Mucoadhesive Electrospun Nanofiber-Based Hybrid System with Controlled and Unidirectional Release of Desmopressin.

The sublingual mucosa is an attractive route for drug delivery, although challenged by a continuous flow of saliva that leads to a loss of drug by swallowing. It is of great benefit that drugs absorbed across the sublingual mucosa avoid exposure to the harsh environment of the gastro-intestinal lumen; this is especially beneficial for drugs of low physicochemical stability such as therapeutic peptides. In this study, a two-layered hybrid drug delivery system was developed for the sublingual delivery of the therapeutic peptide desmopressin. It consisted of peptide-loaded mucoadhesive electrospun chitosan/polyethylene oxide-based nanofibers (mean diameter of 183 ± 20 nm) and a saliva-repelling backing film to promote unidirectional release towards the mucosa. Desmopressin was released from the nanofiber-based hybrid system (approximately 80% of the loaded peptide was released within 45 min) in a unidirectional manner in vitro. Importantly, the nanofiber-film hybrid system protected the peptide from wash-out, as demonstrated in an ex vivo flow retention model with porcine sublingual mucosal tissue. Approximately 90% of the loaded desmopressin was retained at the surface of the ex vivo porcine sublingual mucosa after 15 min of exposure to flow rates representing salivary flow.

Stereochemistry as a determining factor for the effect of a cell-penetrating peptide on cellular viability and epithelial integrity.

Cell-penetrating peptides (CPPs) comprise efficient peptide-based delivery vectors. Owing to the inherent poor enzymatic stability of peptides, CPPs displaying partial or full replacement of l-amino acids with the corresponding d-amino acids might possess advantages as delivery vectors. Thus, the present study aims to elucidate the membrane- and metabolism-associated effects of l-Penetratin (l-PEN) and its corresponding all-d analog (d-PEN). These effects were investigated when exerted on hepatocellular (HepG2) or intestinal (Caco-2 and IEC-6) cell culture models. The head-to-head comparison of these enantiomeric CPPs included evaluation of their effects on cell viability and morphology, epithelial membrane integrity, and cellular ultrastructure. In all investigated cell models, a rapid decrease in cell viability, pronounced membrane perturbation and an altered ultrastructure were detected upon exposure to d-PEN. At equimolar concentrations, these observations were less pronounced or even absent for cells exposed to l-PEN. Both CPPs remained stable for at least 2 h during exposure to proliferating cells (cultured for 24 h), although d-PEN exhibited a longer half-life when compared with that of l-PEN when exposed to well-differentiated cell monolayers (cultured for 18-20 days). Thus, the stereochemistry of the CPP penetratin significantly influences its effects on cell viability and epithelial integrity when profiled against a panel of mammalian cells.

In Vitro ADME Properties of Two Novel Antimicrobial Peptoid-Based Compounds as Potential Agents against Canine Pyoderma.

Antimicrobial peptides (AMPs) hold promise as the next generation of antimicrobial agents, but often suffer from rapid degradation in vivo. Modifying AMPs with non-proteinogenic residues such as peptoids (oligomers of N-alkylglycines) provides the potential to improve stability. We have identified two novel peptoid-based compounds, B1 and D2, which are effective against the canine skin pathogen Staphylococcus pseudintermedius, the main cause of antibiotic use in companion animals. We report on their potential to treat infections topically by characterizing their release from formulation and in vitro ADME properties. In vitro ADME assays included skin penetration profiles, stability to proteases and liver microsomes, and plasma protein binding. Both B1 and D2 were resistant to proteases and >98% bound to plasma proteins. While half-lives in liver microsomes for both were >2 h, peptoid D2 showed higher stability to plasma proteases than the peptide-peptoid hybrid B1 (>2 versus 0.5 h). Both compounds were suitable for administration in an oil-in-water cream formulation (50% release in 8 h), and displayed no skin permeation, in the absence or presence of skin permeability modifiers. Our results indicate that these peptoid-based drugs may be suitable as antimicrobials for local treatment of canine superficial pyoderma and that they can overcome the inherent limitations of stability encountered in peptides.

Fluorophore labeling of a cell-penetrating peptide induces differential effects on its cellular distribution and affects cell viability.

Cell-penetrating peptides constitute efficient delivery vectors, and studies of their uptake and mechanism of translocation typically involve fluorophore-labeled conjugates. In the present study, the influence of a number of specific fluorophores on the physico-chemical properties and uptake-related characteristics of penetratin were studied. An array of seven fluorophores belonging to distinct structural classes was examined, and the impact of fluorophore labeling on intracellular distribution and cytotoxicity was correlated to the physico-chemical properties of the conjugates. Exposure of several mammalian cell types to fluorophore-penetratin conjugates revealed a strong structure-dependent reduction in viability (1.5- to 20-fold lower IC50 values as compared to those of non-labeled penetratin). Also, the degree of less severe effects on membrane integrity, as well as intracellular distribution patterns differed among the conjugates. Overall, neutral hydrophobic fluorophores or negatively charged fluorophores conferred less cytotoxicity as compared to the effect exerted by positively charged, hydrophobic fluorophores. The latter conjugates, however, exhibited less membrane association and more clearly defined intracellular distribution patterns. Thus, selection of the appropriate flurophore is critical.

Conjugation of cell-penetrating peptides to parathyroid hormone affects its structure, potency, and transepithelial permeation.

Delivery of therapeutic peptides and proteins by the use of cell-penetrating peptides (CPPs) as carriers has been suggested as a feasible strategy. The aim of the present study was to investigate the effect of conjugating a series of well-known CPPs to the biologically active part of parathyroid hormone, i.e., PTH(1-34), and to evaluate the effect with regard to secondary structure, potency in Saos-2 cells, immunogenicity, safety, as well as the transepithelial permeation across monolayers by using the Caco-2 cell culture model. Further, co-administration of CPP and PTH(1-34) as an alternative to covalent conjugation was compared with regard to the transepithelial permeation. CPP-conjugated PTH(1-34) fusion peptides were successfully expressed in Escherichia coli and purified from inclusion bodies. No clear correlation between the degree of secondary structure of the CPP-conjugated PTH(1-34) fusion peptides and their potency was found, albeit a general decrease in permeation was observed for both N- and C-terminally CPP-conjugated PTH(1-34) as compared to native PTH(1-34). However, attachment of CPP to the N-terminus significantly increased permeation across Caco-2 cell monolayers as compared to the corresponding C-terminally CPP-conjugated PTH(1-34). In addition, the nonaarginine sequence proved to be the only CPP capable of increasing permeation when conjugated to PTH(1-34) as compared to co-administration of CPP and PTH(1-34). This enhancement effect was, however, associated with an unacceptably low level of cell viability. In conclusion, covalent conjugation of CPPs to PTH(1-34) influenced the secondary structure, potency, and transepithelial permeation efficiency of the resulting conjugate, and hence this approach appears not to be favorable as compared to co-administration when optimizing CPP-mediated permeation of PTH(1-34) across an intestinal epithelium.

Antimicrobial and cell-penetrating properties of penetratin analogs: effect of sequence and secondary structure.

Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) show great potential as drug delivery vectors and new antibiotic drug entities, respectively. The current study deals with the properties of a variety of peptide analogs derived from the well-known CPP penetratin as well as octaarginine and different Tat sequences. The effects of peptide length, guanidinium content, and sequence of non-cationic residues were assessed in mammalian and bacterial cells. The arginine (Arg) content in the penetratin analogs was found to influence eukaryotic cell uptake efficiency, antimicrobial activity towards both Gram-positive and Gram-negative bacteria as well as eukaryotic cell viability. All examined analogs retained the ability to cross eukaryotic membranes giving rise to a distribution within the vacuolar apparatus. Interestingly, a series of shuffled analogs of penetratin with the cationic residues in conserved positions, attain the same α-helical conformation as native penetratin in the presence of cholesterol-containing liposomes, while conformational differences were observed in the presence of highly anionic liposomes. While the antibacterial effect of the two groups of peptides was similar, the eukaryotic cellular uptake of the shuffled analogs was noticeably lower than for native penetratin. Moreover, a point substitution of Met to Leu in native penetratin had no influence on eukaryotic cellular uptake and antimicrobial effect, and only a minor effect on cytotoxicity, in contrast to the fact that the same substitution in the shuffled analog gave rise to reduced eukaryotic cellular uptake while increasing the antibacterial effect and cytotoxicity.

Solid lipid particles for oral delivery of peptide and protein drugs II--the digestion of trilaurin protects desmopressin from proteolytic degradation.

PURPOSE: To investigate the in vitro release and degradation of desmopressin from saturated triglyceride microparticles under both lipolytic and proteolytic conditions. METHODS: The release of desmopressin from different solid lipid microparticles in the absence and presence of a microbial lipase and protease was determined. Trilaurin (TG12), trimyristin (TG14), tripalmitin (TG16), and tristearin (TG18) were used as lipid excipients to produce solid lipid microparticles. RESULTS: In the presence of lipase, the rate of drug release from different lipid particles was in the order of TG14 > TG16 > TG18, which is the same rank order as the lipid degradation rate. A reverse rank order was found for the protection of desmopressin from enzymatic degradation due to spatial separation of desmopressin from the protease. TG12 accelerated the release of desmopressin from all lipid particles when added as either drug-free microparticles to the lipolysis medium or incorporated in TG16 particles. Additionally, TG12 particles protected desmopressin from degradation when present in the lipolysis medium with the other lipid microparticles. CONCLUSIONS: TG12 is a very interesting lipid for oral lipid formulations containing peptides and proteins as it alters release and degradation of the incorporated desmopressin. The present study demonstrates the possibility of bio-relevant in vitro evaluation of lipid-based solid particles.

Engineering of an inhalable DDA/TDB liposomal adjuvant: a quality-by-design approach towards optimization of the spray drying process.

PURPOSE: The purpose of this study was to identify and optimize spray drying parameters of importance for the design of an inhalable powder formulation of a cationic liposomal adjuvant composed of dimethyldioctadecylammonium (DDA) bromide and trehalose-6,6'-dibehenate (TDB). METHODS: A quality by design (QbD) approach was applied to identify and link critical process parameters (CPPs) of the spray drying process to critical quality attributes (CQAs) using risk assessment and design of experiments (DoE), followed by identification of an optimal operating space (OOS). A central composite face-centered design was carried out followed by multiple linear regression analysis. RESULTS: Four CQAs were identified; the mass median aerodynamic diameter (MMAD), the liposome stability (size) during processing, the moisture content and the yield. Five CPPs (drying airflow, feed flow rate, feedstock concentration, atomizing airflow and outlet temperature) were identified and tested in a systematic way. The MMAD and the yield were successfully modeled. For the liposome size stability, the ratio between the size after and before spray drying was modeled successfully. The model for the residual moisture content was poor, although, the moisture content was below 3% in the entire design space. Finally, the OOS was drafted from the constructed models for the spray drying of trehalose stabilized DDA/TDB liposomes. CONCLUSIONS: The QbD approach for the spray drying process should include a careful consideration of the quality target product profile. This approach implementing risk assessment and DoE was successfully applied to optimize the spray drying of an inhalable DDA/TDB liposomal adjuvant designed for pulmonary vaccination.

Carrier peptide interactions with liposome membranes induce reversible clustering by surface adsorption and shape deformation.

The cell-penetrating peptide penetratin and its analogues shuffle and penetramax have been used as carrier peptides for oral delivery of therapeutic peptides such as insulin. Their mechanism of action for this purpose is not fully understood but is believed to depend on the interactions of the peptide with the cell membrane. In the present study, peptide-liposome interactions were investigated using advanced biophysical techniques including small-angle neutron scattering and fluorescence lifetime imaging microscopy. Liposomes were used as a model system for the cell membrane. All the investigated carrier peptides induced liposome clustering at a specific peptide/lipid ratio. However, distinctively different types of membrane interactions were observed, as the liposome clustering was irreversible for penetratin, but fully or partly reversible for shuffle and penetramax, respectively. All three peptides were found to adsorb to the surface of the lipid bilayers, while only shuffle and penetramax led to shape deformation of the liposomes. Importantly, the peptide interactions did not disrupt the liposomes under any of the investigated conditions, which is advantageous for their application in drug delivery. This detailed insight on peptide-membrane interactions is important for understanding the mechanism of peptide-based excipients and the influence of peptide sequence modifications.

Heterogeneous and Surface-Catalyzed Amyloid Aggregation Monitored by Spatially Resolved Fluorescence and Single Molecule Microscopy.

Amyloid aggregation is associated with many diseases and may also occur in therapeutic protein formulations. Addition of co-solutes is a key strategy to modulate the stability of proteins in pharmaceutical formulations and select inhibitors for drug design in the context of diseases. However, the heterogeneous nature of this multicomponent system in terms of structures and mechanisms poses a number of challenges for the analysis of the chemical reaction. Using insulin as protein system and polysorbate 80 as co-solute, we combine a spatially resolved fluorescence approach with single molecule microscopy and machine learning methods to kinetically disentangle the different contributions from multiple species within a single aggregation experiment. We link the presence of interfaces to the degree of heterogeneity of the aggregation kinetics and retrieve the rate constants and underlying mechanisms for single aggregation events. Importantly, we report that the mechanism of inhibition of the self-assembly process depends on the details of the growth pathways of otherwise macroscopically identical species. This information can only be accessed by the analysis of single aggregate events, suggesting our method as a general tool for a comprehensive physicochemical characterization of self-assembly reactions.

Mucoadhesive chitosan- and cellulose derivative-based nanofiber-on-foam-on-film system for non-invasive peptide delivery.

Oromucosal administration is an attractive non-invasive route. However, drug absorption is challenged by salivary flow and the mucosa being a significant permeability barrier. The aim of this study was to design and investigate a multi-layered nanofiber-on-foam-on-film (NFF) drug delivery system with unique properties and based on polysaccharides combined as i) mucoadhesive chitosan-based nanofibers, ii) a peptide loaded hydroxypropyl methylcellulose foam, and iii) a saliva-repelling backing film based on ethylcellulose. NFF displays optimal mechanical properties shown by dynamic mechanical analysis, and biocompatibility demonstrated after exposure to a TR146 cell monolayer. Chitosan-based nanofibers provided the NFF with improved mucoadhesion compared to that of the foam alone. After 1 h, >80 % of the peptide desmopressin was released from the NFF. Ex vivo permeation studies across porcine buccal mucosa indicated that NFF improved the permeation of desmopressin compared to a commercial freeze-dried tablet. The findings demonstrate the potential of the NFF as a biocompatible drug delivery system.

Encapsulation of SAAP-148 in Octenyl Succinic Anhydride-Modified Hyaluronic Acid Nanogels for Treatment of Skin Wound Infections.

Chronic wound infections colonized by bacteria are becoming more difficult to treat with current antibiotics due to the development of antimicrobial resistance (AMR) as well as biofilm and persister cell formation. Synthetic antibacterial and antibiofilm peptide (SAAP)-148 is an excellent alternative for treatment of such infections but suffers from limitations related to its cationic peptidic nature and thus instability and possible cytotoxicity, resulting in a narrow therapeutic window. Here, we evaluated SAAP-148 encapsulation in nanogels composed of octenyl succinic anhydride (OSA)-modified hyaluronic acid (HA) to circumvent these limitations. SAAP-148 was efficiently (>98%) encapsulated with high drug loading (23%), resulting in monodispersed anionic OSA-HA nanogels with sizes ranging 204-253 nm. Nanogel lyophilization in presence of polyvinyl alcohol maintained their sizes and morphology. SAAP-148 was sustainedly released from lyophilized nanogels (37-41% in 72 h) upon reconstitution. Lyophilized SAAP-148-loaded nanogels showed similar antimicrobial activity as SAAP-148 against planktonic and biofilm-residing AMR Staphylococcus aureus and Acinetobacter baumannii. Importantly, formulated SAAP-148 showed reduced cytotoxicity against human erythrocytes, primary human skin fibroblasts and human keratinocytes. Additionally, lyophilized SAAP-148-loaded nanogels eradicated AMR S. aureus and A. baumannii colonizing a 3D human epidermal model, without inducing any cytotoxicity in contrast to SAAP-148. These findings indicate that OSA-HA nanogels increase SAAP-148's therapeutic potential for treatment of skin wound infections.

Structure of immune stimulating complex matrices and immune stimulating complexes in suspension determined by small-angle x-ray scattering.

Immune stimulating complex (ISCOM) particles consisting of a mixture of Quil-A, cholesterol, and phospholipids were structurally characterized by small-angle x-ray scattering (SAXS). The ISCOM particles are perforated vesicles of very well-defined structures. We developed and implemented a novel (to our knowledge) modeling method based on Monte Carlo simulation integrations to describe the SAXS data. This approach is similar to the traditional modeling of SAXS data, in which a structure is assumed, the scattering intensity is calculated, and structural parameters are optimized by weighted least-squares methods when the model scattering intensity is fitted to the experimental data. SAXS data from plain ISCOM matrix particles in aqueous suspension, as well as those from complete ISCOMs (i.e., with an antigen (tetanus toxoid) incorporated) can be modeled as a polydisperse distribution of perforated bilayer vesicles with icosahedral, football, or tennis ball structures. The dominating structure is the tennis ball structure, with an outer diameter of 40 nm and with 20 holes 5-6 nm in diameter. The lipid bilayer membrane is 4.6 nm thick, with a low-electron-density, 2.0-nm-thick hydrocarbon core. Surprisingly, in the ISCOMs, the tetanus toxoid is located just below the membrane inside the particles.

Solid-phase route to Fmoc-protected cationic amino acid building blocks.

Diamino acids are commonly found in bioactive compounds, yet only few are commercially available as building blocks for solid-phase peptide synthesis. In the present work a convenient, inexpensive route to multiple-charged amino acid building blocks with varying degree of hydrophobicity was developed. A versatile solid-phase protocol leading to selectively protected amino alcohol intermediates was followed by oxidation to yield the desired di- or polycationic amino acid building blocks in gram-scale amounts. The synthetic sequence comprises loading of (S)-1-(p-nosyl)aziridine-2-methanol onto a freshly prepared trityl bromide resin, followed by ring opening with an appropriate primary amine, on-resin N(ß)-Boc protection of the resulting secondary amine, exchange of the N(α)-protecting group, cleavage from the resin, and finally oxidation in solution to yield the target γ-aza substituted building blocks having an Fmoc/Boc protection scheme. This strategy facilitates incorporation of multiple positive charges into the building blocks provided that the corresponding partially protected di- or polyamines are available. An array of compounds covering a wide variety of γ-aza substituted analogs of simple neutral amino acids as well as analogs displaying high bulkiness or polycationic side chains was prepared. Two building blocks were incorporated into peptide sequences using microwave-assisted solid-phase peptide synthesis confirming their general utility.

Cell-Penetrating Peptides as Carriers for Transepithelial Drug Delivery.

This chapter describes the use of cell-penetrating peptides (CPPs) as carriers for transepithelial delivery of therapeutic peptides. Assessment of transepithelial peptide permeation and the mechanisms of action that permeability enhancing drug carriers exert on the epithelium requires subtle sample preparation and analysis by orthogonal methods. Here, the preparation and use of CPP-insulin physical mixture samples including the quantification of insulin by enzyme-linked immunosorbent assay (ELISA) is described. In addition, effects of CPPs on the epithelium and its barrier properties immediately upon exposure and after a recovery period are evaluated by epithelial cell viability, transepithelial electrical resistance, immunostaining of the tight junction associated zonula occludens (ZO-1) protein, and actin cytoskeleton staining.

Topical niclosamide (ATx201) reduces Staphylococcus aureus colonization and increases Shannon diversity of the skin microbiome in atopic dermatitis patients in a randomized, double-blind, placebo-controlled Phase 2 trial.

BACKGROUND: In patients with atopic dermatitis (AD), Staphylococcus aureus frequently colonizes lesions and is hypothesized to be linked to disease severity and progression. Treatments that reduce S. aureus colonization without significantly affecting the skin commensal microbiota are needed. METHODS AND FINDINGS: In this study, we tested ATx201 (niclosamide), a small molecule, on its efficacy to reduce S. aureus and propensity to evolve resistance in vitro. Various cutaneous formulations were then tested in a superficial skin infection model. Finally, a Phase 2 randomized, double-blind and placebo-controlled trial was performed to investigate the impact of ATx201 OINTMENT 2% on S. aureus colonization and skin microbiome composition in patients with mild-to-severe AD (EudraCT:2016-003501-33). ATx201 has a narrow minimal inhibitory concentration distribution (.125-.5 µg/ml) consistent with its mode of action - targeting the proton motive force effectively stopping cell growth. In murine models, ATx201 can effectively treat superficial skin infections of methicillin-resistant S. aureus. In a Phase 2 trial in patients with mild-to-severe AD (N = 36), twice-daily treatment with ATx201 OINTMENT 2% effectively reduces S. aureus colonization in quantitative colony forming unit (CFU) analysis (primary endpoint: 94.4% active vs. 38.9% vehicle success rate, p = .0016) and increases the Shannon diversity of the skin microbiome at day 7 significantly compared to vehicle. CONCLUSION: These results suggest that ATx201 could become a new treatment modality as a decolonizing agent.

Investigation of the interaction between modified ISCOMs and stratum corneum lipid model systems.

The modified ISCOMs, so-called Posintro nanoparticles, provide an opportunity for altering the surface charge of the particles, which influences their affinity for the negatively charged antigen sites, cell membranes and lipids in the skin. Hypothetically, this increases the passage of the ISCOMs (or their components) and their load through the stratum corneum. The subsequent increase in the uptake by the antigen-presenting cells results in enhanced transcutaneous immunization. To understand the nature of penetration of Posintro nanoparticles into the intercorneocyte space of the stratum corneum, the interaction between the nanoparticles and lipid model systems in form of liposomes and/or supported lipid bilayer was studied. As a lipid model we used Stratum Corneum Lipid (SCL), a mixture similar in composition to the lipids of the intercorneocyte space. By Förster Resonance Energy Transfer (FRET), Atomic Force Microscopy (AFM), Electrochemical Impedance Spectroscopy (EIS) and cryo-Transmission Electron Microscopy (cryo-TEM) it was shown that application of nanoparticles to the SCL bilayers results in lipid disturbance. Investigation of this interaction by means of Isothermal Titration Calorimetry (ITC) confirmed existence of an enthalpically unfavorable reaction. All these methods demonstrated that the strength of electrostatic repulsion between the negatively charged SCL and the nanoparticles affected their interaction, as decreasing the negative charge of the Posintro nanoparticles leads to enhanced disruption of lipid organization.

Incorporation of the TLR4 agonist monophosphoryl lipid A into the bilayer of DDA/TDB liposomes: physico-chemical characterization and induction of CD8+ T-cell responses in vivo.

PURPOSE: The combination of delivery systems like cationic liposomes and immunopotentiators such as Toll-like receptor (TLR) ligands is a promising approach for rational vaccine adjuvant design. The purpose of this study was to investigate how the incorporation of the poorly soluble TLR4 agonist monophosphoryl lipid A (MPL) into cationic liposomes based on dimethyldioctadecylammonium (DDA) and trehalose 6,6'-dibehenate (TDB) influenced the physicochemical and immunological properties of the liposomes. METHODS: The DDA/TDB/MPL liposomes were characterized with regard to particle size, poly dispersity, surface charge, stability and thermodynamic properties. The adjuvant formulations were tested in vivo in mice using ovalbumin (OVA) as model antigen. RESULTS: Integration of MPL into the bilayer structure of DDA/TDB liposomes was evident from a decreased phase transition temperature, an improved membrane packing, and a reduction in surface charge. The particle size and favorable liposome storage stability were not affected by MPL. In mice, DDA/TDB/MPL liposomes induced an antigen-specific CD8(+) T-cell response and a humoral response. CONCLUSIONS: Enhancing the solubility of MPL by inclusion into the bilayer of DDA/TDB liposomes changes the membrane characteristics of the adjuvant system and provides the liposomes with CD8(+) T-cell inducing properties without compromising humoral responses.

Nanoparticle-mediated pulmonary drug delivery: state of the art towards efficient treatment of recalcitrant respiratory tract bacterial infections.

Recalcitrant respiratory tract infections caused by bacteria have emerged as one of the greatest health challenges worldwide. Aerosolized antimicrobial therapy is becoming increasingly attractive to combat such infections, as it allows targeted delivery of high drug concentrations to the infected organ while limiting systemic exposure. However, successful aerosolized antimicrobial therapy is still challenged by the diverse biological barriers in infected lungs. Nanoparticle-mediated pulmonary drug delivery is gaining increasing attention as a means to overcome the biological barriers and accomplish site-specific drug delivery by controlling release of the loaded drug(s) at the target site. With the aim to summarize emerging efforts in combating respiratory tract infections by using nanoparticle-mediated pulmonary delivery strategies, this review provides a brief introduction to the bacterial infection-related pulmonary diseases and the biological barriers for effective treatment of recalcitrant respiratory tract infections. This is followed by a summary of recent advances in design of inhalable nanoparticle-based drug delivery systems that overcome the biological barriers and increase drug bioavailability. Finally, challenges for the translation from exploratory laboratory research to clinical application are also discussed and potential solutions proposed.

Revealing the importance of carrier-cargo association in delivery of insulin and lipidated insulin.

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.