Physical and Chemical Factors Influencing the Printability of Hydrogel-based Extrusion Bioinks.

Bioprinting researchers agree that "printability" is a key characteristic for bioink development, but neither the meaning of the term nor the best way to experimentally measure it has been established. Furthermore, little is known with respect to the underlying mechanisms which determine a bioink's printability. A thorough understanding of these mechanisms is key to the intentional design of new bioinks. For the purposes of this review, the domain of printability is defined as the bioink requirements which are unique to bioprinting and occur during the printing process. Within this domain, the different aspects of printability and the factors which influence them are reviewed. The extrudability, filament classification, shape fidelity, and printing accuracy of bioinks are examined in detail with respect to their rheological properties, chemical structure, and printing parameters. These relationships are discussed and areas where further research is needed, are identified. This review serves to aid the bioink development process, which will continue to play a major role in the successes and failures of bioprinting, tissue engineering, and regenerative medicine going forward.

Nanogels Derived from Fish Gelatin: Application to Drug Delivery System.

The gelatin extracted from mammals of porcine and bovine has been prominently used in pharmaceutical, medical, and cosmetic products. However, there have been some concerns for their usage due to religious, social and cultural objections, and animal-to-human infectious disease. Recently, gelatin from marine by-products has received growing attention as an alternative to mammalian gelatin. In this study, we demonstrate the formation of nanogels (NGs) using fish gelatin methacryloyl (GelMA) and their application possibility to the drug delivery system. The fabrication of fish GelMA NGs is carried out by crosslinking through the photopolymerization of the methacryloyl substituent present in the nanoemulsion droplets, followed by purification and redispersion. There were different characteristics depending on the aqueous phase in the emulsion and the type of solvent used in redispersion. The PBS-NGs/D.W., which was prepared using PBS for the aqueous phase and D.W. for the final dispersion solution, had a desirable particle size (<200 nm), low PdI (0.16), and high drug loading efficiency (77%). Spherical NGs particles were observed without aggregation in TEM images. In vitro release tests of doxorubicin (DOX)-GelMA NGs showed the pH-dependent release behavior of DOX. Also, the MTT experiments demonstrated that DOX-GelMA NGs effectively inhibited cell growth, while only GelMA NGs exhibit higher percentages of cell viability. Therefore, the results suggest that fish GelMA NGs have a potential for nano-carrier as fine individual particles without the aggregation and cytotoxicity to deliver small-molecule drugs.

The ethanolic extract of Aralia continentalis ameliorates cognitive deficits via modifications of BDNF expression and anti-inflammatory effects in a rat model of post-traumatic stress disorder.

BACKGROUND: Post-traumatic stress disorder (PTSD) is a disease associated with that the experience of traumatic stress. The traumatic experience results in the development of a prolonged stress response that causes impaired memory function and increased inflammation in the hippocampus. Currently, antidepressants are the only approved therapy for PTSD. However, the efficacy of antidepressants in the treatment of PTSD is marginal. The ethanol extract of Aralia continentalis (AC) is traditionally used in oriental medicine, and has been showed to possess pharmacological properties, including anti-inflammatory, anti-cancer, anti-atherosclerotic, and anti-diabetic effects. Nevertheless, the effects of AC on cognitive memory and its mechanism of action in PTSD remain unclear. Given the necessity of further treatment options for PTSD, we investigated the effect of AC on the spatial cognitive impairment caused by single prolonged stress (SPS) in a rat model of PTSD. METHODS: Male rats were treated with various intraperitoneal (i.p.) doses of AC for 21 consecutive days after inducing chronic stress with the SPS procedure. RESULTS: Cognitive impairment caused by SPS were inhibited after treatment with 100 mg/kg AC, as measured by the Morris water maze test and an object recognition test. Additionally, AC treatment significantly alleviated memory-related decreases in brain-derived neurotrophic factor (BDNF) mRNA and protein levels in the hippocampus. Our results suggest that AC significantly inhibited the cognitive deficits caused by SPS via increased expression of pro-inflammatory cytokines, including tumor necrosis factor-α and interleukin-6, in the rat brain. CONCLUSIONS: AC reversed the behavioral impairments and inflammation triggered by SPS-derived traumatic stress and should be further evaluated as a potential therapeutic drug for PTSD.

Osteogenic Effect of Inducible Nitric Oxide Synthase (iNOS)-Loaded Mineralized Nanoparticles on Embryonic Stem Cells.

BACKGROUND/AIMS: This study investigated the effect of inducible nitric oxide synthase-loaded mineralized nanoparticles (iNOS-MNPs) on the osteogenic differentiation of mouse embryonic stem cells (ESCs). METHODS: We prepared iNOS-MNPs using an anionic block copolymer template-mediated calcium carbonate (CaCO3) mineralization process in the presence of iNOS. iNOS-MNPs were spherical and had a narrow size distribution. iNOS was stably loaded within MNPs without denaturation. In order to confirm the successful introduction of iNOS-MNPs into the cytosol of ESCs, intracellular levels of nitric oxide (NO) was determined with a fluorometric analysis. A NO effector molecule, cyclic guanosine 3',5' monophosphate (cGMP) was also quantified with a competitive enzyme immunoassay. Cell viability in response to iNOS-MNP treatment was determined using the cell counting kit-8 (CCK-8) assay. Alkaline phosphatase (ALP) activity assay, intracellular calcium quantification assay, and Alizarin red S staining for matrix mineralization were performed to investigate osteogenic differentiation of ESCs. The protein levels of Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osterix (OSX) as osteogenic-related factors were also assessed by immunofluorescence staining and Western blot analysis. The complex pathways associated with iNOS-MNP-derived osteogenic differentiation of ESCs were evaluated by network-based analysis. RESULTS: Cells with iNOS-MNPs displayed a significant increase in NO and cGMP concentration compared with the control group. When cells were exposed to iNOS-MNPs, there were no adverse effects on cell viability. Importantly, iNOS-MNP uptake promoted the osteogenic differentiation of ESCs. Using transcriptome profiling, we obtained 1,836 differentially-induced genes and performed functional enrichment analysis with ClueGO and KEGG. These analyses identified significantly enriched and interconnected molecular pathways such as protein kinase activity, estrogen receptor activity, bone morphogenetic protein (BMP) receptor binding, ligand-gated ion channel activity, and phosphatidylinositol 3-phosphate binding. CONCLUSION: These findings suggest that iNOS-MNPs can induce osteogenic differentiation in ESCs by integrating complex signaling pathways.

The Anti-Stress Effect of Mentha arvensis in Immobilized Rats.

Stress can lead to inflammation, accelerated aging, and some chronic diseases condition. Mentha arvensis (MA) is a traditional medicine having antioxidant and anti-inflammatory activities. The present study investigated the anti-stress role of MA and fermented MA (FMA) extract in immobilized rats. We studied the lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells and rats were immobilized for 2 h per day for 14 days using a restraining cage. MA (100 mg/kg) and FMA (100 mg/kg) were orally administered to rats 1 h prior to immobilization. Using high-performance liquid chromatography (HPLC) analysis, we determined the rosmarinic acid content of MA and FMA. The generation of malondialdehyde (MDA) and nitric oxide (NO) in RAW 246.7 cells were suppressed by both MA and FMA. In rats, MA and FMA notably improved the body weight, daily food intake, and duodenum histology. MDA and NO level were gradually decreased by MA and FMA treatment. MA and FMA significantly controlled the stress-related hormones by decreasing corticosterone and ß-endorphin and increasing serotonin level. Moreover, protein expression levels of mitogen activated protein kinases (MAPK) and cyclooxygenase-2 (COX-2) were markedly downregulated by MA and FMA. Taken together, MA and FMA could ameliorate immobilized-stress by reducing oxidative stress, regulating stress-related hormones, and MAPK/COX-2 signaling pathways in rats. Particularly, FMA has shown greater anti-stress activities than MA.

Anti-inflammatory effects and corresponding mechanisms of cirsimaritin extracted from Cirsium japonicum var. maackii Maxim.

In this study, we investigated the anti-inflammatory effects and mechanisms of cirsimaritin isolated from an ethanol extract of the aerial parts of Cirsium japonicum var. maackii Maxim. using RAW264.7 cells. The extract and its flavonoid cirsimaritin inhibited nitric oxide (NO) production and inducible nitric oxide synthase expression in RAW264.7 cells. Cirsimaritin inhibited interleukin-6, tumor necrosis factor-α, and NO production in a concentration-dependent manner in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. From a western blot study, pretreatment with cirsimaritin inhibited phosphorylation/degradation of IκBα and phosphorylation of Akt in LPS-stimulated RAW264.7 cells. Moreover, cirsimaritin suppressed activation of LPS-induced transcription factors, such as c-fos and signal transducer and activator of transcription 3 (STAT3), in RAW264.7 cells. Collectively, these results show that cirsimaritin possesses anti-inflammatory activity, which is regulated by inhibition of c-fos and STAT3 phosphorylation in RAW264.7 cells.

Mitochondrial function contributes to oxysterol-induced osteogenic differentiation in mouse embryonic stem cells.

Oxysterols, oxidized derivatives of cholesterol, are biologically active molecules. Specific oxysterols have potent osteogenic properties that act on osteoprogenitor cells. However, the molecular mechanisms underlying these osteoinductive effects on embryonic stem cells (ESCs) are unknown. This study investigated the effect of an oxysterol combination of 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol (SS) on osteogenic differentiation of ESCs and the alterations to mitochondrial activity during differentiation. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, matrix mineralization, mRNA expression of osteogenic factors, runt-related transcription factor 2, osterix, and osteocalcin, and protein levels of collagen type IA (COLIA) and osteopontin (OPN). Treatment of cells with SS increased osteoinductive activity compared to the control group. Intracellular reactive oxygen species production, intracellular ATP content, mitochondrial membrane potential, mitochondrial mass, mitochondrial DNA copy number, and mRNA expression of peroxisome proliferator-activated receptor-γ coactivators 1α and ß, transcription factors involved in mitochondrial biogenesis, were significantly increased during osteogenesis, indicating upregulation of mitochondrial activity. Oxysterol combinations also increased protein levels of mitochondrial respiratory complexes I-V. We also found that SS treatment increased hedgehog signaling target genes, Smo and Gli1 expression. Inhibition of Hh signaling by cyclopamine suppressed mitochondrial biogenesis and ESC osteogenesis. Subsequently, oxysterol-induced Wnt/ß-catenin pathways were inhibited by repression of Hh signaling and mitochondrial biogenesis. Transfection of ß-catenin specific siRNA decreased the protein levels of COLIA and OPN, as well as ALP activity. Collectively, these data suggest that lipid-based oxysterols enhance differentiation of ESCs toward the osteogenic lineage by regulating mitochondrial activity, canonical Hh/Gli, and Wnt/ß-catenin signaling.

HIF-2 Inhibition Supresses Inflammatory Responses and Osteoclastic Differentiation in Human Periodontal Ligament Cells.

Recent reports suggest that hypoxia inducible factor-2α (HIF-2α) is a key regulator of osteoarthritis cartilage destruction. However, the precise role of HIF-2α in the inflammatory response and osteoclast differentiation remains unclear. The purpose of this study was to investigate the effect of HIF-2α on inflammatory cytokines, extracellular matrix (ECM) destruction enzymes, and osteoclastic differentiation in nicotine and lipopolysaccharide (LPS)-stimulated human periodontal ligament cells (PDLCs). HIF-2α was upregulated in chronically inflamed PDLCs of periodontitis patients, and in nicotine- and LPS-exposed PDLC in dose- and time-dependent manners. HIF-2α inhibitor and HIF-2α siRNA attenuated the nicotine- and LPS- induced production of NO and PGE2 , upregulation of iNOS, COX-2, pro-inflammatory cytokines (IL-1ß, TNF-α, IL-1ß, IL-6, IL-8, IL-10, IL-11, and IL-17), and matrix metalloproteinases (MMPs; MMP-1, -8, -13, -2 and -9), and reversed the effect on TIMPs (TIMP-1 and -2) in PDLCs. The conditioned medium produced by nicotine and LPS-treated PDLCs increased the number of TRAP-stained osteoclasts, TRAP activity and osteoclast-specific genes, which has been blocked by HIF-2α inhibition and silencing. HIF-2α inhibitor and HIF-2α siRNA inhibited the effects of nicotine and LPS on the activation of Akt, JAK2 and STAT3, ERK and JNK MAPK, nuclear factor-κB, c-Jun, and c-Fos. Taken together, this study is the first to demonstrate that HIF-2α inhibition exhibits anti-inflammatory activity through the inhibition of inflammatory cytokines and impairment of ECM destruction, as well as blocking of osteoclastic differentiation in a nicotine- and periodontopathogen-stimulated PDLCs model. Thus, HIF-2α inhibition may be a novel molecular target for therapeutic approaches in periodontitis.

Lignin phenolation by graft copolymerization to boost its reactivity.

Lignin is the most abundant phenolic biopolymer and a renewable resource of aromatics. It can be used as a phenol substitute in the synthesis of phenolic resins. However, lignin is not as reactive as phenol, so phenolation is generally carried out to improve lignin reactivity. In this work, we suggest a solution to circumvent the limitations of traditional phenolation (e.g., high temperature, strong acids/bases, limited reactivity, and phenol toxicity). We first attempt new lignin phenolation by graft copolymerization in which polymeric phenol, instead of toxic phenol, is introduced to lignin. Organosolv lignin from hardwood was modified with 2-bromoisobutyryl bromide to act as a lignin macroinitiator (L-Br). A protected phenolic monomer, 4-acetoxystyrene, was graft copolymerized onto L-Br using CuBr2/tris[2-(dimethylamino)ethyl]amine as a catalyst/ligand, after which the resultant lignin copolymer was deacetylated to produce lignin grafted with poly(4-hydroxystyrene). This poly-phenolation process was conducted at room temperature without the strong acids/bases and toxic phenol required in conventional phenolation. The poly-phenolated lignin was analyzed using 1H-, 13C-, and 31P NMR spectroscopy and gel permeation chromatography. This novel phenolation process enhanced the reactive sites of lignin more than tenfold. It also reduced the dark color of technical lignins significantly, thereby overcoming a serious obstacle to their applicability.

Ferritin-nanocaged copper arsenite minerals with oxidative stress-amplifying activity for targeted cancer therapy.

We report copper(II) arsenite-encapsulated ferritin nanoparticles (CuAS-FNs) as oxidative stress-amplifying anticancer agents. The CuAS-FNs were fabricated through CuAS mineralization in the cavity of the FNs. The formation of crystalline CuAS complex minerals in the FNs was systematically identified using various analytical tools, including X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM)-associated energy-dispersive X-ray spectroscopy (TEM-EDS). The CuAS-FNs showed pH-dependent release behavior, in which the CuAS mineral was effectively retained at physiological pH, in contrast, at lysosomal pH, the CuAS complex was dissociated to release arsenite and Cu2+ ions. At lysosomal pH, the release rate of arsenite (HAsO32-) and Cu2+ ions from the CuAS-FNs more accelerated than at physiological pH. Upon transferrin receptor-1-mediated endocytosis, the CuAS-FNs simultaneously released arsenite and Cu2+ ions in cells. The released arsenite ions can increase the intracellular concentration of hydrogen peroxide (H2O2), with which the Cu2+ ions can elevate the level of hydroxyl radicals (·OH) via Fenton-like reaction. Thus, the CuAS-FNs could target cancer cell through the recognizing ability of FNs and kill cancer cells by amplifying the ·OH level through the synergistic activity of Cu2+ and arsenic ions. Importantly, MCF-7 tumors were effectively suppressed by CuAS-FNs without systemic in vivo toxicity. Therefore, the CuAS-FNs is a promising class of Fenton-like catalytic nanosystem for cancer treatment.

Multifunctional bone substitute using carbon dot and 3D printed calcium-deficient hydroxyapatite scaffolds for osteoclast inhibition and fluorescence imaging.

Multifunctional bone substitute materials (BSM) have gained considerable attention with the exponential increase in aging populations. The development of hybrid materials for diagnosis and therapy of bone-related diseases and dysfunctions, especially, has been a significant challenge in the biological and the biomedical field, due to the shortage of agents with specificity and selectivity toward bone. In this study, a hybrid material, referred as Alen-CDs@CDHA, fabricated from alendronate-conjugated carbon dots (Alen-CDs) and calcium-deficient hydroxyapatite (CDHA, the mineral component of bones) scaffolds is offered as a novel multifunctional BSM for in vivo osteoclasts deactivation and fluorescence imaging. The fluorescent Alen-CDs were hydrothermally prepared using phytic acid as carbon source, followed by conjugating alendronate, for controlled alendronate release and fluorescent imaging under acidic conditions. As-prepared fluorescent Alen-CDs were consecutively immobilized on surfaces of CDHA scaffolds, exhibiting high affinity by bisphosphonate group, easily fabricated from α-tricalcium phosphate (α-TCP) paste using three-dimensional (3D) printing system. The resultant Alen-CDs@CDHA caused a significant decrease (> 50%) in viability of osteoclasts at 7 days after in vitro treatment. Furthermore, when Alen-CDs@CDHA was implanted in balb/c nude mice for in vivo evaluation, we found Alen-CDs@CDHA to be suitable for bone imaging through fluorescence signals, without necrosis or inflammatory symptoms in the epidermal tissues. Thus, these observations offer new opportunities for a novel and revolutionary use of Alen-CDs@CDHA as highly specific multifunctional BSM for bone diagnosis and imaging, and as bone-specific drug delivery materials, eventually providing anti-osteoclastogenic treatments solution for degenerative bone disorders. STATEMENT OF SIGNIFICANCE: Alen-CDs@CDHA significantly reduced the viability of osteoclasts and fluorescently imaged in vivo after transplantation, releasing drug via pH modulation. The development of fluorescence materials for bone imaging remains still a major challenge in the biomedical field owing to the shortage of selectivity and specificity. The results could lead to improvements in bone treatment strategies, as it could reduce the invasiveness of procedures and the associated negative outcomes, and increase the precision of strategies. Further, we believe that this study will be of interest to the readership of your journal as clearly focuses on the advancement of a biomaterial, where we have engineered a substance to substitute bone and integrate with a living system.

MnCO3-mineralized polydopamine nanoparticles as an activatable theranostic agent for dual-modality imaging-guided photothermal therapy of cancers.

Background: Single imaging modality is still insufficient to evaluate the biological and anatomical structures of tumors with high accuracy and reliability. Generation of non-specific contrast, leading to a low target-to-background signal ratio, results in low imaging resolution and accuracy. Tumor environment-specific activatable multifunctional contrast agents need to maximize the contrast signals, representing a dual imaging-guided photothermal therapy (PTT) at target tumor sites. Methods: Cellular uptake, cytotoxicity assay, and in vitro photothermal conversion efficiency of MnCO3-mineralized fluorescent polydopamine nanoparticles (MnCO3-FPNPs) were evaluated using 4T1 breast cancer cells. In vivo dual-modality imaging was performed using IVIS imaging and a 4.7 T animal MRI systems after injection into 4T1 tumor-bearing nude mice. The effects of photothermal therapeutic through PTT were measured after irradiation with an 808 nm laser (1.5 W/cm2) for 10 min, measuring the size of the tumors every 2 days. Results: At physiological pH (7.4), MnCO3-FPNP is efficiently quenched. Conversely, at acidic pH (5.4), the strong fluorescence (FL) is recovered due to the dissociation of Mn2+ from the FPNPs. At pH 7.4, MnCO3-FPNP activity is silenced to enhance water proton relaxation due to unionized MnCO3 maintenance; conversely, at acidic pH (5.4), MnCO3-FPNPs efficiently release Mn2+ ions, thereby resulting in T 1-weighted magnetic resonance (MR) contrast enhancement. MnCO3-FPNPs display a promising diagnostic ability for 4T1 breast cancer xenograft models, as well as exhibit a high photothermal conversion efficiency. A successful tumor treatment via their photothermal activity is accomplished within 14 days. Conclusions: Our studies exhibited unique "OFF-ON" activation abilities in FL/MR dual imaging and PTT functions. This approach suggests that the MnCO3-FPNPs may serve as a useful platform for various mineralization-based multimodal imaging-guided PTT models for many cancer theranostic applications.

Copper arsenite-complexed Fenton-like nanoparticles as oxidative stress-amplifying anticancer agents.

We report copper(II) arsenite (CuAS)-integrated polymer micelles (CuAS-PMs) as a new class of Fenton-like catalytic nanosystem that can display reactive oxygen species (ROS)-manipulating anticancer therapeutic activity. CuAS-PMs were fabricated through metal-catechol chelation-based formation of the CuAS complex on the core domain of poly (ethylene glycol)-b-poly(3,4-dihydroxy-L-phenylalanine) (PEG-PDOPA) copolymer micelles. CuAS-PMs maintained structural robustness under serum conditions. The insoluble state of the CuAS complex was effectively retained at physiological pH, whereas, at endosomal pH, the CuAS complex was ionized to release arsenite and cuprous Fenton catalysts (Cu+ ions). Upon endocytosis, CuAS-PMs simultaneously released hydrogen peroxide (H2O2)-generating arsenite and Fenton-like reaction-catalyzing Cu+ ions in cancer cells, which synergistically elevated the level of highly cytotoxic hydroxyl radicals (•OH), thereby preferentially killing cancer cells. Animal experiments demonstrated that CuAS-PMs could effectively suppress the growth of solid tumors without systemic in vivo toxicity. The design rationale of CuAS-PMs may provide a promising strategy to develop diverse oxidative stress-amplifying agents with great potential in cancer-specific therapy.

Ketal cross-linked poly(ethylene glycol)-poly(amino acid)s copolymer micelles for efficient intracellular delivery of doxorubicin.

A biocompatible, robust polymer micelle bearing pH-hydrolyzable shell cross-links was developed for efficient intracellular delivery of doxorubicin (DOX). The rationally designed triblock copolymer of poly(ethylene glycol)-poly(L-aspartic acid)-poly(L-phenylalanine) (PEG-PAsp-PPhe) self-assembled to form polymer micelles with three distinct domains of the PEG outer corona, the PAsp middle shell, and the PPhe inner core. Shell cross-linking was performed by the reaction of ketal-containing cross-linkers with Asp moieties in the middle shells. The shell cross-linking did not change the micelle size and the spherical morphology. Fluorescence quenching experiments confirmed the formation of shell cross-linked diffusion barrier, as judged by the reduced Stern-Volmer quenching constant (K(SV)). Dynamic light scattering and fluorescence spectroscopy experiments showed that shell cross-linking improved the micellar physical stability even in the presence of micelle disrupting surfactants, sodium dodecyl sulfate (SDS). The hydrolysis kinetics study showed that the hydrolysis half-life (t(1/2)) of ketal cross-links was estimated to be 52 h at pH 7.4, whereas 0.7 h at pH 5.0, indicating the 74-fold faster hydrolysis at endosomal pH. Ketal cross-linked micelles showed the rapid DOX release at endosomal pH, compared to physiological pH. Confocal laser scanning microscopy (CLSM) showed that ketal cross-linked micelles were taken up by the MCF-7 breast cancer cells via endocytosis and transferred into endosomes to hydrolyze the cross-links by lowered pH and finally facilitate the DOX release to inhibit proliferation of cancer cells. This ketal cross-linked polymer micelle is promising for enhanced intracellular delivery efficiency of many hydrophobic anticancer drugs.

Injectable hydrogel derived from chitosan with tunable mechanical properties via hybrid-crosslinking system.

Thermo-sensitive injectable hydrogels that spontaneously react to physiological temperature have been widely studied to be used in biomedical fields. However, several challenges on their unstable structures with large-sized pores and low mechanical strength under physiological conditions must be addressed to enable their practical applications. We synthesized the hydroxybutyl methacrylated chitosan (HBC-MA) hydrogel that possesses both thermo-sensitive and photo-crosslinkable properties. The HBC-MA showed effective sol-gel transition under physiological temperature as well as a sensitive photo-crosslinkable property with visible light capable of skin penetration. The co-nonsolvency property and thermo-sensitivity of HBC-MA prevented unintended loss of the hydrogel graft after being subcutaneously injected in mice. Subsequently applied visible light on the skin beneath which the hydrogel was injected significantly improved the mechanical strength and stability of the graft. The injectable HBC-MA hydrogel developed in this study can be applicable to a wide range of biomedical fields such as drug delivery system and tissue engineering.

A mesoporous silica nanoparticle with charge-convertible pore walls for efficient intracellular protein delivery.

We report a smart mesoporous silica nanoparticle (MSN) with a pore surface designed to undergo charge conversion in intracellular endosomal condition. The surface of mesopores in the silica nanoparticles was engineered to have pH-hydrolyzable citraconic amide. Solid-state nuclear magnetic resonance (NMR), Fourier-transform infrared (FT-IR) spectroscopy, and Brunauer-Emmett-Teller (BET) analyses confirmed the successful modification of the pore surfaces. MSNs (MSN-Cit) with citraconic amide functionality on the pore surfaces exhibited a negative zeta potential (-10 mV) at pH 7.4 because of the presence of carboxylate end groups. At cellular endosomal pH (approximately 5.0), MSN-Cit have a positive zeta potential (16 mV) indicating the dramatic charge conversion from negative to positive by hydrolysis of surface citraconic amide. Cytochrome c (Cyt c) of positive charges could be incorporated into the pores of MSN-Cit by electrostatic interactions. The release of Cyt c can be controlled by adjusting the pH of the release media. At pH 7.4, the Cyt c release was retarded, whereas, at pH 5.0, MSN-Cit facilitated the release of Cyt c. The released Cyt c maintained the enzymatic activity of native Cyt c. Hemolytic activity of MSN-Cit over red blood cells (RBCs) was more pronounced at pH 5.0 than at pH 7.0, indicating the capability of intracellular endosomal escape of MSN carriers. Confocal laser scanning microscopy (CLSM) studies showed that MSN-Cit effectively released Cyt c in endosomal compartments after uptake by cancer cells. The MSN developed in this work may serve as efficient intracellular carriers of many cell-impermeable therapeutic proteins.

Preparation and Application of Light-Colored Lignin Nanoparticles for Broad-Spectrum Sunscreens.

Recently, natural sun blockers have been drawing considerable attention because synthetic UV filters could have adverse effects not only on humans but also on the environment. Even though lignin, the second most abundant renewable resource on earth, is a natural UV-absorbing polymer, its unfavorable dark color hampers its applications in sunscreens. In this work, we obtained light-colored lignin (CEL) from rice husks through cellulolytic enzyme treatment and subsequent solvent extraction under mild conditions and compared CEL to technical lignin from rice husks using the International Commission on Illumination L*a*b* (CIELAB) color space. Spherical nanoparticles of CEL (CEL-NP) were also prepared using a solvent shifting method and evaluated for broad-spectrum sunscreens. A moisturizing cream blended with CEL-NP exhibited higher sun protection factor (SPF) and UVA PF (protection factor) values than that with CEL. In addition, CEL-NP had synergistic effects when blended with an organic UV-filter sunscreen: CEL-NP enhanced the SPF and UVA PF values of the sunscreen greatly. However, there was no synergistic effect between CEL-NP and inorganic sunscreens. We expect nanoparticles of light-colored lignin to find high-value-added applications as a natural UV-blocking additive in sunscreens and cosmetics.

Supplement of nitric oxide through calcium carbonate-based nanoparticles contributes osteogenic differentiation of mouse embryonic stem cells.

This study investigated the delivery of S-nitrosothiol (GSNO) as a nitric oxide (NO) donor loaded into calcium carbonate-based mineralized nanoparticles (GSNO-MNPs) to regulate cell signaling pathways for the osteogenic differentiation of mouse embryonic stem cells (ESCs). GSNO-MNPs were prepared by an anionic block copolymer template-mediated calcium carbonate (CaCO3) mineralization process in the presence of GSNO. GSNO-MNPs were spherical and had a narrow size distribution. GSNO was stably loaded within the MNPs without denaturation. TEM analysis also demonstrated the localization of GSNO-MNPs within membrane-bound structures in the cell, indicating the successful introduction of GSNO-MNPs into the cytosol of ESCs. Intracellular levels of NO and cGMP were significantly increased upon treatment with GSNO-MNPs, compared with the control group. When cells were exposed to GSNO-MNPs, the effects of nanoparticles on cell viability were not statistically significant. GSNO-MNPs treatment increased ALP activity assay and intracellular calcium levels. Real-time RT-PCR also revealed highly increased expression levels of the osteogenic target genes ALP, osteocalcin (OCN), and osterix (OSX) in GSNO-MNP-treated ESCs. The protein levels of OSX and Runt-related transcription factor 2 (RUNX2) showed similar patterns of expression based on real-time RT-PCR. These results indicate that GSNO-MNPs influenced the osteogenic differentiation of ESCs. Transcriptome profiling identified several significantly enriched and involved biological networks, such as RAP1, RAS, PI3K-AKT, and MAPK signaling pathways. These findings suggest that GSNO-MNPs can modulate osteogenic differentiation in ESCs via complex molecular pathways.

Optimization of Extraction Conditions of Continentalic and Kaurenoic Acids from Aralia continentalis by HPLC/UV and Their Validation.

Aralia continentalis (AC) is a perennial herb that has long been used as a traditional medicine for many diseases. Continentalic acid (CA) and kaurenoic acid (KA) are major diterpenoids in AC, which are known to exert various pharmacological activities. This study focuses on the optimization of the extraction of CA and KA from dried AC roots by evaluating the influence of different extraction conditions on their yield. Five extraction variables were examined: sample weight, solvent concentration, extraction time, solid matrix and the number of repeated extractions. The analytical method used in this study was also validated in terms of linearity, limit of detection, limit of quantification, precision and accuracy. The CA and KA yields were measured by high-performance liquid chromatography analysis. The results show that CA and KA were the highest when unpulverized samples (3.75 g) were subjected to a single extraction for 5 h using 50% ethanol (300 mL) as the solvent. These conditions are proposed for the optimization of the extraction of CA and KA from AC.

Oral Soft Tissue Regeneration Using Nano Controlled System Inducing Sequential Release of Trichloroacetic Acid and Epidermal Growth Factor.

BACKGROUND: The effect of nano controlled sequential release of trichloroacetic acid (TCA) and epidermal growth factor (EGF) on the oral soft tissue regeneration was determined. METHODS: Hydrophobically modified glycol chitosan (HGC) nano controlled system was developed for the sequential release of TCA and EGF, and the release pattern was identified. The HGC-based nano controlled release system was injected into the critical-sized defects created in beagles' palatal soft tissues. The palatal impression and its scanned body was obtained on various time points post-injection, and the volumetric amount of soft tissue regeneration was compared among the three groups: CON (natural regeneration control group), EXP1 (TCA-loaded nano controlled release system group), EXP2 (TCA and EGF individually loaded nano controlled release system). DNA microarray analysis was performed and various soft tissue regeneration parameters in histopathological specimens were measured. RESULTS: TCA release was highest at Day 1 whereas EGF release was highest at Day 2 and remained high until Day 3. In the volumetric measurements of impression body scans, no significant difference in soft tissue regeneration between the three groups was shown in two-way ANOVA. However, in the one-way ANOVA at Day 14, EXP2 showed a significant increase in soft tissue regeneration compared to CON. High correlation was determined between the histopathological results of each group. DNA microarray showed up-regulation of various genes and related cell signaling pathways in EXP2 compared to CON. CONCLUSION: HGC-based nano controlled release system for sequential release of TCA and EGF can promote regeneration of oral soft tissue defects.