Investigation of Hydrocolloid Plant Polysaccharides as Potential Candidates to Mimic the Functions of MUC5B in Saliva.

The successful substitution of complex physiological fluids, such as human saliva, remains a major challenge in drug development. Although there are a large number of saliva substitutes on the market, their efficacy is often inadequate due to short residence time in the mouth, unpleasant mouthfeel, or insufficient protection of the teeth. Therefore, systems need to be identified that mimic the functions of saliva, in particular the salivary mucin MUC5B and the unique physiological properties of saliva. To this end, plant extracts known to contain hydrocolloid polysaccharides and to have mucus-forming properties were studied to evaluate their suitability as saliva substitutes. The aqueous plant extracts of Calendula officinalis, Fucus sp. thalli, and lichenan from Lichen islandicus were examined for composition using a range of techniques, including GC-MS, NMR, SEC, assessment of pH, osmolality, buffering capacity, viscoelasticity, viscoelastic interactions with human saliva, hydrocolloid network formation, and in vitro cell adhesion. For this purpose, a physiologically adapted adhesive test was developed using human buccal epithelial cells. The results show that lichenan is the most promising candidate to mimic the properties of MUC5B. By adjusting the pH, osmolality, and buffering capacity with K2HPO4, it was shown that lichenan exhibited high cell adhesion, with a maximum detachment force that was comparable to that of unstimulated whole mouth saliva.

Poly(Allylamine Hydrochloride) and ZnO Nanohybrid Coating for the Development of Hydrophobic, Antibacterial, and Biocompatible Textiles.

In healthcare facilities, infections caused by Staphylococcus aureus (S. aureus) from textile materials are a cause for concern, and nanomaterials are one of the solutions; however, their impact on safety and biocompatibility with the human body must not be neglected. This study aimed to develop a novel multilayer coating with poly(allylamine hydrochloride) (PAH) and immobilized ZnO nanoparticles (ZnO NPs) to make efficient antibacterial and biocompatible cotton, polyester, and nylon textiles. For this purpose, the coated textiles were characterized with profilometry, contact angles, and electrokinetic analyzer measurements. The ZnO NPs on the textiles were analyzed by scanning electron microscopy and inductively coupled plasma mass spectrometry. The antibacterial tests were conducted with S. aureus and biocompatibility with immortalized human keratinocyte cells. The results demonstrated successful PAH/ZnO coating formation on the textiles, demonstrating weak hydrophobic properties. Furthermore, PAH multilayers caused complete ZnO NP immobilization on the coated textiles. All coated textiles showed strong growth inhibition (2-3-log reduction) in planktonic and adhered S. aureus cells. The bacterial viability was reduced by more than 99%. Cotton, due to its better ZnO NP adherence, demonstrated a slightly higher antibacterial performance than polyester and nylon. The coating procedure enables the binding of ZnO NPs in an amount (<30 µg cm-2) that, after complete dissolution, is significantly below the concentration causing cytotoxicity (10 µg mL-1).

Microplastics encapsulation in aragonite: efficiency, detection and insight into potential environmental impacts.

Plastic pollution in aquatic ecosystems has become a significant problem especially microplastics which can encapsulate into the skeletons of organisms that produce calcium carbonates, such as foraminifera, molluscs and corals. The encapsulation of microplastics into precipitated aragonite, which in nature builds the coral skeleton, has not yet been studied. It is also not known how the dissolved organic matter, to which microplastics are constantly exposed in aquatic ecosystems, affects the encapsulation of microplastics into aragonite and how such microplastics affect the mechanical properties of aragonite. We performed aragonite precipitation experiments in artificial seawater in the presence of polystyrene (PS) and polyethylene (PE) microspheres, untreated and treated with humic acid (HA). The results showed that the efficiency of encapsulating PE and PE-HA microspheres in aragonite was higher than that for PS and PS-HA microspheres. The mechanical properties of resulting aragonite changed after the encapsulation of microplastic particles. A decrease in the hardness and indentation modulus of the aragonite samples was observed, and the most substantial effect occurred in the case of PE-HA microspheres encapsulation. These findings raise concerns about possible changes in the mechanical properties of the exoskeleton and endoskeleton of calcifying marine organisms such as corals and molluscs due to the incorporation of pristine microplastics and microplastics exposed to dissolved organic matter.

A comprehensive overview of advanced dynamic in vitro intestinal and hepatic cell culture models.

Orally administered drugs pass through the gastrointestinal tract before being absorbed in the small intestine and metabolised in the liver. To test the efficacy and toxicity of drugs, animal models are often employed; however, they are not suitable for investigating drug-tissue interactions and making reliable predictions, since the human organism differs drastically from animals in terms of absorption, distribution, metabolism and excretion of substances. Likewise, simple static in vitro cell culture systems currently used in preclinical drug screening often do not resemble the native characteristics of biological barriers. Dynamic models, on the other hand, provide in vivo-like cell phenotypes and functionalities that offer great potential for safety and efficacy prediction. Herein, current microfluidic in vitro intestinal and hepatic models are reviewed, namely single- and multi-tissue micro-bioreactors, which are associated with different methods of cell cultivation, i.e., scaffold-based versus scaffold-free.

Exploring the anti-inflammatory potential of topical hyaluronic acid for vocal fold injury in a rat model.

PURPOSE: Vocal fold injuries are associated with fibrosis and dysphonia, which is a major obstacle to surgical treatment. The aim of this study is to evaluate the effect of topical hyaluronic acid with or without diclofenac on the inflammatory phase of vocal fold wound healing. METHODS: Forty-one male Sprague-Dawley rats were randomly assigned to four groups: an uninjured control group, an injured control group without any treatment, and two intervention groups in which hyaluronic acid with or without diclofenac was applied to the injured vocal fold. Gene expression of inflammatory markers and ECM-related molecules were examined. RESULTS: Vocal fold injury resulted in a significant upregulation of inflammatory parameters [Ptgs2, Il1b and Il10] and Has1. Tgfb1, Has3 and Eln gene expression were significantly downregulated by the topical application of hyaluronic acid. The combination of hyaluronic acid and diclofenac did not result in any significant changes. CONCLUSIONS: Vocal fold wound healing was significantly improved by a single post-operative topical application of hyaluronic acid. The addition of diclofenac may provide no additional benefit.

Development and Characterization of an In Vitro Intestinal Model Including Extracellular Matrix and Macrovascular Endothelium.

In vitro intestinal models are used to study biological processes, drug and food absorption, or cytotoxicity, minimizing the use of animals in the laboratory. They usually consist of enterocytes and mucus-producing cells cultured for 3 weeks, e.g., on Transwells, to obtain a fully differentiated cell layer simulating the human epithelium. Other important components are the extracellular matrix (ECM) and strong vascularization. The former serves as structural support for cells and promotes cellular processes such as differentiation, migration, and growth. The latter includes endothelial cells, which coordinate vascularization and immune cell migration and facilitate the transport of ingested substances or drugs to the liver. In most cases, animal-derived hydrogels such as Matrigel or collagen are used as ECM in in vitro intestinal models, and endothelial cells are only partially considered, if at all. However, it is well-known that animal-derived products can lead to altered cell behavior and incorrect results. To circumvent these limitations, synthetic and modifiable hydrogels (Peptigel and Vitrogel) were studied here to mimic xenofree ECM, and the data were compared with Matrigel. Careful rheological characterization was performed, and the effect on cell proliferation was investigated. The results showed that Vitrogel exhibited shear-thinning behavior with an internal structure recovery of 78.9 ± 11.2%, providing the best properties among the gels investigated. Therefore, a coculture of Caco-2 and HT29-MTX cells (ratio 7:3) was grown on Vitrogel, while simultaneously endothelial cells were cultured on the basolateral side by inverse cultivation. The model was characterized in terms of cell proliferation, differentiation, and drug permeability. It was found that the cells cultured on Vitrogel induced a 1.7-fold increase in cell proliferation and facilitated the formation of microvilli and tight junctions after 2 weeks of cultivation. At the same time, the coculture showed full differentiation indicated by high alkaline phosphatase release of Caco-2 cells (95.0 ± 15.9%) and a mucus layer produced by HT29-MTX cells. Drug tests led to ex vivo comparable permeability coefficients (Papp) (i.e., Papp; antipyrine = (33.64 ± 5.13) × 10-6 cm/s, Papp; atenolol = (0.59 ± 0.16) × 10-6 cm/s). These results indicate that the newly developed intestinal model can be used for rapid and efficient assessment of drug permeability, excluding unexpected results due to animal-derived materials.

Small volume rapid equilibrium dialysis (RED) measures effects of interstitial parameters on the protein-bound fraction of topical drugs.

The importance of plasma protein binding in the early stages of drug development is well recognized. Free and bound drug fractions in plasma are routinely determined with well-established methods. However, for physiological fluids with a small accessible volume and low protein concentrations, such as dermal interstitial fluid (dISF) validated methods are currently missing. Due to the low protein concentration and highly dynamic processes in the dermis, protein binding data obtained from plasma samples may underestimate in-vivo efficacy. This study aimed to validate a small volume rapid equilibrium dialysis (RED) for low protein samples, as a tool to examine drug-protein binding directly in the biological fluid at the site of action. The sample volume required for RED was successfully downscaled to 50 µl and plasma protein binding values of the four model drugs were consistent with previous studies with an average recovery of 88 ± 8% which makes all tested drugs suitable for small volume RED. Inter- and intra-batch variability showed sufficient reproducibility across RED plates. Small volume RED was successfully applied to assess the effects of interstitial parameters, including the evaluation of the major binding protein and the effects of binding protein concentration, drug concentration, and pH on the protein-bound drug fraction using 2% HSA and/or diluted human plasma as a surrogate for dISF.

A multi-step machine learning approach for accelerating QbD-based process development of protein spray drying.

This study proposes a new material-efficient multi-step machine learning (ML) approach for the development of a design space (DS) for spray drying proteins. Typically, a DS is developed by performing a design of experiments (DoE) with the spray dryer and the protein of interest, followed by deriving the DoE models via multi-variate regression. This approach was followed as a benchmark to the ML approach. The more complex the process and required accuracy of the final model is, the more experiments are necessary. However, most biologics are expensive and thus experiments should be kept to a minimum. Therefore, the suitability of using a surrogate material and ML for the development of a DS was investigated. To this end, a DoE was performed with the surrogate and the data used for training the ML approach. The ML and DoE model predictions were compared to measurements of three protein-based validation runs. The suitability of using lactose as surrogate was investigated and advantages of the proposed approach were demonstrated. Limitations were identified at protein concentrations >35 mg/ml and particle sizes of x50>6 µm. Within the investigated DS protein secondary structure was preserved, and most process settings, resulted in yields >75% and residual moisture <10 wt%.

Investigation of the Influence of Wound-Treatment-Relevant Buffer Systems on the Colloidal and Optical Properties of Gold Nanoparticles.

Biocompatible gold nanoparticles (AuNPs) are used in wound healing due to their radical scavenging activity. They shorten wound healing time by, for example, improving re-epithelialization and promoting the formation of new connective tissue. Another approach that promotes wound healing through cell proliferation while inhibiting bacterial growth is an acidic microenvironment, which can be achieved with acid-forming buffers. Accordingly, a combination of these two approaches appears promising and is the focus of the present study. Here, 18 nm and 56 nm gold NP (Au) were prepared with Turkevich reduction synthesis using design-of-experiments methodology, and the influence of pH and ionic strength on their behaviour was investigated. The citrate buffer had a pronounced effect on the stability of AuNPs due to the more complex intermolecular interactions, which was also confirmed by the changes in optical properties. In contrast, AuNPs dispersed in lactate and phosphate buffer were stable at therapeutically relevant ionic strength, regardless of their size. Simulation of the local pH distribution near the particle surface also showed a steep pH gradient for particles smaller than 100 nm. This suggests that the healing potential is further enhanced by a more acidic environment at the particle surface, making this strategy a promising approach.

Assessment of Carbon Nanotubes on Barrier Function, Ciliary Beating Frequency and Cytokine Release in In Vitro Models of the Respiratory Tract.

The exposure to inhaled carbon nanotubes (CNT) may have adverse effects on workers upon chronic exposure. In order to assess the toxicity of inhaled nanoparticles in a physiologically relevant manner, an air-liquid interface culture of mono and cocultures of respiratory cells and assessment in reconstructed bronchial and alveolar tissues was used. The effect of CNT4003 reference particles applied in simulated lung fluid was studied in bronchial (Calu-3 cells, EpiAirway™ and MucilAir™ tissues) and alveolar (A549 +/-THP-1 and EpiAlveolar™ +/-THP-1) models. Cytotoxicity, transepithelial electrical resistance, interleukin 6 and 8 secretion, mucociliary clearance and ciliary beating frequency were used as readout parameters. With the exception of increased secretion of interleukin 6 in the EpiAlveolar™ tissues, no adverse effects of CNT4003 particles, applied at doses corresponding to the maximum estimated lifetime exposure of workers, in the bronchial and alveolar models were noted, suggesting no marked differences between the models. Since the doses for whole-life exposure were applied over a shorter time, it is not clear if the interleukin 6 increase in the EpiAlveolar™ tissues has physiological relevance.

In Vitro and In Vivo Evaluation of Carbopol 71G NF-Based Mucoadhesive Minitablets as a Gastroretentive Dosage Form.

Gastroretentive dosage forms are intended to stay inside the stomach for a long period of time while releasing an active pharmaceutical ingredient. Such systems may offer significant benefits for numerous drugs compared to other sustained release systems, such as improved pharmacokinetics/bioavailability and reduced intake frequency and thereby improved adherence to the medical therapy. However, there is no gastroretentive product on the market with proven reliable gastroretentive properties in humans. A major obstacle is the motility pattern of the stomach in the fasting state in humans, which reliably ensures gastric emptying of even large indigestible objects into the small intestine. One promising approach to avoid gastric emptying is adhesion of the drug delivery system to the gastric mucosa. In order to achieve mucoadhesive properties, minitablets containing Carbopol 71G NF were developed and compared to minitablets without adhesive properties. In a specialized mucoadhesive test system, the adhesion time was prolonged for adhesive minitablets (240 min) compared to non-adhesive minitablets (30 min). The in vivo transit behavior was investigated using magnetic resonance imaging in 11 healthy volunteers in fasted state in a crossover setup. It was found that the gastric residence time (GRT) of the adhesive minitablets (median of 37.5 min with IQR = 22.5-52.5) was statistically significantly prolonged compared to the non-adhesive minitablets (median of 7.5 with IQR = 7.5-22.5), indicating a delay in gastric emptying by adhesion to the gastric mucosa. However, the system needs further improvement to create a clinical benefit. Furthermore, it was observed that for 9 of 22 administrations (three minitablets were given simultaneously with every administration), the minitablets were not emptied together but showed different GRTs.

Needles to Spheres: Evaluation of inkjet printing as a particle shape enhancement tool.

Active pharmaceutical ingredients (APIs) often reveal shapes challenging to process, e.g. acicular structures, and exhibit reduced bioavailability induced by slow dissolution rate. Leveraging the API particles' surface and bulk properties offers an attractive pathway to circumvent these challenges. Inkjet printing is an attractive processing technique able to tackle these limitations already in initial stages when little material is available, while particle properties are maintained over the entire production scale. Additionally, it is applicable to a wide range of formulations and offers the possibility of co-processing with a variety of excipients to improve the API's bioavailability. This study addresses the optimization of particle shapes for processability enhancement and demonstrates the successful application of inkjet printing to engineer spherical lacosamide particles, which are usually highly acicular. By optimizing the ink formulation, adapting the substrate-liquid interface and tailoring the heat transfer to the particle, spherical particles in the vicinity of 100 µm, with improved flow properties compared to the bulk material, were produced. Furthermore, the particle size was tailored reproducibly by adjusting the deposited ink volume per cycle and the number of printing cycles. Therefore, the present study shows a novel, reliable, scalable and economical strategy to overcome challenging particle morphologies by co-processing an API with suitable excipients.

Development of a Workflow to Engineer Tailored Microparticles Via Inkjet Printing.

PURPOSE: New drug development and delivery approaches result in an ever-increasing demand for tailored microparticles with defined sizes and structures. Inkjet printing technologies could be promising new processes to engineer particles with defined characteristics, as they are created to precisely deliver liquid droplets with high uniformity. METHODS: D-mannitol was used as a model compound alone or co-processed with the pore former agent ammonium bicarbonate, and the polymer polyethylene glycol 200. Firstly, a drop shape analyzer was used to characterize and understand ink/substrate interactions, evaporation, and solidification kinetics. Consequently, the process was transferred to a laboratory-scale inkjet printer and the resulting particles collected, characterized and compared to others obtained via an industrial standard technique. RESULTS: The droplet shape analysis allowed to understand how 3D structures are formed and helped define the formulation and process parameters for inkjet printing. By adjusting the drop number and process waveform, spherical particles with a mean size of approximately 100 µm were obtained. The addition of pore former and polymer allowed to tailor the crystallization kinetics, resulting in particles with a different surface (i.e., spike-like surface) and bulk (e.g. porous and non-porous) structure. CONCLUSION: The workflow described enabled the production of 3D structures via inkjet printing, demonstrating that this technique can be a promising approach to engineer microparticles.

Preparation and detailed characterization of the thiomer chitosan-cysteine as a suitable mucoadhesive excipient for nasal powders.

The therapeutic application of nasal powders requires the development of novel mucoadhesive excipients. Thiolated polymers exhibit significant potential for this purpose based on their increased mucoadhesion attributable to the formation of disulfide bonds between the polymer and mucus surface. A chitosan-cysteine (chit-cyst) conjugate was synthesized using 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in aqueous solution. The synthetic yield and synthesis conditions were optimized, and the efficiency of the reaction was evaluated. Rheological measurements revealed that the polymer derivative exhibited increased mucoadhesive properties in comparison to chitosan powder. To characterize the polymer, a novel purity investigation method was developed and verified to investigate the residual l-cysteine content. The results revealed that l-cysteine was not detectable in the resultant polymer matrix. Based on the cytotoxicity studies, chit-cyst was found to be safe for nasal application. Thereafter, nasal powder formulations were prepared using the polymer and the antiparkinsonian drug levodopa methyl ester hydrochloride by freeze-drying to investigate their nasal applicability. Based on the in vitro studies, these powders might be suitable for reducing the off periods of Parkinson's disease because of their expected higher in vivo mucoadhesion.

In-vial printing and drying of biologics as a personalizable approach.

This study addressed the need for a flexible (personalizable) production of biologics, allowing their stabilization in the solid state and processing of small batch volumes. Therefore, inkjet printing into vials followed by a gentle vacuum drying step at ambient temperature was investigated by screening different formulations with a 22-full factorial design of experiments regarding printability. Human Serum Albumin (HSA) was used as a model protein in a wide range of concentrations (5 to 50 mg/ml), with (10 w/v%) and without the surfactant polysorbate 80 (PS80). PS80 was identified to positively affect the formulations by increasing the Ohnesorge number and stabilizing the printing process. The dispensed volumes with a target dose of 0.5 mg HSA were dried and analyzed concerning their residual moisture (RM) and protein aggregation. All investigated formulations showed an RM < 10 wt% and no significant induced protein aggregation as confirmed by Size Exclusion Chromatography (<2.5%) and Dynamic Light Scattering (Aggregation Index ≤ 2.5). Additionally, long-term printability and the available final dose after reconstitution were investigated for two optimized formulations. A promising formulation providing ∼93% of the targeted dose and a reconstitution time of 30 s was identified.

Toward a new generation of vaginal pessaries via 3D-printing: Concomitant mechanical support and drug delivery.

To improve patient adherence, vaginal pessaries - polymeric structures providing mechanical support to treat stress urinary incontinence (SUI) - greatly benefit from 3D-printing through customization of their mechanics, e.g. infill modifications. However, currently only limited polymers provide both flawless printability and controlled drug release. The current study closes this gap by exploring 3D-printing, more specifically fused filament fabrication, of pharmaceutical grade thermoplastic polyurethanes (TPU) of different hardness and hydrophilicity into complex pessary structures. Next to the pessary mechanics, drug incorporation into such a device was addressed for the first time. Mechanically, the soft hydrophobic TPU was the most promising candidate for pessary customization, as pessaries made thereof covered a broad range of the key mechanical parameter, while allowing self-insertion. From the drug release point of view, the hydrophobic TPUs were superior over the hydrophilic one, as the release levels of the model drug acyclovir were closer to the target value. Summarizing, the fabrication of TPU-based pessaries via 3D-printing is an innovative strategy to create a customized pessary combination product that simultaneously provides mechanical support and pharmacological therapy.

Thiolated Chitosan Conjugated Liposomes for Oral Delivery of Selenium Nanoparticles.

This study aimed to design a hybrid oral liposomal delivery system for selenium nanoparticles (Lip-SeNPs) to improve the bioavailability of selenium. Thiolated chitosan, a multifunctional polymer with mucoadhesive properties, was used for surface functionalization of Lip-SeNPs. Selenium nanoparticle (SeNP)-loaded liposomes were manufactured by a single step microfluidics-assisted chemical reduction and assembling process. Subsequently, chitosan-N-acetylcysteine was covalently conjugated to the preformed Lip-SeNPs. The Lip-SeNPs were characterized in terms of composition, morphology, size, zeta potential, lipid organization, loading efficiency and radical scavenging activity. A co-culture system (Caco-2:HT29-MTX) that integrates mucus secreting and enterocyte-like cell types was used as a model of the human intestinal epithelium to determine adsorption, mucus penetration, release and transport properties of Lip-SeNPs in vitro. Thiolated Lip-SeNPs were positively charged with an average size of about 250 nm. Thiolated Lip-SeNPs tightly adhered to the mucus layer without penetrating the enterocytes. This finding was consistent with ex vivo adsorption studies using freshly excised porcine small intestinal tissues. Due to the improved mucoadhesion and retention in a simulated microenvironment of the small intestine, thiolated Lip-SeNPs might be a promising tool for oral selenium delivery.

The Role of Local Inflammation and Hypoxia in the Formation of Hypertrophic Scars-A New Model in the Duroc Pig.

Hypertrophic scars continue to be a major burden, especially after burns. Persistent inflammation during wound healing appears to be the precipitating aspect in pathologic scarring. The lack of a standardized model hinders research from fully elucidating pathophysiology and therapy, as most therapeutic approaches have sparse evidence. The goal of this project was to investigate the mechanisms of scar formation after prolonged wound inflammation and to introduce a method for generating standardized hypertrophic scars by inducing prolonged inflammation. Four wound types were created in Duroc pigs: full-thickness wounds, burn wounds, and both of them with induced hyperinflammation by resiquimod. Clinical assessment (Vancouver Scar Scale), tissue oxygenation by hyperspectral imaging, histologic assessment, and gene expression analysis were performed at various time points during the following five months. Native burn wounds as well as resiquimod-induced full-thickness and burn wounds resulted in more hypertrophic scars than full-thickness wounds. The scar scale showed significantly higher scores in burn- and resiquimod-induced wounds compared with full-thickness wounds as of day 77. These three wound types also showed relative hypoxia compared with uninduced full-thickness wounds in hyperspectral imaging and increased expression of HIF1a levels. The highest number of inflammatory cells was detected in resiquimod-induced full-thickness wounds with histologic features of hypertrophic scars in burn and resiquimod-induced wounds. Gene expression analysis revealed increased inflammation with only moderately altered fibrosis markers. We successfully created hypertrophic scars in the Duroc pig by using different wound etiologies. Inflammation caused by burns or resiquimod induction led to scars similar to human hypertrophic scars. This model may allow for the further investigation of the exact mechanisms of pathological scars, the role of hypoxia and inflammation, and the testing of therapeutic approaches.

The effect of ethanol on the habit and in vitro aerodynamic results of dry powder inhalation formulations containing ciprofloxacin hydrochloride.

In the case of dry powder inhalation systems (DPIs), the development of carrier-free formulations has gained increased attention. Thereby, spray-drying is a promising technology and is widely used to produce carrier-free DPIs. Numerous works have been published about the co-spray-drying of active ingredients with various solid excipients and their effect on the physicochemical characteristics and aerodynamic properties of the formulations. However, only a few studies have been reported about the role of the solvents used in the stock solutions of spray-dried formulations. In the present work, DPI microcomposites containing ciprofloxacin hydrochloride were prepared by spray-drying in the presence of different ethanol concentrations. The work expresses the roughness, depth and width of the dimples for particle size as a novel calculation possibility, and as a correlation between the MMAD/D0.5 ratio and correlating it with cohesion work, these new terms and correlations have not been published - to the best of our knowledge - which has resulted in gap-filling findings. As a result, different proportions of solvent mixtures could be interpreted and placed in a new perspective, in which the influence of different concentrations of ethanol on the habit of the DPI formulations, and thus on in vitro aerodynamic results. Based on these, it became clear why we obtained the best in vitro aerodynamic results for DPI formulation containing 30% ethanol in the stock solution.

Comparing freeze drying and spray drying of interleukins using model protein CXCL8 and its variants.

Spray-dried products, such as synthetic peptides and hormones, have already been approved by the U.S. Food and Drug Agency and the European Medicines Agency, while spray-dried antibodies or interleukins, are not yet available on the market. Concerning the latter group, knowledge on whether and how spray-drying (SD) can be performed without adversely affecting their biological activity is lacking. Accordingly, this study aimed at establishing a SD process (Büchi B-90 spray dryer) using three Interleukin-8 based proteins (7-74 kDa) that were dispersed in phosphate buffered saline to maintain their stability. A Box-Behnken Design of Experiments was conducted to identify the appropriate process parameters taking into account the thermal stability of interleukin-8. In parallel, a FD process was developed. Both powders were stored for up to 12 weeks. Powder characterization included residual moisture evaluation and the mean particle size of the SD powder was investigated with Laser Diffraction Analysis. The hydrodynamic volume was measured via size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The secondary structure of the model proteins in the solid state was assessed with Fourier-transformation infrared spectroscopy for detecting the protein folding patterns and reconstituted with Circular Dichroism Spectroscopy. Finally, the binding affinity was studied with Surface Plasmon Resonance and Isothermal Fluorescence Titration, the protein stability with Chaotropic Unfolding, and the activity studies were carried out with the chemotaxis assay. The results showed that SD and FD powders with a residual moisture of less than 5 wt% were obtained. The interleukins showed no unfolding upon processing, neither in solid state nor reconstituted. Oligomerization was observed for FD, but not for SD interleukins. However, the unfolding, binding affinity and activity of all interleukins examined did not decrease in neither SD nor FD powders, even after 12 weeks of storage. Thus, it can be concluded that SD of interleukin formulations at outlet temperatures close to ambient temperature is a promising process for transferring them into a stable powder.