Record-keeping in intravenous sedation: Are we meeting the standards?

As primary care dental services continue to offer conscious sedation in practice, this article presents the findings from a record-keeping audit carried out at a dental teaching hospital in the UK. National guidance was used to set out the gold standards for record-keeping. Various shortcomings in terms of documentation were identified. This article enables dental practitioners involved in dental sedation to review their own sedation record-keeping to meet current national standards and ultimately improve clinical practice and the quality of patient care.

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.

Managing direct oral anticoagulants in accordance with the Scottish Dental Clinical Effectiveness Programme guidance for patients undergoing dentoalveolar surgery.

Introduction The Scottish Dental Clinical Effectiveness Programme (SDCEP) guidance on the management of dental patients taking anticoagulant or antiplatelet drugs provides recommendations on the management of patients taking direct oral anticoagulants (DOACs). This guidance was developed by a multidisciplinary Guidance Development Group, based on available resources at the time of publication. We aim to describe our experience of managing a cohort of adult patients prescribed DOACs, undergoing dentoalveolar procedures in accordance with the SDCEP guidance, between April 2017 and March 2020.Methods As part of our routine practice, patients received a telephone consultation one week following treatment, to assess any post-operative bleeding. Review of the clinical notes was used to assess clinician adherence to the guidance recommendations.Results In total, 98 patients underwent 119 dentoalveolar procedures. Persistent bleeding followed 17 (14.3%) procedures, of which 11 (9.2%) procedures required specific intervention. Absolute compliance with the recommendations was 43.7%, supporting the recommendation for audit and staff education.Discussion A diagnosis of heart failure and advanced age were identified as contributory factors to post-operative bleeding.Conclusions The SDCEP guidance is safe to follow, with no patients experiencing major haemorrhage. Consulting a medical specialist for patients with heart failure of likely significance, based on the medical or drug history, in addition to those who report an advanced heart failure diagnosis and the frail/older person, could reduce the incidence of post-operative bleeding.

Retrospective evaluation of sensory neuropathies after extraction of mandibular third molars with confirmed "high-risk" features on cone beam computed topography scans.

OBJECTIVES: The objective of this study was to identify the incidence of inferior alveolar nerve (IAN) injury after extraction of mandibular third molars with confirmed "high-risk" features on cone beam computed topography scans and establish any risk factors that significantly increase the chances of sustaining an IAN injury. STUDY DESIGN: This study is a retrospective analysis of outcomes after surgical extraction of 500 mandibular third molars over a 5-year period. All teeth showed signs of contact with or compression of the IAN on cone beam computed topography scans. RESULTS: The overall incidence of IAN injury was 6.6%, with permanent neuropathies accounting for 1.8% within this high-risk cohort. Statistically significant factors shown to increase the risk of nerve injury included increasing age (P = .002), compression of the nerve evident on cone beam computed topography scan (P = .005), and buccal or interradicular position of the nerve (P = .042). CONCLUSIONS: Results indicate a low incidence of IAN injury, particularly given the high-risk nature of all teeth that were extracted. It highlights the benefits of cone beam computed topography scans in surgical planning. These data are an important contribution to the existing literature and valuable in the consent procedure for patients undergoing surgical removal of mandibular third molars in contact with the IAN.

Taste buds are not derived from neural crest in mouse, chicken, and zebrafish.

Our lineage tracing studies using multiple Cre mouse lines showed a concurrent labeling of abundant taste bud cells and the underlying connective tissue with a neural crest (NC) origin, warranting a further examination on the issue of whether there is an NC derivation of taste bud cells. In this study, we mapped NC cell lineages in three different models, Sox10-iCreERT2/tdT mouse, GFP+ neural fold transplantation to GFP- chickens, and Sox10-Cre/GFP-RFP zebrafish model. We found that in mice, Sox10-iCreERT2 specifically labels NC cell lineages with a single dose of tamoxifen at E7.5 and that the labeled cells were widely distributed in the connective tissue of the tongue. No labeled cells were found in taste buds or the surrounding epithelium in the postnatal mice. In the GFP+/GFP- chicken chimera model, GFP+ cells migrated extensively to the cranial region of chicken embryos ipsilateral to the surgery side but were absent in taste buds in the base of oral cavity and palate. In zebrafish, Sox10-Cre/GFP-RFP faithfully labeled known NC-derived tissues but did not label taste buds in lower jaw or the barbel. Our data, together with previous findings in axolotl, indicate that taste buds are not derived from NC cells in rodents, birds, amphibians or teleost fish.

Horizontal ridge augmentation of a single atrophic site in the anterior maxilla using hydroxyapatite and rhBMP-2 / Augmento óseo horizontal de reborde alveolar en sitio atrófico único en maxila anterior usando hidroxiapatita y rhBMP-2

SUMMARY: Bone morphogenetic protein (rhBMP-2) is a powerful osteo-inductive growth factor widely used in bone reconstruction and both the vehicle used to administer it and the scaffold substrate could determine its success in clinical situations. The aim was to analyse the clinical behaviour of dental implants placed in single alveolar ridges with a horizontal deficiency in the maxillary anterior region that were reconstructed horizontally with rhBMP-2 and porous hydroxyapatite (HA). Inclusion criteria were both males and females, between the ages of 18 and 29 with single tooth loss of one upper incisor. Cone Beam Computed Tomography (CBCT) was used to take measurements prior to bone augmentation and again prior to the implant insertion. Surgery was carried out under local anaesthetic. In the primary procedure, bone substitute was introduced using porous HA and rhBMP-2; after 4 to 5 months, dental implant surgery was carried out and the implant placed; after 3 months of consolidation the provisional prosthesis was placed and then a definitive restoration was placed. Variables were analysed using the t-test with a p-value of < 0.05 in order to assess statistical significance. Thirteen subjects were included (6 females and 7 males). Bone augmentation resulted in a bone gain of 4.15mm (p=0.023), which was shown to be statistically significant. All of the grafts placed were successful and 13 implants were placed, using torques between 30 and 70N, without complications. For the final prostheses, 11 were screw retained and 2 were cemented in place. The horizontal bone augmentation using HA and rhBMP-2 is an efficient technique for single bone defects in the anterior maxillary area; clinical trials on a larger scale are needed to confirm these results.

RESUMEN: La proteína ósea morfogenética (BMP-2) es un potente osteoinductor utilizado ampliamente en técnicas reconstructivas; el vehículo de instalación es determinante en su evolución. El objetivo fue analizar el comportamiento clínico de implantes dentales instalados en rebordes alveolares únicos con deficiencia horizontal del sector anterior reconstruida horizontalmente con BMP-2 e hidroxiapatita (HA) porosa. Fueron incluidos sujetos de ambos sexos de entre 18 y 29 años, con pérdida dentaria unitaria a nivel de incisivos superiores. Se utilizó tomografía computadorizada para realizar mediciones en las etapas previa a la instalación del injerto y previo a la instalación del implante. Las cirugías fueron realizadas bajo anestesia local. En la primera intervención se realizó la instalación del injerto óseo utilizando HA porosa y BMP-2; después de 4 a 5 meses se realizó la instalación del implante dental; 3 meses después se realizó la conexión protésica y rehabilitación final. Las variables fueron estudiadas con la prueba t test considerando el valor de p< 0,05 para considerar significancia estadística. Trece sujetos fueron incluidos (6 mujeres y 7 hombres); con la reconstrucción ósea se obtuvo una ganancia ósea de 4,15mm (p=0.023) que fue estadísticamente significativo. No existió pérdida en ningún injerto realizado; se instalaron 13 implantes con torques entre 30 y 70N sin complicaciones; se realizaron prótesis fijas atornilladas en 11 casos y cementadas en 2 casos. La técnica con HA y BMP- 2 es eficiente para reconstruir defectos horizontales en perdidas unitarias del sector anterior maxilar; ensayos clínicos de mayor escala son necesarios para confirmar estos resultados.

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.

CRISPR-Cas9 Gene Editing in Lizards through Microinjection of Unfertilized Oocytes.

CRISPR-Cas9-mediated gene editing has enabled the direct manipulation of gene function in many species. However, the reproductive biology of reptiles presents unique barriers for the use of this technology, and there are no reptiles with effective methods for targeted mutagenesis. Here, we demonstrate that the microinjection of immature oocytes within the ovaries of Anolis sagrei females enables the production of CRISPR-Cas9-induced mutations. This method is capable of producing F0 embryos and hatchlings with monoallelic or biallelic mutations. We demonstrate that these mutations can be transmitted through the germline to establish genetically modified strains of lizards. Direct tests of gene function can now be performed in Anolis lizards, an important model for studies of reptile evolution and development.

Imaging neural events in zebrafish larvae with linear structured illumination light sheet fluorescence microscopy.

Light sheet fluorescence microscopy (LSFM) is a powerful tool for investigating model organisms including zebrafish. However, due to scattering and refractive index variations within the sample, the resulting image often suffers from low contrast. Structured illumination (SI) has been combined with scanned LSFM to remove out-of-focus and scattered light using square-law detection. Here, we demonstrate that the combination of LSFM with linear reconstruction SI can further increase resolution and contrast in the vertical and axial directions compared to the widely adopted root-mean square reconstruction method while using the same input images. We apply this approach to imaging neural activity in 7-day postfertilization zebrafish larvae. We imaged two-dimensional sections of the zebrafish central nervous system in two colors at an effective frame rate of 7 frames per second.

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.

Bypassing Glutamic Acid Decarboxylase 1 (Gad1) Induced Craniofacial Defects with a Photoactivatable Translation Blocker Morpholino.

γ-Amino butyric acid (GABA) mediated signaling is critical in the central and enteric nervous systems, pancreas, lungs, and other tissues. It is associated with many neurological disorders and craniofacial development. Glutamic acid decarboxylase (GAD) synthesizes GABA from glutamate, and knockdown of the gad1 gene results in craniofacial defects that are lethal in zebrafish. To bypass this and enable observation of the neurological defects resulting from knocking down gad1 expression, a photoactivatable morpholino oligonucleotide (MO) against gad1 was prepared by cyclization with a photocleavable linker rendering the MO inactive. The cyclized MO was stable in the dark and toward degradative enzymes and was completely linearized upon brief exposure to 405 nm light. In the course of investigating the function of the ccMOs in zebrafish, we discovered that zebrafish possess paralogous gad1 genes, gad1a and gad1b. A gad1b MO injected at the 1-4 cell stage caused severe morphological defects in head development, which could be bypassed, enabling the fish to develop normally, if the fish were injected with a photoactivatable, cyclized gad1b MO and grown in the dark. At 1 day post fertilization (dpf), light activation of the gad1b MO followed by observation at 3 and 7 dpf led to increased and abnormal electrophysiological brain activity compared to wild type animals. The photocleavable linker can be used to cyclize and inactivate any MO, and represents a general strategy to parse the function of developmentally important genes in a spatiotemporal manner.

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.

Chitosan films with improved tensile strength and toughness from N-acetyl-cysteine mediated disulfide bonds.

To improve the mechanical properties of chitosan (Ct) materials without the use of cytotoxic crosslinkers, disulfide cross-linkable Ct was synthesized by grafting N-acetyl-cysteine (NAC) to Ct using carbodiimide chemistry. Cast films of NAC-Ct conjugates were prepared with degrees of substitution (DS) of 0%, 6%, 15%, and 20%, and the disulfide bond formation was induced by increasing the reaction media pH to 11. The tensile strength, breaking strain, elastic moduli and toughness of disulfide cross-linked polymers were analyzed by monotonic tensile testing of hydrated NAC-Ct films. Crystallinity was determined via XRD. Results demonstrated that NAC incorporation and crosslinking in chitosan produced tougher polymer films with 4-fold higher tensile strength (10 MPa) and 6-fold greater elongation (365%), but reduced crystallinity, compared to unmodified chitosan. The resilience of NAC-Ct films was evaluated by cyclic testing, and results demonstrate that increasing NAC content produced a more resilient material that dissipated less energy when deformed. These improved mechanical properties broaden chitosan's applicability towards the construction of mechanically robust implantable scaffolds for tissue regeneration.

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.