Abstract Poly(N-vinylcaprolactam) (PNVCL) is a thermoresponsive polymer of particular interest for stimuli-responsive drug delivery due to its physiologically relevant lower critical solution temperature (LCST). However, temperature-induced conformational changes vary significantly with polymer concentration, making the polymer’s hydration state a crucial design parameter. In this study, we investigated the thermoresponsive properties of PNVCL at hydration states representative of various drug delivery applications, extending current knowledge on dissolved PNVCL to hydration states also relevant for cutaneous application. PNVCL with low molecular weight (<5000 g/mol) and low dispersity (1.2–1.8) was synthesized via free radical polymerization, and the molecular weight was characterized using a multimethod approach. UV–vis spectrophotometry, Raman spectroscopy, and small- and wide-angle X-ray scattering (SAXS/WAXS) were employed to probe conformational changes at multiple length scales. We found distinct structural transitions around the LCST for all hydration states, consistent with a coil-to-globule transition. The LCST exhibited a significant dependence on hydration state, with transitions occurring at 27 °C for powder PNVCL and at 48 °C for highly diluted PNVCL (10 mg/mL in water). These findings highlight the importance of accounting for concentration-dependent shifts in LCST when designing PNVCL-based drug delivery systems, particularly for applications involving changes in local polymer concentration upon administration.

Peptide drugs have gained considerable attention as new molecular entities, due to their high target specificity. However, oral delivery of peptides remains a major challenge, and current formulation approaches typically achieve only low single-digit bioavailability, prompting exploration of synergistic strategies to improve absorption. In this work, a self-unfolding foil device, designed for unidirectional drug release in close proximity to the intestinal mucosa, is combined with a potent permeation enhancer-based ionogel formulation for exploitation of synergies from two different oral delivery approaches. The choline decanoate ionogel is loaded into the foil, and insulin release optimized through hydrophilic surface modification and tailored cavity design. In vivo evaluation in rats demonstrate a bioavailability of 12.5

Sodium decanoate (C10) is a medium-chain fatty acid widely studied as a permeation enhancer and considered a benchmark compound for improving the intestinal permeability of poorly absorbed drugs. However, although the mechanism of action of C10 has been extensively investigated, the effect of its colloidal structure on this mechanism remains incompletely understood. This study investigates how pH-dependent colloidal forms of C10, i.e., micelles (pH 8.5) and vesicles (pH 6.5), modulate drug permeation across intestinal mucosae. Ex vivo permeation studies using rat intestinal mucosa were performed with atenolol and 4 kDa fluorescein isothiocyanate (FITC)-dextran as paracellular markers, and metoprolol as a transcellular marker. A pH EpiStat unit was integrated with the Ussing chamber system to allow continuous pH monitoring and precise titration. C10 micelles, at 30 mM, enhanced marker permeation across the intestinal mucosa, particularly metoprolol, whereas vesicles caused minimal enhancement. Using an epithelial-mimetic artificial membrane, micelles were found to cause rapid and complete barrier disruption, while vesicles slowly increased membrane permeability, resulting in a sustained increase in marker permeation. Furthermore, C10 vesicles displayed stronger interactions with mucin and promoted greater retention of marker compounds within the mucosa. Collectively, these findings suggest that both micellar and vesicular C10 enhance permeation primarily via the transcellular route but through different mechanisms: micelles disrupt lipid membrane integrity, while vesicles fluidize membranes without compromising barrier function and interacting with the mucus layer. This work provides new mechanistic insight into C10-mediated permeation enhancement and informs the rational design of more effective oral delivery systems.

Immunotherapy is conventionally managed via subcutaneous injections, yet recently sublingual immunotherapy (SLIT) has been established as an effective and safer route. However, short retention times at the mucosa, in addition to insufficient presentation to antigen-presenting cells can lead to inefficient performance of SLIT. To overcome these challenges, we aimed to co-deliver 33-mer gliadin peptide, the primary trigger of celiac disease, with immunostimulatory Lactococcus lactis Gram-positive enhancer matrix (GEM) particles in a mucoadhesive patch. Accordingly, 33-mer gliadin peptide was co-loaded with GEMs in a two-layer mucoadhesive patch prepared by electrospinning. The delivery system consisted of i) a mucoadhesive layer based on chitosan/polyethylene oxide (PEO) nanofibers to ensure adherence to the mucosa and ii) an active layer of PEO nanofibers for fast co-release of 33-mer gliadin peptide and GEMs. Both layers displayed smooth nanofibers with the GEMs appearing as ‘beads on a string’. Upon wetting, 33-mer gliadin peptide and GEMs were released within minutes in vitro, with no changes in structure or morphology. The two-layer patch showed optimal mechanical properties, maintaining flexibility and shape during handling. Prolonged residence time at the oral mucosa is crucial for SLIT efficacy by enhancing antigen presentation. Strong adhesion of the two-layer patch was demonstrated by measuring adhesion to ex vivo porcine oral mucosa. Furthermore, biocompatibility was demonstrated by exposure to human oral epithelial cell monolayers in vitro. In conclusion, the two-layer patch displayed appropriate properties to serve as a dosage form for SLIT, capable of co-presentation of immunostimulatory actives and enhancing retention at the mucosal surface.

The permeation enhancers (PEs) sodium salcaprozate (SNAC) and sodium decanoate (C10) are reported to enhance gastric and intestinal absorption of different macromolecules. While previous preclinical and clinical studies report effects of these PEs, no head-to-head comparison between their effect in different dosage forms administered by oral gavage in rats is reported. For that purpose, we developed mini-tablets containing octreotide and SNAC or C10 as the PE. The disintegration behavior of the mini-tablets was evaluated using biorelevant liquid volumes of 0.1-0.5 mL in different experimental setups. The efficacy of the PEs co-administered in solution and as a solid dosage form was compared in rats after oral gavage and subsequent assessment of the octreotide pharmacokinetics (PK). While the type of PE and the volume only slightly affected the disintegration time for the mini-tablets, significant differences were observed to depend on the exact experimental setup. Upon oral gavage, plasma octreotide concentrations were enhanced in presence of a PE for both solution and mini-tablet formulations compared to control formulations without PEs, as reflected by the higher area under the plasma concentration curves (AUC). SNAC produced a significantly greater relative bioavailability than both the control and the C10-containing formulations. The relative bioavailability increased 2.0- and 9.4-fold for C10 and SNAC solutions, respectively. Notably, the mini-tablets both with and without PE showed higher numerical octreotide absorption than their solution counterparts and the mini-tablets resulted in a 1.6- and 1.8-fold increase for C10, and a 4.8- and 5.4-fold increase for SNAC in relative bioavailability after co-administration with low and high liquid volumes, respectively. In conclusion, the study shows that the PK profiles for octreotide are different after oral gavage administration of solutions compared to mini-tablets containing the PEs SNAC and C10 and that SNAC outperformed C10 as a permeation enhancer when administered by oral gavage.

Needle patches are commonly used for administration of therapeutics via several routes such as transdermal and buccal delivery. Researchers have further used this strategy for oral delivery of poorly permeable therapeutics by direct injections in the gastrointestinal epithelium, which raises safety concerns about the long-term adverse effects. This study aims to investigate the use of microneedles for oral delivery of compounds with a wide range of molecular weights. For this purpose, we produced fully enteric microneedle patches for unidirectional release by casting. The microneedles presented a length of 175 µm (minimum mucus thickness in the small intestine) to minimize the risk of epithelial damage. A homogeneous drug distribution in the patches was verified by confocal Raman spectroscopy and fluorescence microscopy. Microneedle patches appeared to increase the force displacement in porcine small intestine compared to negative controls (4.4 ± 2.1 mN and 1.8 ± 0.9 mN, respectively), which suggest that microneedles could increase the retention time of oral devices in the gastrointestinal tract. Additionally, the length and integrity of the needles were not affected during the mechanical test studies. In vitro studies at gastric and intestinal pH confirmed successful enteric release and permeation of the drug compounds through biosimilar mucus independently of the molecular weight. The proximity of the patch to the membrane increased the permeation rate, which indicates the potential advantage of microneedles to increase the retention time of more complex devices such as self-unfolding foils, which can ensure drug release in close proximity to the epithelium.

Oral delivery of biopharmaceuticals can be enhanced by some cell-penetrating peptides applied as carriers. The objective here was to improve the permeability enhancing properties of such two peptides by branching into dimer and trimer variants. The transepithelial permeation of cargos including insulin, dextran, mannitol, and metoprolol mediated by penetratin and its analogue penetramax in their linear and branched versions was evaluated in the Caco-2 cell culture model. The enhancing properties of penetramax and particularly penetratin were significantly increased in accordance with the degree of branching, overall increasing the potency of the molecule. The enhanced cargo permeation was associated with peptide-induced effects on the Caco-2 cell monolayers including immediate and reversible decrease in integrity and alterations of the cell cytoskeleton. Overall, the results of the in vitro studies display that the paracellular pathway is involved in the enhanced cargo permeation and that peptide modification by branching indeed increases the permeation of cargos. In vivo pharmacokinetic and histological assessment with insulin as a cargo confirmed the potential of dimeric penetramax as a carrier in comparison to its linear counterpart.

Double emulsions are potential oral delivery systems for the simultaneous administration of hydrophilic drugs and hydrophobic permeation enhancers to enable effective intestinal absorption of macromolecular drugs. Emulsions stabilized by solid particles, i.e., Pickering stabilizers, have shown potential to form gastric-stable emulsions that can protect their cargo from release under gastric conditions. Here, we use acylated cellulose nanocrystals to facilitate the formation of stable double emulsions for intestinal drug delivery. Water-in-oil-in-water double emulsions were obtained by a two-step emulsification process and found to be colloidally stable over 6 months allowing permanent encapsulation of a high molecular weight compound (4 kDa fluorescein isothiocyanate-labelled dextran, FD4) in the inner water phase at >90 % encapsulation efficiency. Exposure to simulated gastric conditions and gastric lipase did not affect the emulsion structure or trigger FD4 release. Double emulsions underwent a pronounced restructuring under simulated intestinal conditions due to the presence of bile, yet without triggering excessive FD4 release (<10 %). Digested emulsions reduced the transepithelial electrical resistance of an intestinal in vitro Caco-2 cell culture model by hydrolysis of the emulsion oil phase into medium chain fatty acids that act as intrinsic permeation enhancers. The double emulsions facilitated permeation of FD4 across the intestinal in vitro model at similar levels as non-formulated FD4 and C10. Hence, double Pickering emulsions stabilized by acylated cellulose nanocrystals comprise a novel gastric stable oral delivery system that can co-deliver large hydrophilic macromolecules and permeation enhancers to the small intestine towards effective intestinal absorption.

Oligonucleotides represent a class of molecules that exhibit remarkable therapeutic potential due to their unparalleled target specificity, yet they suffer from limited cellular uptake and lack of tissue selectivity. Extensive research is conducted with cell-penetrating peptides (CPPs) as delivery excipients due to their ability to translocate across cellular membranes and deliver cargo into cells. This study aims to investigate an innovative approach to rapidly, and with small amounts of compound, analyze and compare complexation of CPPs to oligonucleotides. The study applies surface plasmon resonance (SPR) to evaluate a comprehensive library of CPPs regarding their interaction with a double-stranded oligonucleotide to assess their potential as complexing molecules or whether the CPP should be chemically linked to the negatively charged oligonucleotide to ensure proximity. Specifically, a small interfering RNA (siRNA) was immobilized on a biotinylated chip, and solutions of 66 CPPs were subsequently injected to determine their binding stoichiometry with the siRNA. The most influential molecular properties of the CPPs were determined to be the positive charge-to-length ratio, the total number of positive charges, and the overall hydrophobicity of the CPP. These findings demonstrate the effectiveness and utility of SPR as a high throughput screening tool for selecting peptide/oligonucleotide pairs intended for complexation or conjugation.

Antimicrobial peptides (AMPs) constitute the first line of defense in the human body and exogenous application of AMPs is a desirable therapeutic strategy to combat bacterial infections. However, the antibacterial properties of AMPs are often time limited due to fast degradation by host and bacterial proteases, and administration of the needed high doses may result in local inflammation, as well as nephro- and hepatotoxicity. In this study, we assessed the possibility of using nanogels composed of hyaluronic acid modified with octenyl succinic anhydride (HA-OSA) as a drug delivery system to improve the pharmacokinetics and safety profile of LL37, a naturally occurring AMP, when administered to the mucosal surface of the lungs. The peptide LL37 and the polymer HA-OSA were radiolabeled with 67gallium and 111indium, respectively, allowing for non-invasive tracking over time in mice following intratracheal administration. When non-formulated LL37 was administered, approximately 85 % of the peptide dose was cleared from the lungs over 48 h, whereas encapsulation of LL37 in HA-OSA nanogels increased peptide retention in the lungs by 36 %. Additionally, the amount of peptide in excretory organs was reduced, decreasing potential liver and kidney toxicity known to be associated with AMP-based therapies. The findings in this study indicate that encapsulation of LL37 in nanogels provides beneficial pharmacokinetic effects.

Oral absorption is limited for many small-molecule drugs due to their poor aqueous solubility as well as, for some, poor membrane permeation. One such is levosulpiride (LSP), used to treat psychotic and other conditions. The present study aims to explore the effect of nanostructured lipid carriers (NLCs) for the delivery of LSP. The permeation of LSP in vitro and ex vivo as well as effects on the epithelium and mucosa was monitored. In vitro and ex vivo permeation studies exhibited an 8-fold and 1.6-fold increase in the Papp of LSP respectively, as compared to unformulated LSP applied as a suspension. Transepithelial electrical resistance (TEER) measured in real-time by impedance spectroscopy decreased during exposure yet recovered upon removal of the NLCs. Together with the increased passage of the paracellular markers [14C]-mannitol and FD4 applied together with blank NLCs, but not the transcellular marker [3H]-metoprolol, this indicates permeation of LSP via the paracellular pathway. The reversible effect on integrity was associated with altered cell morphology confirmed by occludin and f-actin localization with insignificant effect on metabolic activity. These results suggest that the NLCs and/or components thereof can mediate improved absorption of drugs by increasing the permeability of the intestinal epithelial membrane, further facilitated by increased drug solubilization.

The increased focus on peptide therapeutics has created an unmet need for technologies and materials enabling therapeutic efficacy after oral administration. Currently, permeation enhancers are the gold standard for oral peptide delivery, with sodium decanoate being one of the most widely tested in preclinical and clinical studies. This study aims to develop and investigate a decanoate-based ionic liquid (IL) inspired by the reported absorption enhancing effect of choline geranate (CAGE) IL. The delivery system is prepared by loading choline decanoate with insulin and upon optimization of the ratio, the chC10 1:2 lead formulation shows gel-like rheological properties. Its high viscosity and hydrophobicity results in slow dissolution in vitro and sustained absorption in vivo. In vivo data show that it can mediate a 7- and a 13-fold higher oral bioavailability of insulin (6.5%) compared to CAGE (0.9%) and sodium decanoate (0.5%), respectively. Histological evaluation reveals that exposure to chC10 1:2 does not affect villi morphology, while 15 min exposure to CAGE significantly reduces villi height. The villi erosion observed is transient and not significantly different from that observed with sodium decanoate. These results showcase the high potential of the chC10 1:2 as an oral drug delivery vehicle.

Oral administration of solid dosage forms for delivery of therapeutic peptides is highly desired. Preclinical investigations on co-administration with permeation enhancers (PEs) to enable sufficient oral bioavailabilities are, however, predominantly done using liquid formulations despite the commercial end-goal being a solid dosage form. Given the amounts needed of PE, this will typically result in a compacted tablet with high amounts of the PE of choice. The aim of this study was to compare the pharmacokinetics (PK) and pharmacodynamics (PD) of insulin after co-formulation with a fixed dose of sodium caprate (C10) in solid dosage forms versus liquid dosage forms. PK/PD parameters in rats were evaluated after dosing mini-tablets and liquid formulations with different amounts of insulin and 26 mg/kg C10 after intestinal administration. Absorption of insulin was dose-dependent in the presence of the PE for both types of dosage forms, which was also reflected in the blood glucose levels. A significant absorption enhancing effect of C10 was found when dosing a 75 IU/kg insulin mini-tablet, resulting in a 26-fold increase in bioavailability. The effect of C10 on the rat intestinal tissue was investigated by histomorphological assessment evaluating erosion and villi height. Effects caused by the C10 mini-tablets and the liquid formulations were similar and shown to be transient. Overall, the findings in this study suggest that mini-tablets can be used to assess peptide bioavailability and the effect of PEs in rats as a preclinical model, and PK data may be nominally different from those obtained with liquid formulations.

Herein, we describe the design and synthesis of 16 neo-glycolipids that are potential permeation enhancers for oral drug delivery of peptide therapeutics. These amphiphilic neo-glycolipids are composed of fatty acids and various carbohydrates ( d -glucose, lactose, cellobiose, maltose) via an oxime linker. The ability of the synthesized neo-glycolipids to enhance permeation of fluorescein-labelled dextran (4 kDa) or 3 H-mannitol across intestinal epithelium was investigated in vitro using monolayers of human epithelial Caco-2 cells. Their effects were compared with (pre-)clinically known enhancers as reference compounds; sodium salts of octanoic, decanoic, and dodecanoic acid, and sodium salcaprozate (SNAC). Most neo-glycolipids increased the permeation of the model compounds, proving that neo-glycolipids, which possess vastly different properties from the reference compounds, e. g ., in terms of clogD and polar surface area, are effective permeation enhancers. The neo-glycolipid based on decanoic acid and glucose was more potent than related compounds based on disaccharides. Significant differences in solubility and cellular compatibility were found for neo-glyolipids based on different carbohydrates. Finally, neo-glycolipids were evaluated as permeation enhancers for the peptide hormone PYY 3-36 . Glucose- and maltose-derived neo-glycolipids based on decanoic and dodecanoic acid showed promising enhancements in PYY 3-36 permeation in vitro while maintaining good cellular compatibility, relevant for oral delivery of obesity treatments.

Human cathelicidin LL-37, a cationic host defense peptide (CHDP), has several important physiological roles, including antimicrobial activity, immune modulation, and wound healing, and is a being investigated as a therapeutic candidate for several indications. While the effects of endogenously produced LL-37 are well studied, the biodistribution of exogenously administered LL-37 are less known. Here we assess the biodistribution of a gallium-67 labeled variant of LL-37 using nuclear imaging techniques over a 48 h period in healthy mice. When administered as an intravenous bolus just over 20 mu g, the LL-37-based radiotracer was rapidly cleared from the blood, largely by the liver, while an appreciable fraction of the dose temporarily distributed to the lungs. When administered subcutaneously at the same dose level, the radiotracer was absorbed systemically following a twophase kinetic model and was predominately cleared renally. Uptake into sites rich in immune cells, such as the lymph nodes and the spleen, was observed for both routes of administration. Scans of free gallium-67 were also performed as controls. Important preclinical insights into the biodistribution of exogenously administered LL-37 were gained from this study, which can aid in the understanding of this and related cationic host-defense peptides.

Self-assembled hyaluronic acid-based nanogels are versatile drug carriers due to their biodegradable nature and gentle preparation conditions, making them particularly interesting for delivery of peptide therapeutics. This study aims to elucidate the relation between peptide structure and encapsulation in a nanogel. Key peptide properties that affect encapsulation in octenyl succinic anhydride-modified hyaluronic acid nanogels were identified as we explored the effect on nanogel characteristics using 12 peptides with varying charge and hydrophobicity. The size and surface properties of the microfluidics-assembled peptide-loaded nanogels were evaluated using dynamic light scattering, laser Doppler electrophoresis, and small angle neutron scattering. Additionally, the change in peptide secondary structure upon encapsulation in nanogels, their release from the nanogels, and the in vitro antimicrobial activity were assessed. In conclusion, the more hydrophobic peptides showed stronger binding to the nanogel carrier and localized internally rather than on the surface of the nanogel, resulting in more spherical nanogels with smoother surfaces and slower release profiles. In contrast, cationic and hydrophilic peptides localized at the nanogel surface resulting in fluffier nanogel structures and quick and more complete release in biorelevant medium. These findings emphasize that the advantages of nanogel delivery systems for different applications depend on the therapeutic peptide properties.

Native mucus is heterogeneous, displays high inter-individual variation and is prone to changes during harvesting and storage. To overcome the lack of reproducibility and availability of native mucus, commercially available purified mucins, porcine gastric mucin (PGM) and mucin from bovine submaxillary gland (BSM), have been widely used. However, the question is to which extent the choice of mucin matters in studies of their interaction with polymers as their composition, structure and hence physicochemical properties differ. Accordingly, the interactions between PGM or BSM with two widely used polymers in drug delivery, polyethylene oxide and chitosan, was studied with orthogonal methods: turbidity, dynamic light scattering, and quartz crystal microbalance with dissipation monitoring. Polymer binding and adsorption to the two commercially available and purified mucins, PGM and BSM, is different depending on the mucin type. PEO, known to interact weakly with mucin, only displayed limited interaction with both mucins as confirmed by all employed methods. In contrast, chitosan was able to bind to both PGM and BSM. Interestingly, the results suggest that chitosan interacts with BSM to a greater extent than with PGM indicating that the choice of mucin, PGM or BSM, can affect the outcome of studies of mucin interactions with polymers.

The integrity of the intestinal mucus barrier is crucial for human health, as it serves as the body's first line of defense against pathogens. However, postnatal development of the mucus barrier and interactions between maturity and its ability to adapt to external challenges in neonatal infants remain unclear. In this study, we unveil a distinct developmental trajectory of the mucus barrier in preterm piglets, leading to enhanced mucus microstructure and reduced mucus diffusivity compared to term piglets. Notably, we found that necrotizing enterocolitis (NEC) is associated with increased mucus diffusivity of our large pathogen model compound, establishing a direct link between the NEC condition and the mucus barrier. Furthermore, we observed that addition of sodium decanoate had varying effects on mucus diffusivity depending on maturity and health state of the piglets. These findings demonstrate that regulatory mechanisms governing the neonatal mucosal barrier are highly complex and are influenced by age, maturity, and health conditions. Therefore, our results highlight the need for specific therapeutic strategies tailored to each neonatal period to ensure optimal gut health.