Improved miscibility and toughness of biological poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/poly(lactic acid) blends via melt-blending-induced thermal degradation.

Polymer blending has been a facile method to resolve the brittle issue of poly(lactic acid) (PLA). Yet, miscibility becomes the primary concern that would affect the synergy effect of polymer blending. This study aimed to improve the miscibility of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB) and PLA by lowering their molecular weights via a melt-blending-induced thermal degradation during mechanical mixing to form m-P34HB/PLA blends. The molecular weight of the P34HB was significantly reduced after blending, thereby improving the miscibility of the blends, as evidenced by the shift of glass transition temperatures. Also, simulation based on Flory-Huggins theory demonstrated increased miscibility with decreasing molecular weight of the polymers. Moreover, the thermal gravimetric analysis revealed that the PLA provided a higher shielding effect to the P34HB in the blends prepared by melt-blending than those by solution-blending, that the addition of PLA could retard the chain scission of P34HB and delay its degradation. The addition of m-P34HB at 20 wt% in the blend contributed to a 60-fold enhancement in the elongation at break and an increment of 4.6 folds in the Izod impact strength. The enzymatic degradation using proteinase K revealed the preferential to degrade the PLA in the blends and followed the surface erosion mechanism.

In Situ Encapsulation of Camptothecin by Self-Assembly of Poly(acrylic acid)-b-Poly(N-Isopropylacrylamide) and Chitosan for Controlled Drug Delivery.

Camptothecin (CPT) has been shown to exhibit anticancer activity against several cancers. Nevertheless, CPT is very hydrophobic with poor stability, and thus its medical application is limited. Therefore, various drug carriers have been exploited for effectively delivering CPT to the targeted cancer site. In this study, a dual pH/thermo-responsive block copolymer of poly(acrylic acid-b-N-isopropylacrylamide) (PAA-b-PNP) was synthesized and applied to encapsulate CPT. At temperatures above its cloud point, the block copolymer self-assembled to form nanoparticles (NPs) and in situ encapsulate CPT, owing to their hydrophobic interaction as evidenced by fluorescence spectrometry. Chitosan (CS) was further applied on the surface through the formation of a polyelectrolyte complex with PAA for improving biocompatibility. The average particle size and zeta potential of the developed PAA-b-PNP/CPT/CS NPs in a buffer solution were 168 nm and -30.6 mV, respectively. These NPs were still stable at least for 1 month. The PAA-b-PNP/CS NPs exhibited good biocompatibility toward NIH 3T3 cells. Moreover, they could protect the CPT at pH 2.0 with a very slow-release rate. At pH 6.0, these NPs could be internalized by Caco-2 cells, followed by intracellular release of the CPT. They became highly swollen at pH 7.4, and the released CPT was able to diffuse into the cells at higher intensity. Among several cancer cell lines, the highest cytotoxicity was observed for H460 cells. As a result, these environmentally-responsive NPs have the potential to be applied in oral administration.

In Situ Release of Ulvan from Crosslinked Ulvan/Chitosan Complex Films and Their Evaluation as Wound Dressings.

When a wound forms due to any injuries, it should be covered with a functional wound dressing for accelerating wound healing and reducing infection. In this study, crosslinked ulvan/chitosan complex films were prepared with or without the addition of glycerol and chlorophyll, and their wound healing properties were evaluated for potential application in wound dressing. The results showed that the tensile strength and elongation at break of the prepared ulvan/chitosan complex films were 2.23-2.48 MPa and 83.8-108.5%, respectively. Moreover, their water vapor transmission rates (WVTRs) were in the range of 1791-2029 g/m2-day, providing suitable environment for wound healing. Particularly, these complex films could release ulvan in situ in a short time, and the film with chlorophyll added had the highest release rate, reaching 62.8% after 20 min of releasing. In vitro studies showed that they were biocompatible toward NIH 3T3 and HaCaT cells, and promoted the migration of NIH 3T3 cells. These complex films could protect HaCaT cells from oxidative damage and reduce the production of reactive oxygen species (ROS); the addition of chlorophyll also effectively reduced the inflammatory response induced by LPS as found in the reduction in both NO and IL-6. Animal models showed that the complex films added with glycerol and chlorophyll could promote wound healing in the early stage, while accelerating the regeneration of dermal glands and collagen production. Briefly, these ulvan/chitosan complex films had good physiochemical properties and biological activity, and could accelerate wound healing both in vitro and in vivo.

Theophylline-Loaded Pectin/Chitosan Hydrochloride Submicron Particles Prepared by Spray Drying with a Continuous Feeding Ultrasonic Atomizer.

Pectin/chitosan hydrochloride (CHC) particles containing theophylline were prepared by a spray-drying apparatus coupled with a continuous feeding ultrasonic atomizer and a heating column. The formation of the submicron particles was investigated at various compositions of pectin solutions added with a chitosan hydrochloride or calcium chloride solution as a crosslinking agent. Scanning electron microscopic (SEM) images showed the pectin/chitosan hydrochloride particles had spherical and smooth surfaces. Depending on the feeding concentrations, the produced particles had diameters in the range of 300 to 800 nm with a narrow size distribution. Furthermore, the theophylline (TH)-loaded pectin/CHC particles were also prepared by the same apparatus. The TH release from the submicron particles in phosphate-buffered saline at 37 °C was monitored in real-time by a UV-Visible spectrophotometer. The Ritger-Peppas model could well describe the TH release profiles. All the diffusional exponents (n) of the release systems were greater than 0.7; thus, the transport mechanism was not a simple Fickian diffusion. Particularly, the n value was 1.14 for the TH-loaded particles at a pectin/CHC weight ratio of 5/2, which was very close to the zero-order drug delivery (n = 1). Therefore, the constant drug-release rate could be achieved by using the spray-dried pectin/CHC particles as the drug carrier.

Preparation and characterization of fast dissolving ulvan microneedles for transdermal drug delivery system.

Dissolving microneedles made from natural polymers recently have gained much attention as an efficient transdermal drug delivery system (TDDS). For the first time, ulvan, a sulfated polysaccharide extracted from Ulva lactuca, was applied to fabricate dissolving microneedles through a two-step casting method. The ulvan microneedles (UMNs) made from 4% ulvan solution were in a pyramidal shape with an average height of 655 µm and an aspect ratio of 2.63. The in vitro skin insertion study showed the UMNs could totally penetrate into the porcine skin to the dermis layer and rapidly dissolved as the needle height was reduced by 90.3% after post-insertion of only 2 min. The rapid dissolution of UMNs in situ thus could release the loaded model drugs of rhodamine 6G (R6G) and bovine serum albumin-fluorescein isothiocyanate conjugate (FITC-BSA) in the skin tissue. The in vitro drug release profiles through porcine skin revealed the UMNs markedly enhanced the cumulative release of FITC-BSA. In addition, the UMNs had good biocompatibility towards normal cells of HaCaT and NIH3T3. Briefly, this study demonstrates the rapidly dissolving UMNs could effectively carry the drug into skin and thus can be developed as a potential TDDS in the pharmaceutical and cosmeceutical fields.

Curcumin-laden dual-targeting fucoidan/chitosan nanocarriers for inhibiting brain inflammation via intranasal delivery.

Curcumin can reduce the production of brain inflammatory mediators and symptoms of brain diseases. However, a large amount of free curcumin needs to be administered to achieve an effective level in the brain because of its poor water-solubility. Fucoidan and chitosan were reported to respectively target P-selectin and acidic microenvironment expressed by pathologically inflammatory cells/tissues. Herein, the self-assembly of chitosan and fucoidan which could encapsulate curcumin was developed to form the multi-stimuli-responsive nanocarriers, and their pathological pH- and P-selectin-responsive aspects were characterized. Through intranasal delivery to the brain, these curcumin-containing chitosan/fucoidan nanocarriers with dual pH-/P-selectin-targeting properties to the brain lesions improved drug delivery, distribution, and accumulation in the inflammatory brain lesions as evidenced by an augmented inhibitory effect against brain inflammation. This promising multifunctional nanocarrier with a novel drug-delivery route should allow potential clinical biomedical uses by neurosurgeon in the future.

Carboxymethyl chitosan has sensitive two-way CO2-responsive hydrophilic/hydrophobic feature.

Carboxymethyl chitosans (CMC) with various degrees of carboxymethyl substitution were prepared and investigated on their changes in water solubility in response to bubbling of CO2 or N2 as a function of the relative concentrations of COOH and NH2 side groups. When having similar concentrations of COOH and NH2, the produced CMC was water soluble at pH 10 and consecutively experienced peculiar dissolution-to-precipitation-to-dissolution during bubbling of CO2, and experienced reverse dissolution-to-precipitation-to-dissolution process during subsequently bubbling of N2. With the concentration of COOH much higher than that of NH2, the water soluble CMC at pH 10 exhibited no phase changes in response to bubbling of CO2 and N2. This newly developed CMC solution system with novel CO2 responsive amphiphilic feature has a potential use as a CO2 switchable surfactant to control interface of mixtures of hydrophilic and hydrophobic species in emulsification/demulsification applications.

Guided bone regeneration activity of different calcium phosphate/chitosan hybrid membranes.

To fulfill the properties of membrane for guided bone tissue regeneration, chitosan (CS) and calcium phosphates were blended to produce porous hybrid membranes by lyophilization. We synthesized three different calcium phosphates: calcium deficient hydroxyapatite (CDHA), biphasic calcium phosphate (BCP) and ß­tricalcium phosphate (TCP) by a reverse emulsion method followed by calcination, and compared their efficacy on bone regeneration. The CDHA/CS, BCP/CS, and TCP/CS membranes had an interconnected pore structure with porosity of 91-95% and pore size of 102-147 µm. These hybrid membranes could promote the permeability and adhesiveness to bone cells as demonstrated by in-vitro cell culture of primary osteoblast. Particularly, the CDHA/CS and BCP/CS could further increase the cell attachment and differentiation, whereas the BCP/CS and TCP/CS could enhance cell proliferation. Finally, these hybrid membranes were assessed for guided bone regeneration in the critical-size calvarial bone defects created in SD rats. Histological and histomorphometric analyses revealed that the BCP/CS membrane had the most effective bone regeneration compared to the other two hybrid membranes. At three-week post-surgery, the BCP/CS membrane could enhance new bone generation up to 57% of the original bone defect area. The BCP/CS membrane thus has the potential to be applied for guided bone regeneration.

Temperature/pH/Enzyme Triple-Responsive Cationic Protein/PAA-b-PNIPAAm Nanogels for Controlled Anticancer Drug and Photosensitizer Delivery against Multidrug Resistant Breast Cancer Cells.

The tumor microenvironments are often acidic and overexpress specific enzymes. In this work, we synthesized a poly(AA-b-NIPAAm) copolymer (PAA-b-PNIPAAm) using a reversible addition-fragmentation chain transfer (RAFT) polymerization method. PAA-b-PNIPAAm and a cationic protein (protamine) were self-assembled into nanogels, which effectively reduced the cytotoxicity of protamine. The protamine/PAA-b-PNIPAAm nanogels were responsive to the stimuli including temperature, pH, and enzyme due to disaggregation of PAA-b-PNIPAAm, change in random coil/α-helix conformation of protamine, and enzymatic hydrolysis of the protein. Changing the pH from 7.4 to a lowered pHe (6.5-5.0) resulted in an increase in mean particle size and smartly converted surface charge from negative to positive. The cationic nanogels easily passed through the cell membrane and enhanced intracellular localization and accumulation of doxorubicin-loaded nanogels in multidrug resistant MCF-7/ADR breast cancer cells. Cold shock treatment triggered rapid intracellular release of doxorubicin against P-glycoprotein (Pgp)-mediated drug efflux, showing significantly improved anticancer efficacy as compared with free DOX. Furthermore, the nanogels were able to carry a rose bengal photosensitizer and caused significant damage to the multidrug resistant cancer cells under irradiation. The cationic nanogels with stimuli-responsive properties show promise as drug carrier for chemotherapy and photodynamic therapy against cancers.

Delivery of berberine using chitosan/fucoidan-taurine conjugate nanoparticles for treatment of defective intestinal epithelial tight junction barrier.

Bacterial-derived lipopolysaccharides (LPS) can cause defective intestinal barrier function and play an important role in the development of inflammatory bowel disease. In this study, a nanocarrier based on chitosan and fucoidan was developed for oral delivery of berberine (Ber). A sulfonated fucoidan, fucoidan-taurine (FD-Tau) conjugate, was synthesized and characterized by Fourier transform infrared (FTIR) spectroscopy. The FD-Tau conjugate was self-assembled with berberine and chitosan (CS) to form Ber-loaded CS/FD-Tau complex nanoparticles with high drug loading efficiency. Berberine release from the nanoparticles had fast release in simulated intestinal fluid (SIF, pH 7.4), while the release was slow in simulated gastric fluid (SGF, pH 2.0). The effect of the berberine-loaded nanoparticles in protecting intestinal tight-junction barrier function against nitric oxide and inflammatory cytokines released from LPS-stimulated macrophage was evaluated by determining the transepithelial electrical resistance (TEER) and paracellular permeability of a model macromolecule fluorescein isothiocyanate-dextran (FITC-dextran) in a Caco-2 cells/RAW264.7 cells co-culture system. Inhibition of redistribution of tight junction ZO-1 protein by the nanoparticles was visualized using confocal laser scanning microscopy (CLSM). The results suggest that the nanoparticles may be useful for local delivery of berberine to ameliorate LPS-induced intestinal epithelia tight junction disruption, and that the released berberine can restore barrier function in inflammatory and injured intestinal epithelial.

Preparation of water-resistant antifog hard coatings on plastic substrate.

A novel water resistant antifog (AF) coating for plastic substrates was developed, which has a special hydrophilic/hydrophobic bilayer structure. The bottom layer, acting both as a mechanical support and a hydrophobic barrier against water penetration, is an organic-inorganic composite comprising colloidal silica embedded in a cross-linked network of dipentaethritol hexaacrylate (DPHA). Atop this layer, an AF coating is applied, which incorporates a superhydrophilic species synthesized from Tween-20 (surfactant), isophorone diisocyanate (coupling agent), and 2-hydroxyethyl methacrylate (monomer). Various methods, e.g., FTIR, SEM, AFM, contact angle, and steam test, were employed to characterize the prepared AF coatings. The results indicated that the size and the continuity of the hydrophilic domains on the top surface increased with increasing added amount of T20, however, at the expense of hardness, adhesiveness, and water resistivity. The optimal T20 content was found to be 10 wt %, at which capacity the resultant AF coating was transparent and wearable (5H, hardness) and could be soaked in water for 7 days at 25 °C without downgrading of its AF capability.

Asymmetric composite membranes from chitosan and tricalcium phosphate useful for guided bone regeneration.

To fulfill the properties of barrier membranes useful for guided bone tissue regeneration in the treatment of periodontitis, in this study a simple process combining lyophilization with preheating treatment to produce asymmetric barrier membranes from biodegradable chitosan (CS) and functional ß-tricalcium phosphate (TCP) was proposed. By preheating TCP/CS (3:10, w/w) in an acetic acid solution at 40°C, a skin layer that could greatly increase the mechanical properties of the membrane was formed. The asymmetric membrane with a skin layer had a modulus value almost 4-times that of the symmetric porous membrane produced only by lyophilization. This is beneficial for maintaining a secluded space for the bone regeneration, as well as to prevent the invasion of other tissues. The subsequent lyophilization at -20°C then gave the rest of material an interconnected pore structure with high porosity (83.9-90.6%) and suitable pore size (50-150 µm) which could promote the permeability and adhesiveness to bone cells, as demonstrated by the in vitro cell-culture of hFOB1.19 osteoblasts. Furthermore, the TCP particles added to CS could further increase the rigidity and the cell attachment and proliferation of hFOB1.19. The TCP/CS asymmetric composite membrane thus has the potential to be used as the barrier membrane for guided bone regeneration.

Preparation and characterization of poly(hydroxyalkanoate) from the fermentation of Haloferax mediterranei.

In this study, fed-batch fermentation of Haloferax mediterranei using glucose and yeast extract as carbon and nitrogen source, respectively, was carried out to produce poly(hydroxyalkanoate) (PHA). After fermentation for 117 h, the concentration of H. mediterranei and PHA content reached 85.8 g/l and 48.6%, respectively. 1H- and 13C-NMR spectra proved that the produced PHA was poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) co-polymer. However, further fractionation using chloroform/acetone revealed that the produced PHA consisted of at least two compositionally different co-polymers (P1 and P2). One P(3HB-co-3HV) co-polymer (P1, 93.4 wt%) contains 10.7 mol% of 3-HV unit in the chain structure and has a high molecular weight of 569.5 kg/mol. The other one (P2, 6.6 wt%) has a slightly higher 3-HV content, ca. 12.3 mol%, but its molecular weight is relatively low, 78.2 kg/mol. Both fractions exhibit two overlapped melting peaks measured by differential scanning calorimetry when the heating rate is at and below 20 degrees C/min. For example, at a heating rate of 10 degrees C/min, the two melting peaks occur at 134.8 degrees C and 144.3 degrees C for P1, and 131.1 degrees C and 140.6 degrees C for P2. Through observing the variation of relative intensity of these two melting peaks by changing the heating rate, it was proven that the phenomenon is caused by a melt/recrystallization process. Glass-transition temperature, crystallization temperature and thermal degradation behavior of these co-polymers were also discussed.

Preparation and antibacterial test of chitosan/PAA/PEGDA bi-layer composite membranes.

Chitosan/poly(acrylic acid)/poly(ethylene glycol) diacrylate (PEGDA) composite membranes with a bi-layer configuration were prepared and their potential application as an antibacterial material was examined. A two-step process was adopted. A dope consisting of PEGDA, acrylic acid (AA) and a photoinitiator was cast and then UV-cured on a glass substrate to form a mechanically strong, dense membrane. Subsequently, the membrane was coated with a layer of solution composed of chitosan, AA and water. As the majority of AA diffused downwards into the supporting layer underneath, chitosan coagulated with residual AA to form a nano-layer on the top surface by means of UV irradiation. Low-voltage field-emission scanning electron microscopy was used to observe the membrane morphology and to measure the thickness of the top layer. Contact angle measurements indicated a top layer mixed with chitosan and poly(acrylic acid), which was confirmed by chemical composition analysis of X-ray photon spectroscopy. The antibacterial activities of the formed membranes were tested both with respect to a Gram-negative (Escherichia coli) and a Gram-positive (Staphylococcus aureus) bacteria.