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Abstract [ABSTRACT FROM AUTHOR]

Drug delivery systems (DDS) for oral delivery of peptide drugs contain excipients that facilitate and enhance absorption. However, little knowledge exists on how DDS excipients such as permeation enhancers interact with the gastrointestinal mucus barrier. This study aimed to investigate interactions of the permeation enhancer sodium 8-[(2-hydroxybenzoyl)amino]octanoate (SNAC) with ex vivo porcine intestinal mucus (PIM), ex vivo porcine gastric mucus (PGM), as well as with in vitro biosimilar mucus (BM) by profiling their physical and barrier properties upon exposure to SNAC. Bulk mucus permeability studies using the peptides cyclosporine A and vancomycin, ovalbumin as a model protein, as well as fluorescein-isothiocyanate dextrans (FDs) of different molecular weights and different surface charges were conducted in parallel to mucus retention force studies using a texture analyzer, rheological studies, cryo-scanning electron microscopy (cryo-SEM), and single particle tracking of fluorescence-labelled nanoparticles to investigate the effects of the SNAC-mucus interaction. The exposure of SNAC to PIM increased the mucus retention force, storage modulus, viscosity, increased nanoparticle confinement within PIM as well as decreased the permeation of cyclosporine A and ovalbumin through PIM. Surprisingly, the viscosity of PGM and the permeation of cyclosporine A and ovalbumin through PGM was unaffected by the presence of SNAC, thus the effect of SNAC depended on the regional site that mucus was collected from. In the absence of SNAC, the permeation of different molecular weight and differently charged FDs through PIM was comparable to that through BM. However, while bulk permeation of neither of the FDs through PIM was affected by SNAC, the presence of SNAC decreased the permeation of FD4 and increased the permeation of FD150 kDa through BM. Additionally, and in contrast to observations in PIM, nanoparticle confinement within BM remained unaffected by the presence of SNAC. In conclusion, the present study showed that SNAC altered the physical and barrier properties of PIM, but not of PGM. The effects of SNAC in PIM were not observed in the BM in vitro model. Altogether, the study highlights the need for further understanding how permeation enhancers influence the mucus barrier and illustrates that the selected mucus model for such studies should be chosen with care., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: To investigate the effect of polyquaternium-1 (PQ)-preserved and benzalkonium chloride (BAK)-preserved travoprost eye drops on viability of primary human conjunctival goblet cell (GC) cultures and on secretion of mucin and cytokines. Furthermore, to evaluate the physicochemical properties of the branded travoprost eye drop Travatan® and available generics., Methods: The effect of travoprost eye drops was evaluated on GC cultures. Cell viability was assessed through lactate dehydrogenase (LDH) and tetrazolium dye (MTT) colorimetric assays. Mucin secretion was evaluated by immunohistochemical staining. Secretion of interleukin (IL)-6 and IL-8 was measured using BD Cytometric Bead Arrays. pH, viscosity, droplet mass, osmolality and surface tension were measured for all included eye drops., Results: In the LDH assay, BAK travoprost caused significant GC loss after 2 hrs of incubation compared to the control. PQ travoprost caused no GC loss at any time point. Both PQ- and BAK travoprost caused secretion of mucin to the cytoplasma. No difference in IL-6 and IL-8 secretion was identified compared to controls. The pH values for the generics were lower (pH 6.0) than the pH value for Travatan (pH 6.7; p < 0.0001). The viscosity was lowest for Travatan, while the mean droplet mass was higher for Travatan (35 mg) than the generics (28-30 mg; p ≤ 0.0318). The osmolality and surface tension did not differ between the eye drops investigated., Conclusion: BAK travoprost caused GC loss, indicating that PQ preservation may be preferable in treatment of glaucoma. Furthermore, physicochemical properties of branded and generic travoprost eye drops can not be assumed to be identical., (© 2022 The Authors. Acta Ophthalmologica published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation.)

Porcine intestinal mucus (PIM) is often utilized as an ex vivo mucus model in mucus interaction studies. However, numerous isolation procedures and storage conditions for PIM are reported, yet their potential impact on preserving the critical properties of PIM remains unknown. This study investigated the effect of isolation procedures (rinsing and anatomical site of mucus isolation) and storage conditions (-20 °C, -80 °C, snap frozen in li-quid nitrogen prior to storage at -80 °C, or freeze-dried followed by storage at room temperature and reconstitution) of PIM in regard to the permeation of fluorescein-isothiocyanate-labelled dextran (FD) macromolecules of 4, 40 and 150 kDa, rheological properties as well as pH, osmolality, protein and water content. Rinsing intestines with tap water or phosphate-buffered saline as well as isolating PIM from different regions of the first five meters of the proximal jejunum did not affect the pH or osmolality of isolated PIM. The permeation of FD4, FD40 and FD150 through stored PIM was similar to permeation through fresh PIM. The rheological properties of stored PIM were similar to properties of fresh PIM. Osmolality, protein and water content were similar in stored and fresh PIM whereas pH decreased with 0.3 unit for all stored PIM. Overall, PIM samples stored at -20 °C, -80 °C, snap frozen or freeze-dried were found to have similar properties to freshly isolated PIM and can all be consi-dered good alternatives to fresh PIM for mucus studies., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Delivery of therapeutic peptides upon oral administration is highly desired and investigations report that the cell-penetrating peptide (CPP) penetratin and its analogues shuffle and penetramax show potential as carriers to enhance insulin delivery. Exploring this, the specific aim of the present study was to understand the impact that their complexation with a lipidated or non-lipidated therapeutic cargo would have on the delivery, to evaluate the effect of differences in membrane interactions in vitro and in vivo, as well as to deduce the mode of action leading to enhanced delivery. Fundamental biophysical aspects were studied by a range of orthogonal methods. Transepithelial permeation of therapeutic peptide was evaluated using the Caco-2 cell culture model supplemented with epithelial integrity measurements, real-time assessment of the carrier peptide effects on cell viability and on mode of action. Pharmacokinetic and pharmacodynamic (PK/PD) parameters were evaluated following intestinal administration to rats and tissue effects were investigated by histology. The biophysical studies revealed complexation of insulin with shuffle and penetramax, but not with penetratin. This corresponded to enhanced transepithelial permeation of insulin, but not of lipidated insulin, when in physical mixture with shuffle or penetramax. The addition of shuffle and penetramax was associated with a lowering of Caco-2 cell monolayer integrity and viability, where the lowering of cell viability was immediate, but reversible. Insulin delivery in rats was enhanced by shuffle and penetramax and accompanied by a 10-20-fold decrease in blood glucose with immediate effect on the intestinal mucosa. In conclusion, shuffle and penetramax, but not penetratin, demonstrated to be potential candidates as carriers for transmucosal delivery of insulin upon oral administration, and their effect depended on association with both cargo and cell membrane. Interestingly, the present study provides novel mechanistic insight that peptide carrier-induced cargo permeation points towards enhancement via the paracellular route in the tight epithelium. This is different from the anticipated belief being that it is the cell-penetrating capability that facilitate transepithelial cargo permeation via a transcellular route., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

To generate the desired effect in the human body, the active pharmaceutical ingredient usually needs to interact with a receptor located on the cell membrane or inside the cell. Thus, understanding membrane interactions is of great importance when it comes to the development and testing of new drug molecules or new drug delivery systems. Nowadays, there is a tremendous selection of both model cell membranes and of techniques that can be used to characterize interactions between selected model cell membranes and a drug molecule, an excipient, or a drug delivery system. Having such a wide selection of model cell membranes and techniques available makes it sometimes challenging to select the optimal combination for a specific study. Furthermore, it is difficult to compare results obtained using different model cell membranes and techniques, and not all in vitro studies translate as well to an estimation of the in vivo biological activity or understanding of mode of action. This review provides an overview of the available lipid bilayer-based model cell membranes and of the most widely employed techniques for studying membrane interactions. Finally, the need for employing complimentary characterization techniques in order to acquire more reliable and in-depth information is highlighted., (Copyright © 2020. Published by Elsevier B.V.)

Oral drug delivery is an attractive noninvasive alternative to injectables. However, oral delivery of biopharmaceuticals is highly challenging due to low stability during transit in the gastrointestinal tract (GIT), resulting in low systemic bioavailability. Thus, novel formulation strategies are essential to overcome this challenge. An interesting approach is increasing retention in the GIT by utilizing mucoadhesive biomaterials as excipients. Here, we explored the potential of the GRAS excipient sucrose acetate isobutyrate (SAIB) to obtain mucoadhesion in vivo. Mucoadhesive properties of a 90% SAIB/10% EtOH (w/w) drug delivery system (DDS) were assessed using a biosimilar mucus model and evaluation of rheological behavior after immersion in biosimilar intestinal fluid. To ease readability of this manuscript, we will refer to this as SAIB DDS. The effect of SAIB DDS on cell viability and epithelial membrane integrity was tested in vitro prior to in vivo studies that were conducted using SPECT/CT imaging in rats. When combining SAIB DDS with biosimilar mucus, increased viscosity was observed due to secondary interactions between biosimilar mucus and sucrose ester predicting considerable mucoadhesion. Mucoadhesion was confirmed in vivo, as radiolabeled insulin entrapped in SAIB DDS, remained in the small intestine for up to 22 h after administration. Moreover, the integrity of the system was investigated using the dynamic gastric model under conditions simulating the chemical composition of stomach fluid and physical shear stress in the antrum under fasted conditions. In conclusion, SAIB is an interesting and safe biomaterial to promote high mucoadhesion in the GIT after oral administration.

The generalized phase contrast method is employed as an efficient "phase-only" laser beam-shaping technique in an optical setup built for catapulting microspheres through simple mucus models. The influence of the laser power and mucin concentration on the motion of the microspheres is investigated in terms of instant and average velocities on a 250-μm trajectory, corresponding to the mucus thickness in the human gastrointestinal tract. Increasing the laser power leads to higher velocities in all the tested samples, while increasing the mucin concentration leads to significant velocity decrease for similar laser input power. However, velocities of up to 95  μm  ·  s  -  1 are demonstrated in a 5% mucin simple mucus model using our catapulting system. This study contributes to understanding and overcoming the challenges of optical manipulation in mucus models. This paves the way for efficient optical manipulation of three-dimensional-printed light-controlled microtools with the ability to penetrate the mucus biobarrier for in vitro drug-delivery studies.

Group B Streptococcus (GBS) is a leading cause of serious bacterial neonatal infections worldwide, which provides an unmet medical need for a globally effective vaccine. The recombinant GBS fusion antigen GBS-NN contains the N-terminal regions of the GBS Rib and Alpha C proteins. It shows promising immunogenicity eliciting protective immunity in mice and encouraging results in early human clinical trials. Understanding the physical stability of GBS-NN containing conformational B-cell epitopes is crucial to ensure optimal vaccine stability, efficacy, and safety. We initially discovered that GBS-NN is prone to form higher-order structures at elevated temperatures. We therefore investigated the self-assembly behavior of GBS-NN and characterized the higher-order conformational structures as a function of temperature. In the native state, GBS-NN exists as a monomer and has a secondary structure containing α-helix and β-sheet. Langmuir studies demonstrated that the native protein is highly surface-active and forms a monolayer film at the air-water interface because of its amphipathic properties. The conformational stability of GBS-NN was measured as a function of temperature. GBS-NN has an unusual thermal behavior with a phase transition of approximately 61 °C, which is not accompanied by any major changes in the secondary structure. However, the antigen showed irreversible self-assembly as a function of temperature into higher-order structures with a hydrodynamic diameter of approximately 100 nm. Cryo-transmission electron microscopy analyses demonstrated that these self-assemblies consist of vesicular, ring-like structures with a hollow aqueous interior. Therefore, GBS-NN is a physically stable monomeric protein but is prone to temperature-induced self-assembly above 61 °C.

Biopharmaceuticals are increasingly important for patients and the pharmaceutical industry due to their ability to treat and, in some cases, even cure chronic and potentially life-threatening diseases. Most biopharmaceuticals are administered by injection, but intensive focus on development of systems for oral delivery of biopharmaceuticals may result in new treatment modalities to increase the patient compliance and reduce product cost. In the preclinical development phase, use of experimental animal models is essential for evaluation of new formulation designs. In general, the limited oral bioavailability of biopharmaceuticals, of just a few percent, is expected, and therefore, the animal models and the experimental settings must be chosen with utmost care. More knowledge and focus on this topic is highly needed, despite experience from the numerous studies evaluating animal models for oral drug delivery of small molecule drugs. This review highlights and discusses pros and cons of the most currently used animal models and settings. Additionally, it also looks into the influence of anesthetics and sampling methods for evaluation of drug delivery systems for oral delivery of biopharmaceuticals primarily with examples on insulin., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Purpose: Induction of cell-mediated immune (CMI) responses is crucial for vaccine-mediated protection against difficult vaccine targets, e.g., Chlamydia trachomatis (Ct). Adjuvants are included in subunit vaccines to potentiate immune responses, but many marketed adjuvants stimulate predominantly humoral immune responses. Therefore, there is an unmet medical need for new adjuvants, which potentiate humoral and CMI responses. The purpose was to design an oil-in-water nanoemulsion adjuvant containing a synthetic CMI-inducing mycobacterial monomycoloyl glycerol (MMG) analogue to concomitantly induce humoral and CMI responses., Methods: The influence of emulsion composition was analyzed using a systematic approach. Three factors were varied: i) saturation of the oil phase, ii) type and saturation of the applied surfactant mixture, and iii) surfactant mixture net charge., Results: The emulsions were colloidally stable with a droplet diameter of 150-250 nm, and the zeta-potential correlated closely with the net charge of the surfactant mixture. Only cationic emulsions containing the unsaturated surfactant mixture induced concomitant humoral and CMI responses upon immunization of mice with a Ct antigen, and the responses were enhanced when squalene was applied as the oil phase. In contrast, emulsions with neutral and net negative zeta-potentials did not induce CMI responses. The saturation degree of the oil phase did not influence the adjuvanticity., Conclusion: Cationic, MMG analogue-containing nanoemulsions are potential adjuvants for vaccines against pathogens for which both humoral and CMI responses are needed.

Background: Autologous serum has been advocated for the treatment of persistent corneal epithelial defects and other ocular surface disorders which may be a local manifestation of a systemic disease. Many of these underlying disorders are cytokine-mediated and require immunosuppressive therapy. The systemic disease and medication could potentially influence the epitheliotrophic capacity of serum eye drops. We compared the effect of serum from healthy and immunosuppressed donors in a human corneal epithelial cell culture model., Methods: Serum was prepared under standardised conditions from full blood samples of 10 healthy donors and 10 patients suffering from rheumatoid arthritis. All patients were treated with prednisolone and methotrexate, one also with azathioprine. In these serum samples EGF, FGF, HGF, PDGF-AB, TGF-beta1, fibronectin, vitamin A and E as well as IL-6 were quantified by means of routine ELISA or HPLC technology. SV-40 immortalised human corneal keratinocytes were cultured in 96-well plates at 37 degrees C, 5 % CO (2) with a fully defined culture medium. At 30 % confluency the culture medium was substituted by one of the test preparations. Proliferation of cell cultures was quantified by means of a luminescence-based ATP assay in dose-response experiments. A colony dispersion assay was used to examine the effect on cell migration and differentiation was assessed by means of scanning electron microscopy., Results: Serum from healthy donors differed from serum of immunosuppressed individuals suffering from rheumatoid arthritis only in that it contained significantly higher amounts of fibronectin and TGF-beta1. Support of proliferation, migration and differentiation of corneal epithelial cells was dose-dependent, but no significant difference was observed between the serum of the two different groups of donors. At a dilution of 25 % serum of healthy donors showed a significantly higher stimulation of migration than serum of immunosuppressed patients., Conclusion: The effect of serum on migration but not proliferation is affected by systemic diseases requiring immunosuppression. If an epithelial defect of a patient with rheumatoid arthritis does not respond to treatment with diluted autologous serum, undiluted serum should be tried since the positive effect of serum on cell migration is positively correlated with dose.

Purpose: Serum eye drops have been successfully used in the treatment of severe ocular surface disorders. Fresh frozen plasma (FFP) and platelet concentrates have not yet been tested for use as eye drops, although they are easily available as quality-controlled products from blood banks and are routinely used for transfusion. To test whether FFP or platelet-derived growth factor solutions could be used for ocular surface diseases, we compared the epitheliotrophic capacity of platelet releasate and FFP with that of serum in cell culture models., Methods: The concentrations of EGF, TGF-beta1, PDGF-AB, fibronectin, vitamin A and vitamin E in serum, FFP, and platelet releasate were evaluated with ELISA and HPLC. Corneal epithelial cells were incubated with the various preparations and cell proliferation, migration, and differentiation were evaluated by means of a luminescence-based adenosine triphosphate (ATP) assay, a colony dispersion assay, and scanning electron microscopy., Results: Growth factor concentrations were significantly higher in platelet releasate than in serum and were lowest in FFP. Fibronectin and vitamins were found in higher concentrations in serum than in FFP and were lowest in platelet releasate. Cell proliferation was best supported by platelet releasate followed by serum and FFP; however, cell migration and differentiation were better supported by serum than by platelet releasate and FFP. The reduced nutrient capacity of FFP was in part found to be due to an antiproliferative effect of citrate used as an anticoagulant in the production process., Conclusions: Platelet releasate but not FFP may offer additional potential for the treatment of severe ocular surface disease. Platelet releasate may be suitable as a novel treatment option for ocular surface disease with a superior effect on cell growth.

Background: Serum eyedrops have been successfully used in the treatment of severe dry eye, persistent epithelial defects and other severe ocular surface disorders. A number of clinical studies showed a variable efficacy of this approach, but the parameters for the production of this blood product varied significantly. In order to establish an optimised protocol for the production of serum eyedrops, we examined the effect of various clotting times, centrifugation forces, types of diluent and dilutions on the concentration of growth factors, fibronectin, and vitamins in serum and tested the epitheliotrophic capacity of these serum modifications in a cell culture model of human SV-40-immortalised corneal epithelial cells (HCE-T)., Methods: Serum samples were prepared with a clotting time of 20, 60 or 120 min, a centrifugation force of 500 xg or 3,000 xg, and diluted with BSS or isotonic saline. The concentrations of EGF, TGF-beta1, PDGF-AB, FGF, HGF, fibronectin, vitamin A and vitamin E in these samples were evaluated with ELISA and HPLC. HCE-T cells were incubated for 24, 48, 72, 96 and 144 h with 100, 50, 25, 12.5, 6.25 and 3.125% serum in diluent, and cell proliferation, migration and differentiation were evaluated by means of a luminescence-based ATP assay, a colony-dispersion assay and scanning electron microscopy., Results: Using a longer clotting time resulted in an increased concentration of all the epitheliotrophic factors examined in serum; the difference was statistically significant for EGF, TGF-beta1 and HGF. Increasing the g force of centrifugation from 500 xg to 3,000 xg resulted in significantly less TGF-beta1, but more EGF and vitamin A. Cell proliferation was better supported by serum prepared with 3,000 xg and diluted with BSS. Serum prepared with a longer clotting time yielded better cell migration and differentiation., Conclusion: Clotting time, centrifugation and diluents have a significant impact on the composition and epitheliotrophic effects of serum. A long clotting time (>or=120 min), a sharp centrifugation (3,000 xg for 15 min) and dilution with BSS improve the ability of serum eyedrops to support proliferation, migration and differentiation of corneal epithelial cells.

Background: Topical application of serum eye drops has been reported to accelerate healing of persistent ocular surface defects. It is supposed that growth factors in serum support the wound healing process. Platelets (PLTs) are rich in growth factors and easily available as PLT concentrates (PCs) from blood banks. Therefore, growth factor preparations from PCs may serve as a new and superior therapeutic agent for such defects., Study Design and Methods: After thrombin stimulation for growth factor release, the cell-free supernatant (PLT releasate) of washed PCs (n = 8) was analyzed for epitheliotrophic factors and its wound healing capacity in comparison to serum (n = 8). Human corneal epithelial cells were used as a model to investigate cell growth, migration, and differentiation in response to both blood products., Results: PLT releasate contains more epithelial growth factor, PLT-derived growth factor, and transforming growth factor-beta, but less hepatocyte growth factor, fibronectin, and vitamins. Cell growth was significantly better in response to PLT releasate. Migration and differentiation were slightly better supported by serum., Conclusion: Possibly owing to its high content of growth factors, PLT releasate has a distinct superior effect on cell growth. Stimulation of migration and differentiation was slightly inferior but still acceptable. PLT releasate could therefore be a novel treatment option for ocular surface defects.