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.

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.

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.

Impact of chain length on antibacterial activity and hemocompatibility of quaternary N-alkyl and n,n-dialkyl chitosan derivatives.

A highly efficient method for chemical modification of chitosan biopolymers by reductive amination to yield N,N-dialkyl chitosan derivatives was developed. The use of 3,6-O-di-tert-butyldimethylsilylchitosan as a precursor enabled the first 100% disubstitution of the amino groups with long alkyl chains. The corresponding mono N-alkyl derivatives were also synthesized, and all the alkyl compounds were then quaternized using an optimized procedure. These well-defined derivatives were studied for antibacterial activity against Gram positive S. aureus, E. faecalis, and Gram negative E. coli, P. aeruginosa, which could be correlated to the length of the alkyl chain, but the order was dependent on the bacterial strain. Toxicity against human red blood cells and human epithelial Caco-2 cells was found to be proportional to the length of the alkyl chain. The most active chitosan derivatives were found to be more selective for killing bacteria than the quaternary ammonium disinfectants cetylpyridinium chloride and benzalkonium chloride, as well as the antimicrobial peptides melittin and LL-37.

The effect of substituent, degree of acetylation and positioning of the cationic charge on the antibacterial activity of quaternary chitosan derivatives.

A series of water-soluble cationic chitosan derivatives were prepared by chemoselective functionalization at the amino group of five different parent chitosans having varying degrees of acetylation and molecular weight. The quaternary moieties were introduced at different alkyl spacer lengths from the polymer backbone (C-0, C-2 and C-6) with the aid of 3,6-di-O-tert-butyldimethylsilyl protection of the chitosan backbone, thus allowing full (100%) substitution of the free amino groups. All of the derivatives were characterized using 1H-NMR, 1H-1H COSY and FT-IR spectroscopy, while molecular weight was determined by GPC. Antibacterial activity was investigated against Gram positive S. aureus and Gram negative E. coli. The relationship between structure and activity/toxicity was defined, considering the effect of the cationic group's structure and its distance from the polymer backbone, as well as the degree of acetylation within a molecular weight range of 7-23 kDa for the final compounds. The N,N,N-trimethyl chitosan with 100% quaternization showed the highest antibacterial activity with moderate cytotoxicity, while increasing the spacer length reduced the activity. Trimethylammoniumyl quaternary ammonium moieties contributed more to activity than 1-pyridiniumyl moieties. In general, no trend in the antibacterial activity of the compounds with increasing molecular weight or degree of acetylation up to 34% was observed.

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.

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.

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.

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.

Drug delivery characteristics of the progenitor bronchial epithelial cell line VA10.

PURPOSE: To determine the integrity and permeability properties of the immortalized human VA10 bronchial epithelial cell line for its suitability as an in vitro drug permeation model. METHODS: Cells were grown under liquid-covered culture (LCC) or air-liquid interface (ALI) culture, characterized using electron microscopy and immunostaining. Integrity was measured using transepithelial electrical resistance (TER) and permeability of fluorescein sodium (Flu-Na). General permeability was established with dextrans and model drugs and P-glycoprotein (P-gp) function determined with bidirectional flux of rhodamine-123. RESULTS: ALI culture resulted in 2-3 cell layers with differentiation towards ciliated cells but LCC showed undifferentiated morphology. VA10 cells formed TJ, with higher TER in LCC than ALI (∼2500 vs. ∼1200 Ω*cm(2)) and Flu-Na permeability ∼1-2 × 10(-7) cm/s. ALI cultured cells expressed P-gp and distinguished between compounds depending on lipophilicity and size, consistent with previous data from Calu-3 and 16HBE14o-cell lines. CONCLUSIONS: ALI cultured cell layers capture the in vivo-like phenotype of bronchial epithelium and form functional cell barrier capable of discriminating between compounds depending on physiochemical properties. The VA10 cell line is an important alternative to previously published cell lines and a relevant model to study airway drug delivery in vitro.

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.

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.

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.

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.

Studies on curcumin and curcuminoids. XXXIX. Photophysical properties of bisdemethoxycurcumin.

The steady-state absorption and fluorescence, as well as the time-resolved fluorescence properties of bisdemethoxycurcumin dissolved in several solvents differing in polarity and H-bonding capability were measured. The photodegradation quantum yield of the compound in acetonitrile and methanol was determined. The bisdemethoxycurcumin decay mechanisms from the S(1) state were discussed and compared with those of curcumin. The differences in S(1) dynamics observed between bisdemethoxy-curcumin and curcumin could be ascribed to a difference in H-bond acceptor/donor properties of the phenolic OH and a difference in strength of the intramolecular H-bond in the keto-enol moiety within the two molecules.

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.