The quantitative molecular weight-antimicrobial activity relationship for chitosan polymers, oligomers, and derivatives.

Chitosan and chitosan derivatives can kill pathogenic microorganisms including bacteria and fungi. The antimicrobial activity is dependent on the degree of acetylation, substituent structure, and molecular weight. Over the past four decades, numerous studies have endeavored to elucidate the relationship between molecular weight and the activity against microorganisms. However, investigators have reported divergent and, at times, conflicting conclusions. Here a bilinear equation is proposed, delineating the relationship between antimicrobial activity, defined as log (1/MIC), and the molecular weight of chitosan and chitosan derivatives. Three constants AMin, AMax, and CMW govern the shape of the curve determined by the equation. The constant AMin denotes the minimal activity expected as the molecular weight tends towards zero while AMax represents the maximal activity observed for molecular weights exceeding CMW, the critical molecular weight required for max activity. This equation was applied to analyze data from seven studies conducted between 1984 and 2019, which reported MIC (Minimum Inhibitory Concentration) values against bacteria and fungi for various molecular weights of chitosan and its derivatives. All the 29 datasets exhibited a good fit (R2 ≥ 0.5) and half excellent (R2 ≥ 0.95) fit to the equation. The CMW generally ranged from 4 to 10 KD for datasets with an excellent fit to the equation.

Optimizing N,N,N-trimethyl chitosan synthesis: A design of experiments (DoE) approach.

This study aimed to optimize the synthesis of trimethyl chitosan (TMC) with a high degree of N,N,N-trimethylation (DTM) through a one-step procedure, minimizing reagent use, reaction time, and avoiding O-methylation, using the Design of Experiments (DoE) approach. Initially, sequential designs were done. Following the determination of the initial conditions a Fractional Factorial Design was used, investigating methyl iodide (MeI) and NaHCO3 molar ratios, temperature, and reaction time on DTM. MeI and NaHCO3 molar ratios were found to be significant (p-values equal to 0.02 and 0.02, respectively), the reaction temperature (p = 0.04) displayed a non-linear effect, while the reaction time was found to be non-significant (p = 0.93). Finally, a Full Factorial Design was done to optimize temperature and base addition methods. Incremental addition of the base was determined to be feasible without affecting the DTM, thereby preventing any viscosity-related problems. DTM was achieved up to 72 % in a one-step procedure, with no O-methylation. These optimized conditions offer a cost-effective, one-step synthesis method for TMC production, holding significant promise for industrial applications by avoiding multistep reactions, ensuring minimal reagent use, and preventing O-methylation. The findings mark a substantial advancement in TMC synthesis, presenting a streamlined and efficient approach with substantial practical implications for process development.

Water-Soluble Quaternary and Protonable Basic Chitotriazolans: Synthesis by Click Chemistry Conversion of Chitosan Azides and Investigation of Antibacterial Activity.

The azide transfer reaction and copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) can be used to convert the amino groups in chitosan to triazole 1,2,3-moieties. The resulting polymer has been named chitotriazolan. This synthesis was performed with six different quaternary ammonium alkynes and three amine alkynes to obtain a series of nine water-soluble chitotriazolan derivatives. The structure and complete conversion of the azide were confirmed by FT-IR and proton NMR spectroscopy. The derivatives were investigated for antibacterial activity against S. aureus, E. faecalis, E. coli, and P. aeruginosa. The activity of the quaternized chitotriazolan derivatives varied depending on the structure of the quaternary moiety and the species of bacteria. The basic protonable derivatives were less active or inactive against the bacteria.

The European Polysaccharide Network of Excellence (EPNOE) research roadmap 2040: Advanced strategies for exploiting the vast potential of polysaccharides as renewable bioresources.

Polysaccharides are among the most abundant bioresources on earth and consequently need to play a pivotal role when addressing existential scientific challenges like climate change and the shift from fossil-based to sustainable biobased materials. The Research Roadmap 2040 of the European Polysaccharide Network of Excellence (EPNOE) provides an expert's view on how future research and development strategies need to evolve to fully exploit the vast potential of polysaccharides as renewable bioresources. It is addressed to academic researchers, companies, as well as policymakers and covers five strategic areas that are of great importance in the context of polysaccharide related research: (I) Materials & Engineering, (II) Food & Nutrition, (III) Biomedical Applications, (IV) Chemistry, Biology & Physics, and (V) Skills & Education. Each section summarizes the state of research, identifies challenges that are currently faced, project achievements and developments that are expected in the upcoming 20 years, and finally provides outlines on how future research activities need to evolve.

Nano-based formulations of curcumin: elucidating the potential benefits and future prospects in skin cancer.

Skin cancer refers to any malignant lesions that occur in the skin and are observed predominantly in populations of European descent. Conventional treatment modalities such as excision biopsy, chemotherapy, radiotherapy, immunotherapy, electrodesiccation, and photodynamic therapy (PDT) induce several unintended side effects which affect a patient's quality of life and physical well-being. Therefore, spice-derived nutraceuticals like curcumin, which are well tolerated, less expensive, and relatively safe, have been considered a promising agent for skin cancer treatment. Curcumin, a chemical constituent extracted from the Indian spice, turmeric, and its analogues has been used in various mammalian cancers including skin cancer. Curcumin has anti-neoplastic activity by triggering the process of apoptosis and preventing the multiplication and infiltration of the cancer cells by inhibiting some signaling pathways and thus subsequently preventing the process of carcinogenesis. Curcumin is also a photosensitizer and has been used in PDT. The major limitations associated with curcumin are poor bioavailability, instability, limited permeation into the skin, and lack of solubility in water. This will constrain the use of curcumin in clinical settings. Hence, developing a proper formulation that can ideally release curcumin to its targeted site is important. So, several nanoformulations based on curcumin have been established such as nanogels, nanoemulsions, nanofibers, nanopatterned films, nanoliposomes and nanoniosomes, nanodisks, and cyclodextrins. The present review mainly focuses on curcumin and its analogues as therapeutic agents for treating different types of skin cancers. The significance of using various nanoformulations as well non-nanoformulations loaded with curcumin as an effective treatment modality for skin cancer is also emphasized.

Asymmetric Phenyl Substitution: An Effective Strategy to Enhance the Photosensitizing Potential of Curcuminoids.

Curcumin has been demonstrated to exhibit photosensitized bactericidal activity. However, the full exploitation of curcumin as a photo-pharmaceutical active principle is hindered by fast deactivation of the excited state through the transfer of the enol proton to the keto oxygen. Introducing an asymmetry in the molecular structure through acting on the phenyl substituents is expected to be a valuable strategy to impair this undesired de-excitation mechanism competing with the therapeutically relevant ones. In this study, two asymmetric curcumin analogs were synthesized and characterized as to their electronic-state transition spectroscopic properties. Fluorescence decay distributions were also reconstructed. Their analysis confirmed the substantial stabilization of the fluorescent state with respect to the parent compound. Nuclear magnetic resonance experiments were performed with the aim of determining the structural features of the keto-enol ring and the strength of the keto-enol hydrogen bond. Electronic structure calculations were also undertaken to elucidate the effects of substitution on the features of the keto-enol semi-aromatic system and the proneness to proton transfer. Finally, their singlet oxygen-generation efficiency was compared to that of curcumin through the 9,10-dimethylanthracene fluorescent assay.

Antibacterial properties of poly (N,N-dimethylaminoethyl methacrylate) obtained at different initiator concentrations in solution polymerization.

The samples of poly(N,N-dimethylaminoethyl methacrylate) were synthesized by radical polymerization. The amount of monomer and solvent was constant as opposed to an amount of initiator which was changing. No clear relationship between polymerization conditions and the molecular weight of the polymer was found, probably due to the branched configuration of produced polymer. Bactericidal interactions in all samples against Gram-positive and Gram-negative bacteria have been demonstrated. However, the observed effect has various intensities, depending on the type of bacteria and the type of sample.

Chitosan-hydroxycinnamic acid conjugates: Optimization of the synthesis and investigation of the structure activity relationship.

A new synthesis method was developed and optimized by a full factorial design for conjugating hydroxycinnamic acids (HCA-s) to chitosan. Cinnamic acid and tert-butyldimethylsilyl protected HCA-s were converted to their corresponding acyl chlorides and reacted with 3,6-di-O-tert-butyldimethylsilyl-chitosan to selectively form amide linkages, resulting in water-soluble conjugates after deprotection. Nineteen conjugates were obtained with various degrees of substitution (DS) ranging from 3% to 60%. The conjugates were found to be bactericidal against Staphylococcus aureus and Escherichia coli, with their activities equal to chitosan at low DS but an increase in the DS correlated with reduced activity. DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging assay was performed to determine the EC50 values. Chitosan only exhibited low antioxidant activity, whereas the HCA-chitosan conjugates exhibited higher antioxidant activities correlating with the DS. One caffeic acid conjugate (21%) was 4000 times more active than chitosan and more active than free caffeic acid.

Chitotriazolan (poly(ß(1-4)-2-(1H-1,2,3-triazol-1-yl)-2-deoxy-d-glucose)) derivatives: Synthesis, characterization, and evaluation of antibacterial activity.

Here we describe the first synthesis of a new type of polysaccharides derived from chitosan. In these structures, the 2-amino group on the pyranose ring was quantitively replaced by an aromatic 1,2,3-triazole moiety. The 2-amino group of chitosan and di-TBDMS chitosan was converted into an azide by diazo transfer reaction. The chitosan azide and TBDMS-chitosan azide were poorly soluble but could be fully converted to triazoles by "copper-catalysed Huisgen cycloaddition" in DMF or DMSO. The reaction could be done with different alkynes but derivatives lacking cationic or anionic groups were poorly soluble or insoluble in tested aqueous and organic solvents. Derivatives with N,N-dimethylaminomethyl, N,N,N-trimethylammoniummethyl, sulfonmethyl, and phosphomethyl groups linked to the 4-position of the triazole moiety were soluble in water at neutral or basic conditions and could be analyzed by 1H, 13C APT, COSY, and HSQC NMR. The quaternized cationic chitotriazolan's had high activity against S. aureus and E. coli, whereas the anionic chitotriazolan's lacked activity.

In vitro biological response of human osteoblasts in 3D chitosan sponges with controlled degree of deacetylation and molecular weight.

We have studied the effect of chitosan sponges, produced from chitosan batches with distinct degree of deacetylation (DDA) and molecular weight (Mw), on the adhesion, growth and differentiation of primary human osteoblasts with an aim to offer a suitable tool for guided bone regeneration. All the chitosan sponges revealed similar microstructure, irrespective of the DDA (58, 73, 82, 88, and 91 %) and Mw (749, 547, 263, 215, and 170 kDa, respectively). Cell spreading was higher on sponges having a higher DDA. Higher DDA induced a more pronounced increase in alkaline phosphatase activity, osteopontin (OPN), vascular endothelial growth factor-A (VEGF), interleukin-6 (IL-6), and reduction in monocyte chemoattractant protein-1 (MCP-1), sclerostin (SOST) and dickkopf related protein-1 as compared to lower DDA. Lower DDA induced the increased secretion of osteoprotegerin and SOST as compared to higher DDA. The combination of higher DDA and Mw induced an increased secretion of VEGF and IL-6, however reduced the secretion of OPN as compared to chitosan with similar DDA but with lower Mw. In summary, the variations in cellular responses to the different chitosan sponges indicate a potential for individual tailoring of desired responses in guided bone regeneration.

The antibacterial structure-activity relationship for common chitosan derivatives.

The relationship between the degree of substitution and antibacterial activity was studied for six common chitosan derivatives N, N,N-trimethyl chitosan (TMCNH2/TM and TMCTM/DM) N-(2-(N,N,N-trimethylammoniumyl)acetyl)-chitin (TACin), N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan (HTC), hydroxypropyl chitosan (HPC), thioglycolic chitosan (TGC) and carboxymethyl chitosan (CMC). The degree of substitution (DS) in the 36 studied samples ranged from 0.02 to 1.1 as determined by 1H NMR. The activity was determined as the minimum inhibitory concentration (MIC) against S. aureus and E. coli at pH 7.2 and 5.5. The antibacterial effect of TMC and TACin increased with DS. Samples of these derivatives with high DS were more active than chitosan at pH 7.2. HTC was more active than chitosan against S. aureus, but this activity was not affected by DS. In other cases, the activity of HTC decreased with an increase in DS. The DS for the TGC was very low and the activity was similar to unmodified chitosan. The activity of HPC decreased with DS. CMC was not active in this study.

Selective synthesis of N,N,N-trimethylated chitosan derivatives at different degree of substitution and investigation of structure-activity relationship for activity against P. aeruginosa and MRSA.

Two new cationic chitosan derivatives were synthesized using a combination of Boc and TBDMS protection strategies. This included a series of six samples of the TMCNH2/TM derivative, where some of the amino groups were N,N,N-trimethylated and the remaining was in the primary state. A series of six samples of the TACin derivative, where some of the amino groups were N-acetylated with quaternary 2-(N,N,N-trimethylammoniumyl) acetyl group and the remaining fully N-acetylated, were also synthesized. The degree of substitution (DS) for quaternary amino groups in these series ranged from 0.06-0.89. TMCDM/TM derivatives with a mix of N,N,N-trimethylated and N,N-dimethylated groups were also synthesized according to a published procedure but in this case, it was more difficult to control the DS than with the TBDMS protection strategy. Broth microdilution assay revealed a markedly different structure-activity relationship (SAR) for the two derivatives. The activity for the TMC derivatives reached a plateau above 0.2-0.3 DS whereas the activity increased continuously with DS for TACin. The highest DS TMCNH2/TM was more active than the highest DS, TACin, against Gram-positive MRSA but less active against the Gram-negative P. aeruginosa.

Drug-Loaded Photosensitizer-Chitosan Nanoparticles for Combinatorial Chemo- and Photodynamic-Therapy of Cancer.

In this study we have developed biodegradable polymeric nanoparticles (NPs) containing the cytostatic drugs mertansine (MRT) or cabazitaxel (CBZ). The NPs are based on chitosan (CS) conjugate polymers synthesized with different amounts of the photosensitizer tetraphenylchlorin (TPC). These TPC-CS NPs have high loading capacity and strong drug retention due to π-π stacking interactions between the drugs and the aromatic photosensitizer groups of the polymers. CS polymers with 10% of the side chains containing TPC were found to be optimal in terms of drug loading capacity and NP stability. The TPC-CS NPs loaded with MRT or CBZ displayed higher cytotoxicity than the free form of these drugs in the breast cancer cell lines MDA-MB-231 and MDA-MB-468. Furthermore, light-induced photochemical activation of the NPs elicited a strong photodynamic therapy effect on these breast cancer cells. Biodistribution studies in mice showed that most of the TPC-CS NPs accumulated in liver and lungs, but they were also found to be localized in tumors derived from HCT-116 cells. These data suggest that the drug-loaded TPC-CS NPs have a potential in combinatory anticancer therapy and as contrast agents.

Moxifloxacin-loaded acrylic intraocular lenses: In vitro and in vivo performance.

PURPOSE: To assess the possibility of using acrylic intraocular lenses (IOLs) to ensure controlled and sustained release of moxifloxacin, an antibiotic commonly used for endophthalmitis prophylaxis after cataract surgery. SETTING: Academic, industrial, and clinical partners from Portugal, Belgium, Iceland, and the United States. DESIGN: Experimental study. METHODS: The physical properties of IOLs loaded with moxifloxacin by soaking were characterized. In vitro drug-release studies were performed under hydrodynamic conditions similar to those of the eye, and the activity of the released drug was tested. In vitro cytotoxicity was evaluated, and the in vivo efficacy of the devices was assessed through rabbit experiments in which the effects of topical moxifloxacin drops (control) and moxifloxacin-loaded IOLs were compared. RESULTS: The presence of moxifloxacin in the IOLs had little effect on the evaluated physical properties and did not induce cytotoxicity. In vitro drug release experiments showed that the IOLs provided controlled release of moxifloxacin for approximately 2 weeks. The drug remained active against the tested microorganisms during that period. Moxifloxacin-loaded IOLs and the control treatment induced similar in vivo behavior in terms of inflammatory reactions, capsular bag opacification scores, and uveal and capsule biocompatibility. The drug concentration in the aqueous humor after 1 week was similar in both groups; however, the concentration with the loaded IOLs was less variable. CONCLUSION: The moxifloxacin-loaded IOLs released the drug in a controlled manner, providing therapeutic levels.

The Effect of Molecular Weight on the Antibacterial Activity of N,N,N-Trimethyl Chitosan (TMC).

N,N,N-trimethyl chitosan (TMC) with 93% degree of trimethylation was synthesized. TMC and the chitosan starting material were subjected to acidic hydrolysis to produce 49 different samples with a reduced average molecular weight (Mw) ranging from 2 to 144 kDa. This was done to allow the investigation of the relationship between antibacterial activity and Mw over a wide Mw range. NMR investigation showed that hydrolysis did not affect the degree of trimethylation (DSTRI) or the structure of the polymer backbone. The activity of TMC against Staphylococcus aureus (S. aureus) increased sharply with Mw until a certain Mw value (critical Mw for high activity, CMW) was reached. After the CMW, the activity was not affected by a further increase in the Mw. A similar pattern of activity was observed for chitosan. The CMW was determined to be 20 kDa for TMC and 50 kDa for chitosan.

Quaternary Ammoniumyl Chitosan Derivatives for Eradication of Staphylococcus aureus Biofilms.

Bacterial biofilms tolerate extreme levels of antibiotics. Treatment of biofilm infections therefore requires the development of new or modified antimicrobials that can penetrate biofilms and are effective against dormant persistent cells. One such new approach uses the biodegradable biopolymer chitosan and its derivatives as antimicrobials. In this study, we performed synthetic modification of chitosan to selectively introduce different cationic and hydrophobic moieties at varying ratios on chitosan. This improved its aqueous solubility and antimicrobial activity toward bacterial biofilms. Initial evaluation of the chitosan derivatives showed increased activity toward planktonic Staphylococcus aureus. The effect of the quaternary ammoniumyl chitosan derivatives against Staphylococcus aureus biofilms was more variable. The most effective derivatives contained hydrophobic groups, and their efficacy against biofilms depended on the ratio and length of the alkyl chains. Three-dimensional imaging of biofilms confirmed the accessibility and antimicrobial effect of chitosan derivatives with alkyl chains in the full depth of the biofilms.

A numerical framework for drug transport in a multi-layer system with discontinuous interlayer condition.

Discontinuous boundary conditions arise naturally when describing various physical phenomena and numerically modelling such conditions can prove difficult. In the field of pharmaceutical sciences, two such cases are the partitioning of a compound between different materials and a flux rate membrane controlling mass transfer between materials which both result in a discontinuous jump in concentration across adjacent materials. In this study, we introduce a general one-dimensional finite element drug delivery framework, which along with diffusion, reversible binding and dissolution within material layers, incorporates the partitioning and mass transfer conditions between layers of material. We apply the framework to construct models of experiments, which along with experimental data, allow us to infer pharmacokinetic properties of potential material for drug delivery. Understanding such material properties is the key to optimising the therepeutic effect of a targeted drug delivery system.

Antimicrobial Chitosan and Chitosan Derivatives: A Review of the Structure-Activity Relationship.

This review gives an updated overview of the current state-of-the-art for antimicrobial chitosan and chitosan derivatives and the effects of structural modifications on activity and toxicity. The various synthetic routes introduced for chemical modification of chitosan are discussed, and the most common functional groups are highlighted. Different analytical techniques used for structural characterization of the synthesized chitosan derivatives are discussed and critically evaluated. For the purpose of this review, the antimicrobial chitosan derivatives have been classified on the basis of the type of functional group conjugated to the polymer backbone. In each case, the influence of the degree of substitution on the biological properties has been examined. Finally, we have summarized the collective information and suggested future directions for further research to improve our understanding of the bioactivity and to develop more useful chitosan conjugates.

Endosome Targeting meso-Tetraphenylchlorin-Chitosan Nanoconjugates for Photochemical Internalization.

Four amphiphilic covalently linked meso-tetraphenylchlorin-chitosan nanoconjugates were synthesized and evaluated for use in photochemical internalization (PCI) in vitro and in vivo. The synthetic protocol for the preparation of two different hydrophobic chlorin photosensitizers, 5-(4-aminophenyl)-10,15,20-triphenylchlorin and 5-(4-carboxyphenyl)-10,15,20-triphenylchlorin, was optimized. These monofunctional photosensitizers were covalently attached to carrier chitosan via silyl-protected 3,6-di-O-tert-butyldimethylsilyl-chitosan (Di-TBDMS-chitosan) with 0.10 degree of substitution per glucosamine (DS). Hydrophilic moieties such as trimethylamine and/or 1-methylpiperazine were incorporated with 0.9 DS to give fully water-soluble conjugates after removal of the TBDMS groups. A dynamic light scattering (DLS) study confirmed the formation of nanoparticles with a 140-200 nm diameter. These nanoconjugates could be activated at 650 nm (red region) light, with a fluorescence quantum yield (ΦF) of 0.43-0.45, and are thus suitable candidates for use in PCI. These nanoconjugates were taken up and localized in the endocytic vesicles of HCT116/LUC human colon carcinoma cells, and upon illumination they substantially enhanced plasmid DNA transfection. The nanoconjugates were also evaluated in preliminary in vivo experiments in tumor-bearing mice, showing that the nanoconjugates could induce a strong photodynamic therapy (PDT) and also PCI effects in treatment with bleomycin.

Experimental design for determining quantitative structure activity relationship for antibacterial chitosan derivatives.

Experimental design approach was successfully used to guide the synthesis and determine the structure-activity relationship for antimicrobial derivatives of the biopolymer chitosan. Specialized software with D-optimal design capabilities was used to create a library of chitosan derivatives with optimal structural variation in order to conduct a detailed investigation of the structure-activity relationship. The derivatives contain three substituents: N,N,N-trimethylamine, N-acetyl and N-stearoyl at different degrees of substitution (DS) on the 2-amino group of chitosan. The design matrix consisted of 14 target materials that were synthesized in 'one-pot synthesis' using TBDMS-chitosan as the precursor to allow precise control of the DS. The antibacterial activity (MIC) towards the Gram positive bacteria Staphylococcus aureus and the Gram negative bacteria Escherichia coli, hemolytic activity (HC50) towards human red blood cells and solubility of the chitosan derivatives were used as the responses in the model. The response surface model was refined by removing the interaction terms to improve the statistical significance and predictive power of the model. The investigation showed that materials with DS for trimethylation in the range 0.45-0.65, acetylation in the range 0.08-0.33 and stearoylation in the range 0.22-0.29 were capable of showing high antimicrobial activity, high solubility and low hemolytic activity.