Hydroxybutyl chitosan thermo-sensitive hydrogel: a potential drug delivery system

Drug delivery mediated by hydrogel has shown great promise in controlled drug release field. We report here the development of a hydroxybutyl chitosan (HBC) thermo-sensitive gel to deliver doxorubicine hydrochloride (DOX·HCl) for cancer treatment. Concentrated HBC aqueous solution could transform into hydrogel within 30 s after injection under physiological temperature in non-chemical fashion. The properties of the HBC gels including chemical structure, surface morphology, and rheologic properties were studied. Gelation temperature and gelation time of HBC could be adjusted by HBC concentrations. The gel erosion rate in vivo was faster than solubilization rate in vitro. The mild inflammatory response caused by implantation of the hydrogel was acceptable. The DOX·HCl (1 mg/ml) loaded HBC gels displayed slow release rates that were independent of the HBC concentration, and significantly reduced viability of 4T-1 cells compared with the HBC gels after 1 day incubation. These results indicate that thermo-sensitive HBC hydrogels have promising potential as an injectable drug carrier for pharmaceutical applications.     

Elevated anti-inflammatory effects of eicosapentaenoic acid based self-aggregated glycol chitosan nanoparticles

The formation of nanoparticles from eicosapentaenoic acid (EPA) is crucial to improving EPA's bioavailability and pharmacological properties, and widening its use in biomedical fields. In this study, we report EPA-conjugated glycol chitosan (GC) that can self-aggregate into core-shell nanoparticles. The EPA-GC nanoparticles were internalized into the cytosol of RAW 264.7 cells by endocytosis, which results in effective delivery of EPA to the cells. There were no differences in the cell viability after the treatment with EPA-GC nanoparticles. In the anti-inflammatory studies, the EPA-GC nanoparticles significantly inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production and interleukin-1 beta (IL-1 beta) secretion in RAW 264.7 cells. The anti-inflammatory effects of the EPA-GC nanoparticles were far better than those seen for EPA only. Given their excellent bio-physicochemical properties, it is expected that EPA-GC nanoparticles may have a potential for widening the use of EPA in biomedical fields and, in particular, the treatment of inflammatory diseases.     

Role of Curvature-Sensing Proteins in the Uptake of Nanoparticles with Different Mechanical Properties

Nanoparticles of different properties, such as size, charge, and rigidity, are used for drug delivery. Upon interaction with the cell membrane, because of their curvature, nanoparticles can bend the lipid bilayer. Recent results show that cellular proteins capable of sensing membrane curvature are involved in nanoparticle uptake; however, no information is yet available on whether nanoparticle mechanical properties also affect their activity. Here liposomes and liposome-coated silica are used as a model system to compare uptake and cell behavior of two nanoparticles of similar size and charge, but different mechanical properties. High-sensitivity flow cytometry, cryo-TEM, and fluorescence correlation spectroscopy confirm lipid deposition on the silica. Atomic force microscopy is used to quantify the deformation of individual nanoparticles at increasing imaging forces, confirming that the two nanoparticles display distinct mechanical properties. Uptake studies in HeLa and A549 cells indicate that liposome uptake is higher than for the liposome-coated silica. RNA interference studies to silence their expression show that different curvature-sensing proteins are involved in the uptake of both nanoparticles in both cell types. These results confirm that curvature-sensing proteins have a role in nanoparticle uptake, which is not restricted to harder nanoparticles, but includes softer nanomaterials commonly used for nanomedicine applications.     

Enhanced In Vitro Percutaneous Absorption and In Vivo Anti-Inflammatory Effect of a Selective Cyclooxygenase Inhibitor Using Microemulsion

Celecoxib, a specific COX-2 inhibitor, was recently approved for the treatment of rheumatoid and osteoarthritis, acute pain, familial adenomatous polyposis and primary dysmenorrhea. Oral administration of celecoxib is effective against ultraviolet B radiation (UVB)-induced skin carcinogenesis; however, its clinical use is restricted because of its failure to block the characteristic cutaneous inflammatory response and lower availability at the site of inflammation. Topical application of celecoxib has been effective compared with oral in certain clinical conditions. The present study was undertaken to develop and investigate the development of microemulsion system (isopropyl myristate/medium-chain glyceride/polysorbate 80/water) for topical delivery of celecoxib. The pseudoternary phase diagram was constructed with constant surfactant concentration, and several compositions were identified and characterized by using dynamic light scattering. The in vitro permeation rate of celecoxib through rat skin was determined for microemulsions, microemulsion gel, and cream by using the modified Franz-type diffusion cell. In all formulations tested, celecoxib permeated more quickly, and the microemulsions increased the permeation rate of celecoxib up to 5 and 11 times compared with those of microemulsion gel and cream, respectively. Increasing the concentration of medium-chain mono-/di-glyceride in microemulsion imparted increased droplet size and viscosity and decreased diffusion coefficient. In vivo anti-inflammatory study suggested that the developed microemulsion formulations might serve as potential drug vehicle for the prevention of UVB-induced skin cancer.     

Enhanced in vitro percutaneous absorption and in vivo anti-inflammatory effect of a selective cyclooxygenase inhibitor using microemulsion

Celecoxib, a specific COX-2 inhibitor, was recently approved for the treatment of rheumatoid and osteoarthritis, acute pain, familial adenomatous polyposis and primary dysmenorrhea. Oral administration of celecoxib is effective against ultraviolet B radiation (UVB)-induced skin carcinogenesis; however, its clinical use is restricted because of its failure to block the characteristic cutaneous inflammatory response and lower availability at the site of inflammation. Topical application of celecoxib has been effective compared with oral in certain clinical conditions. The present study was undertaken to develop and investigate the development of microemulsion system (isopropyl myristate/medium-chain glyceride/polysorbate 80/water) for topical delivery of celecoxib. The pseudoternary phase diagram was constructed with constant surfactant concentration, and several compositions were identified and characterized by using dynamic light scattering. The in vitro permeation rate of celecoxib through rat skin was determined for microemulsions, microemulsion gel, and cream by using the modified Franz-type diffusion cell. In all formulations tested, celecoxib permeated more quickly, and the microemulsions increased the permeation rate of celecoxib up to 5 and 11 times compared with those of microemulsion gel and cream, respectively. Increasing the concentration of medium-chain mono-/di-glyceride in microemulsion imparted increased droplet size and viscosity and decreased diffusion coefficient. In vivo anti-inflammatory study suggested that the developed microemulsion formulations might serve as potential drug vehicle for the prevention of UVB-induced skin cancer.     

Enhanced In Vitro Percutaneous Absorption and In Vivo Anti-Inflammatory Effect of a Selective Cyclooxygenase Inhibitor Using Microemulsion

Celecoxib, a specific COX-2 inhibitor, was recently approved for the treatment of rheumatoid and osteoarthritis, acute pain, familial adenomatous polyposis and primary dysmenorrhea. Oral administration of celecoxib is effective against ultraviolet B radiation (UVB)-induced skin carcinogenesis; however, its clinical use is restricted because of its failure to block the characteristic cutaneous inflammatory response and lower availability at the site of inflammation. Topical application of celecoxib has been effective compared with oral in certain clinical conditions. The present study was undertaken to develop and investigate the development of microemulsion system (isopropyl myristate/medium-chain glyceride/polysorbate 80/water) for topical delivery of celecoxib. The pseudoternary phase diagram was constructed with constant surfactant concentration, and several compositions were identified and characterized by using dynamic light scattering. The in vitro permeation rate of celecoxib through rat skin was determined for microemulsions, microemulsion gel, and cream by using the modified Franz-type diffusion cell. In all formulations tested, celecoxib permeated more quickly, and the microemulsions increased the permeation rate of celecoxib up to 5 and 11 times compared with those of microemulsion gel and cream, respectively. Increasing the concentration of medium-chain mono-/di-glyceride in microemulsion imparted increased droplet size and viscosity and decreased diffusion coefficient. In vivo anti-inflammatory study suggested that the developed microemulsion formulations might serve as potential drug vehicle for the prevention of UVB-induced skin cancer.     

Excessive sodium ions delivered into cells by nanodiamonds: implications for tumor therapy

Nanodiamonds (NDs) possess many excellent physical and chemical properties that make them attractive materials for applications in biomedicine. In this paper, the adsorption and delivery of a large amount of sodium ions into the cell interior by NDs in serum-free medium is demonstrated. The excess sodium ions inside the cells induce osmotic stresses followed by cell swelling and an increase in the intracellular levels of calcium and reactive oxygen species (ROS), which leads to severe cellular damage. In complete culture medium, however, serum proteins wrapped around the NDs effectively prevent the sodium ions from adsorbing onto the NDs, and thus the NDs show no cytotoxicity. This work is the first to elaborate on the correlation between the sodium ions adsorbed on the nanomaterials and their bio-effects. Excessive ions delivered into cells by NDs might have potential applications in tumor therapy.     

Collagen gel three-dimensional matrices combined with adhesive proteins stimulate neuronal differentiation of mesenchymal stem cells

Three-dimensional gel matrices provide specialized microenvironments that mimic native tissues and enable stem cells to grow and differentiate into specific cell types. Here, we show that collagen three-dimensional gel matrices prepared in combination with adhesive proteins, such as fibronectin (FN) and laminin (LN), provide significant cues to the differentiation into neuronal lineage of mesenchymal stem cells (MSCs) derived from rat bone marrow. When cultured within either a three-dimensional collagen gel alone or one containing either FN or LN, and free of nerve growth factor (NGF), the MSCs showed the development of numerous neurite outgrowths. These were, however, not readily observed in two-dimensional culture without the use of NGF. Immunofluorescence staining, western blot and fluorescence-activated cell sorting analyses demonstrated that a large population of cells was positive for NeuN and glial fibrillary acidic protein, which are specific to neuronal cells, when cultured in the three-dimensional collagen gel. The dependence of the neuronal differentiation of MSCs on the adhesive proteins containing three-dimensional gel matrices is considered to be closely related to focal adhesion kinase (FAK) activation through integrin receptor binding, as revealed by an experiment showing no neuronal outgrowth in the FAK-knockdown cells and stimulation of integrin β1 gene. The results provided herein suggest the potential role of three-dimensional collagen-based gel matrices combined with adhesive proteins in the neuronal differentiation of MSCs, even without the use of chemical differentiation factors. Furthermore, these findings suggest that three-dimensional gel matrices might be useful as nerve-regenerative scaffolds.     

Biomaterials for Personalized Cell Therapy

Cell therapy has already had an important impact on healthcare and provided new treatments for previously intractable diseases. Notable examples include mesenchymal stem cells for tissue regeneration, islet transplantation for diabetes treatment, and T cell delivery for cancer immunotherapy. Biomaterials have the potential to extend the therapeutic impact of cell therapies by serving as carriers that provide 3D organization and support cell viability and function. With the growing emphasis on personalized medicine, cell therapies hold great potential for their ability to sense and respond to the biology of an individual patient. These therapies can be further personalized through the use of patient-specific cells or with precision biomaterials to guide cellular activity in response to the needs of each patient. Here, the role of biomaterials for applications in tissue regeneration, therapeutic protein delivery, and cancer immunotherapy is reviewed, with a focus on progress in engineering material properties and functionalities for personalized cell therapies.     

The effect of starch-based biomaterials on leukocyte adhesion and activation <Emphasis Type="Italic">in vitro</Emphasis>

Leukocyte adhesion to biomaterials has long been recognised as a key element to determine their inflammatory potential. Results regarding leukocyte adhesion and activation are contradictory in some aspects of the material's effect in determining these events. It is clear that together with the wettability or hydrophilicity/hydrophobicity, the roughness of a substrate has a major effect on leukocyte adhesion. Both the chemical and physical properties of a material influence the adsorbed proteins layer which in turn determines the adhesion of cells. In this work polymorphonuclear (PMN) cells and a mixed population of monocytes/macrophages and lymphocytes (mononuclear cells) were cultured separately with a range of starch-based materials and composites with hydroxyapatite (HA). A combination of both reflected light microscopy and scanning electron microscopy (SEM) was used in order to study the leukocyte morphology. The quantification of the enzyme lactate dehydrogenase (LDH) was used to determine the number of viable cells adhered to the polymers. Cell adhesion and activation was characterised by immunocytochemistry based on the expression of several adhesion molecules, crucial in the progress of an inflammatory response. This work supports previous in vitro studies with PMN and monocytes/macrophages, which demonstrated that there are several properties of the materials that can influence and determine their biological response. From our study, monocytes/macrophages and lymphocytes adhere in similar amounts to more hydrophobic (SPCL) and to moderately hydrophilic (SEVA-C) surfaces and do not preferentially adhere to rougher substrates (SCA). Contrarily, more hydrophilic surfaces (SCA) induced higher PMN adhesion and lower activation. In addition, the hydroxyapatite reinforcement induces changes in cell behaviour for some materials but not for others. The observed response to starch-based biodegradable polymers was not significantly different from the control materials. Thus, the results reported herein indicate the low potential of the starch-based biodegradable polymers to induce inflammation especially the HA reinforced composite materials.     

Intracellular Labeling with Extrinsic Probes: Delivery Strategies and Applications

Extrinsic probes have outstanding properties for intracellular labeling to visualize dynamic processes in and of living cells, both in vitro and in vivo. Since extrinsic probes are in many cases cell‐impermeable, different biochemical, and physical approaches have been used to break the cell membrane barrier for direct delivery into the cytoplasm. In this Review, these intracellular delivery strategies are discussed, briefly explaining the mechanisms and how they are used for live‐cell labeling applications. Methods that are discussed include three biochemical agents that are used for this purpose—purpose‐different nanocarriers, cell penetrating peptides and the pore‐foraming bacterial toxin streptolysin O. Most successful intracellular label delivery methods are, however, based on physical principles to permeabilize the membrane and include electroporation, laser‐induced photoporation, micro‐ and nanoinjection, nanoneedles or nanostraws, microfluidics, and nanomachines. The strengths and weaknesses of each strategy are discussed with a systematic comparison provided. Finally, the extrinsic probes that are reported for intracellular labeling so‐far are summarized, together with the delivery strategies that are used and their performance. This combined information should provide for a useful guide for choosing the most suitable delivery method for the desired probes.     

Therapeutic‐Gas‐Responsive Hydrogel

Nitric oxide (NO) is a crucial signaling molecule with various functions in physiological systems. Due to its potent biological effect, the preparation of responsive biomaterials upon NO having temporally transient properties is a challenging task. This study represents the first therapeutic‐gas (i.e., NO)‐responsive hydrogel by incorporating a NO‐cleavable crosslinker. The hydrogel is rapidly swollen in response to NO, and not to other gases. Furthermore, the NO‐responsive gel is converted to enzyme‐responsive gels by cascade reactions from an enzyme to NO production for which the NO precursor is a substrate of the enzyme. The application of the hydrogel as a NO‐responsive drug‐delivery system is proved here by revealing effective protein drug release by NO infusion, and the hydrogel is also shown to be swollen by the NO secreted from the cultured cells. The NO‐responsive hydrogel may prove useful in many applications, for example drug‐delivery vehicles, inflammation modulators, and as a tissue scaffold.     

Small mesh size hydrogel for functional photocontrol of encapsulated enzymes and small probe molecules

Previously, we developed the "protein activation and release from cage by external light" (PARCEL) method for controlling the function of proteins by encapsulating them in a photodegradable hydrogel and subsequently releasing them by ultraviolet (UV) irradiation of the gel. However, controlling small proteins is difficult because small proteins can leak from the gap (ca. 12.4 nm) of the mesh structure of the hydrogel without irradiation. Here, we developed a photodegradable gel with a smaller mesh size (~3.6 nm) and used the new gel to control the function of three small enzymes (trypsin, chymotrypsin, and elastase) and several small nonprotein molecules. The new gel showed reduced leakage of the proteins without irradiation, and tryptic activity increased approximately 78-fold upon irradiation of gel-encapsulated trypsin. The new gel also permitted encapsulation and release of 4',6-diamidino-2-phenylindole (DAPI, molecular weight 277), a small DNA-specific fluorescent probe. After irradiation to the gel-encapsulated DAPI and subsequent addition of DNA, strong fluorescence of the DAPI-DNA complex was observed. Our results indicate that reducing the gel mesh size from 12.4 to 3.6 nm should allow the encapsulation of various proteins and small molecules in an inactive state and their subsequent light-induced release. We expect this method to be useful in preparation of photoactivated biosensors, drug delivery systems, and catalysis.     

Proteome Analysis of Thyroid Cancer Cells After Long-Term Exposure to a Random Positioning Machine

Annulling gravity during cell culturing triggers various types of cells to change their protein expression in a time dependent manner. We therefore decided to determine gravity sensitive proteins and their period of sensitivity to the effects of gravity. In this study, thyroid cancer cells of the ML-1 cell line were cultured under normal gravity (1?g) or in a random positioning machine (RPM), which simulated near weightlessness for 7 and 11?days. Cells were then sonicated and proteins released into the supernatant were separated from those that remained attached to the cell fragments. Subsequently, both types of proteins were fractionated by free-flow isoelectric focussing (FF-IEF). The fractions obtained were further separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to which comparable FF-IEF fractions derived from cells cultured either under 1?g or on the RPM had been applied side by side. The separation resulted in pairs of lanes, on which a number of identical bands were observed. Selected gel pieces were excised and their proteins determined by mass spectrometry. Equal proteins from cells cultured under normal gravity and the RPM, respectively, were detected in comparable gel pieces. However, many of these proteins had received different Mascot scores. Quantifying heat shock cognate 71?kDa protein, glutathione S-transferase P, nucleoside diphosphate kinase A and annexin-2 by Western blotting using whole cell lysates indicated usefulness of Mascot scores for selecting the most efficient antibodies.     

Sustained release of a p38 inhibitor from non-inflammatory microspheres inhibits cardiac dysfunction

Cardiac dysfunction following acute myocardial infarction is a major cause of death in the world and there is a compelling need for new therapeutic strategies. In this report we demonstrate that a direct cardiac injection of drug-loaded microparticles, formulated from the polymer poly(cyclohexane-1,4-diylacetone dimethylene ketal) (PCADK), improves cardiac function following myocardial infarction. Drug-delivery vehicles have great potential to improve the treatment of cardiac dysfunction by sustaining high concentrations of therapeutics within the damaged myocardium. PCADK is unique among currently used polymers in drug delivery in that its hydrolysis generates neutral degradation products. We show here that PCADK causes minimal tissue inflammatory response, thus enabling PCADK for the treatment of inflammatory diseases, such as cardiac dysfunction. PCADK holds great promise for treating myocardial infarction and other inflammatory diseases given its neutral, biocompatible degradation products and its ability to deliver a wide range of therapeutics.     

An insight on human skin penetration of diflunisal: lipogel versus hydrogel microemulsion

Diflunisal is a NSAID used in acute and long term management of pain and inflammation associated with osteoarthritis, rheumatoid arthritis and symptoms of primary dysmenorrhea. However, its oral use is associated with side effects such as peptic ulceration, dyspepsia, gastrointestinal disturbances and bleeding. The aim of this work was to develop lecithin organogels (LO) transdermal delivery system for diflunisal and to study its human skin penetration ability in comparison with an optimized microemulsion-based hydrogel. Ternary phase diagrams were constructed using butyl lactate as an organic solvent and two commercial grades of lecithin. The formation of gel phase was lecithin concentration dependent with Phosholipion 85?G being capable of forming organogels at lower lecithin concentration than Lipoid S75. The gels prepared using butyl lactate were able to tolerate higher amounts of water than could be incorporated in the lipogels prepared with other organic solvents. All the investigated gels possessed acceptable physical properties and were able to deliver diflunisal through human skin. The lipogels delivered higher total drug amount through the skin than the hydrogel. The composition of lecithin seemed to have some effect on the skin permeability enhancement ability of the lipogel. Lecithin containing higher amount of phosphatidyl ethanolamine could provide better transdermal delivery. The elaborated lecithin organogels are potential carriers that create a good opportunity for transdermal delivery of diflunisal overcoming the side effects associating its oral route.     

Electronic control of Ca2+ signalling in neuronal cells using an organic electronic ion pump

Cells and tissues use finely regulated ion fluxes for their intra- and intercellular communication. Technologies providing spatial and temporal control for studies of such fluxes are however, limited. We have developed an electrophoretic ion pump made of poly(3,4-ethylenedioxythiophene) doped with poly(styrene sulphonate) (PEDOT:PSS) to mediate electronic control of the ion homeostasis in neurons. Ion delivery from a source reservoir to a receiving electrolyte via a PEDOT:PSS thin-film channel was achieved by electronic addressing. Ions are delivered in high quantities at an associated on/off ratio exceeding 300. This induces physiological signalling events that can be recorded at the single-cell level. Furthermore, miniaturization of the device to a 50-microm-wide channel allows for stimulation of individual cells. As this technology platform allows for electronic control of ion signalling in individual cells with proper spatial and temporal resolution, it will be useful in further studies of communication in biological systems.     

Biocompatible SU-8-Based Microprobes for Recording Neural Spike Signals From Regenerated Peripheral Nerve Fibers

A biocompatible neural microprobe constructed using well-established SU-8 microfabrication techniques is described that was designed to record fiber spike signals from regenerated axons within peripheral nerves. These microprobes features bipolar longitudinal gold electrodes recessed below the surface within “grooves” designed to guide the growth of regenerating axons along the length of the grooves and limit the number of fibers that come in contact with the longitudinal electrodes. In addition, screening microprobe toxicity using cultures of human skin fibroblasts, the biocompatibility of these SU-8 microprobes for neural interface applications, in particular, was specifically verified using primary cultures of two sensitive cell types found in peripheral nerves: purified Schwann cells and explanted dorsal root ganglion (DRG) neurons and their fibers. The SU-8 microprobes were surgically implanted into transected rat Sciatic nerves within a unique peripheral nerve regeneration tube. Long-term fiber spike signals were recorded with these SU-8 microprobes in 13 chronically implanted rats for periods from 4 to 51 weeks without any signs of tissue damage or inflammatory reaction.      

Nanostructured Mineral Coatings Stabilize Proteins for Therapeutic Delivery

Proteins tend to lose their biological activity due to their fragile structural conformation during formulation, storage, and delivery. Thus, the inability to stabilize proteins in controlled‐release systems represents a major obstacle in drug delivery. Here, a bone mineral inspired protein stabilization strategy is presented, which uses nanostructured mineral coatings on medical devices. Proteins bound within the nanostructured coatings demonstrate enhanced stability against extreme external stressors, including organic solvents, proteases, and ethylene oxide gas sterilization. The protein stabilization effect is attributed to the maintenance of protein conformational structure, which is closely related to the nanoscale feature sizes of the mineral coatings. Basic fibroblast growth factor (bFGF) released from a nanostructured mineral coating maintains its biological activity for weeks during release, while it maintains activity for less than 7 d during release from commonly used polymeric microspheres. Delivery of the growth factors bFGF and vascular endothelial growth factor using a mineral coated surgical suture significantly improves functional Achilles tendon healing in a rabbit model, resulting in increased vascularization, more mature collagen fiber organization, and a two fold improvement in mechanical properties. The findings of this study demonstrate that biomimetic interactions between proteins and nanostructured minerals provide a new, broadly applicable mechanism to stabilize proteins in the context of drug delivery and regenerative medicine.