS induced 50% Factor VII silencing at siRNA doses of 0.01 and 0.03 mg/kg, respectively. Together, these studies demonstrate potent co-delivery of siRNA and mRNA and show that inclusion of a negatively charged "helper polymer" enhances the efficacy of LNP delivery systems.

Oral delivery of siRNA lipid nanoparticles: Fate in the GI tract.

Oral delivery, a patient-friendly means of drug delivery, is preferred for local administration of intestinal therapeutics. Lipidoid nanoparticles, which have been previously shown to deliver siRNA to intestinal epithelial cells, have potential to treat intestinal disease. It is unknown, however, whether the oral delivery of these particles is possible. To better understand the fate of lipid nanoparticles in the gastrointestinal (GI) tract, we studied delivery under deconstructed stomach and intestinal conditions in vitro. Lipid nanoparticles remained potent and stable following exposure to solutions with pH values as low as 1.2. Efficacy decreased following exposure to "fed", but not "fasting" concentrations of pepsin and bile salts. The presence of mucin on Caco-2 cells also reduced potency, although this effect was mitigated slightly by increasing the percentage of PEG in the lipid nanoparticle. Mouse biodistribution studies indicated that siRNA-loaded nanoparticles were retained in the GI tract for at least 8 hours. Although gene silencing was not initially observed following oral LNP delivery, confocal microscopy confirmed that nanoparticles entered the epithelial cells of the mouse small intestine and colon. Together, these data suggest that orally-delivered LNPs should be protected in the stomach and upper intestine to promote siRNA delivery to intestinal epithelial cells.

ATRP-grown protein-polymer conjugates containing phenylpiperazine selectively enhance transepithelial protein transport.

Despite its patient-friendliness, the oral route is not yet a viable strategy for the delivery of biomacromolecular therapeutics. This is, in part, due to the large size of proteins, which greatly limits their absorption across the intestinal epithelium. Although chemical permeation enhancers can improve macromolecular transport, their positive impact is often accompanied by toxicity. One element potentially contributing to this toxicity is the lack of co-localization of the enhancer with the protein drug, which can result in non-specific permeation of the intestine as well as enhancer overdosing in some areas due to non-uniform distribution. To circumvent these issues, this study describes a new way of increasing protein permeability via a polymer conjugation process that co-localizes permeation enhancer with the protein. Based on previous reports demonstrating the utility of 1-phenylpiperazine as an intestinal permeation enhancer, we synthesized protein-polymer conjugates with a phenylpiperazine-containing polymer using polymer-based protein engineering. A novel phenylpiperazine acrylamide monomer was synthesized and chain extended using atom transfer radical polymerization from the model protein bovine serum albumin (BSA). At non-cytotoxic doses, the protein-polymer conjugates induced a dose dependent reduction in the trans-epithelial electrical resistance of Caco-2 monolayers and an impressive ~30-fold increase in BSA permeability. Furthermore, this permeability increase was selective, as the permeability of the small molecule calcein co-incubated with the protein-polymer conjugate increased only 5-fold. Together, these data represent an important first step in the development of protein polymer conjugates that facilitate selective protein transport across membranes that are typically impermeable to macromolecules.

Achieving long-term stability of lipid nanoparticles: examining the effect of pH, temperature, and lyophilization.

The broadest clinical application of siRNA therapeutics will be facilitated by drug-loaded delivery systems that maintain stability and potency for long times under ambient conditions. In the present study, we seek to better understand the stability and effect of storage conditions on lipidoid nanoparticles (LNPs), which have been previously shown by our group and others to potently deliver RNA to various cell and organ targets both in vitro and in vivo. Specifically, this study evaluates the influence of pH, temperature, and lyophilization on LNP efficacy in HeLa cells. When stored under aqueous conditions, we found that refrigeration (2°C) kept LNPs the most stable over 150 days compared to storage in the -20°C freezer or at room temperature. Because the pH of the storage buffer was not found to influence stability, it is suggested that the LNPs be stored under physiologically appropriate conditions (pH 7) for ease of use. Although aggregation and loss of efficacy were observed when LNPs were subjected to freeze-thaw cycles, their stability was retained with the use of the cryoprotectants, trehalose, and sucrose. Initially, lyophilization of the LNPs followed by reconstitution in aqueous buffer also led to reductions in efficacy, most likely due to aggregation upon reconstitution. Although the addition of ethanol to the reconstitution buffer restored efficacy, this approach is not ideal, as LNP solutions would require dialysis prior to use. Fortunately, we found that the addition of trehalose or sucrose to LNP solutions prior to lyophilization facilitated room temperature storage and reconstitution in aqueous buffer without diminishing delivery potency.

Lipidoid Nanoparticles for siRNA Delivery to the Intestinal Epithelium: In Vitro Investigations in a Caco-2 Model.

Short interfering ribonucleic acid (siRNA) therapeutics show promise for the treatment of intestinal diseases by specifically suppressing the expression of disease relevant proteins. Recently, a class of lipid-like materials termed "lipidoids" have been shown to potently deliver siRNA to the liver and immune cells. Here, we seek to establish the utility of lipidoid nanoparticles (LNPs) in the context of siRNA delivery to the intestinal epithelium. Initial studies demonstrated that the siRNA-loaded LNPs mediated potent, dose dependent, and durable gene silencing in Caco-2 intestinal epithelial cells, with a single 10 nM dose depressing GAPDH mRNA expression for one week. Transfection with siRNA-loaded LNPs did not induce significant cytotoxicity in Caco-2 cells or alter intestinal barrier function. Protein silencing was confirmed by Western blotting, with the lowest levels of GAPDH protein expression observed five days post-transfection. Together, these data underscore the potential of LNPs for the treatment of intestinal disorders.

Endoscopic diagnosis of a pylorogastric intussusception with spontaneous resolution.

A 7 mo old intact female golden retriever was evaluated for acute vomiting. Abdominal radiographs revealed a possible gastric foreign body. Upper gastrointestinal endoscopy revealed an edematous, tubular antral mass, which on further evaluation was determined to be a pylorogastric intussusception based on radiographic, endoscopic, and surgical findings. Spontaneous resolution of the intussusception occurred upon surgical exploration of the abdomen. Histopathology of a full-thickness gastric biopsy revealed vascular congestion consistent with an intussusception, but did not indicate the primary cause. The dog recovered uneventfully from surgery and had no further vomiting during the 6 mo follow-up period. This case was significant as it was the first report of pylorogastric intussusception diagnosed using endoscopy. This description of the unique endoscopic appearance of pylorogastric intussusception will be useful for the veterinary endoscopist.

Ovarian remnant syndrome in dogs and cats: 21 cases (2000-2007).

OBJECTIVE: To describe signalment, clinical findings, diagnostic tests, and results of treatment of dogs and cats with ovarian remnant syndrome (ORS). DESIGN: Retrospective case series. ANIMALS: 19 dogs and 2 cats with ORS. PROCEDURES: Medical records for animals examined between June 2000 and October 2007 were reviewed for signalment, clinical signs, age at time of ovariohysterectomy (OHE), surgical findings during OHE, experience of the surgeon (veterinary student vs veterinarian), interval from OHE until diagnosis of ORS, results of diagnostic tests, surgical findings, and results of histologic examination of excised tissues. RESULTS: 21 animals (19 dogs and 2 cats) with ORS were identified. The most common clinical signs were those associated with proestrus and estrus. More dogs than cats were affected, and all residual ovarian tissues were found in the region of the ovarian pedicles. The right ovary in dogs was affected significantly more often than the left ovary. Seven animals had neoplasms of the reproductive system. These animals had a significantly longer interval between OHE and diagnosis of ORS than did the 14 animals without neoplasms. Long-term follow-up of 18 animals revealed resolution of clinical signs following exploratory laparotomy. CONCLUSIONS AND CLINICAL RELEVANCE: Ovarian remnants were found in typical locations for ovaries and were not considered ectopic tissue; thus, surgical error during OHE was suspected as the cause of ORS. Anatomic differences may account for differences between species, and clinical signs may not be recognized until years after OHE. Surgical removal of residual ovarian tissue resulted in resolution of clinical signs.