Improving Determination of Pigment Contents in Microalgae Suspension with Absorption Spectroscopy: Light Scattering Effect and Bouguer-Lambert-Beer Law.

The Bouguer-Lambert-Beer (BLB) law serves as the fundamental basis for the spectrophotometric determination of pigment content in microalgae. Although it has been observed that the applicability of the BLB law is compromised by the light scattering effect in microalgae suspensions, in-depth research concerning the relationship between the light scattering effect and the accuracy of spectrophotometric pigment determination remains scarce. We hypothesized that (1) the precision of spectrophotometric pigment content determination using the BLB law would diminish with increasing nonlinearity of absorbance, and (2) employing the modified version of the BLB (mBLB) law would yield superior performance. To assess our hypotheses, we cultivated Phaeodactylum tricornutum under varying illumination conditions and nitrogen supplies in controlled indoor experiments, resulting in suspensions with diverse pigment contents. Subsequently, P. tricornutum samples were diluted into subsamples, and spectral measurements were conducted using different combinations of biomass concentrations and path lengths. This was carried out to assess the applicability of the BLB law and the nonlinearity of absorbance. The chlorophyll a and fucoxanthin contents in the samples were analyzed via high-performance liquid chromatography (HPLC) and subsequently used in our modeling. Our findings confirm our hypotheses, showing that the modified BLB law outperforms the original BLB law in terms of the normalized root mean square error (NRMSE): 6.3% for chlorophyll a and 5.8% for fucoxanthin, compared to 8.5% and 7.9%, respectively.

Publisher Correction: The choice of biopolymer is crucial to trigger angiogenesis with vascular endothelial growth factor releasing coatings.

A Correction to this paper has been published: https://doi.org/10.1007/s10856-020-06463-w.

The choice of biopolymer is crucial to trigger angiogenesis with vascular endothelial growth factor releasing coatings.

Bio-based coatings and release systems for pro-angiogenic growth factors are of interest to overcome insufficient vascularization and bio-integration of implants. This study compares different biopolymer-based coatings on polyethylene terephthalate (PET) membranes in terms of coating homogeneity and stability, coating thickness in the swollen state, endothelial cell adhesion, vascular endothelial growth factor (VEGF) release and pro-angiogenic properties. Coatings consisted of carbodiimide cross-linked gelatin type A (GelA), type B (GelB) or albumin (Alb), and heparin (Hep), or they consisted of radically cross-linked gelatin methacryloyl-acetyl (GM5A5) and heparin methacrylate (HepM5). We prepared films with thicknesses of 8-10 µm and found that all coatings were homogeneous after washing. All gelatin-based coatings enhanced the adhesion of primary human endothelial cells compared to the uncoated membrane. The VEGF release was tunable with the loading concentration and dependent on the isoelectric points and hydrophilicities of the biopolymers used for coating: GelA-Hep showed the highest releases, while releases were indistinguishable for GelB-Hep and Alb-Hep, and lowest for GM5A5-HepM5. Interestingly, not only the amount of VEGF released from the coatings determined whether angiogenesis was induced, but a combination of VEGF release, metabolic activity and adhesion of endothelial cells. VEGF releasing GelA-Hep and GelB-Hep coatings induced angiogenesis in a chorioallantoic membrane assay, so that these coatings should be considered for further in vivo testing.

Quantification of Substitution of Gelatin Methacryloyl: Best Practice and Current Pitfalls.

Cross-linkable gelatin methacryloyl (GM) is widely used for the generation of artificial extracellular matrix (ECM) in tissue engineering. However, the quantification of modified groups in GM is still an unsolved issue, although this is the key factor for tailoring the physicochemical material properties. In this contribution, 1H-13C-HSQC NMR spectra are used to gain detailed structural information on GMs and of 2-fold modified gelatin containing methacryloyl and acetyl groups (GMAs). Distinctive identification of methacrylate, methacrylamide, and acetyl groups present in GMs and GMAs revealed an overlap of methacrylamide and modified hydroxyproline signals in the 1H NMR spectrum. Considering this, we suggest a method to quantify methacrylate and methacrylamide groups in GMs precisely based on simple 1H NMR spectroscopy with an internal standard. Quantification of acetylation in GMAs is also possible, yet, 2D NMR spectra are necessary. The described methods allow direct quantification of modified groups in gelatin derivatives, making them superior to other, indirect methods known so far.

Soft Sub-Structured Multi-Material Biosensor Hydrogels with Enzymes Retained by Plant Viral Scaffolds.

An all-soft multi-material combination consisting of a hydrogel based on poly(ethylene glycol) (PEG) coated with spatially defined spots of gelatin methacryloyl (GM) containing selectively addressable viral nanorods is presented, and its basic application as a qualitative biosensor with reporter enzymes displayed on the tobacco mosaic virus (TMV) bioscaffolds within the GM is demonstrated. Biologically inert PEG supports are equipped with GM spots serving as biological matrix for enzymes clustered on TMV particles preventing diffusion out of the gel. For this multi-material combination, i) the PEG-based hydrogel surface is modified to achieve a clear boundary between coated and non-coated regions by introducing either isothiouronium or thiol groups. ii) Cross-linking of the GM spots is studied to achieve anchoring to the hydrogel surface. iii) The enzymes horseradish peroxidase or penicillinase (Pen) are conjugated to TMV and integrated into the GM matrix. In contrast to free enzymes, enzyme-decorated TMVs persist in GM spots and show sustained enzyme activity as evidenced by specific color reaction after 7 days of washing, and for Pen after 22 months after dry storage. Therefore, the integration of enzyme-coupled TMV into hydrogel matrices is a promising and versatile approach to obtaining reusable and analyte-specific sensor components.

Influence of light conditions on the production of chrysolaminarin using Phaeodactylum tricornutum in artificially illuminated photobioreactors.

The light conditions are of utmost importance in any microalgae production process especially involving artificial illumination. This also applies to a chrysolaminarin (soluble 1,3-ß-glucan) production process using the diatom Phaeodactylum tricornutum. Here we examine the influence of the amount of light per gram biomass (specific light availability) and the influence of two different biomass densities (at the same amount of light per gram biomass) on the accumulation of the storage product chrysolaminarin during nitrogen depletion in artificially illuminated flat-panel airlift photobioreactors. Besides chrysolaminarin, other compounds (fucoxanthin, fatty acids used for energy storage [C16 fatty acids], and eicosapentaenoic acid) are regarded as well. Our results show that the time course of C-allocation between chrysolaminarin and fatty acids, serving as storage compounds, is influenced by specific light availability and cell concentration. Furthermore, our findings demonstrate that with increasing specific light availability, the maximal chrysolaminarin content increases. However, this effect is limited. Beyond a certain specific light availability (here: 5 µmolphotons gDW -1 s-1 ) the maximal chrysolaminarin content no longer increases, but the rate of increase becomes faster. Furthermore, the conversion of light to chrysolaminarin is best at the beginning of nitrogen depletion. Additionally, our results show that a high biomass concentration has a negative effect on the maximal chrysolaminarin content, most likely due to the occurring self-shading effects.

A novel model extended from the Bouguer-Lambert-Beer law can describe the non-linear absorbance of potassium dichromate solutions and microalgae suspensions.

Introduction: The Bouguer-Lambert-Beer law is widely used as the fundamental equation for quantification in absorption spectroscopy. However, deviations from the Bouguer-Lambert-Beer law have also been observed, such as chemical deviation and light scattering effect. While it has been proven and shown that the Bouguer-Lambert-Beer law is valid only under very restricted limitations, there are only a few alternatives of analytical models to this law. Based on the observation in the experiments, we propose a novel model to solve the problem of chemical deviation and light scattering effect. Methods: To test the proposed model, a systematic verification was conducted using potassium dichromate solutions and two types of microalgae suspensions with varying concentrations and path lengths. Results: Our proposed model demonstrated excellent performance, with a correlation coefficient ( R 2 ) exceeding 0.995 for all tested materials, significantly surpassing the Bouguer-Lambert-Beer law, which had an R 2 as low as 0.94. Our results confirm that the absorbance of pure pigment solutions follows the Bouguer-Lambert-Beer law, while the microalgae suspensions do not due to the light scattering effect. We also show that this scattering effect leads to huge deviations for the commonly used linear scaling of the spectra, and we provide a better solution based on the proposed model. Discussion: This work provides a powerful tool for chemical analysis and especially for the quantification of microorganisms, such as the concentration of biomass or intracellular biomolecules. Not only the high accuracy but also the simplicity of the model makes it a practical alternative to the existing Bouguer-Lambert-Beer law.

Improving microalgae growth modeling of outdoor cultivation with light history data using machine learning models: A comparative study.

Accurate prediction of microalgae growth is crucial for understanding the impacts of light dynamics and optimizing production. Although various mathematical models have been proposed, only a few of them have been validated in outdoor cultivation. This study aims to investigate the use of machine learning algorithms in microalgae growth modeling. Outdoor cultivation data of Phaeodactylum tricornutum in flat-panel airlift photobioreactors for 50 days were used to compare the performance of Long Short-Term Memory (LSTM) and Support Vector Regression (SVR) with traditional models, namely Monod and Haldane. The results indicate that the machine learning models outperform the traditional models due to their ability to utilize light history as input. Moreover, the LSTM model shows an excellent ability to describe the light acclimation effect. Last, two potential applications of these models are demonstrated: 1) use as a biomass soft sensor and 2) development of an optimal harvest strategy for outdoor cultivation.

Preparation of multifunctional hydrogels with accessible isothiouronium groups via radical cross-linking copolymerization.

Hydrogels can be equipped with functional groups for specific purposes. Isothiouronium groups can enhance adsorptivity, or allow coupling of other functional groups through mild reactions after transformation to thiol groups. Here we present a method to prepare multifunctional hydrogels by introducing isothiouronium groups into poly(ethylene glycol) diacrylate (PEGDA) hydrogels, and convert them into thiol-functionalized hydrogels by the reduction of the isothiouronium groups. For this purpose, the amphiphilic monomer 2-(11-(acryloyloxy)-undecyl)isothiouronium bromide (AUITB), containing an isothiouronium group, was synthesized and copolymerized with PEGDA. In this convenient way, it was possible to incorporate up to 3 wt% AUITB into the hydrogels without changing their equilibrium swelling degree. The successful functionalization was demonstrated by surface analysis of the hydrogels with water contact angle measurements and increased isoelectric points of the hydrogel surfaces from 4.5 to 9.0 due to the presence of the isothiouronium groups. The hydrogels showed a suitability as an adsorbent, as exemplified by the pronounced adsorption of the anionic drug diclofenac. The potential of the functionalization for (bio)conjugation reactions was demonstrated by the reduction of isothiouronium groups to thiols and subsequent immobilization of the functional enzyme horseradish peroxidase on the hydrogels. The results show that fully accessible isothiouronium groups can be introduced into radically cross-linked hydrogels.

On the reproducibility of extrusion-based bioprinting: round robin study on standardization in the field.

The outcome of three-dimensional (3D) bioprinting heavily depends, amongst others, on the interaction between the developed bioink, the printing process, and the printing equipment. However, if this interplay is ensured, bioprinting promises unmatched possibilities in the health care area. To pave the way for comparing newly developed biomaterials, clinical studies, and medical applications (i.e. printed organs, patient-specific tissues), there is a great need for standardization of manufacturing methods in order to enable technology transfers. Despite the importance of such standardization, there is currently a tremendous lack of empirical data that examines the reproducibility and robustness of production in more than one location at a time. In this work, we present data derived from a round robin test for extrusion-based 3D printing performance comprising 12 different academic laboratories throughout Germany and analyze the respective prints using automated image analysis (IA) in three independent academic groups. The fabrication of objects from polymer solutions was standardized as much as currently possible to allow studying the comparability of results from different laboratories. This study has led to the conclusion that current standardization conditions still leave room for the intervention of operators due to missing automation of the equipment. This affects significantly the reproducibility and comparability of bioprinting experiments in multiple laboratories. Nevertheless, automated IA proved to be a suitable methodology for quality assurance as three independently developed workflows achieved similar results. Moreover, the extracted data describing geometric features showed how the function of printers affects the quality of the printed object. A significant step toward standardization of the process was made as an infrastructure for distribution of material and methods, as well as for data transfer and storage was successfully established.

Optical sensors with molecularly imprinted nanospheres: a promising approach for robust and label-free detection of small molecules.

Molecularly imprinted nanospheres obtained by miniemulsion polymerization have been applied as the sensitive layer for label-free direct optical sensing of small molecules. Using these particles as the sensitive layer allowed for improving response times in comparison to sensors using MIP layers. As a model compound, well-characterized nanospheres imprinted against L-Boc-phenylalanine anilide (L-BFA) were chosen. For immobilization, a simple concept based on electrostatic adsorption was used, showing its applicability to different types of surfaces, leading to a good surface coverage. The sensor showed short response times, good selectivity, and high reversibility with a limit of detection down to 60 µM and a limit of quantitation of 94 µM. Furthermore, reproducibility, selectivity, and long-term stability of the sensitive layers were tested. The best results were achieved with an adsorption on aminopropylsilane layers, showing a chip-to-chip reproducibility of 22%. Furthermore, the sensors showed no loss in signal after a storage time of 1 year.

Stiff gelatin hydrogels can be photo-chemically synthesized from low viscous gelatin solutions using molecularly functionalized gelatin with a high degree of methacrylation.

Gelatin is a very promising matrix material for in vitro cell culture and tissue engineering, e.g. due to its native RGD content. For the generation of medical soft tissue implants chemical modification of gelatin improves the mechanical properties of gelatin hydrogels and the viscous behavior of gelatin solutions for liquid handling. We present a systematic study on the influence of high degrees of methacrylation on the properties of gelatin solutions and photo-chemically crosslinked hydrogels. Changes from shear thinning to shear thickening behavior of gelatin solutions were observed depending on mass fraction and degree of methacrylation. Degrees of swelling of crosslinked hydrogels ranged from 194 to 770 % and storage moduli G' from 368 to 5 kPa, comparable to various natural tissues including several types of cartilage. Crosslinked gels proofed to be cytocompatible according to extract testings based on DIN ISO 10933-5 and in contact with porcine chondrocytes.

Evaluation of novel biomaterials for cartilage regeneration based on gelatin methacryloyl interpenetrated with extractive chondroitin sulfate or unsulfated biotechnological chondroitin.

Gelatin is widely proposed as scaffold for cartilage tissue regeneration due to its high similarities to the extracellular matrix. However, poor mechanical properties and high sensitivity to enzymatic degradation encouraged the scientific community to develop strategies to obtain better performing hydrogels. Gelatin networks, specifically gelatin-methacryloyl (GM), have been coupled to hyaluronan or chondroitin sulfate (CS). In this study, we evaluated the biophysical properties of an innovative photocross-linked hydrogel based on GM with the addition of CS or a new unsulfated biotechnological chondroitin (BC). Biophysical, mechanical, and biochemical characterization have been assessed to compare GM hydrogels to the chondroitin containing networks. Moreover, mesenchymal stem cells (MSCs) were seeded on these biomaterials in order to evaluate the differentiation toward the chondrocyte phenotype in 21 days. Rheological characterization showed that both CS and BC increased the stiffness (G' was about 2-fold), providing a stronger rigid matrix, with respect to GM alone. The biological tests confirmed the onset of MSCs differentiation process starting from 14 days of in vitro culture. In particular, the combination GM + BC resulted to be more effective than GM + CS in the up-regulation of key genes such as collagen type 2A1 (COLII), SOX-9, and aggrecan). In addition, the scanning microscope analyses revealed the cellular adhesion on materials and production of extracellular vesicles. Immunofluorescence staining confirmed an increase of COLII in presence of both chondroitins. Finally, the outcomes suggest that BC entangled within cross-linked GM matrix may represent a promising new biomaterial with potential applications in cartilage regeneration.

Analytical and toxicological aspects of nanomaterials in different product groups: Challenges and opportunities.

The widespread integration of engineered nanomaterials into consumer and industrial products creates new challenges and requires innovative approaches in terms of design, testing, reliability, and safety of nanotechnology. The aim of this review article is to give an overview of different product groups in which nanomaterials are present and outline their safety aspects for consumers. Here, release of nanomaterials and related analytical challenges and solutions as well as toxicological considerations, such as dose-metrics, are discussed. Additionally, the utilization of engineered nanomaterials as pharmaceuticals or nutraceuticals to deliver and release cargo molecules is covered. Furthermore, critical pathways for human exposure to nanomaterials, namely inhalation and ingestion, are discussed in the context of risk assessment. Analysis of NMs in food, innovative medicine or food contact materials is discussed. Specific focus is on the presence and release of nanomaterials, including whether nanomaterials can migrate from polymer nanocomposites used in food contact materials. With regard to the toxicology and toxicokinetics of nanomaterials, aspects of dose metrics of inhalation toxicity as well as ingestion toxicology and comparison between in vitro and in vivo conclusions are considered. The definition of dose descriptors to be applied in toxicological testing is emphasized. In relation to potential exposure from different products, opportunities arising from the use of advanced analytical techniques in more unique scenarios such as release of nanomaterials from medical devices such as orthopedic implants are addressed. Alongside higher product performance and complexity, further challenges regarding material characterization and safety, as well as acceptance by the general public are expected.

Azido-functionalized gelatin via direct conversion of lysine amino groups by diazo transfer as a building block for biofunctional hydrogels.

Gelatin is one of the most prominent biopolymers in biomedical material research and development. It is frequently used in hybrid hydrogels, which combine the advantageous properties of bio-based and synthetic polymers. To prevent the biological component from leaching out of the hydrogel, the biomolecules can be equipped with azides. Those groups can be used to immobilize gelatin covalently in hydrogels by the highly selective and specific azide-alkyne cycloaddition. In this contribution, we functionalized gelatin with azides at its lysine residues by diazo transfer, which offers the great advantage of only minimal side-chain extension. Approximately 84-90% of the amino groups are modified as shown by 1 H-NMR spectroscopy, 2,4,6-trinitrobenzenesulfonic acid assay as well as Fourier-transform infrared spectroscopy, rheology, and the determination of the isoelectric point. Furthermore, the azido-functional gelatin is incorporated into hydrogels based on poly(ethylene glycol) diacrylate (PEG-DA) at different concentrations (0.6, 3.0, and 5.5%). All hydrogels were classified as noncyctotoxic with significantly enhanced cell adhesion of human fibroblasts on their surfaces compared to pure PEG-DA hydrogels. Thus, the new gelatin derivative is found to be a very promising building block for tailoring the bioactivity of materials.

Differentiation of physical and chemical cross-linking in gelatin methacryloyl hydrogels.

Gelatin methacryloyl (GM) hydrogels have been investigated for almost 20 years, especially for biomedical applications. Recently, strengthening effects of a sequential cross-linking procedure, whereby GM hydrogel precursor solutions are cooled before chemical cross-linking, were reported. It was hypothesized that physical and enhanced chemical cross-linking of the GM hydrogels contribute to the observed strengthening effects. However, a detailed investigation is missing so far. In this contribution, we aimed to reveal the impact of physical and chemical cross-linking on strengthening of sequentially cross-linked GM and gelatin methacryloyl acetyl (GMA) hydrogels. We investigated physical and chemical cross-linking of three different GM(A) derivatives (GM10, GM2A8 and GM2), which provided systematically varied ratios of side-group modifications. GM10 contained the highest methacryloylation degree (DM), reducing its ability to cross-link physically. GM2 had the lowest DM and showed physical cross-linking. The total modification degree, determining the physical cross-linking ability, of GM2A8 was comparable to that of GM10, but the chemical cross-linking ability was comparable to GM2. At first, we measured the double bond conversion (DBC) kinetics during chemical GM(A) cross-linking quantitatively in real-time via near infrared spectroscopy-photorheology and showed that the DBC decreased due to sequential cross-linking. Furthermore, results of circular dichroism spectroscopy and differential scanning calorimetry indicated gelation and conformation changes, which increased storage moduli of all GM(A) hydrogels due to sequential cross-linking. The data suggested that the total cross-link density determines hydrogel stiffness, regardless of the physical or chemical nature of the cross-links.

Influence of Hard Segment Content and Diisocyanate Structure on the Transparency and Mechanical Properties of Poly(dimethylsiloxane)-Based Urea Elastomers for Biomedical Applications.

The effect of hard segment content and diisocyanate structure on the transparency and mechanical properties of soft poly(dimethylsiloxane) (PDMS)-based urea elastomers (PSUs) was investigated. A series of PSU elastomers were synthesized from an aminopropyl-terminated PDMS (M¯n: 16,300 g·mol-1), which was prepared by ring chain equilibration of the monomers octamethylcyclotetrasiloxane (D4) and 1,3-bis(3-aminopropyl)-tetramethyldisiloxane (APTMDS). The hard segments (HSs) comprised diisocyanates of different symmetry, i.e., 4,4'-methylenebis(cyclohexyl isocyanate) (H12MDI), 4,4'-methylenebis(phenyl isocyanate) (MDI), isophorone diisocyanate (IPDI), and trans-1,4-cyclohexane diisocyanate (CHDI). The HS contents of the PSU elastomers based on H12MDI and IPDI were systematically varied between 5% and 20% by increasing the ratio of the diisocyanate and the chain extender APTMDS. PSU copolymers of very low urea HS contents (1.0-1.6%) were prepared without the chain extender. All PSU elastomers and copolymers exhibited good elastomeric properties and displayed elongation at break values between 600% and 1100%. The PSUs with HS contents below 10% were transparent and became increasingly translucent at HS contents of 15% and higher. The Young's modulus (YM) and ultimate tensile strength values of the elastomers increased linearly with increasing HS content. The YM values differed significantly among the PSU copolymers depending on the symmetry of the diisocyanate. The softest elastomer was that based on the asymmetric IPDI. The elastomers synthesized from H12MDI and MDI both exhibited an intermediate YM, while the stiffest elastomer, i.e., that comprising the symmetric CHDI, had a YM three-times higher than that prepared with IPDI. The PSUs were subjected to load-unload cycles at 100% and 300% strain to study the influence of HS morphology on 10-cycle hysteresis behavior. At 100% strain, the first-cycle hysteresis values of the IPDI- and H12MDI-based elastomers first decreased to a minimum of approximately 9-10% at an HS content of 10% and increased again to 22-28% at an HS content of 20%. A similar, though less pronounced, trend was observed at 300% strain. First-cycle hysteresis among the PSU copolymers at 100% strain was lowest in the case of CHDI and highest in the IPDI-based elastomer. However, this effect was reversed at 300% strain, with CHDI displaying the highest hysteresis in the first cycle. In vitro cytotoxicity tests performed using HaCaT cells did not show any adverse effects, revealing their potential suitability for biomedical applications.

Nano-in-Micro-Particles Consisting of PLGA Nanoparticles Embedded in Chitosan Microparticles via Spray-Drying Enhances Their Uptake in the Olfactory Mucosa.

Intranasal delivery has gained prominence since 1990, when the olfactory mucosa was recognized as the window to the brain and the central nervous system (CNS); this has enabled the direct site specific targeting of neurological diseases for the first time. Intranasal delivery is a promising route because general limitations, such as the blood-brain barrier (BBB) are circumvented. In the treatment of multiple sclerosis (MS) or Alzheimer's disease, for example, future treatment prospects include specialized particles as delivery vehicles. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are well known as promising delivery systems, especially in the area of nose-to-brain (N2B) delivery. Chitosan is also broadly known as a functional additive due to its ability to open tight junctions. In this study, we produced PLGA nanoparticles of different sizes and revealed for the first time their size-time-dependent uptake mechanism into the lamina propria of porcine olfactory mucosa. The intracellular uptake was observed for 80 and 175 nm within only 5 min after application to the epithelium. After 15 min, even 520 nm particles were detected, associated with nuclei. Especially the presence of only 520 nm particles in neuronal fibers is remarkable, implying transcellular and intracellular transport via the olfactory or the trigeminal nerve to the brain and the CNS. Additionally, we developed successfully specialized Nano-in-Micro particles (NiMPs) for the first time via spray drying, consisting of PLGA nanoparticles embedded into chitosan microparticles, characterized by high encapsulation efficiencies up to 51%, reproducible and uniform size distribution, as well as smooth surface. Application of NiMPs accelerated the uptake compared to purely applied PLGA nanoparticles. NiMPs were spread over the whole transverse section of the olfactory mucosa within 15 min. Faster uptake is attributed to additional paracellular transport, which was examined via tight-junction-opening. Furthermore, a separate chitosan penetration gradient of ∼150 µm caused by dissociation from PLGA nanoparticles was observed within 15 min in the lamina propria, which was demonstrated to be proportional to an immunoreactivity gradient of CD14. Due to the beneficial properties of the utilized chitosan-derivative, regarding molecular weight (150-300 kDa), degree of deacetylation (80%), and particle size (0.1-10 µm) we concluded that M2-macrophages herein initiated an anti-inflammatory reaction, which seems to already take place within 15 min following chitosan particle application. In conclusion, we demonstrated the possibility for PLGA nanoparticles, as well as for chitosan NiMPs, to take all three prominent intranasal delivery pathways to the brain and the CNS; namely transcellular, intracellular via neuronal cells, and paracellular transport.

Eclectic characterisation of chemically modified cell-derived matrices obtained by metabolic glycoengineering and re-assessment of commonly used methods.

Azide-bearing cell-derived extracellular matrices ("clickECMs") have emerged as a highly exciting new class of biomaterials. They conserve substantial characteristics of the natural extracellular matrix (ECM) and offer simultaneously small abiotic functional groups that enable bioorthogonal bioconjugation reactions. Despite their attractiveness, investigation of their biomolecular composition is very challenging due to the insoluble and highly complex nature of cell-derived matrices (CDMs). Yet, thorough qualitative and quantitative analysis of the overall material composition, organisation, localisation, and distribution of typical ECM-specific biomolecules is essential for consistent advancement of CDMs and the understanding of the prospective functions of the developed biomaterial. In this study, we evaluated frequently used methods for the analysis of complex CDMs. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and (immune)histochemical staining methods in combination with several microscopic techniques were found to be highly eligible. Commercially available colorimetric protein assays turned out to deliver inaccurate information on CDMs. In contrast, we determined the nitrogen content of CDMs by elementary analysis and converted it into total protein content using conversion factors which were calculated from matching amino acid compositions. The amount of insoluble collagens was assessed based on the hydroxyproline content. The Sircol™ assay was identified as a suitable method to quantify soluble collagens while the Blyscan™ assay was found to be well-suited for the quantification of sulphated glycosaminoglycans (sGAGs). Eventually, we propose a series of suitable methods to reliably characterise the biomolecular composition of fibroblast-derived clickECM.

Azide-Functional Extracellular Matrix Coatings as a Bioactive Platform for Bioconjugation.

In recent years, the development and application of decellularized extracellular matrices (ECMs) for use as biomaterials have grown rapidly. These cell-derived matrices (CDMs) represent highly bioactive and biocompatible materials consisting of a complex assembly of biomolecules. Even though CDMs mimic the natural microenvironment of cells in vivo very closely, they still lack specifically addressable functional groups, which are often required to tailor a biomaterial functionality by bioconjugation. To overcome this limitation, metabolic glycoengineering has emerged as a powerful tool to equip CDMs with chemical groups such as azides. These small chemical handles are known for their ability to undergo bioorthogonal click reactions, which represent a desirable reaction type for bioconjugation. However, ECM insolubility makes its processing very challenging. In this contribution, we isolated both the unmodified ECM and azide-modified clickECM by osmotic lysis. In a first step, these matrices were concentrated to remove excessive water from the decellularization step. Next, the hydrogel-like ECM and clickECM films were mechanically fragmentized, resulting in easy to pipette suspensions with fragment sizes ranging from 7.62 to 31.29 µm (as indicated by the mean d90 and d10 values). The biomolecular composition was not impaired as proven by immunohistochemistry. The suspensions were used for the reproducible generation of surface coatings, which proved to be homogeneous in terms of ECM fragment sizes and coating thicknesses (the mean coating thickness was found to be 33.2 ± 7.3 µm). Furthermore, they were stable against fluid-mechanical abrasion in a laminar flow cell. When primary human fibroblasts were cultured on the coated substrates, an increased bioactivity was observed. By conjugating the azides within the clickECM coatings with alkyne-coupled biotin molecules, a bioconjugation platform was obtained, where the biotin-streptavidin interaction could be used. Its applicability was demonstrated by equipping the bioactive clickECM coatings with horseradish peroxidase as a model enzyme.