Anti-biofilm peptides are a subset of antimicrobial peptides and represent promising broad-spectr... more Anti-biofilm peptides are a subset of antimicrobial peptides and represent promising broad-spectrum agents for the treatment of bacterial biofilms, though some display host toxicity in vivo. Here we evaluated nanogels composed of modified hyaluronic acid for the encapsulation of the anti-biofilm peptide DJK-5 in vivo. Nanogels of 174 to 194 nm encapsulating 33-60% of peptide were created. Efficacy and toxicity of the nanogels were tested in vivo employing a murine abscess model of a Pseudomonas aeruginosa LESB58 high bacterial density infection. The dose of DJK-5 that could be administered intravenously to mice without inducing toxicity was more than doubled after encapsulation in nanogels. Upon subcutaneous administration, the toxicity of the DJK-5 in nanogels was decreased four-fold compared to non-formulated peptide, without compromising the anti-abscess effect of DJK-5. These findings support the use of nanogels to increase the safety of antimicrobial and anti-biofilm peptides after intravenous and subcutaneous administration.
This study aimed to investigate the effect of a novel kind of immune-stimulating complexes (ISCOM... more This study aimed to investigate the effect of a novel kind of immune-stimulating complexes (ISCOMs) on human skin penetration of model compounds in vitro to evaluate their potential as a delivery system, ultimately for transcutaneous vaccination. Special focus was on elucidating the mechanisms of penetration. Preparation of ISCOMs was done by dialysis and subsequent purification in a sucrose density gradient. The penetration pathways of acridine-labeled ISCOMs were visualized using confocal laser scanning microscopy (CLSM). Transmission electron microscopy (TEM) was used to evaluate the ultrastructural changes in the skin after application of the ISCOMs with or without hydration. Transcutaneous permeation of the model compound, methyl nicotinate, was evaluated in diffusion cells. The prepared ISCOMs were 42-52 nm in diameter as evaluated by dynamic light scattering with zeta potentials of -33 to -26.1 mV. TEM investigations verified the presence of ISCOM structures. Penetration of acridine into skin was greatly increased by incorporation into ISCOMs as visualized by CLSM. Permeation of methyl nicotinate was enhanced in the presence of ISCOMs. Ultrastructural changes of the intercellular space in the stratum corneum after exposure of ISCOMs were observed on micrographs, especially for hydrated skin. In conclusion, cutaneous application of ISCOMs leads to increased penetration of hydrophobic model compounds through human stratum corneum and thus shows potential as a transcutaneous delivery system. The increased penetration seems to be reflected by a change in the intercellular space between the corneocytes, and the effect is most likely caused by the components of the ISCOMs rather than intact ISCOMs.
Drug Delivery and Translational Research, Mar 10, 2021
Recalcitrant respiratory tract infections caused by bacteria have emerged as one of the greatest ... more 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 nanoparticlebased 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.
20 Nicotine replacement therapy (NRT) formulations for oromucosal administration induce a delayed... more 20 Nicotine replacement therapy (NRT) formulations for oromucosal administration induce a delayed 21 rise in nicotine blood levels as opposed to the immediate nicotine increase obtained from cigarette 22 smoking; this being a shortcoming of the therapy. Here, we demonstrate that α-23 lactalbumin/polyethylene oxide (ALA/PEO) electrospun nanofibers constitute an efficient 24 oromucosal delivery system for fast-onset nicotine delivery of high relevance for acute dosing NRT 25 applications. In vitro, nicotine-loaded nanofibers showed fast disintegration in water, with a weight 26 loss up to 40% within minutes, and a faster nicotine release (26.1±4.6% after 1 min of incubation) 27 of the loaded nicotine compared to two relevant marketed NRT formulations with a comparable 28 nicotine dose (i.e. 7.9±5.1% and 2.2±0.3% nicotine was released from a lozenge and a sublingual 29 tablet, respectively). Model-fitting of the release data indicated that the release mechanism of 30 nicotine from the hydrophilic nanofibers was possibly governed by more than one type of release 31 phenomena. Remarkably, ex vivo studies using porcine buccal mucosa demonstrated a more 32 efficient permeation of the nicotine released from the nanofibers (flux of 1.06±0.22 33 nmol/(cm 2 ×min)) compared to when dosing even a ten-fold concentrated nicotine solution (flux of 34 0.17±0.14 nmol/(cm 2 ×min)). Moreover, MALDI MS imaging of ex vivo porcine buccal mucosa 35 exposed to nicotine-loaded nanofibers clearly revealed higher amounts of nicotine throughout the 36 epithelium, as well as in the lamina propria and submucosa of the tissue. Our findings suggest that 37 nicotine-loaded ALA/PEO nanofibers have potential as a mucosal, fast-releasing and 38 biocompatible delivery system for nicotine, which can overcome the limitations of current 39 marketed NRTs.
Inhaled antibiotic treatment of cystic fibrosis (CF)-related bacterial biofilm infections is chal... more Inhaled antibiotic treatment of cystic fibrosis (CF)-related bacterial biofilm infections is challenging due to the pathological environment of the lungs. Here, we present an 'environment adaptive' nanoparticle composed of a solid poly lactic-co-glycolic acid (PLGA) core and a mucus-inert, enzymatically cleavable shell of d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) for sitespecific delivery of antibiotics to bacterial biofilms via aerosol administration. The hybrid nanoparticles with ultra-small size were self-assembled via a nanoprecipitation process by using a facile microfluidic method. The interactions of the nanoparticles with the biological barriers were comprehensively investigated by using cutting-edge techniques (e.g. quartz crystal microbalance with dissipation monitoring, total internal reflection fluorescence microscopy-based particle tracking, in vitro biofilm model cultured in a flow-chamber system, and quantitative imaging analysis). Our results suggest that the mucus-inert, enzymatically cleavable TPGS shell enables the nanoparticles to penetrate through the mucus, accumulate in the deeper layer of the biofilms, and serve as sustained release depot, thereby, improving the killing efficacy of azithromycin (a macrolide antibiotic) against biofilm-forming Pseudomonas aeruginosa. In conclusion, the ultra-small TPGS-PLGA hybrid nanoparticles represent an efficient delivery system to overcome the multiple barriers and release antibiotics with a sustained manner in the vicinity of the biofilm-forming bacteria.
Background: Advances in biomedical nanotechnology raise hopes in patient populations but may also... more Background: Advances in biomedical nanotechnology raise hopes in patient populations but may also raise questions regarding biodistribution and biocompatibility, especially during pregnancy. Special consideration must be given to the placenta as a biological barrier because a pregnant woman's exposure to nanoparticles could have significant effects on the fetus developing in the womb. Therefore, the purpose of this study is to optimize an in vitro model for characterizing the transport of nanoparticles across human placental trophoblast cells. Methods: The growth of BeWo (clone b30) human placental choriocarcinoma cells for nanoparticle transport studies was characterized in terms of optimized Transwell ® insert type and pore size, the investigation of barrier properties by transmission electron microscopy, tight junction staining, transepithelial electrical resistance, and fluorescein sodium transport. Following the determination of nontoxic concentrations of fluorescent polystyrene nanoparticles, the cellular uptake and transport of 50 nm and 100 nm diameter particles was measured using the in vitro BeWo cell model. Results: Particle size measurements, fluorescence readings, and confocal microscopy indicated both cellular uptake of the fluorescent polystyrene nanoparticles and the transcellular transport of these particles from the apical (maternal) to the basolateral (fetal) compartment. Over the course of 24 hours, the apparent permeability across BeWo cells grown on polycarbonate membranes (3.0 µm pore size) was four times higher for the 50 nm particles compared with the 100 nm particles. Conclusion: The BeWo cell line has been optimized and shown to be a valid in vitro model for studying the transplacental transport of nanoparticles. Fluorescent polystyrene nanoparticle transport was size-dependent, as smaller particles reached the basal (fetal) compartment at a higher rate.
Journal of Colloid and Interface Science, Oct 1, 2020
Bacterial biofilm represents a protected mode of bacterial growth that significantly enhances the... more Bacterial biofilm represents a protected mode of bacterial growth that significantly enhances the resistance to antibiotics. Poly lactic-co-glycolic acid (PLGA)-based nanoparticle delivery systems have been intensively investigated to combat the bacterial biofilms-associated infections. However, some drawbacks associated with current PLGA-based nanoformulations (e.g. the relatively low drug loading capability, premature burst release and/or incapability of on-demand release of cargos at the site of action) restrict the transition from the lab research to the clinical applications. One potent strategy to overcome the above-mentioned limitations is exploiting the unique properties of carbon quantum dots (CQDs) and combining CQDs with the conventional PLGA nanoparticles. In the present study, the CQDs were innovatively incorporated into PLGA nanoparticles by using a microfluidic method. The resulting CQD-PLGA hybrid nanoparticles presented good loading capability of azithromycin (a macrolide antibiotic, AZI) and tobramycin (an aminoglycoside antibiotic, TOB), and stimuli-responsive release of the cargos upon laser irradiation. Consequently, AZI-loaded CQD-PLGA hybrid nanoparticles showed chemo-photothermally synergistic anti-biofilm effects against P. aeruginosa biofilms. Additionally, the CQD-PLGA hybrid nanoparticles demonstrated good biocompatibility with the eukaryotic cells. Overall, the proof-of-concept of CQD-PLGA hybrid nanoparticles may open a new possibility in chemo-photothermal therapy against bacterial biofilms.
Chronic wound infections colonized by bacteria are becoming more difficult to treat with current ... more 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.
Oral delivery of therapeutic peptides is hampered by their large molecular size and labile nature... more Oral delivery of therapeutic peptides is hampered by their large molecular size and labile nature, thus limiting their permeation across the intestinal epithelium. Promising approaches to overcome the latter include co-administration with carrier peptides. In this study, the cell-penetrating peptide penetratin was employed to investigate effects of co-administration with insulin and the pharmacologically active part of parathyroid hormone (PTH(1-34)) at pH 5, 6.5, and 7.4 with respect to complexation, enzymatic stability, and transepithelial permeation of the therapeutic peptide in vitro and in vivo. Complex formation between insulin or PTH(1-34) and penetratin was pH-dependent. Micron-sized complexes dominated in the samples prepared at pH-values at which penetratin interacts electrostatically with the therapeutic peptide. The association efficiency was more pronounced between insulin and penetratin than between PTH(1-34) and penetratin. Despite the high degree of complexation, penetratin retained its membrane activity when applied to liposomal structures. The enzymatic stability of penetratin during incubation on polarized Caco-2 cell monolayers was pH-dependent with a prolonged half-live determined at pH 5 when compared to pH 6.5 and 7.4. Also, the penetratin-mediated transepithelial permeation of insulin and PTH(1-34) was increased in vitro and in vivo upon lowering the sample pH from 7.4 or 6.5 to 5. Thus, the formation of penetratin-cargo complexes with several molecular entities is not prerequisite for penetratin-mediated transepithelial permeation a therapeutic peptide. Rather, a sample pH, which improves the penetratin stability, appears to optimize the penetratin-mediated transepithelial permeation of insulin and PTH(1-34).
Mucosal administration of drugs and drug delivery systems has gained increasing interest. However... more Mucosal administration of drugs and drug delivery systems has gained increasing interest. However, nanoparticles intended to protect and deliver drugs to epithelial surfaces require transport through the surface-lining mucus. Translation from bench to bedside is particularly challenging for mucosal administration since a variety of parameters will influence the specific barrier properties of the mucus including the luminal fluids, the microbiota, the mucus composition and clearance rate, and the condition of the underlying epithelia. Besides, after administration, nanoparticles interact with the mucosal components, forming a biomolecular corona that modulates their behavior and fate after mucosal administration. These interactions are greatly influenced by the nanoparticle properties and therefore different designs and surfaceengineering strategies have been proposed. Overall, it is essential to evaluate these biomoleculenanoparticle interactions by complementary techniques using complex and relevant mucus barrier matrices.
ABSTRACTAmyloid aggregation is associated with many diseases and may also occur in therapeutic pr... more ABSTRACTAmyloid 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 multi-component system in terms of structures and mechanisms poses a number of challenges for the analysis of the chemical reaction. Combining a spatially resolved fluorescence approach with single molecule microscopy and machine learning approaches, we disentangle the different contributions from multiple species within a single aggregation experiment. Moreover, 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, providing a general tool for a comprehensive analysis of self-assembly reactions.Table of Contents
The sublingual mucosa is an attractive route for drug delivery, although challenged by a continuo... more 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 th...
Membrane-active peptides have been extensively studied to probe protein–membrane interactions, to... more Membrane-active peptides have been extensively studied to probe protein–membrane interactions, to act as antimicrobial agents and cell-penetrating peptides (CPPs) for the delivery of therapeutic agents to cells. Hundreds of membrane-active sequences acting as CPPs have now been described including bioportides that serve as single entity modifiers of cell physiology at the intracellular level. Translation of promising CPPs in pre-clinical studies have, however, been disappointing as only few identified delivery systems have progressed to clinical trials. To search for novel membrane-active peptides a sequence from the EGFR juxtamembrane region was identified (named EJP18), synthesised, and examined in its L- and D-form for its ability to mediate the delivery of a small fluorophore and whole proteins to cancer cell lines. Initial studies identified the peptide as being highly membrane-active causing extensive and rapid plasma membrane reorganisation, blebbing, and toxicity. At lower, ...
Many oral mucosal conditions cause considerable and prolonged pain that to date has been difficul... more Many oral mucosal conditions cause considerable and prolonged pain that to date has been difficult to alleviate via topical delivery, and the use of injection causes many patients dental anxiety and needle-prick pain. Therefore, developing a noninjectable drug delivery system as an alternative administration procedure may vastly improve the health and wellbeing of these patients. Recent advances in the development of mucoadhesive electrospun patches for the direct delivery of therapeutics to the oral mucosa offer a potential solution, but as yet, the release of local anesthetics from this system and their uptake by oral tissue have not been demonstrated. Here, we demonstrate the fabrication of lidocaine-loaded electrospun fiber patches, drug release, and subsequent uptake and permeation through the porcine buccal mucosa. Lidocaine HCl and lidocaine base were incorporated into the electrospun patches to evaluate the difference in drug permeation for the two drug compositions. Lidocaine released from the lidocaine HCl-containing electrospun patches was significantly quicker than from the lidocaine base patches, with double the amount of drug released from the lidocaine HCl patches in the first 15 min (0.16 ± 0.04 mg) compared to that from the lidocaine base patches (0.07 ± 0.01 mg). The permeation of lidocaine from the lidocaine HCl electrospun patches through ex vivo porcine buccal mucosa was also detected in 15 min, whereas permeation of lidocaine from the lidocaine base patch was not detected. Matrix-assisted laser desorption ionization-mass spectrometry imaging was used to investigate localization of lidocaine within the oral tissue. Lidocaine in the solution as well as from the mucoadhesive patch penetrated into the buccal mucosal tissue in a time-dependent manner and was detectable in the lamina propria after only 15 min. Moreover, the lidocaine released from lidocaine HCl electrospun patches retained biological activity, inhibiting veratridine-mediated opening of voltage-gated sodium channels in SH-SY5Y neuroblastoma cells. These data suggest that a mucoadhesive electrospun patch may be used as a vehicle for rapid uptake and sustained anesthetic drug delivery to treat or prevent oral pain.
The antimicrobial effects of laponite nanoparticles with or without loading of the antimicrobial ... more The antimicrobial effects of laponite nanoparticles with or without loading of the antimicrobial peptide LL-37 was investigated along with their membrane interactions. The study combines data from ellipsometry, circular dichroism, fluorescence spectroscopy, particle size/ζ-potential measurements, and confocal microscopy. As a result of the net negative charge of laponite, loading of net positively charged LL-37 increases with increasing pH. The peptide was found to bind primarily to the outer surface of the laponite nanoparticles in a predominantly helical conformation, leading to charge reversal. Despite
Users may download and print one copy of any publication from the public portal for the purpose... more Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Antimicrobial peptides (AMPs) hold promise as the next generation of antimicrobial agents, but of... more 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-alkylglycines) provides the potential to improve stability. We have identified two novel peptoid-based compounds,and, which are effective against the canine skin pathogen, 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. Bothandwere resistant to proteases and >98% bound to plasma proteins. While half-lives in liver microsomes for both were >2 h, peptoidshowed higher stability to plasma proteases than the peptide-peptoid hybrid B1 (>2 versus 0.5 h). Both compounds were suitable for administra...
Quantification of pharmaceutical peptides in human plasma by RP-LC-ICP-MS and post column isotope... more Quantification of pharmaceutical peptides in human plasma by RP-LC-ICP-MS and post column isotope dilution.
Anti-biofilm peptides are a subset of antimicrobial peptides and represent promising broad-spectr... more Anti-biofilm peptides are a subset of antimicrobial peptides and represent promising broad-spectrum agents for the treatment of bacterial biofilms, though some display host toxicity in vivo. Here we evaluated nanogels composed of modified hyaluronic acid for the encapsulation of the anti-biofilm peptide DJK-5 in vivo. Nanogels of 174 to 194 nm encapsulating 33-60% of peptide were created. Efficacy and toxicity of the nanogels were tested in vivo employing a murine abscess model of a Pseudomonas aeruginosa LESB58 high bacterial density infection. The dose of DJK-5 that could be administered intravenously to mice without inducing toxicity was more than doubled after encapsulation in nanogels. Upon subcutaneous administration, the toxicity of the DJK-5 in nanogels was decreased four-fold compared to non-formulated peptide, without compromising the anti-abscess effect of DJK-5. These findings support the use of nanogels to increase the safety of antimicrobial and anti-biofilm peptides after intravenous and subcutaneous administration.
This study aimed to investigate the effect of a novel kind of immune-stimulating complexes (ISCOM... more This study aimed to investigate the effect of a novel kind of immune-stimulating complexes (ISCOMs) on human skin penetration of model compounds in vitro to evaluate their potential as a delivery system, ultimately for transcutaneous vaccination. Special focus was on elucidating the mechanisms of penetration. Preparation of ISCOMs was done by dialysis and subsequent purification in a sucrose density gradient. The penetration pathways of acridine-labeled ISCOMs were visualized using confocal laser scanning microscopy (CLSM). Transmission electron microscopy (TEM) was used to evaluate the ultrastructural changes in the skin after application of the ISCOMs with or without hydration. Transcutaneous permeation of the model compound, methyl nicotinate, was evaluated in diffusion cells. The prepared ISCOMs were 42-52 nm in diameter as evaluated by dynamic light scattering with zeta potentials of -33 to -26.1 mV. TEM investigations verified the presence of ISCOM structures. Penetration of acridine into skin was greatly increased by incorporation into ISCOMs as visualized by CLSM. Permeation of methyl nicotinate was enhanced in the presence of ISCOMs. Ultrastructural changes of the intercellular space in the stratum corneum after exposure of ISCOMs were observed on micrographs, especially for hydrated skin. In conclusion, cutaneous application of ISCOMs leads to increased penetration of hydrophobic model compounds through human stratum corneum and thus shows potential as a transcutaneous delivery system. The increased penetration seems to be reflected by a change in the intercellular space between the corneocytes, and the effect is most likely caused by the components of the ISCOMs rather than intact ISCOMs.
Drug Delivery and Translational Research, Mar 10, 2021
Recalcitrant respiratory tract infections caused by bacteria have emerged as one of the greatest ... more 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 nanoparticlebased 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.
20 Nicotine replacement therapy (NRT) formulations for oromucosal administration induce a delayed... more 20 Nicotine replacement therapy (NRT) formulations for oromucosal administration induce a delayed 21 rise in nicotine blood levels as opposed to the immediate nicotine increase obtained from cigarette 22 smoking; this being a shortcoming of the therapy. Here, we demonstrate that α-23 lactalbumin/polyethylene oxide (ALA/PEO) electrospun nanofibers constitute an efficient 24 oromucosal delivery system for fast-onset nicotine delivery of high relevance for acute dosing NRT 25 applications. In vitro, nicotine-loaded nanofibers showed fast disintegration in water, with a weight 26 loss up to 40% within minutes, and a faster nicotine release (26.1±4.6% after 1 min of incubation) 27 of the loaded nicotine compared to two relevant marketed NRT formulations with a comparable 28 nicotine dose (i.e. 7.9±5.1% and 2.2±0.3% nicotine was released from a lozenge and a sublingual 29 tablet, respectively). Model-fitting of the release data indicated that the release mechanism of 30 nicotine from the hydrophilic nanofibers was possibly governed by more than one type of release 31 phenomena. Remarkably, ex vivo studies using porcine buccal mucosa demonstrated a more 32 efficient permeation of the nicotine released from the nanofibers (flux of 1.06±0.22 33 nmol/(cm 2 ×min)) compared to when dosing even a ten-fold concentrated nicotine solution (flux of 34 0.17±0.14 nmol/(cm 2 ×min)). Moreover, MALDI MS imaging of ex vivo porcine buccal mucosa 35 exposed to nicotine-loaded nanofibers clearly revealed higher amounts of nicotine throughout the 36 epithelium, as well as in the lamina propria and submucosa of the tissue. Our findings suggest that 37 nicotine-loaded ALA/PEO nanofibers have potential as a mucosal, fast-releasing and 38 biocompatible delivery system for nicotine, which can overcome the limitations of current 39 marketed NRTs.
Inhaled antibiotic treatment of cystic fibrosis (CF)-related bacterial biofilm infections is chal... more Inhaled antibiotic treatment of cystic fibrosis (CF)-related bacterial biofilm infections is challenging due to the pathological environment of the lungs. Here, we present an 'environment adaptive' nanoparticle composed of a solid poly lactic-co-glycolic acid (PLGA) core and a mucus-inert, enzymatically cleavable shell of d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) for sitespecific delivery of antibiotics to bacterial biofilms via aerosol administration. The hybrid nanoparticles with ultra-small size were self-assembled via a nanoprecipitation process by using a facile microfluidic method. The interactions of the nanoparticles with the biological barriers were comprehensively investigated by using cutting-edge techniques (e.g. quartz crystal microbalance with dissipation monitoring, total internal reflection fluorescence microscopy-based particle tracking, in vitro biofilm model cultured in a flow-chamber system, and quantitative imaging analysis). Our results suggest that the mucus-inert, enzymatically cleavable TPGS shell enables the nanoparticles to penetrate through the mucus, accumulate in the deeper layer of the biofilms, and serve as sustained release depot, thereby, improving the killing efficacy of azithromycin (a macrolide antibiotic) against biofilm-forming Pseudomonas aeruginosa. In conclusion, the ultra-small TPGS-PLGA hybrid nanoparticles represent an efficient delivery system to overcome the multiple barriers and release antibiotics with a sustained manner in the vicinity of the biofilm-forming bacteria.
Background: Advances in biomedical nanotechnology raise hopes in patient populations but may also... more Background: Advances in biomedical nanotechnology raise hopes in patient populations but may also raise questions regarding biodistribution and biocompatibility, especially during pregnancy. Special consideration must be given to the placenta as a biological barrier because a pregnant woman's exposure to nanoparticles could have significant effects on the fetus developing in the womb. Therefore, the purpose of this study is to optimize an in vitro model for characterizing the transport of nanoparticles across human placental trophoblast cells. Methods: The growth of BeWo (clone b30) human placental choriocarcinoma cells for nanoparticle transport studies was characterized in terms of optimized Transwell ® insert type and pore size, the investigation of barrier properties by transmission electron microscopy, tight junction staining, transepithelial electrical resistance, and fluorescein sodium transport. Following the determination of nontoxic concentrations of fluorescent polystyrene nanoparticles, the cellular uptake and transport of 50 nm and 100 nm diameter particles was measured using the in vitro BeWo cell model. Results: Particle size measurements, fluorescence readings, and confocal microscopy indicated both cellular uptake of the fluorescent polystyrene nanoparticles and the transcellular transport of these particles from the apical (maternal) to the basolateral (fetal) compartment. Over the course of 24 hours, the apparent permeability across BeWo cells grown on polycarbonate membranes (3.0 µm pore size) was four times higher for the 50 nm particles compared with the 100 nm particles. Conclusion: The BeWo cell line has been optimized and shown to be a valid in vitro model for studying the transplacental transport of nanoparticles. Fluorescent polystyrene nanoparticle transport was size-dependent, as smaller particles reached the basal (fetal) compartment at a higher rate.
Journal of Colloid and Interface Science, Oct 1, 2020
Bacterial biofilm represents a protected mode of bacterial growth that significantly enhances the... more Bacterial biofilm represents a protected mode of bacterial growth that significantly enhances the resistance to antibiotics. Poly lactic-co-glycolic acid (PLGA)-based nanoparticle delivery systems have been intensively investigated to combat the bacterial biofilms-associated infections. However, some drawbacks associated with current PLGA-based nanoformulations (e.g. the relatively low drug loading capability, premature burst release and/or incapability of on-demand release of cargos at the site of action) restrict the transition from the lab research to the clinical applications. One potent strategy to overcome the above-mentioned limitations is exploiting the unique properties of carbon quantum dots (CQDs) and combining CQDs with the conventional PLGA nanoparticles. In the present study, the CQDs were innovatively incorporated into PLGA nanoparticles by using a microfluidic method. The resulting CQD-PLGA hybrid nanoparticles presented good loading capability of azithromycin (a macrolide antibiotic, AZI) and tobramycin (an aminoglycoside antibiotic, TOB), and stimuli-responsive release of the cargos upon laser irradiation. Consequently, AZI-loaded CQD-PLGA hybrid nanoparticles showed chemo-photothermally synergistic anti-biofilm effects against P. aeruginosa biofilms. Additionally, the CQD-PLGA hybrid nanoparticles demonstrated good biocompatibility with the eukaryotic cells. Overall, the proof-of-concept of CQD-PLGA hybrid nanoparticles may open a new possibility in chemo-photothermal therapy against bacterial biofilms.
Chronic wound infections colonized by bacteria are becoming more difficult to treat with current ... more 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.
Oral delivery of therapeutic peptides is hampered by their large molecular size and labile nature... more Oral delivery of therapeutic peptides is hampered by their large molecular size and labile nature, thus limiting their permeation across the intestinal epithelium. Promising approaches to overcome the latter include co-administration with carrier peptides. In this study, the cell-penetrating peptide penetratin was employed to investigate effects of co-administration with insulin and the pharmacologically active part of parathyroid hormone (PTH(1-34)) at pH 5, 6.5, and 7.4 with respect to complexation, enzymatic stability, and transepithelial permeation of the therapeutic peptide in vitro and in vivo. Complex formation between insulin or PTH(1-34) and penetratin was pH-dependent. Micron-sized complexes dominated in the samples prepared at pH-values at which penetratin interacts electrostatically with the therapeutic peptide. The association efficiency was more pronounced between insulin and penetratin than between PTH(1-34) and penetratin. Despite the high degree of complexation, penetratin retained its membrane activity when applied to liposomal structures. The enzymatic stability of penetratin during incubation on polarized Caco-2 cell monolayers was pH-dependent with a prolonged half-live determined at pH 5 when compared to pH 6.5 and 7.4. Also, the penetratin-mediated transepithelial permeation of insulin and PTH(1-34) was increased in vitro and in vivo upon lowering the sample pH from 7.4 or 6.5 to 5. Thus, the formation of penetratin-cargo complexes with several molecular entities is not prerequisite for penetratin-mediated transepithelial permeation a therapeutic peptide. Rather, a sample pH, which improves the penetratin stability, appears to optimize the penetratin-mediated transepithelial permeation of insulin and PTH(1-34).
Mucosal administration of drugs and drug delivery systems has gained increasing interest. However... more Mucosal administration of drugs and drug delivery systems has gained increasing interest. However, nanoparticles intended to protect and deliver drugs to epithelial surfaces require transport through the surface-lining mucus. Translation from bench to bedside is particularly challenging for mucosal administration since a variety of parameters will influence the specific barrier properties of the mucus including the luminal fluids, the microbiota, the mucus composition and clearance rate, and the condition of the underlying epithelia. Besides, after administration, nanoparticles interact with the mucosal components, forming a biomolecular corona that modulates their behavior and fate after mucosal administration. These interactions are greatly influenced by the nanoparticle properties and therefore different designs and surfaceengineering strategies have been proposed. Overall, it is essential to evaluate these biomoleculenanoparticle interactions by complementary techniques using complex and relevant mucus barrier matrices.
ABSTRACTAmyloid aggregation is associated with many diseases and may also occur in therapeutic pr... more ABSTRACTAmyloid 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 multi-component system in terms of structures and mechanisms poses a number of challenges for the analysis of the chemical reaction. Combining a spatially resolved fluorescence approach with single molecule microscopy and machine learning approaches, we disentangle the different contributions from multiple species within a single aggregation experiment. Moreover, 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, providing a general tool for a comprehensive analysis of self-assembly reactions.Table of Contents
The sublingual mucosa is an attractive route for drug delivery, although challenged by a continuo... more 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 th...
Membrane-active peptides have been extensively studied to probe protein–membrane interactions, to... more Membrane-active peptides have been extensively studied to probe protein–membrane interactions, to act as antimicrobial agents and cell-penetrating peptides (CPPs) for the delivery of therapeutic agents to cells. Hundreds of membrane-active sequences acting as CPPs have now been described including bioportides that serve as single entity modifiers of cell physiology at the intracellular level. Translation of promising CPPs in pre-clinical studies have, however, been disappointing as only few identified delivery systems have progressed to clinical trials. To search for novel membrane-active peptides a sequence from the EGFR juxtamembrane region was identified (named EJP18), synthesised, and examined in its L- and D-form for its ability to mediate the delivery of a small fluorophore and whole proteins to cancer cell lines. Initial studies identified the peptide as being highly membrane-active causing extensive and rapid plasma membrane reorganisation, blebbing, and toxicity. At lower, ...
Many oral mucosal conditions cause considerable and prolonged pain that to date has been difficul... more Many oral mucosal conditions cause considerable and prolonged pain that to date has been difficult to alleviate via topical delivery, and the use of injection causes many patients dental anxiety and needle-prick pain. Therefore, developing a noninjectable drug delivery system as an alternative administration procedure may vastly improve the health and wellbeing of these patients. Recent advances in the development of mucoadhesive electrospun patches for the direct delivery of therapeutics to the oral mucosa offer a potential solution, but as yet, the release of local anesthetics from this system and their uptake by oral tissue have not been demonstrated. Here, we demonstrate the fabrication of lidocaine-loaded electrospun fiber patches, drug release, and subsequent uptake and permeation through the porcine buccal mucosa. Lidocaine HCl and lidocaine base were incorporated into the electrospun patches to evaluate the difference in drug permeation for the two drug compositions. Lidocaine released from the lidocaine HCl-containing electrospun patches was significantly quicker than from the lidocaine base patches, with double the amount of drug released from the lidocaine HCl patches in the first 15 min (0.16 ± 0.04 mg) compared to that from the lidocaine base patches (0.07 ± 0.01 mg). The permeation of lidocaine from the lidocaine HCl electrospun patches through ex vivo porcine buccal mucosa was also detected in 15 min, whereas permeation of lidocaine from the lidocaine base patch was not detected. Matrix-assisted laser desorption ionization-mass spectrometry imaging was used to investigate localization of lidocaine within the oral tissue. Lidocaine in the solution as well as from the mucoadhesive patch penetrated into the buccal mucosal tissue in a time-dependent manner and was detectable in the lamina propria after only 15 min. Moreover, the lidocaine released from lidocaine HCl electrospun patches retained biological activity, inhibiting veratridine-mediated opening of voltage-gated sodium channels in SH-SY5Y neuroblastoma cells. These data suggest that a mucoadhesive electrospun patch may be used as a vehicle for rapid uptake and sustained anesthetic drug delivery to treat or prevent oral pain.
The antimicrobial effects of laponite nanoparticles with or without loading of the antimicrobial ... more The antimicrobial effects of laponite nanoparticles with or without loading of the antimicrobial peptide LL-37 was investigated along with their membrane interactions. The study combines data from ellipsometry, circular dichroism, fluorescence spectroscopy, particle size/ζ-potential measurements, and confocal microscopy. As a result of the net negative charge of laponite, loading of net positively charged LL-37 increases with increasing pH. The peptide was found to bind primarily to the outer surface of the laponite nanoparticles in a predominantly helical conformation, leading to charge reversal. Despite
Users may download and print one copy of any publication from the public portal for the purpose... more Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Antimicrobial peptides (AMPs) hold promise as the next generation of antimicrobial agents, but of... more 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-alkylglycines) provides the potential to improve stability. We have identified two novel peptoid-based compounds,and, which are effective against the canine skin pathogen, 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. Bothandwere resistant to proteases and >98% bound to plasma proteins. While half-lives in liver microsomes for both were >2 h, peptoidshowed higher stability to plasma proteases than the peptide-peptoid hybrid B1 (>2 versus 0.5 h). Both compounds were suitable for administra...
Quantification of pharmaceutical peptides in human plasma by RP-LC-ICP-MS and post column isotope... more Quantification of pharmaceutical peptides in human plasma by RP-LC-ICP-MS and post column isotope dilution.
Background: Cell penetrating peptides have gained much recognition as a versatile transport vehic... more Background: Cell penetrating peptides have gained much recognition as a versatile transport vehicle for the intracellular delivery of wide range of cargoes (i.e. oligonucelotides, small molecules, proteins, etc.), that otherwise lack bioavailability, thus offering great potential as future therapeutics. Keeping in mind the therapeutic importance of these peptides, we have developed in silico methods for the prediction of cell penetrating peptides, which can be used for rapid screening of such peptides prior to their synthesis. Methods: In the present study, support vector machine (SVM)-based models have been developed for predicting and designing highly effective cell penetrating peptides. Various features like amino acid composition, dipeptide composition, binary profile of patterns, and physicochemical properties have been used as input features. The main dataset used in this study consists of 708 peptides. In addition, we have identified various motifs in cell penetrating peptides, and used these motifs for developing a hybrid prediction model. Performance of our method was evaluated on an independent dataset and also compared with that of the existing methods. Results: In cell penetrating peptides, certain residues (e.g. Arg, Lys, Pro, Trp, Leu, and Ala) are preferred at specific locations. Thus, it was possible to discriminate cell-penetrating peptides from non-cell penetrating peptides based on amino acid composition. All models were evaluated using five-fold cross-validation technique. We have achieved a maximum accuracy of 97.40% using the hybrid model that combines motif information and binary profile of the peptides. On independent dataset, we achieved maximum accuracy of 81.31% with MCC of 0.63. Conclusion: The present study demonstrates that features like amino acid composition, binary profile of patterns and motifs, can be used to train an SVM classifier that can predict cell penetrating peptides with higher accuracy. The hybrid model described in this study achieved more accuracy than the previous methods and thus may complement the existing methods. Based on the above study, a user-friendly web server CellPPD has been developed to help the biologists, where a user can predict and design CPPs with much ease. CellPPD web server is freely accessible at http://crdd.osdd.net/raghava/cellppd/.
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2019 by Hanne Nielsen
Papers by Hanne Nielsen