A Critical View at the Analysis of Chitosan Characteristics: Towards Biomedical Application.

Event Abstract Back to Event Human macrophages release higher IL-1ra over IL-1beta when stimulated by block acetylated chitosan microparticles and not by random acetylated chitosans or water-soluble oligomers David Fong1, Pascal Grégoire-Gélinas2, Marc Lavertu1, 2, Sachiko Sato3 and Caroline D. Hoemann1, 2 1 École Polytechnique de Montréal, Institute of Biomedical Engineering, Canada 2 École Polytechnique de Montréal, Department of Chemical Engineering, Canada 3 Laval University, Department of Microbiology and Immunology, and Research Centre for Infectious Diseases, Canada Introduction: Human macrophages were previously shown to release excess IL-1ra over IL-1β when stimulated by chitosan microparticles (140 kDa, 80% glucosamine and 20% block N-acetyl glucosamine, 80% degree of deacetylation (DDA))[1]. These data suggest the potential of using chitosan to treat knee joint inflammation by guiding the ­in situ release of immunomodulatory cytokines. The purpose of this study was to identify the minimal chitosan motif required to induce higher IL-1ra release, using a U937 human macrophage model and a novel chitosan library with distinct DDA, acetylation patterns, and array of 5 different number-average molecular weights (Mn). Materials and Methods: 15 chitosans with different Mn, DDA, and block (B) or random (R) acetylation pattern were generated, including acid-soluble ≥ 140 kDa and 10 kDa chitosans, and water-soluble 1, 3, 5 kDa oligomers (Table 1). B-acetylated (80% DDA, Mn=190 kDa) or fully deacetylated (98% DDA, Mn=140 kDa) chitosans were nitrous acid depolymerized. 98% DDA chitosans were reacetylated to 60% or 80% DDA to obtain R-acetylated chitosans. Chitosan Mn and polydispersity were determined by size exclusion chromatography and DDA by H1 NMR. Phorbol ester-differentiated U937 macrophages were stimulated for 24 hours with 5, 50 or 150 µg/mL chitosans that form particles in culture medium at an Mn ≥ 10 kDa , or with LPS and IL-4 as controls. Cell culture medium was analyzed for cytokine release by ELISA (N=3). Cell cytotoxicity was determined by lactate dehydrogenase release. Results: Macrophages with no stimulation released high levels of IL-1ra (5364±1720 pg/mL) and negligible levels of IL-1β (56±6 pg/mL). Amongst all chitosans, only the B-acetylated 80% DDA 190 kDa chitosan (80-190K-B) stimulated a reproducible 2 to 3-fold increase in IL-1ra release at selected doses (Fig. 1A). The B-acetylated 80% DDA 10 and 190 kDa chitosans stimulated a 2 to 5-fold increase in IL-1β release (Fig. 1B). In contrast, R-acetylated 80% DDA 140 kDa (80-140K-R) chitosan induced a modest 2-fold increase in IL-1β without further enhancing IL-1ra release (Fig. 1). All other chitosans had no effects on cytokine release at any dose. Cell viability remained over 80% under all conditions. Discussion: The failure of the 1 to 5 kDa B-acetylated chitosans to induce any cytokine release indicates that there is a minimal Mn required for chitosan to stimulate IL-1β in macrophages and suggests full cytocompatibility of chitosan oligomers at 150 µg/mL. At 80% DDA and above 10 kDa Mn, the B-acetylation pattern contributes to stimulating IL-1β release. These data are consistent with previous report suggesting that chitosan microparticle formation is necessary to activate the inflammasome and stimulate IL-1β liberation from macrophages[2]. The ability of the 80-190K-B, and not the 80-140K-R, to enhance IL-1ra release indicates that the B-acetylation pattern is necessary to shift macrophages towards a more anabolic phenotype. Conclusion: A novel, comprehensive chitosan library was successfully generated to evaluate the impact of chitosan Mn, DDA and acetylation pattern on cytokine release in human macrophages. Water-soluble chitosan oligomers had no influence on IL-1ra and IL-1β release. Above 100 kDa, 80% DDA B-acetylated but not R-acetylated chitosans stimulated more IL-1ra, suggesting that B-acetylated chitosans are more useful for inducing the release of anti-inflammatory factors that preserve joint health. This work was funded by the Canadian Institutes of Health Research. Salary support was from the Fonds de recherche du Québec - Santé (FRQ-S, CDH), Fonds de recherche du Québec - Nature et Technologies (FRQ-NT, DF) and the Natural Sciences and Engineering Research Council (NSERC, PGG).

Chitosan is a cationic polysaccharide that has been evaluated as an adjuvant due to its biocompatible and biodegradable nature. The polysaccharide can enhance antibody responses and cell-mediated immunity following vaccination by injection or mucosal routes. However, the optimal polymer characteristics for activation of dendritic cells (DCs) and induction of antigen-specific cellular immune responses have not been resolved. Here, we demonstrate that only chitin-derived polymers with a high degree of deacetylation (DDA) enhance generation of mitochondrial reactive oxygen species (mtROS), leading to cGAS-STING mediated induction of type I IFN. Additionally, the capacity of the polymers to activate the NLRP3 inflammasome was strictly dependent on the degree and pattern of deacetylation and mtROS generation. Polymers with a DDA below 80% are poor adjuvants while a fully deacetylated polyglucosamine polymer is most effective as a vaccine adjuvant. Furthermore, this polyglucosamine polymer enhanced antigen-specific Th1 responses in a NLRP3 and STING-type I IFN-dependent manner. Overall these results indicate that the degree of chitin deacetylation, the acetylation pattern and its regulation of mitochondrial ROS are the key determinants of its immune enhancing effects.

Chitosan is a natural polymer revealing an increased potential to be used in different biomedical applications, including drug delivery systems, and tissue engineering. It implies the evaluation of the organism response to the biomaterial implantation. Low-molecular degradation products, the chito-oligomers, are resulting mainly from the influence of enzymes, which are found in the organism fluids. Within this study, we have performed the computational assessment of pharmacological profiles and toxicological effects on human health of small chito-oligomers with distinct molecular weights, deacetylation degrees, and acetylation patterns. Our approach is based on the fact that regulatory agencies and researchers in the drug development field rely on the use of modeling to predict biological effects and to guide decision making. To be considered as valid for regulatory purposes, every model that is used for predictions should be associated with a defined toxicological endpoint and has appropriate robustness and predictivity. Within this context, we have used FAF-Drugs4, SwissADME, and PreADMET tools to predict the oral bioavailability of chito-oligomers and SwissADME, PreADMET, and admetSAR2.0 tools to predict their pharmacokinetic profiles. The organs and genomic toxicities have been assessed using admetSAR2.0 and PreADMET tools but specific computational facilities have been also used for predicting different toxicological endpoints: Pred-Skin for skin sensitization, CarcinoPred-EL for carcinogenicity, Pred-hERG for cardiotoxicity, ENDOCRINE DISRUPTOME for endocrine disruption potential and Toxtree for carcinogenicity and mutagenicity. Our computational assessment showed that investigated chito-oligomers reflect promising pharmacological profiles and limited toxicological effects on humans, regardless of molecular weight, deacetylation degree, and acetylation pattern. According to our results, there is a possible inhibition of the organic anion transporting peptides OATP1B1 and/or OATP1B3, a weak potential of cardiotoxicity, a minor probability of affecting the androgen receptor, and phospholipidosis. Consequently, these results may be used to guide or to complement the existing in vitro and in vivo toxicity tests, to optimize biomaterials properties and to contribute to the selection of prototypes for nanocarriers.

Nanomaterials offer promising solutions for chemotherapy challenges, addressing issues like cytotoxicity and biocompatibility. In cancer clinical protocols, biomedical imaging is vital, providing insights into tumor morphology. Luminescent nanomaterials or nanoparticles (LNPs), particularly effective for diseases like cancer, possess controllable properties like size (usually <100 nm), surface charge, and external functionalization. LNPs interact with biological systems at systemic and cellular levels. Cellular uptake is crucial, allowing selective targeting of cancer cells through overexpressed surface receptors such as transferrin receptor (TfR), G-protein coupled receptor (GPCR), folate receptor (FR), epidermal growth factor receptor (EGFR), lectins, and low-density lipoprotein receptor (LDLR). LNPs can accumulate in subcellular compartments, playing a pivotal role in drug delivery. Studies explore LNPs’ internalization into cells, investigating their potential to deliver cargoes like DNA, siRNA, miRNA, and small-molecule drugs. This review highlights the latest advancements in LNPs and their biomedical applications. Despite these promising developments, comprehensive nanotoxicological assessments are crucial for a better understanding of LNPs’ behavior in biological systems, paving the way for future clinical applications.

Modeling acclimatization by hybrid systems: condition changes alter biological system behavior models.

Since biopolymers are so ecologically benign, sustainable, and biocompatible, they have emerged as a significant class of polymers in the field of polymer sciences, especially for a variety of biological applications. In aqueous acidic circumstances, Chitosan (CS) is generated when 50% of the 1, 4-glycosidic-linked d-glucosamine and N-acetyl-d-glucosamine units are deacetylated with different molecular weights (Mw), acetylation patterns (PA), and degrees of deacetylation (DD). Because of its remarkable chemical and biological properties, Chitosan nanoparticles (CNPs) have gained significant importance in a range of pharmaceutical and biomedical applications, such as drug delivery, gene transport, tissue engineering, biosensing, etc. CS can be utilized in applications where the sensor may come into touch with biological materials or living tissues because it is non-toxic and biocompatible. The manuscript reviews the newer studies of Chitosan-based nanoparticles (CNPs) in the form of hydrogels, micelles, nanospheres, selenium nanoparticles, etc. In order to improve overall analytical performance, this review paper highlights the production and recent applications, current state, and challenges of CS-derived nanoparticles in the fields of drug delivery for treating different types of cancers, diabetes management, anti-microbial activity, and moreover for biosensing of blood lactate, histamine, and glucose. Preparation and applications of Chitosan-based nanoparticles in drug delivery and biosensing.

Carbon materials and their hybrid metal composites have garnered significant attention in biomedical applications due to their exceptional biocompatibility. This biocompatibility arises from their inherent chemical stability and low toxicity within biological systems. This review offers a comprehensive overview of carbon nanomaterials and their metal composites, emphasizing their biocompatibility-focused applications, including drug delivery, bioimaging, biosensing, and tissue engineering. The paper outlines advancements in surface modifications, coatings, and functionalization techniques designed to enhance the biocompatibility of carbon materials, ensuring minimal adverse effects in biological systems. A comprehensive investigation into hybrid composites integrating carbon nanomaterials is conducted, categorizing them as fullerenes, carbon quantum dots, carbon nanotubes, carbon nanofibers, graphene, and diamond-like carbon. The concluding section addresses regulatory considerations and challenges associated with integrating carbon materials into medical devices. This review culminates by providing insights into current achievements, challenges, and future directions, underscoring the pivotal role of carbon nanomaterials and their metal composites in advancing biocompatible applications.

Chitosan is a biomaterial with a range of current and potential biomedical applications. Manipulation of chitosan degree of deacetylation (DDA) to achieve specific properties appears feasible, but studies investigating its influence on properties are often contradictory. With a view to the potential of chitosan in the regeneration of nerve tissue, the influence of DDA on the growth and health of olfactory ensheathing cells (OECs) was investigated. There was a linear increase in OEC proliferation as the DDA increased from 72 to 85%. This correlated with linear increases in average surface roughness (0.62 to 0.78 μm) and crystallinity (4.3 to 10.1%) of the chitosan films. Mitochondrial activity and membrane integrity of OECs was significantly different for OECs cultivated on chitosan with DDAs below 75%, while those on films with DDAs up to 85% were similar to cells in asynchronous growth. Apoptotic indices and cell cycle analysis also suggested that chitosan films with DDAs below 75% were cytocompatible but induced cellular stress, while OECs grown on films fabricated from chitosan with DDAs above 75% showed no significant differences compared to those in asynchronous growth. Tensile strength and elongation to break varied with DDA from 32.3 to 45.3 MPa and 3.6 to 7.1% respectively. DDA had no significant influence on abiotic and biotic degradation profiles of the chitosan films which showed approximately 8 and 20% weight loss respectively. Finally, perceived patterns in property changes are subject to change based on potential variations in DDA analysis. NMR examination of the chitosan samples here revealed significant differences depending upon which peaks were selected for integration; 6 to 13% in DDA values within individual samples. Furthermore, differences between DDA values determined here and those reported by the commercial suppliers were significant and this may also be a source of concern when selecting commercial chitosans for biomaterial research.

Tuning chitosan’s chemical structure for enhanced biological functions

Modeling framework for membrane computing in biological systems: Evaluation with a case study

A biological-like controller using improved spiking neural networks

An important influence of new technology on medical practice is derived from the development and application of biomaterials. The close association between the development of biomaterials and that of blood detoxification devices (Klinkmann, 1984) means that a continuing influence of new technology on extracorporeal blood purification must be expected. Successful utilisation of new technology could enable the extension of the clinical application of extracorporeal blood purification from established procedures such as the artificial kidney into other areas. One such area is that of artificial liver support.

Nanoparticles are known to have a high specific surface area, which accounts for an increased probability of their interaction with bacterial cells. Therefore, the application of silver(I) nanoparticles (AgNPs) and their nanocomposites as antimicrobial agents against drug-resistant bacterial strains appears to be prospective. A critical point for the advancement of AgNPs into clinical practice is a fundamental understanding of their behavior in biological systems, including protein binding and interaction with blood components, which reflects their toxicity. The latter is primarily determined by the physicochemical properties of AgNPs, namely their size, shape, surface chemistry, etc. Therefore, nanotoxicity may be substantially reduced through the manipulation of certain physicochemical characteristics of AgNPs, increasing their biocompatibility and hence paving the way for possible biomedical applications. In this study we have focused on estimating the binding affinity of the synthesized Ag(I) complexes of 2-(4,6-di-tert-butyl-2,3-dihydroxyphenylsulfanyl)-acetic acid and 4,6-di-tert-butyl-2,3-dihydroxybenzaldehyde isonicotinoyl hydrazone, as well as AgNPs derived thereof to bovine serum albumin (BSA) and hemoglobin by the fluorimetric method. Furthermore, cellular toxicity of the AgNPs towards human erythrocytes was measured in a hemolysis assay. Organosols formed by the Ag(I) complexes upon their reduction to AgNPs in acetonitrile and DMSO were characterized by the transmission electron microscopy (TEM) method and atomic force microscopy (AFM).

Background: Macrophages are essential in maintaining tissue homeostasis. However, their functionality and phenotype can be impaired by senescence, which impacts immune competence and inflammatory responses. Despite the growing use of engineered nanoparticles (NPs) in biomedical applications or chronic exposure to environmental NPs, their impact on macrophage senescence and immune function remains poorly understood, particularly in the context of prolonged NP exposure. Results: Here, we first confirm that human monocyte-derived macrophages (MDMs) undergo senescence in vitro, as indicated by a panel of senescence-associated markers that increased within 10 days of culture. Then, we investigate how gold (AuNPs), silica (SiO2 NPs), and polyethylene terephthalate (PET NPs) influence senescence-associated markers, and immunocompetence in MDMs. By examining key markers such as senescence-associated beta-galactosidase (SA-β-gal), CDKN2A (p16), and senescence-associated secretory phenotype (SASP) cytokines, e.g., interleukin (IL)-6 and IL-8, over extended exposure periods up to 10 days, we revealed material-specific effects: AuNPs induce a strong pro-inflammatory response, SiO2 NPs demonstrate low inflammatory potential, and PET NPs modulate the gene expression level of CDKN2A involved in cell cycle regulation. Conclusion: Our findings underscore the need to characterize long-term NP behavior in biological systems and reveal material-specific effects on macrophage senescence-associated traits and immune function. Rather than assessing NP-induced senescence, this study defines how prolonged NP exposure modulates selected senescence-associated signatures in non-proliferative macrophages, offering valuable insights into the functional consequences of chronic NP accumulation.

Nanoparticle (NP) aggregation can not only change the unique properties of NPs but also affect NP transport and membrane penetration behavior in biological systems. Coarse-grained (CG) molecular dynamics (MD) simulations were performed in this work to investigate the aggregation behavior of NPs with different properties in ionic solutions under different temperature conditions. Four types of NPs and NP aggregates were modeled to analyze the effects of NP aggregation on NP translocation across the cell membrane at different temperatures. Hydrophilic modification and surface charge modification inhibited NP aggregation, whereas stronger hydrophobicity and higher temperature resulted in a higher degree of NP aggregation and a denser structure of NP aggregates. The final aggregation percentage of hydrophobic NPs in the NaCl solution at 37 °C is 87.5%, while that of hydrophilic NPs is 0%, and the time required for hydrophobic NPs to reach 85% aggregation percentage at 42 °C is 6 ns, while it is 9.2 ns at 25 °C. The counterions in the solution weakened the effect of surface charge modification, thereby realizing good dispersity. High temperature could promote the NP membrane penetration for the same NP, while it also could enhance the NP aggregation which would increase the difficulty in NP translocation across cell membrane, especially for the hydrophobic NPs. Therefore, suitable surface modification of NPs and temperature control should be comprehensively considered in promoting NP membrane penetration in biomedical applications.