Sulfated glycosaminoglycan-based block copolymer: preparation of biocompatible chondroitin sulfate-b-poly(lactic acid) micelles.

Despite a growing interest in amphiphilic polysaccharide-based diblock copolymers as functional polymeric drug delivery nanosystems, biologically relevant sulfated glycosaminoglycan systems were not yet investigated. Here, we report the synthesis and the self-assembly properties in water of chondroitin sulfate-b-poly(lactic acid) (CS-b-PLA(n)). The CS-b-PLA(n) were synthesized using click-grafting onto method implying reducing-end alkynation of low-molecular weight depolymerized CS (M(w) = 5000 g·mol(-1)) and azide-terminated functionalization of PLAn (M(w) = 6500 g·mol(-1) (n = 46) and M(w) = 1700 g·mol(-1) (n = 20)). The diblock copolymer self-assembled in water giving rise to spherical micelles that were characterized in solution using dynamic/static light scattering and at dry state by TEM technique. In vitro assays on healthy cells showed that at high concentrations, up to 10 µg·mL(-1), CS-b-PLA(n) were noncytotoxic. Those preliminary studies are promising in the perspective to use them as biocompatible nanovehicles for anticancer drug delivery.

Preparing silk fibroin nanofibers through electrospinning: further heparin immobilization toward hemocompatibility improvement.

Sodium heparin (HS) was immobilized on the surface of the silk fibroin nanofibers (FS) prepared by electrospinning with the objective of improving the hemocompatibility of the fibers for application as scaffolds in tissue engineering. The nanofiber mats of silk fibroin without (MF-FS) and with (MF-FS/HS) immobilized heparin were characterized through scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR), thermogravimetric analyses (TGA), energy dispersive spectroscopy (EDS), contact angle, chemical analysis, and biological tests. The formation of hydrogen bonds between the silk fibroin and heparin was discussed based on FTIR-ATR spectra. The amount of immobilized heparin was quantified through papain/N-acetyl-l-cysteine digestion followed by dimethylmethylene blue complexation. Furthermore, the samples with immobilized HS showed higher hydrophilic capability compared to samples without HS due to lower contact angles. It was possible to verify that the capillary end-to-collector distance of 8.5 cm and flow rate of 0.35 mL h(-1) used in the electrospinning process at 20 kV are good conditions for obtaining a small average fiber diameter maintaining the amount of immobilized heparin on MF-FS/HS in ca. 4% w/w. Biological analysis showed that no hemolysis is provoked by MF-FS and MF-FS/HS mat fragments and those such mats are not toxic to Vero cells. However, the MF-FS/HS showed higher cell growth and proliferation than MF-FS, indicating an improvement in the hemocompatibility of the material due to heparin immobilization.

Antiadhesive and antibacterial multilayer films via layer-by-layer assembly of TMC/heparin complexes.

N-Trimethyl chitosan (TMC), an antibacterial agent, and heparin (HP), an antiadhesive biopolymer, were alternately deposited on modified polystyrene films, as substrates, to built antiadhesive and antibacterial multilayer films. The properties of the multilayer films were investigated by Fourier transform infrared spectroscopy, atomic force microscopy, scanning electron microscopy, and Kelvin force microscopy. In vitro studies of controlled release of HP were evaluated in simulated intestinal fluid and simulated gastric fluid. The initial adhesion test of E. coli on multilayer films surface showed effective antiadhesive properties. The in vitro antibacterial test indicated that the multilayer films of TMC/HP based on TMC80 can kill the E. coli bacteria. Therefore, antiadhesive and antibacterial multilayer films may have good potential for coatings and surface modification of biomedical applications.

Development of composite hydrogel based on hydroxyapatite mineralization over pectin reinforced with cellulose nanocrystal.

Hydrogels based on pectin and cellulose nanocrystals (CNC) were used in our study to nucleation and growth of hydroxyapatite (HAp) by the biomimetic method. In this study, we evaluated the direct impact of the different percentages of CNC on pectin hydrogel and the influence of HAp obtained through two methods. CNC were obtained from HCl hydrolysis following chemical functionalization through vinyl groups. The percentage of CNC positively induces thermal stability, mechanical properties and HAp mineralization from biomimetic using simulated body fluid (1.5 SBF). Hydrogels with 5% of CNC showed a higher amount of HAp immersed for 14 days, about 28% of HAp. The obtained hydrogels were compared with hydrogels containing 20% of HAp nanoparticles obtained by chemical precipitation. Biocompatibility of the hydrogels was evaluated by cell viability using fibroblasts (L929). In general, the hydrogels obtained through the biomimetic method show slightly larger biocompatibility compared to the hybrid hydrogels obtained from chemical precipitation.

Application of a polyelectrolyte complex based on biocompatible polysaccharides for colorectal cancer inhibition.

Strategies for incorporating water-insoluble photosensitisers (PS) in drug delivery systems have been extensively studied. In this work, we evaluate the formation, characterisation, drug sorption studies, and cytotoxicity of chitosan (CHT)/chondroitin sulphate (CS) polyelectrolyte complexes (PECs) coated with polystyrene-block-poly(acrylic acid) (PS-b-PAA) nanoparticles (NPs) loaded with chloroaluminum phthalocyanine (AlClPc). The PECs were characterised by infrared spectroscopy (FTIR), differential scanning calorimetric (DSC), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The PS-b-PAA NPs on the PEC surface was confirmed by scanning electron microscopy (SEM). Additionally, optical images distinguished the PEC structures containing PS-b-PAA or PS-b-PAA/AlClPc from the unloaded PEC. Kinetic and equilibrium studies investigate the sorption capacity of the PEC/PS-b-PAA toward AlClPc. The encapsulation efficiency reached 95% at 190 µg mL-1 AlClPc after only 15 min. The Brunauer-Emmett-Teller (BET) isotherm and pseudo-second-order kinetic fitted well to the experimental data. The PS-b-PAA NPs on the PEC surfaces increase the AlClPc bioavailability and the PEC structure stabilizes the PS-b-PAA/AlClPc nanostructures. The materials were cytocompatible upon healthy VERO (kidney epithelial cells), and cytotoxic against colorectal cancerous cells (HT-29 cells). For the first time, we associate PS-b-PAA/AlClPc with a hydrophilic and cytocompatible polysaccharide matrix. We suggest the use of these materials in strategies to treat cancer by using photodynamic therapy.

First report of electrospun cellulose acetate nanofibers mats with chitin and chitosan nanowhiskers: Fabrication, characterization, and antibacterial activity.

Physical adsorption has shown to be facile and highly effective to deposit chitosan nanowhiskers (CsNWs, 60 % deacetylated, length: 247 nm, thickness: 4-12 nm, width:15 nm) on electrospun cellulose acetate nanofibers (CANFs, 560 nm) to effect complete surface charge reversal from negatively charged CANFs (-40 mV) to positively charged CsNWs-adsorbed CANFs (+8 mV). The CsNWs coverage did not alter the smooth and homogeneous morphology of fibers, as observed from SEM images. Biological assays showed the CsNWs covered nanofibers were effective against the Gram-negative bacterium E. coli, reducing 99 % of colony forming units (CFU) in 24 h and atoxic to healthy Vero cells. The use of CsNWs to modify cellulose fiber surfaces has been proved to be efficient and may be applied to a broad scope of fields, especially as biomaterials and biomedical applications.

pH-responsive hybrid hydrogels: Chondroitin sulfate/casein trapped silica nanospheres for controlled drug release.

In this study, the materials were synthesized by chemically crosslinking chondroitin sulfate (CS), casein (CAS), and silica nanospheres (SiO2), creating a highly crosslinked network. The hydrogel release profile was adaptable (that is, it could be faster or slower as needed) simply by changing the polymeric proportion. The incorporation of 5% of silica nanospheres, in mass, for all CAS/CS matrices promoted a better-controlled and sustained release of l-dopa, focusing on the matrix based on 70% of CAS, 30% of CS and 5% of silica, whose l-dopa release lasted for 87 h. Besides, hydrogels are cytocompatible. These new hydrogels can be considered highly attractive materials to be used for controlled and sustained drug release purposes, as well as scaffolds and wound dressing systems.

Nanofibrous silica microparticles/polymer hybrid aerogels for sustained delivery of poorly water-soluble camptothecin.

The surface functionalization of nanoporous silica materials with chemical agents opens up numerous possibilities, including improvement in the materials' ability to carry high payloads of drugs. In this study, KCC-1 nanofibrous silica microparticles are functionalized with methyl groups and then combined with poly(vinyl alcohol) (PVA) and poly(acrylic acid) (PAA) to produce hybrid aerogels that can deliver a poorly water-soluble anticancer drug. The synthetic steps involve freeze-drying a polymer solution of PVA and PAA that contains methyl-modified KCC-1 microparticles and then cross-linking the two polymers via a solid-state reaction. Benefiting from the incorporated methyl-modified KCC-1 microparticles, the hybrid aerogels can load and deliver a payload of camptothecin (CPT), an anticancer drug with antitumor activity but limited clinical application due to its hydrophobicity. The aerogels also show a sustained release of CPT for more than two weeks. The drug release profile can further be tuned by varying the relative amounts of PVA, PAA, and methyl-modified KCC-1. The aerogels are biocompatible to healthy cells, such as immortalized human epithelial (HaCaT), African green monkey kidney (Vero) and murine fibroblast (L929) cells. When loaded with CPT, they show potent antitumor activity against HeLa (HPV18-positive), SiHa (HPV16-positive) and C33A (HPV-negative) cancer cells, significantly inhibiting cell growth.

Two-dimensional thermoresponsive sub-microporous substrate for accelerated cell tissue growth and facile detachment.

Thermoresponsive sub-microporous films having a lower critical solution temperature (LCST), promptly obtained by using the breath figure method, were applied to tissue engineering. These sub-microporous films, sized 100-400 nm, were prepared by blending poly(N-isopropylacrylamide) (PNIPAAm) with polystyrene (PS), in addition to applying the dynamic breath figure (BF) method. The thermoresponsive blends were prepared with polyethylene terephthalate (PET) substrate by using a spin coater; the pore size was modulated according to the spin speed. The sub-microporous films, either with pure PNIPAAm or with different PNIPAAm contents were applied as substrates in order to obtain cell growth (Vero cells); moreover, the effect of PNIPAAm use was evaluated. The PNIPAAm sub-microporous films made the cellular viability to be 9-13-fold higher than the control sample commonly used in cell culture. In addition, the thermoresponsive PNIPAAm properties were even noticed at a low PNIPAAm content in the porous films. Such polymer system was successfully applied to detach the Vero cell tissue using temperature variation.

Liposome and polymeric micelle-based delivery systems for chlorophylls: Photodamage effects on Staphylococcus aureus.

Chlorophyll derivatives (Chls), loaded in F-127 polymeric micelles and DPPC liposomes as drug delivery systems (DDS), have been shown to be remarkable photosensitizers for photodynamic inactivation (PDI). Assays of photoinactivation of Staphylococcus aureus bacteria (as biological models) showed that the effectiveness of Chls in these nanocarriers is dependent on photobleaching processes, photosensitizer locations in DDS, singlet oxygen quantum yields, and Chl uptake to bacteria. These are factors related to changes in Chl structure, such as the presence of metals, charge, and the phytyl chain. The photodynamic activity was significantly greater for Chls without the phytyl chain, i.e., phorbides derivatives. Furthermore, the inactivation of S. aureus was increased by the use of liposomes compared to micelles. Therefore, this research details and shows the high significance of the Chl structure and delivery system to enhance the photodynamic activity. It also highlights the chlorophylls (particularly phorbides) in liposomes as promising photosensitizers for PDI.

Heparosan as a potential alternative to hyaluronic acid for the design of biopolymer-based nanovectors for anticancer therapy.

Glycosaminoglycans (GAGs) are important components of the extracellular matrix that have attracted great interest for drug delivery and pharmaceutical applications due to their diverse biological functions. Among GAGs, heparosan (Hep), a biosynthetic precursor of heparin, has recently emerged as a promising building block for the design of nanoparticles with stealth properties. Though this non-sulfated polysaccharide has a chemical structure very close to that of hyaluronic acid (HA), it distinguishes from HA in that it is biologically inert in the extracellular spaces in the body. In this study, we designed Hep- and HA-based nanogels (NGs) that differ only in the chemical nature of the hydrophilic shell. The nanogels were prepared in a very straightforward way from Hep and HA modified with a thermoresponsive copolymer properly designed to induce self-assembly below room temperature. This versatile synthetic approach also enabled further shell-crosslinking allowing an increase in the colloidal stability. After careful characterization of the un-crosslinked and crosslinked Hep and HA NGs in terms of size (Z-average diameters of un-crosslinked and crosslinked NGs ∼110 and 150 nm) and morphology, they were injected intravenously into tumor-bearing mice for biodistribution experiments. Interestingly, these show that the liver uptake of Hep nanogels is remarkably reduced and tumor accumulation significantly improved as compared to HA nanogels (intensity ratios of tumor-to-liver of 2.2 and 1.4 for the un-crosslinked and crosslinked Hep NGs versus 0.11 for the un-crosslinked and crosslinked HA ones). These results highlight the key role played by the shell-forming GAGs on the in vivo fate of nanogels, which correlates with the specific biological properties of Hep and HA.

Multifunctional hybrid aerogels: hyperbranched polymer-trapped mesoporous silica nanoparticles for sustained and prolonged drug release.

In this study, we show the synthesis of novel hybrid organic-inorganic aerogel materials with one-dimensionally aligned pores and demonstrate their use as sustained and prolonged release systems for a hydrophobic drug. The materials are synthesized by trapping mesoporous silica nanoparticles within a hyperbranched polymer network made from poly(vinyl alcohol) (PVA) and poly(acrylic acid) (PAA). The synthetic method involves dispersing mesoporous silica nanoparticles in a polymer solution, then freeze-drying the solution, and finally subjecting the resulting materials to high temperature to activate a solid-state condensation reaction between PVA and PAA. Before trapping the mesoporous silica nanoparticles within the hyperbranched polymeric network, their pores are decorated with hydrophobic groups so that they can serve as good host materials for hydrophobic drugs. The potential application of the hybrid aerogels as drug carriers is demonstrated using the hydrophobic, anti-inflammatory agent dexamethasone (DEX) as a model drug. Due to their hydrophobic pores, the hybrid aerogels show excellent drug loading capacity for DEX, with an encapsulation efficiency higher than 75%. Furthermore, the release pattern of the payloads of DEX encapsulated in the aerogels is highly tailorable (i.e., it can be made faster or slower, as needed) simply by varying the PVA-to-PAA weight ratio in the precursors, and thus the 3-dimensional (3-D) structures of the cross-linked polymers in them. The materials also show sustained drug release, for over 50 days or more. In addition, the aerogels are biocompatible, as demonstrated with Vero cells, and greatly promote the cell proliferation of L929 fibroblasts. Also, the nanoparticles functionalized with quaternary groups and dispersed within the aerogels display bactericidal activity against E. coli, S. aureus, B. subtilis, and P. aeruginosa. These new hybrid aerogels can, thus, be highly appealing biomaterials for sustained and prolonged drug release, such as wound dressing systems.

Cellulose nanowhiskers decorated with silver nanoparticles as an additive to antibacterial polymers membranes fabricated by electrospinning.

Antimicrobial films based on distinct polymer matrices, poly (vinyl alcohol) (PVA) or poly (N-isopropylacrylamide) (PNIPAAm), and silver nanoparticles (AgNPs) immobilized onto cellulose nanowhiskers (CWs) were successfully prepared by either casting or electrospinning. CWs were first functionalized with carboxylate groups (labeled as CWSAc) and later they were immersed in a silver nitrate solution (AgNO3). After Ag+ ions anchored in the COO- groups are chemically reduced to produce AgNPs. The CWSAc/AgNPs biological activity was evaluated against Staphylococcus aureus (S. aureus), Bacillus Subtilis (B. subtilis), Escherichia coli (E. coli), and Candida albicans (C. albicans). The materials were more effective against C. albicans that showed a MIC of 15.6 µg/mL. In the process of AgNPs synthesis, the activity of the stabilizing agent (gelatin) and concentration of precursor and reducing agents were evaluated. The synthesized polymeric films displayed good antimicrobial activity against S. aureus, E. coli, and Pseudomonas aeruginosa (P. aeruginosa) bacteria. The PVA films with CWSAc/AgNPs showed diameter of the inhibition halo of up to 11 mm. The results obtained displayed that the films obtained have a potential application to be used in different fields such as packaging, membrane filtration, wound dressing, clothing and in different biomedical applications.

Antibacterial Performance of a PCL-PDMAEMA Blend Nanofiber-Based Scaffold Enhanced with Immobilized Silver Nanoparticles.

In the present study, nanofiber meshes (NFs), composed of polycaprolactone and poly[(2-dimethylamino)ethyl methacrylate] at 80/20 and 50/50 PCL/PDMAEMA blend ratios, were obtained through electrospinning. Silver nanoparticles (AgNPs) formed in situ were then immobilized on NF surfaces through adsorption processes at different pHs. It was possible to observe that the amount of NF-AgNPs can be tuned by changing the pH of AgNPs immobilization and the PCL/PDMAEMA ratio in the blend. The neat NF and NF-AgNPs were characterized with respect to their morphology and mechanical properties. The effects of AgNPs on the antibacterial activities and cytotoxicity of meshes were also evaluated. The antibacterial performance of such NF was improved by the presence of AgNPs. The NF-AgNPs presented good antibacterial effect against S. aureus and partial toxicity against E. coli and P. aeruginosa. Also, compared with neat PCL/PDMAEMA the NF-AgNPs presented lower cytotoxicity against VERO cells, showing their potential for applications in tissue engineering for different types of cell growth.

Development, characterization and biocompatibility of chondroitin sulfate/poly(vinyl alcohol)/bovine bone powder porous biocomposite.

Chondroitin sulfate (ChS), a sulfated glycosaminoglycan, poly(vinyl alcohol) (PVA) and bovine bone powder (BBP) were blended to form a novel eco-friendly biocomposite through cyclic freeze-thawing under mild conditions. The systematic investigation reveals that the content of BBP has a remarkable effect on the pore size, porosity, mechanical and liquid uptake properties and biodegradability. At 10wt.% BBP the biocomposite exhibited enhanced mechanical properties and biodegradability rate as compared to the pristine sample. Further, different properties of the biocomposite can be tailored according to the content of BBP. In vitro assays showed that ChS/PVA-BBP does not exert cytotoxicity against healthy cells. In vivo and ex vivo experiments revealed that ChS/PVA-BBP biocomposites are biocompatibility materials without exert pro-inflammatory responses. The biocomposite was completely biodegraded and bioresorbed after 15days of treatment. Taken together, BBP is a low-cost source of hydroxyapatite and collagen, which are insurance. All these results suggest that the biocomposite designed in this study is a promising biomaterial for potential skin tissue engineering.

Scaffolds based on chitosan/pectin thermosensitive hydrogels containing gold nanoparticles.

Thermosensitive hydrogels based on chitosan/pectin (CS/Pec) and CS/Pec/gold nanoparticles (CS/Pec/AuNPs) were successfully prepared with different AuNP levels. Using a tilting method, gelation temperature was demonstrated to decrease when the amount of AuNPs increased and pectin concentrations decreased. The presence of AuNPs in the CS/Pec composite was evaluated via WAXS and UV-vis techniques, while SEM analysis assessed the average size of pores (350-600µm). All samples were extremely cytocompatible with many cell types, such as normal kidney epithelial cells (VERO cells), epithelial colorectal adenocarcinoma cells (HT-29 cells), HPV-16 positive human cervical tumour cells (SiHa cells), kidney epithelial cells (LLCMK2 cells) and murine macrophage cells (J774A1 cells). Cell viability assays using the MTT method upon mouse preosteoblastic cells (MC3T3-E1 cells) showed that CS/Pec and CS/Pec/AuNPs composites had the potential to foster proliferation and growth of bone cells, making them possible stimulators for reconstruction of bone tissues.

Controlling cell growth with tailorable 2D nanoholes arrays.

A facile and reproducible route that can lead to two-dimensional arrays of nanopores in thin polymer films is demonstrated. The formation of the pores in the polymer films involves breath figure phenomenon and occurs during the film deposition by spin coating. The formation of nanoporous thin films takes only few seconds, and the method does not require complex equipment or expensive chemicals. This method also constitutes a straightforward approach to control the size of the pores formed in thin films. Besides allowing control over the average pore size of the porous films, the use of dynamic deposition with the breath figure phenomenon causes the reduction in the pore size to nanometer scale. The nanoporous arrays obtained by the breath figure are applied as substrates for cell growth, and the effect of their nanopore size on cell growth was evaluated. Notably, it is found that cell viability is related to pore size, where 2D nanoporous structure is more beneficial for cell culture than 2D microporous structures. The change in the average pore size of the polymer films from 1.22 µm to 346 nm results in a threefold increase in cell viability.

Polyelectrolyte complex containing silver nanoparticles with antitumor property on Caco-2 colon cancer cells.

Polyelectrolyte complex (beads) based on N,N,N-trimethyl chitosan/alginate was successful obtained and silver nanoparticles (AgNPs) were loaded within beads. In vitro cytotoxicity assays using beads/silver nanoparticles (beads/AgNPs) provided results, indicating that this material significantly inhibited the growth of colon cancer cells (Caco-2). In vitro release studies showed that the beads stabilized AgNPs and repressed Ag(0) oxidation under gastric conditions (pH 2.0). On the other hand, at physiological condition (pH 7.4) the beads/AgNPs released 3.3 µg of Ag(+) per each beads milligram. These studies showed that the concentration of Ag(+) released (3.3 µg) was cytotoxic for the Caco-2 cells and was not cytotoxic on healthy VERO cells. This result opens new perspectives for the manufacture of biomaterials based on beads/AgNPs with anti-tumor properties.

Polyelectrolyte complexes of poly[(2-dimethylamino) ethyl methacrylate]/chondroitin sulfate obtained at different pHs: I. Preparation, characterization, cytotoxicity and controlled release of chondroitin sulfate.

For the first time, polyelectrolyte complex based on poly[(2-dimethylamino) ethyl methacrylate] (PDMAEMA) and chondroitin sulfate (CS) was prepared. The properties of novel material and precursors were investigated by WAXS, FTIR, TGA, SEM and DLS analysis. The PDMAEMA/CS PECs presented hydrophilic-hydrophobic transition at pHs 6.0, 7.0 and 8.0 whereas the non-complexed PDMAEMA showed such a transition at pH 8.0 and not at pHs 6.0 and 7.0. Studies of CS release from PECs at pHs 6 and 8 confirmed that the samples possess the potential to release the CS in alkaline and not in acidic conditions. Since PECs are thermo-responsive due to the reduction of LCST caused by the increase in pH, the release of CS was dependent on temperature and pH factors. Cytotoxicity assays using healthy VERO cells showed that the complexation between CS and PDMAEMA increased the PECs' biocompatibility related to PDMAEMA. However, the biocompatibility depends on the amount of CS present in the PECs.

Silver sulfadiazine loaded chitosan/chondroitin sulfate films for a potential wound dressing application.

Silver sulfadiazine (AgSD) loaded chitosan/chondroitin sulfate (CHI/CS) films were formed to be applied as a potential wound dressing material. The liquid uptake capacity of both, CHI/CS and CHI/CS/AgSD, films exhibited a pH-dependent behavior. Tensile tests showed that the amount of CS used to form the films and the further incorporation of AgSD affect the mechanical properties of the films. In vitro AgSD-release assays showed that the CHI/CS mass ratio influences the AgSD release rate. All the investigated CHI/CS/AgSD films sustain the AgSD release up to 96h at physiological pH. Antibacterial activity and cell viability assays showed that all the CHI/CS/AgSD films have activity against Pseudomonas aeruginosa and Staphylococcus aureus but they were not toxic to Vero cells. The results presented in this work indicate that the CHI/CS/AgSD exhibits potential to be applied as a wound dressing material.