Core-sheath gelatin based electrospun nanofibers for dual delivery release of biomolecules and therapeutics.

Coaxial electrospinning with the ability to use simultaneously two separate solvents provides a promising strategy for drug delivery. Nevertheless, controlled release of hydrophilic and sensitive therapeutics from slow biodegradable polymers is still challenging. To address this gap, we fabricated core-sheath fibers for dual delivery of lysozyme, as a model protein, and phenytoin sodium as a small therapeutic molecule. The sheath was processed by a gelatin solution while the core fibers were fabricated from an aqueous gelatin/PVA solution. Microstructural studies by transmission and scanning electron microscopy reveal the formation of homogeneous core-sheath nanofibers with an outer and inner diameter of 180 ± 48 nm and 106 ± 30 nm, respectively. Thermal gravimetric analysis determines that the mass loss of the core-sheath fibers fall between the mass loss values of individual sheath and core fibers. Swelling studies indicate higher water absorption of the core-sheath mat compared to the separate sheath and core membranes. In vitro drug release studies in Phosphate Buffered Saline (PBS) determine sustained release of the therapeutics from the core-sheath structure. The release trails three stages including non-Fickian diffusion at the early stage followed by the Fickian diffusion mechanism. The present study shows a useful approach to design core-sheath nanofibrous membranes with controlled and programmable drug release profiles.

The hepatic-targeted, resveratrol loaded nanoparticles for relief of high fat diet-induced nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the early stage of many metabolic syndromes. The intervention of NAFLD can prevent its further development into severe metabolic syndromes. Given the inefficiency and side effects of chemical drugs for treating NAFLD, the hepatic-targeted nanocarriers loaded with bioactive compounds may offer a more effective and acceptable strategy for eliminating NAFLD. Here we developed hepatic-targeted oxidized starch-lysozyme (OSL) nanocarriers to specifically deliver resveratrol (Res) to liver tissue in order to maximize its therapeutic efficiency. The hepatic targeting was achieved using covalently conjugated galactose (Gal), which is recognized by the asialoglycoprotein receptors specifically expressed in hepatocytes. In steatotic HepG2 cell model, treatment with hepatic-targeted Gal-OSL/Res nanocarriers enhanced the cellular Res uptake and anti-lipogenesis capabilities, and effectively decreased triglyceride accumulation by modulating AMP-activated protein kinase (AMPK)/silent information regulation 2 homolog 1(SIRT1)/fatty acid synthase (FAS)/sterol regulatory element-binding protein-1c (SREBP1c) signaling pathway. In mice, Gal-OSL increased Res delivery into liver tissues and increased their hepatic effective concentration in liver. Most importantly, Gal-OSL/Res nanocarriers effectively reversed NAFLD and recovered hepatic insulin sensitivity of NAFLD mice to the healthy state. Furthermore, Gal-OSL/Res efficiently ameliorated lipid deposition and insulin resistance by modulating AMPK/SIRT1/FAS/SREBP1c signaling pathway and downregulated insulin receptor substrate-1 (IRS-1) phosphorylation at serine 307 in liver. These findings suggested that the hepatic-targeted Gal-OSL nanocarriers delivering Res could potentially serve as a safe and promising platform for NAFLD and other liver related diseases.

Polymeric Lids for Microcontainers for Oral Protein Delivery.

Oral delivery of proteins and peptides is one of the main challenges in pharmaceutical drug development. Microdevices have the possibility to protect the therapeutics until release is desired, avoiding losses by degradation. One type of microdevice is polymeric microcontainers. In this study, lysozyme is chosen as model protein and loaded into microcontainers with the permeation enhancer sodium decanoate (C10). The loaded microcontainers are sealed and functionalized by applying polymeric lids onto the cavity of the devices. The first lid is poly(lactic-co-glycolic) acid (PLGA) and on top of this either polyethylene glycol (PEG) or chitosan is applied (PLGA+PEG or PLGA+chitosan, respectively). The functionalization is evaluated in vitro for morphology, drug release, and mucoadhesive properties. These are coupled with in vitro and ex vivo studies using Caco-2 cells, Caco-2/HT29-MTX-E12 co-cultures, and porcine intestinal tissue. PLGA+chitosan shows slower release compared to PLGA+PEG or only PLGA in buffer and the transport of lysozyme across cell cultures is not enhanced compared to the bulk powder. Microcontainers coated with chitosan or PEG demonstrate a three times stronger adhesion during ex vivo mucoadhesion studies compared to samples without coatings. Altogether, functionalized microcontainers with mucoadhesive properties and tunable release for oral protein delivery are developed and characterized.

Stability of lysozyme incorporated into electrospun fibrous mats for wound healing.

In this study, we investigated the feasibility of incorporating protein drugs into electrospun fibrous mats (EFMs) for wound healing using lysozyme as a model drug. Lysozyme nanoparticles (Lyso- NPs) were first obtained by electrospray. Lysozyme solutions were prepared with a binary solvent mixture of ethanol (EtOH)-water (H2O) at varied volume ratios. Subsequently, Lyso-NPs were suspended in poly(lactic-co-glycolic acid) (PLGA) solutions using trifluoroethanol (TFE) as a solvent. Lyso-NPs loaded EFMs were obtained by electrospinning of the aforementioned suspensions, and the bioactivity of lysozyme in the EFMs was investigated using fluorescence-based assay kit. The electrosprayed Lyso-NPs were spherical with barely altered bioactivity as compared to the untreated raw material when using EtOH- H2O (30:70, v/v) as the solvent. After the subsequent electrospinning process, more than 90% of the bioactivity of lysozyme was retained compared to the raw material. The cytotoxicity of the produced EFMs was evaluated by thiazolyl blue tetrazolium bromide (MTT) study and the proliferation and distribution of mouse fibroblast cells (L929) growing on EFMs were investigated using 4,6-diamidino-2-phenylindol dihydrochloride (DAPI) for nucleic acid staining. Nearly negligible cytotoxicity of all the EFMs was observed according to the MTT study. Furthermore, it was observed that the L929 cells grew well on the Lyso-EFMs, especially those with the modification of polyethylene glycol (PEG) that was added to improve the hydrophilicity of EFMs. This study demonstrated that the electrospray/electrospinning processes are suitable for loading biomacromolecules to produce functionalized wound dressings to promote wound healing.

Quality enhancement of large yellow croaker treated with edible coatings based on chitosan and lysozyme.

The aim of this study was to evaluate the preservation effect of chitosan (CS) and lysozyme (Lys) coating on the quality of large yellow croaker (Larimichthys crocea) via physicochemical analyses, microbiological and sensorial assessments during refrigerated storage. Results showed that CS films incorporated with 0.6 mg·mL-1 of lysozyme exhibited the strongest inhibition effect against S. aureus, whose inhibition zone was 16.10 ±â€¯0.71 mm. The utilization of CS singly, or CS-lysozyme (Lys) coating significantly reduced lipid oxidation (thiobarbituric acid value, TBA) and improved the sensorial scores of fishes. Control and treated groups exceeded the maximum permissible level (30 mg N/100 g) of total volatile basic nitrogen (TVB-N) in fishery products on days 6 (control) and 9 (CS or CS-Lys treated), respectively. Fish samples reached the value of 7.0 Log CFU/g (Total Viable Count, TVC) on days 15 (control), whereas both CS and CS-Lys treated flesh samples never exceed this limit value during the refrigerated storage. Results of our study indicate CS-Lys have high efficiency in inhibiting microorganism growth and lipid oxidation and in maintaining sensorial quality, which is a promising method to maintain the quality and extend the shelf life of refrigerated fishes.

pH-Responsive Coacervate Droplets Formed from Acid-Labile Methylated Polyrotaxanes as an Injectable Protein Carrier.

In prior research it has been demonstrated that methylated ß-cyclodextrins-threaded acid-labile polyrotaxanes (Me-PRXs) exhibit a lower critical solution temperature (LCST) and form coacervate droplets above their LCST. In this study, the encapsulation of proteins in coacervate droplets and the pH-responsive release of proteins, through the acid-induced dissociation of the Me-PRX, were investigated. The coacervate droplets encapsulate various proteins, such as bovine serum albumin (BSA), lysozyme, and ß-galactosidase, at pH 7.4, into their hydrophobic inner phase. Concomitant with the pH-dependent dissociation of the Me-PRXs, the coacervates degraded below pH 6.5 and released encapsulated proteins, with the intrinsic activity of proteins maintained. Additionally, the subcutaneous injection of coacervate droplets encapsulating BSA in mice revealed that the retention time of the BSA at the injection site was prolonged compared to that of free BSA. Altogether, the coacervate droplets of the Me-PRX would be utilized as a new class of pH-responsive and injectable carrier for the controlled release of therapeutic proteins.

Lipid based liquid-crystalline stabilized formulations for the sustained release of bioactive hydrophilic molecules.

Lipid based formulations, endowed of long term stability as a result of the formation of lamellar liquid crystals, were prepared using the natural lipids lecithin and glycerol trioleate in water, and characterized using optical microscopy, SAXRD and NMR. The formulations, designed as possible carriers for lysozyme and caffeine, were evaluated for structural features and stability after the loading of the guest molecules. Release experiments were performed at 37 °C using the PBS medium. No burst release was observed either for lysozyme or caffeine. Although lysozyme released from the lipid formulations does not fully retain its biological activity, the investigated liquid crystal stabilized formulations display a promising potential as drug and cosmetic carriers for topical applications, due to their high biocompatibility.

A pH- and temperature-responsive bioresorbable injectable hydrogel based on polypeptide block copolymers for the sustained delivery of proteins in vivo.

Sustained delivery of protein therapeutics is limited owing to the fragile nature of proteins. Despite its great potential, delivery of proteins without any loss of bioactivity remains a challenge in the use of protein therapeutics in the clinic. To surmount this shortcoming, we report a pH- and temperature-responsive in situ-forming injectable hydrogel based on comb-type polypeptide block copolymers for the controlled delivery of proteins. Polypeptide block copolymers, composed of hydrophilic polyethylene glycol (PEG), temperature-responsive poly(γ-benzyl-l-glutamate) (PBLG), and pH-responsive oligo(sulfamethazine) (OSM), exhibit pH- and temperature-induced sol-to-gel transition behavior in aqueous solutions. Polypeptide block copolymers were synthesized by combining N-carboxyanhydride-based ring-opening polymerization and post-functionalization of the chain-end using N-hydroxy succinimide ester activated OSM. The physical properties of polypeptide-based hydrogels were tuned by varying the composition of temperature- and pH-responsive PBLG and OSM in block copolymers. Polypeptide block copolymers were non-toxic to human embryonic kidney cells at high concentrations (2000 µg mL-1). Subcutaneous administration of polypeptide block copolymer sols formed viscoelastic gel instantly at the back of Sprague-Dawley (SD) rats. The in vivo gels exhibited sustained degradation and were found to be bioresorbable in 6 weeks without any noticeable inflammation at the injection site. Anionic characteristics of hydrogels allow efficient loading of a cationic model protein, lysozyme, through electrostatic interaction. Lysozyme-loaded polypeptide block copolymer sols readily formed a viscoelastic gel in vivo and sustained lysozyme release for at least a week. Overall, the results demonstrate an elegant approach to control the release of certain charged proteins and open a myriad of therapeutic possibilities in protein therapeutics.

Development of a non-toxic and non-denaturing formulation process for encapsulation of SDF-1α into PLGA/PEG-PLGA nanoparticles to achieve sustained release.

Chemokines are known to stimulate directed migration of cancer cells. Therefore, the strategy involving gradual chemokine release from polymeric vehicles for trapping cancer cells is of interest. In this work, the chemokine stromal cell-derived factor-1α (SDF-1α) was encapsulated into nanoparticles composed of poly-(lactic-co-glycolic acid) (PLGA) and a polyethylene glycol (PEG)-PLGA co-polymer to achieve sustained release. SDF-1α, and lysozyme as a model protein, were firstly precipitated to promote their stability upon encapsulation. A novel phase separation method utilising a non-toxic solvent in the form of isosorbide dimethyl ether was developed for the individual encapsulation of SDF-1α and lysozyme precipitates. Uniform nanoparticles of 200-250 nm in size with spherical morphologies were successfully synthesised under mild formulation conditions and conveniently freeze-dried in the presence of hydroxypropyl-ß-cyclodextrin as a stabiliser. The effect of PLGA carboxylic acid terminal capping on protein encapsulation efficiency and release rate was also explored. Following optimisation, sustained release of SDF-1α was achieved over a period of 72 h. Importantly, the novel encapsulation process was found to induce negligible protein denaturation. The obtained SDF-1α nanocarriers may be subsequently incorporated within a hydrogel or other scaffolds to establish a chemokine concentration gradient for the trapping of glioblastoma cells.

Parameters affecting the transscleral delivery of two positively charged proteins of comparable size.

Apart from molecular weight and net surface charge, there are other macromolecule-related factors that could, in principle, influence their diffusion across biological tissues, such as shape, conformability, water solubility and surface charge distribution. Lysozyme and cytochrome c, proteins with comparable molecular weight, isoelectric point and net surface charge in physiological conditions (approx. +7.8), are suitable model compounds for comparative studies, in particular to find out if other properties can have a role in the permeation across the sclera. The comparison between lysozyme and cytochrome c permeability was conducted by studying the permeation across the sclera and the choroid-Bruch's membrane and the diffusion across a hyaluronan gel-matrix. Melanin binding tests and the measurement of the electroosmosis flow during transscleral iontophoresis allowed for the evaluation of macromolecules affinity for the ocular tissues. Finally, anodal iontophoresis was applied to further confirm the interaction of the two proteins with the sclera. The data here collected show that two proteins with very similar MW, p Ka and charge can display very different diffusion properties across biological barriers. In particular, these differences can be attributed to a different interaction with specific components of ocular tissues: while the interaction with melanin and collagen fibers is apparently the same for the two molecules, a relevant difference was found in case of hyaluronic acid. Considering also literature evidences, the important parameters that can be responsible for this different affinity are molecular shape (spherical for cytochrome c vs prolate for lysozyme) and a combination of hydrophobic and electrostatic interactions that depends on the surface charge distribution. The interactions between sclera components and lysozyme are relatively strong and were not altered by the application of electric current.

Enhanced antibacterial activity of lysozyme immobilized on chitin nanowhiskers.

In this paper, the contribution of chitin nanowhiskers (CHNW) to the enzymatic activity and antimicrobial activity of lysozyme adsorbed on CHNW was investigated. An activity assay showed that the lysozyme-CHNW system exhibited significant promotion potency on lysozyme activity, which was approximately 1.5-fold greater than that of free lysozyme. The molecular promotion mechanism of lysozyme immobilized by adsorption onto CHNW was investigated by ultraviolet-visible spectrophotometry, fluorescence spectroscopy, and circular dichroism spectroscopy. The results indicated that changes in the secondary structure of lysozyme adsorption onto CHNW resulted in superior enzymatic activity. Furthermore, the antimicrobial assays indicated that the antimicrobial activity of the lysozyme-CHNW system was greater than that of free lysozyme, whereas its antimicrobial effect on a gram-negative bacterium was better than that on gram-positive bacteria. This research provides a facile and promising approach for increasing the application of chitin-derived and enhancing the antibacterial efficiency on preservation.

Lysozyme loading and release from Se doped hydroxyapatite nanoparticles.

Element-substituted hydroxyapatite (HA) based nanocomposites have become a promising therapeutic material for improving bone defect repair. Selenium substituted HA nanoparticles can both induce apoptosis of bone tumor cells and enhance osteointegration. However, the effect of selenite ions on the proteins in combination with the HA nanoparticles remains to be elucidated. Here, we investigated the influence of selenium doping concentration on the loading and release of lysozyme (LSM) as a model protein drug. The selenium substituted HA-LSM composites with different doping concentrations were synthesized and characterized. The subsequent delivery of lysozyme was studied in a phosphate buffer solution (PBS). We found that selenium substituted HA-LSM composites with Se:P=10% showed the highest amount of lysozyme loading (41.7%), whereas the amount of lysozyme loaded in undoped HA nanoparticles was the lowest (34.1%). The doped selenium interacts with lysozyme molecules, which leads to the increase of ß-sheet and unordered, and the decrease of self-association, α-helix and ß-turns in protein structures. Moreover, selenium addition significantly slows the protein release from HA-LSM composites. The composites with Se:P=10% release lysozyme at the slightly slower rate among the samples with different Se doping concentrations. It also shows that the released lysozyme retains most of its enzymatic activity.

Controlled association and delivery of nanoparticles from jet-sprayed hybrid microfibrillar matrices.

To develop bioactive scaffolds of targeted properties for tissue repair or biomedical applications, hybrid microfiber-nanoparticle (MF-NP) matrices capable of controlled nanoparticle (NP) delivery were prepared through two novel approaches. In a first strategy, the suppleness of the jet-spraying method to produce polymer microfibers (MF) was used to deposit poly(d,l-lactide) (PLA) NP on poly(lactic-co-glycolic acid) (PLGA) MF by direct co-projection. The second approach relied on the post-incubation of PLA NP aqueous dispersion with MF preliminarily prepared by jet-spraying. NP coverage density onto MF and NP release was assessed by scanning electron microscopy and fluorescence measurements using coumarin-6 loaded NP. The first process was shown to allow high coverage density of NP onto MF (300 µg/mg MF) and strong association, with no NP release observed over time. In the second approach, direct incubation of PLA NP with PLA MF led to lower NP coverage density (40 µg/mg MF) with very fast release of NP from MF. The pre-coating of MF with poly-l-lysine (PLL) or the one of NP with lysozyme as a model protein drug afforded a higher coverage density and stronger association, coupled with a more sustained release of NP from MF over time. These results show the possibility to control the immobilization density and release of NP through appropriate preparation process and surface modification, and are of prime interest for the development of complex scaffolds with orchestrated bioactivity.

Development of prilling process for biodegradable microspheres through experimental designs.

The prilling process proposes a microparticle formulation easily transferable to the pharmaceutical production, leading to monodispersed and highly controllable microspheres. PLGA microspheres were used for carrying an encapsulated protein and adhered stem cells on its surface, proposing a tool for regeneration therapy against injured tissue. This work focused on the development of the production of PLGA microspheres by the prilling process without toxic solvent. The required production quality needed a complete optimization of the process. Seventeen parameters were studied through experimental designs and led to an acceptable production. The key parameters and mechanisms of formation were highlighted.

[EFFECT OF DIFFERENT PROTEINS ON THE REABSORPTION OF YELLOW FLUORESCENT PROTEIN IN THE KIDNEY OF BROWN FROG RANA TEMPORARIA].

Protein reabsorption in the proximal tubules (PT) of the frog kidney was studied by the methods of immunohistochemistry, fluorescent and confocal microscopy. Yellow fluorescent protein (YFP) was introduced in combination with other proteins. Reabsorption of YFP introduced simultaneously with ly- sozyme or green fluorescent protein (GFP) did not differ from the result of YFP injection only. Previous lysozyme injection did not change YFP absorption in contrast to YFP uptake reduced after GFP pretreat- ment. Lysozyme loading for 4 days resulted in a significant reduction in YFP absorption. The results show that receptor-mediated endocytosis in the frog kidney depends on the molecular nature of absorbable ligands, conditions of their competitive absorption and lysosomal accumulation in epithelial PT cells.

A novel antimicrobial peptide derived from fish goose type lysozyme disrupts the membrane of Salmonella enterica.

In aquaculture, accumulation of antibiotics resulted in development of resistance among bacterial pathogens. Consequently, it became mandatory to find alternative to synthetic antibiotics. Antimicrobial peptides (AMPs) which are described as evolutionary ancient weapons have been considered as promising alternates in recent years. In this study, a novel antimicrobial peptide had been derived from goose type lysozyme (LyzG) which was identified from the cDNA library of freshwater fish Channa striatus (Cs). The identified lysozyme cDNA contains 585 nucleotides which encodes a protein of 194 amino acids. CsLyzG was closely related to Siniperca chuatsi with 92.8% homology. The depicted protein sequence contained a GEWL domain with conserved GLMQ motif, 7 active residues and 2 catalytic residues. Gene expression analysis revealed that CsLyzG was distributed in major immune organs with highest expression in head kidney. Results of temporal expression analysis after bacterial (Aeromonas hydrophila) and fungal (Aphanomyces invadans) challenges indicated a stimulant-dependent expression pattern of CsLyzG. Two antimicrobial peptides IK12 and TS10 were identified from CsLyzG and synthesized. Antibiogram showed that IK12 was active against Salmonella enterica, a major multi-drug resistant (MDR) bacterial pathogen which produces beta lactamase. The IK12 induced loss of cell viability in the bacterial pathogen. Flow cytometry assay revealed that IK12 disrupt the membrane of S. enterica which is confirmed by scanning electron microscope (SEM) analysis that reveals blebs around the bacterial cell membrane. Conclusively, CsLyzG is a potential innate immune component and the identified antimicrobial peptide has great caliber to be used as an ecofriendly antibacterial substance in aquaculture.

Dye functionalized cryogel columns for reversible lysozyme adsorption.

In this study, poly (methyl methacrylate-glycidyl methacrylate) [poly(MMA-GMA)] cryogels were prepared by radical cryocopolymerization of MMA with GMA as a functional comonomer. Reactive Green 19 dye was then attached to the cryogel by nucleophilic substitution reaction, and this dye-attached cryogel column was used for lysozyme adsorption. Characterization of the cryogel was performed by Fourier transform infrared spectroscopy, environmental scanning electron microscopy, Brunauer-Emmett-Teller, and energy dispersive X-ray analysis. Pore size of the cryogels was 15-30 µm and pores were interconnected structure. Attached amount of Reactive Green 19 to cryogel support was calculated as 106.25 µmol/g cryogel. Lysozyme adsorption studies were carried out by using a continuous system. It was found that the maximum amount of lysozyme adsorption (32 mg/g cryogel) obtained from experimental results was found to be approximately same with the calculated Langmuir adsorption capacity (33 mg/g cryogel). Desorption of adsorbed lysozyme was carried out by using 1.5 M NaCl in pH 4.5 acetate buffer, and desorption yield was found to be 97.4%. Cryogels were very stable, and it was found that there was no remarkable reduction in the adsorption capacity at the end of ten adsorption-desorption cycles. As a result, Reactive Green 19-attached cryogels have great advantages such as easy preparation, rapid adsorption, and desorption, being economic and allowing the direct separation of proteins.

Effect of hydroxyapatite-containing microspheres embedded into three-dimensional magnesium phosphate scaffolds on the controlled release of lysozyme and in vitro biodegradation.

The functionality of porous three-dimensional (3D) magnesium phosphate (MgP) scaffold was investigated for the development of a novel protein delivery system and biomimetic bone tissue engineering scaffold. This enhancement can be achieved by incorporation of hydroxyapatite (HA)-containing polymeric microspheres (MSs) into a bulk MgP matrix, and a paste-extruding deposition (PED) system. In this work, the amount of MS and HA was precisely controlled when manufacturing MS-embedded MgP (MS/MgP) composite scaffolds. The main influence was researched in terms of in vitro lysozyme-release, in vitro biodegradation, mechanical properties, and in vitro calcification. The controlled release of lysozyme was indicated, while showing graded release patterns according to HA content. The composite scaffolds degraded gradually with MS content and degradation time. Due to the effect of HA inclusion, the higher HA-containing MS/MgP scaffolds could, not only delay the biodegradation process but also, compensate for the possible loss of mechanical properties. In this regard, it is reasonable to confirm the inverse relationship between biodegradation and corresponding compressive properties. In order to encourage bioactivity and osteoconductivity, the MS/MgP composite scaffolds were subjected to simulated body fluid treatment. Calcium deposition was, in turn, improved with increasing MS and HA content over time. This quantitative result was also proved using morphological and elemental analysis. In summary, a significant transformation of a monolithic MgP scaffold was directed toward a multifunctional bone tissue engineering scaffold equipped with controlled protein delivery, biodegradability, and bioactivity.

Understanding the poor iontophoretic transport of lysozyme across the skin: when high charge and high electrophoretic mobility are not enough.

The original aim of the study was to investigate the transdermal iontophoretic delivery of lysozyme and to gain further insight into the factors controlling protein electrotransport. Initial experiments were done using porcine skin. Lysozyme transport was quantified by using an activity assay based on the lysis of Micrococcus lysodeikticus and was corrected for the release of endogenous enzyme from the skin during current application. Cumulative iontophoretic permeation of lysozyme during 8h at 0.5mA/cm(2) (0.7mM; pH6) was surprisingly low (5.37±3.46µg/cm(2) in 8h) as compared to electrotransport of cytochrome c (Cyt c) and ribonuclease A (RNase A) under similar conditions (923.0±496.1 and 170.71±92.13µg/cm(2), respectively) - despite its having a higher electrophoretic mobility. The focus of the study then became to understand and explain the causes of its poor iontophoretic transport. Lowering formulation pH to 5 increased histidine protonation in the protein and decreased the ionisation of fixed negative charges in the skin (pI ~4.5) and resulted in a small but statistically significant increase in permeation. Co-iontophoresis of acetaminophen revealed a significant inhibition of electroosmosis; inhibition factors of 12-16 were indicative of strong lysozyme binding to skin. Intriguingly, lidocaine electrotransport, which is due almost exclusively to electromigration, was also decreased (approximately 2.7-fold) following skin pre-treatment by lysozyme iontophoresis (cf. iontophoresis of buffer solution) - suggesting that lysozyme was also able to influence subsequent cation electromigration. In order to elucidate the site of skin binding, different porcine skin models were tested (dermatomed skin with thicknesses of 250 and 750µm, tape-stripped skin and heat-separated dermis). Although no difference was seen between permeation across 250 and 750µm dermatomed skin (13.57±12.20 and 5.37±3.46µg/cm(2), respectively), there was a statistically significant increase across tape-stripped skin and heat-separated dermis (36.86±7.48 and 43.42±13.11µg/cm(2), respectively) - although transport was still much less than that seen across intact skin for Cyt c or RNase A. Furthermore, electroosmotic inhibition factors fell to 2.2 and 1.0 for tape-stripped skin and heat-separated dermis - indicating that lysozyme affected convective solvent flow through interactions with the epidermis and predominantly the stratum corneum. Finally, cation exchange and hydrophobic interaction chromatography confirmed that although lysozyme had greater positive charge than Cyt c or RNase A under the conditions used for iontophoresis, it also possessed the highest surface hydrophobicity, which may have facilitated the interactions with the transport pathways and encouraged aggregation in the skin microenvironment. Thus, high charge and electrophoretic mobility seem to be inadequate descriptors to predict the transdermal iontophoretic transport of proteins whose complex three dimensional structures can facilitate interactions with cutaneous transport pathways.

Intestinal absorption of lysozyme, an egg-white allergen, in rats: kinetics and effect of NSAIDs.

The absorption pathway(s) of a representative food allergen, lysozyme, and the mechanisms of lysozyme absorption facilitated by non-steroidal anti-inflammatory drugs were examined by intestinal closed-loop and re-circulating perfusion methods in rats. The absorption rate of fluorescein isothiocyanate (FITC)-labeled lysozyme in the proximal intestine was higher than that for a marker of non-specific absorption, FD-10, and was suppressed by colchicine (endocytosis inhibitor). Aspirin increased the absorption of FITC-lysozyme in the proximal intestine with no effects on tissue accumulation. Diclofenac facilitated FITC-lysozyme absorption, but meloxicam and loxoprofen exerted no effects on absorption. Co-administration of misoprostol (synthetic prostaglandin-E1 analog) with aspirin significantly ameliorated the aspirin-facilitated absorption of FITC-lysozyme to the same level as that seen with controls. Thus, lysozyme absorption was mediated by endocytic and paracellular pathways in the proximal intestine, and was facilitated by aspirin and diclofenac after impairment of the paracellular pathway. Misoprostol may suppress the allergen absorption facilitated by aspirin.