Enzyme-Catalyzed Polymerization Process: A Novel Approach to the Preparation of Polyaniline Colloidal Dispersions with an Immunomodulatory Effect.

A green, nature-friendly synthesis of polyaniline colloidal particles based on enzyme-assisted oxidation of aniline with horseradish peroxidase and chitosan or poly(vinyl alcohol) as steric stabilizers was successfully employed. Physicochemical characterization revealed formation of particles containing the polyaniline emeraldine salt and demonstrated only a minor effect of polymer stabilizers on particle morphology. All tested colloidal particles showed in vitro antioxidation activity determined via scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. In vitro, they were able to reduce oxidative stress and inhibit the production of reactive oxygen species by neutrophils and inflammatory cytokines by macrophages. The anti-inflammatory effect observed was related to their antioxidant activity, especially in the case of neutrophils. The particles can thus be especially advantageous as active components of biomaterials modulating the early stages of inflammation. In addition to the immunomodulatory effect, the presence of intrinsically conducting polyaniline can impart cell-instructive properties to the particles. The approach to particle synthesis that we employed─an original one using environmentally friendly and biocompatible horseradish peroxidase─represents a smart way of preparing conducting particles with unique properties, which can be further modified by the stabilizers used.

Formulation of Magneto-Responsive Hydrogels from Dually Cross-Linked Polysaccharides: Synthesis, Tuning and Evaluation of Rheological Properties.

Smart hydrogels based on natural polymers present an opportunity to fabricate responsive scaffolds that provide an immediate and reversible reaction to a given stimulus. Modulation of mechanical characteristics is especially interesting in myocyte cultivation, and can be achieved by magnetically controlled stiffening. Here, hyaluronan hydrogels with carbonyl iron particles as a magnetic filler are prepared in a low-toxicity process. Desired mechanical behaviour is achieved using a combination of two cross-linking routes-dynamic Schiff base linkages and ionic cross-linking. We found that gelation time is greatly affected by polymer chain conformation. This factor can surpass the influence of the number of reactive sites, shortening gelation from 5 h to 20 min. Ionic cross-linking efficiency increased with the number of carboxyl groups and led to the storage modulus reaching 103 Pa compared to 101 Pa-102 Pa for gels cross-linked with only Schiff bases. Furthermore, the ability of magnetic particles to induce significant stiffening of the hydrogel through the magnetorheological effect is confirmed, as a 103-times higher storage modulus is achieved in an external magnetic field of 842 kA·m-1. Finally, cytotoxicity testing confirms the ability to produce hydrogels that provide over 75% relative cell viability. Therefore, dual cross-linked hyaluronan-based magneto-responsive hydrogels present a potential material for on-demand mechanically tunable scaffolds usable in myocyte cultivation.

Hierarchically Structured Polystyrene-Based Surfaces Amplifying Fluorescence Signals: Cytocompatibility with Human Induced Pluripotent Stem Cell.

An innovative multi-step phase separation process was used to prepare tissue culture for the polystyrene-based, hierarchically structured substrates, which mimicked in vivo microenvironment and architecture. Macro- (pore area from 3000 to 18,000 µm2; roughness (Ra) 7.2 ± 0.1 µm) and meso- (pore area from 50 to 300 µm2; Ra 1.1 ± 0.1 µm) structured substrates covered with micro-pores (area around 3 µm2) were prepared and characterised. Both types of substrate were suitable for human-induced pluripotent stem cell (hiPSC) cultivation and were found to be beneficial for the induction of cardiomyogenesis in hiPSC. This was confirmed both by the number of promoted proliferated cells and the expressions of specific markers (Nkx2.5, MYH6, MYL2, and MYL7). Moreover, the substrates amplified the fluorescence signal when Ca2+ flow was monitored. This property, together with cytocompatibility, make this material especially suitable for in vitro studies of cell/material interactions within tissue-mimicking environments.

Behaviour of Titanium Dioxide Particles in Artificial Body Fluids and Human Blood Plasma.

The growing application of materials containing TiO2 particles has led to an increased risk of human exposure, while a gap in knowledge about the possible adverse effects of TiO2 still exists. In this work, TiO2 particles of rutile, anatase, and their commercial mixture were exposed to various environments, including simulated gastric fluids and human blood plasma (both representing in vivo conditions), and media used in in vitro experiments. Simulated body fluids of different compositions, ionic strengths, and pH were used, and the impact of the absence or presence of chosen enzymes was investigated. The physicochemical properties and agglomeration of TiO2 in these media were determined. The time dependent agglomeration of TiO2 related to the type of TiO2, and mainly to the type and composition of the environment that was observed. The presence of enzymes either prevented or promoted TiO2 agglomeration. TiO2 was also observed to exhibit concentration-dependent cytotoxicity. This knowledge about TiO2 behavior in all the abovementioned environments is critical when TiO2 safety is considered, especially with respect to the significant impact of the presence of proteins and size-related cytotoxicity.

Modulation of Differentiation of Embryonic Stem Cells by Polypyrrole: The Impact on Neurogenesis.

The active role of biomaterials in the regeneration of tissues and their ability to modulate the behavior of stem cells in terms of their differentiation is highly advantageous. Here, polypyrrole, as a representantive of electro-conducting materials, is found to modulate the behavior of embryonic stem cells. Concretely, the aqueous extracts of polypyrrole induce neurogenesis within embryonic bodies formed from embryonic stem cells. This finding ledto an effort to determine the physiological cascade which is responsible for this effect. The polypyrrole modulates signaling pathways of Akt and ERK kinase through their phosphorylation. These effects are related to the presence of low-molecular-weight compounds present in aqueous polypyrrole extracts, determined by mass spectroscopy. The results show that consequences related to the modulation of stem cell differentiation must also be taken into account when polypyrrole is considered as a biomaterial.

Selectively Oxidized Cellulose with Adjustable Molecular Weight for Controlled Release of Platinum Anticancer Drugs.

The synthesis of selectively oxidized cellulose, 2,3-dicarboxycellulose (DCC), is optimized for preparation of highly oxidized material for biological applications, which includes control over the molecular weight of the product during its synthesis. Conjugates of DCC and cisplatin simultaneously offer a very high drug binding efficiency (>90%) and drug loading capacity (up to 50 wt %), while retaining good aqueous solubility. The adjustable molecular weight of the DCC together with variances in drug feeding ratio allows to optimize cisplatin release profiles from delayed (<2% of cisplatin released during 6 h) to classical burst release with more than 60% of cisplatin released after 24 h. The release rates are also pH-dependent (up to 2 times faster release at pH 5.5 than at pH 7.4), which allows to exploit the acidic nature of tumor microenvironment. Extensive in vitro studies were performed on eight different cell lines for two cisplatin-DCC conjugates with different release profiles. In comparison with free cisplatin, both cisplatin-DCC conjugates demonstrated considerably lower cytotoxicity toward healthy cells. Conjugates with burst release profiles were found more effective against prostate cell lines, while DCC conjugates with slower release were more cytotoxic against ovarian and lung carcinoma cell lines. In vivo studies indicated a significantly longer survival rate, a reduction in tumor volume, and a higher accumulation of platinum in tumors of mice treated with the cisplatin-DCC conjugate in comparison to those treated by free cisplatin.

Adhesion, Proliferation and Migration of NIH/3T3 Cells on Modified Polyaniline Surfaces.

Polyaniline shows great potential and promises wide application in the biomedical field thanks to its intrinsic conductivity and material properties, which closely resemble natural tissues. Surface properties are crucial, as these predetermine any interaction with biological fluids, proteins and cells. An advantage of polyaniline is the simple modification of its surface, e.g., by using various dopant acids. An investigation was made into the adhesion, proliferation and migration of mouse embryonic fibroblasts on pristine polyaniline films and films doped with sulfamic and phosphotungstic acids. In addition, polyaniline films supplemented with poly (2-acrylamido-2-methyl-1-propanesulfonic) acid at various ratios were tested. Results showed that the NIH/3T3 cell line was able to adhere, proliferate and migrate on the pristine polyaniline films as well as those films doped with sulfamic and phosphotungstic acids; thus, utilization of said forms in biomedicine appears promising. Nevertheless, incorporating poly (2-acrylamido-2-methyl-1-propanesulfonic) acid altered the surface properties of the polyaniline films and significantly affected cell behavior. In order to reveal the crucial factor influencing the surface/cell interaction, cell behavior is discussed in the context of the surface energy of individual samples. It was clearly demonstrated that the lesser the difference between the surface energy of the sample and cell, the more cyto-compatible the surface is.

Comparison of dialdehyde polysaccharides as crosslinkers for hydrogels: The case of poly(vinyl alcohol).

A little is known about the link between the macromolecular architecture of dialdehyde polysaccharides (DAPs), their crosslinking capabilities, and the properties of resulting hydrogels. Here, DAPs based on cellulose, dextrin, dextran, and hyaluronate were compared as crosslinkers for poly(vinyl alcohol), PVA. The swelling, network parameters, viscoelastic properties, porosity, and cytotoxicity of PVA/DAP hydrogels were investigated concerning the crosslinker structure, molecular weight, aldehyde group density per macromolecule, and the size of spontaneously formed crosslinker nano-assemblies. Generally, crosslinkers based on linear polysaccharides (cellulose, hyaluronate) performed more reliably, while the presence of branching could be both beneficial (dextran) but also detrimental (dextrin) at lower crosslinker concentrations. For example, the hydrogel swelling differed by up to one-third (600 vs. 400%) and storage modulus even by up to one half (~7000 vs. ~3500 Pa) depending on crosslinker structure and properties. These differences were rationalized by variances in crosslinking modes derived based on obtained data.

Dicarboxylated hyaluronate: Synthesis of a new, highly functionalized and biocompatible derivative.

Sequential periodate-chlorite oxidation of sodium hyaluronate to 2,3-dicarboxylated hyaluronate (DCH), a novel biocompatible and highly functionalized derivative bearing additional pair of COOH groups at C2 and C3 carbons of oxidized ᴅ-glucuronic acid units, is investigated. The impact of various reaction parameters (time, oxidizer concentration, and molar amount) on DCH's composition, molecular weight, degree of oxidation, and cytotoxicity are investigated to guide the synthesis of DCH derivatives of desired properties. Subsequently, fully (99%) and partially (70%) oxidized DCH derivatives were compared to untreated sodium hyaluronate in terms of anticancer drug cisplatin loading efficacy, carrier capacity, drug release rates, and cytotoxicity towards healthy and cancerous cell lines. DCH derivatives were found to be superior in every aspect, having nearly twice the carrier capacity, significantly slower release rates, and higher efficacy. DCH is thus a highly interesting hyaluronate derivative with an adjustable degree of oxidation, molecular weight, and great potential for further modifications.

Self-crosslinked chitosan/dialdehyde xanthan gum blended hypromellose hydrogel for the controlled delivery of ampicillin, minocycline and rifampicin.

The design of improved biopolymeric based hydrogel materials with high load-capacity to serve as biocompatible drug carriers is a challenging task with vital implications in health sciences. In this work, chitosan crosslinked dialdehyde xanthan gum interpenetrated hydroxypropyl methylcellulose gels were developed for the controlled delivery of different antibiotic drugs including ampicillin, minocycline and rifampicin. The prepared hydrogel scaffolds were characterized by rheology method, FTIR, SEM, TGA and compression analysis. In addition, gelation kinetics, swelling, in vitro degradation and drug release rate were studied under simulated gastrointestinal fluid conditions of pH 2.0 and 7.4 at 37 °C. Results demonstrated the gel composition and structure affected drug release kinetics. The release study showed more than 50% cumulative release within 24 h for all investigated antibiotic drugs. In vitro cell cytocompatibility using mouse embryonic fibroblast cell lines depicted ≥80% cell viability, indicating the gels are non-toxic. Finally, the antibacterial activity of loaded gels was evaluated against Gram-negative and positive bacteria (Escherichia coli, Staphylococcus aureus and Klebsiella pneumonia), which correlated well with swelling and drug release results. Overall, the present study demonstrated that the produced hydrogel scaffolds serves as promising material for controlled antibiotic delivery towards microbial growth inhibition.

Plasma Mediated Chlorhexidine Immobilization onto Polylactic Acid Surface via Carbodiimide Chemistry: Antibacterial and Cytocompatibility Assessment.

The development of antibacterial materials has great importance in avoiding bacterial contamination and the risk of infection for implantable biomaterials. An antibacterial thin film coating on the surface via chemical bonding is a promising technique to keep native bulk material properties unchanged. However, most of the polymeric materials are chemically inert and highly hydrophobic, which makes chemical agent coating challenging Herein, immobilization of chlorhexidine, a broad-spectrum bactericidal cationic compound, onto the polylactic acid surface was performed in a multistep physicochemical method. Direct current plasma was used for surface functionalization, followed by carbodiimide chemistry to link the coupling reagents of N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) and N-Hydroxysuccinimide (NHs) to create a free bonding site to anchor the chlorhexidine. Surface characterizations were performed by water contact angle test, X-ray photoelectron spectroscopy (XPS) and scanning electron microscope (SEM). X-ray photoelectron spectroscopy (XPS) and scanning electron microscope (SEM). The antibacterial activity was tested using Staphylococcus aureus and Escherichia coli. Finally, in vitro cytocompatibility of the samples was studied using primary mouse embryonic fibroblast cells. It was found that all samples were cytocompatible and the best antibacterial performance observed was the Chlorhexidine immobilized sample after NHs activation.

Oxidized polysaccharides for anticancer-drug delivery: What is the role of structure?

Study provides an in-depth analysis of the structure-function relationship of polysaccharide anticancer drug carriers and points out benefits and potential drawbacks of differences in polysaccharide glycosidic bonding, branching and drug binding mode of the carriers. Cellulose, dextrin, dextran and hyaluronic acid have been regioselectively oxidized to respective dicarboxylated derivatives, allowing them to directly conjugate cisplatin, while preserving their major structural features intact. The structure of source polysaccharide has crucial impact on conjugation effectiveness, carrier capacity, drug release rates, in vitro cytotoxicity and cellular uptake. For example, while branched structure of dextrin-based carrier partially counter the undesirable initial burst release, it also attenuates the cellular uptake and the cytotoxicity of carried drug. Linear polysaccharides containing ß-(1→4) glycosidic bonds and oxidized at C2 and C3 (cellulose and hyaluronate) have the best overall combination of structural features for improved drug delivery applications including potentiation of the cisplatin efficacy towards malignances.

Conducting composite films based on chitosan or sodium hyaluronate. Properties and cytocompatibility with human induced pluripotent stem cells.

Novel composite films combining biocompatible polysaccharides with conducting polyaniline (PANI) were prepared via the in-situ polymerization of aniline hydrochloride in the presence of sodium hyaluronate (SH) or chitosan (CH). The composite films possess very good cytocompatibility in terms of adhesion and proliferation of two lines of human induced pluripotent stem cells (hiPSC). Moreover, the cardiomyogenesis and even formation of beating clusters were successfully induced on the films. The proportion of formed cardiomyocytes demonstrated excellent properties of composites for tissue engineering of stimuli-responsive tissues. The testing also demonstrated antibacterial activity of the films against E. coli and PANI-SH was able to reduce bacterial growth from 2 × 105 to < 1 cfu cm-2. Physicochemical characterization revealed that the presence of polysaccharides did not notably influence conductivities of the composites being ∼1 and ∼2 S cm-1 for PANI-SH and PANI-CH respectively; however, in comparison with neat PANI, it modified their topography making the films smoother with mean surface roughness of 4 (PANI-SH) and 14 nm (PANI-CH). The combination of conductivity, antibacterial activity and mainly cytocompatibility with hiPSC opens wide application potential of these polysaccharide-based composites.

Effect of Iron-Oxide Nanoparticles Impregnated Bacterial Cellulose on Overall Properties of Alginate/Casein Hydrogels: Potential Injectable Biomaterial for Wound Healing Applications.

In this study we report the preparation of novel multicomponent hydrogels as potential biomaterials for injectable hydrogels comprised of alginate, casein and bacterial cellulose impregnated with iron nanoparticles (BCF). These hydrogels demonstrated amide cross-linking of alginate-casein, ionic cross-linking of alginate and supramolecular interaction due to incorporation of BCF. Incorporation of BCF into the hydrogels based on natural biopolymers was done to reinforce the hydrogels and impart magnetic properties critical for targeted drug delivery. This study aimed to improve overall properties of alginate/casein hydrogels by varying the BCF loading. The physico-chemical properties of gels were characterized via FTIR, XRD, DSC, TGA, VSM and mechanical compression. In addition, swelling, drug release, antibacterial activity and cytotoxicity studies were also conducted on these hydrogels. The results indicated that incorporation of BCF in alginate/casein hydrogels led to mechanically stronger gels with magnetic properties, increased porosity and hence increased swelling. A porous structure, which is essential for migration of cells and biomolecule transportation, was confirmed from microscopic analysis. The porous internal structure promoted cell viability, which was confirmed through MTT assay of fibroblasts. Moreover, a hydrogel can be useful for the delivery of essential drugs or biomolecules in a sustained manner for longer durations. These hydrogels are porous, cell viable and possess mechanical properties that match closely to the native tissue. Collectively, these hybrid alginate-casein hydrogels laden with BCF can be fabricated by a facile approach for potential wound healing applications.

Design of dialdehyde cellulose crosslinked poly(vinyl alcohol) hydrogels for transdermal drug delivery and wound dressings.

2,3-Dialdehyde cellulose (DAC) was used as an efficient and low-toxicity crosslinker to prepare thin PVA/DAC hydrogel films designed for topical applications such as drug-loaded patches, wound dressings or cosmetic products. An optimization of hydrogel properties was achieved by the variation of two factors - the amount of crosslinker and the weight-average molecular weight (Mw) of the source PVA. The role of each factor to network parameters, mechanical, rheological and surface properties, hydrogel porosity and transdermal absorption is discussed. The best results were obtained for hydrogel films prepared using 0.25 wt% of DAC and PVA with Mw = 130 kDa, which had a high porosity and drug-loading capacity (high water content), mechanical properties allowing easy handling, best adherence to the skin from all tested samples and improved transdermal drug-delivery. Hydrogel films are biocompatible, show no cytotoxicity and have no negative impact on cell growth and morphology in their presence. Furthermore, hydrogels do not support cell migration and attachment to their surface, which should ensure easy removal of hydrogel patches even from wounded or damaged skin after use.

The biocompatibility of polyaniline and polypyrrole 2: Doping with organic phosphonates.

Conducting polymers (CP) can be used as pH- and/or electro-responsive components in various bioapplications, for example, in 4D smart scaffolds. The ability of CP to maintain conductivity under physiological conditions is, therefore, their crucial property. Unfortunately, the conductivity of the CP rapidly decreases in physiological environment, as their conducting salts convert to non-conducting bases. One of the promising solutions how to cope with this shortcoming is the use of alternative "doping" process that is not based on the protonation of CP with acids but on interactions relying in acidic hydrogen bonding. Therefore, the phosphonates (dimethyl phosphonate, diethyl phosphonate, dibutyl phosphonate, or diphenyl phosphonate) were used to re-dope two most common representatives of CP, polyaniline (PANI) and polypyrrole (PPy) bases. As a result, PANI doped with organic phosphonates proved to have significantly better stability of conductivity under different pH. It has also been shown that cytotoxicity of studied materials determined on embryonic stem cells and their embryotoxicity, determined as the impact on cardiomyogenesis and erythropoiesis, depend both on the polymer and phosphonate types used. With the exception of PANI doped with dibutyl phosphonate, all PPy-based phosphonates showed better biocompatibility than the phosphonates based on PANI.

Biocompatible dialdehyde cellulose/poly(vinyl alcohol) hydrogels with tunable properties.

Solubilized dialdehyde cellulose (DAC), an efficient crosslinking agent for poly(vinyl alcohol) (PVA), provides less toxic alternative to current synthetic crosslinking agents such as glutaraldehyde, while simultaneously allowing for the preparation of hydrogels with comparably better characteristics. PVA/DAC hydrogels prepared using 0.5, 1 and 1.5 wt% of DAC were analyzed in terms of mechanical, swelling and cytotoxicity characteristics. Materials properties of PVA/DAC hydrogels range from stiff substances to soft viscoelastic gels capable of holding large amounts of water. Superior mechanical properties, porosity and surface area in comparison with analogical PVA/glutaraldehyde hydrogels were observed. Biological studies showed low toxicity and good biocompatibility of PVA/DAC hydrogels. Potential of PVA/DAC in mesh-controlled release of biologically active compounds was investigated using ibuprofen, rutin and phenanthriplatin. Hydrogel loaded with anticancer drug phenantriplatin was found effective against alveolar cancer cell line A549 under in vitro conditions.

The use of fractionated Kraft lignin to improve the mechanical and biological properties of PVA-based scaffolds.

The mechanical properties of poly(vinyl alcohol) (PVA)-based scaffolds were successfully improved. The improvements in mechanical properties correlated with the amount of Kraft lignin in PVA matrices. The critical property for any scaffold is its capacity to allow cells to ingrow and survive within its internal structure. The ingrowth of cells was tested using bioreactors creating simulated in vivo conditions. In the context of all the mentioned parameters, the most advantageous properties were exhibited by the scaffold containing 99 wt% PVA and 1 wt% Kraft lignin. The composites with 1 wt% Kraft lignin exhibited sufficient mechanical stability, a lack of cytotoxicity, and mainly the ability to allow the ingrowth of cells into the scaffold in a rotation bioreactor.

Exploring the Critical Factors Limiting Polyaniline Biocompatibility.

Today, the application of polyaniline in biomedicine is widely discussed. However, information about impurities released from polyaniline and about the cytotoxicity of its precursors aniline, aniline hydrochloride, and ammonium persulfate are scarce. Therefore, cytotoxicity thresholds for the individual precursors and their combinations were determined (MTT assay) and the type of cell death caused by exposition to the precursors was identified using flow-cytometry. Tests on fibroblasts revealed higher cytotoxicity of ammonium persulfate than aniline hydrochloride. Thanks to the synergic effect, both monomers in combination enhanced their cytotoxicities compared with individual substances. Thereafter, cytotoxicity of polyaniline doped with different acids (sulfuric, nitric, phosphoric, hydrochloric, and methanesulfonic) was determined and correlated with impurities present in respective sample (HPLC). The lowest cytotoxicity showed polyaniline doped with phosphoric acid (followed by sulfuric, methanesulfonic, and nitric acid). Cytotoxicity of polyaniline was mainly attributed to the presence of residual ammonium persulfate and low-molecular-weight polar substances. This is crucial information with respect to the purification of polyaniline and production of its cytocompatible form.