Chemo-photothermal therapeutic effect of chitosan-gelatin hydrogels containing methotrexate and melanin on a collagen-induced arthritis mouse model.

Heat stimulation can promote osteoblast differentiation and bone formation. Combining photothermal therapy and chemotherapy is an effective strategy for treating rheumatoid arthritis (RA). Herein, we prepared chitosan/gelatin/ß-glycerophosphate-melanin-methotrexate (CMM) hydrogel that could be used to perform simultaneous chemotherapy and photothermal therapy for patients with RA. The CMM solution was successfully converted to a gel state at body temperature. Due to intrinsic photothermal properties of melanin, CMM hydrogel exhibited effective temperature increase both in vitro and in vivo with increasing time of near-infrared (NIR) laser irradiation. After NIR laser irradiation, 50 % of methotrexate was rapidly released from the hydrogel within 3 h. Its release rate showed an instantaneous increase with additional NIR laser irradiation. After CMM hydrogel was injected directly into the paw joint of each collagen-induced arthritis (CIA) mouse followed by irradiation with a NIR laser (808 nm, 0.5 W/cm2, 3 min), swelling and redness at the inflamed area were significantly alleviated at 14 days after treatment. Micro-CT analysis confirmed that treated joints of mice were similar to normal joints. Hence, CMM hydrogel could be used as an attractive RA therapeutic agent for simultaneous chemo-photothermal therapy.

Alginate nanocapsules by water-in-oil emulsification and external gelation for drug delivery to fine dust stimulated keratinocytes.

Methodologies for synthesizing drug-loaded alginate nanocapsules were optimized and indomethacin and phloroglucinol loading capacities were studied. Their biological effects were studied for ameliorating fine dust (FD) induced detrimental effects in keratinocytes. The 1 % alginate to oil phase ratio of 1:20 was the optimal parameter for water in oil emulsification. The oil phase was optimized to contain sunflower oil: span 80 ratios of 17:3. Nanocapsule drug encapsulation efficiencies were 36.91 ± 5.56 and 32.41 ± 4.05 % respectively for phloroglucinol (EG2P) and indomethacin (EG2I) while the loading capacities were 25.28 ± 3.36 and 23.15 ± 2.84 %. Dried nanocapsules indicated a 40-140 nm diameter range while their hydrodynamic diameter was 989.69 nm at pH 7.0. Nanocapsules swelling was pH-dependent and in releasing media of pH values 4.5, 7.4, and 8.5, the drug release indicated a complex mechanism of swelling, diffusion, and erosion while at pH 2.0 the drug release followed the non-Fickian release. EG2P and EG2I treatment dose-dependently lowered FD-induced intracellular ROS production, apoptosis and inflammatory responses mediated through the NF-κB pathway in FD stimulated HaCaT keratinocytes and reduced epidermal barrier degradation. Further research could investigate the use of this technique in formulating cosmeceuticals containing drug-loaded alginate nanocapsules for achieving controlled release.

NIR-Mediated drug release and tumor theranostics using melanin-loaded liposomes.

BACKGROUND: Heat generation in a drug delivery carrier by exposure to near-infrared (NIR) light with excellent tissue transmittance is an effective strategy for drug release and tumor therapy. Because liposomes have amphiphilic properties, they are useful as drug carriers. Liposomes are also very suitable for drug delivery strategies using heat generation by NIR laser because lipid bilayers are easily broken by heat. Thermally generated bubbles from liposomes not only induce drug release, but also enable ultrasound imaging. METHODS: Melanin, perfluorohexane (PFH), and 5-fluorouracil (5-FU)-loaded liposomes (melanin@PFH@5-FU-liposomes) that can generate heat and bubble by NIR laser irradiation were prepared by a thin film method. Conversion of light to heat and bubble generation of melanin@PFH@5-FU-liposomes were evaluated using an infrared (IR) thermal imaging camera and an ultrasound imaging system both in vitro and in vivo. To investigate tumor therapeutic effect, NIR laser of 808 nm was used to irradiate tumor site for 10 min after injecting melanin@PFH@5-FU-liposome into tail veins of CT26-bearing mice. RESULTS: Melanin@PFH@5-FU-liposomes showed a spherical shape with a size of 209.6 ± 4.3 nm. Upon NIR laser irradiation, melanin@PFH@5-FU-liposomes exhibited effective temperature increase both in vitro and in vivo. In this regard, temperature increase caused a phase transition of PFH to induce bubble generation dramatically, resulting in effective drug release behavior and ultrasound imaging. The temperature of the tumor site was increased to 52 t and contrast was greatly enhanced during ultrasound imaging due to the generation of bubble. More importantly, tumor growth was effectively inhibited by injection of melanin@PFH@5-FU-liposomes with laser irradiation. CONCLUSIONS: Based on intrinsic photothermal properties of melanin and phase transition properties of PFH, melanin@PFH@5-FU-liposomes exhibited effective heat and bubble generation upon NIR laser irradiation. The elevated temperature induced bubble generation, resulting in contrast enhancement of ultrasound imaging. Melanin@PFH@5-FU-liposomes under NIR laser irradiation induced the death of cancer cells, thereby effectively inhibiting tumor growth. These results suggest that melanin@PFH@5-FU-liposomes can be utilized as a promising agent for photothermal tumor therapy and ultrasound imaging.

Biomedical Membrane of Fish Collagen/Gellan Gum Containing Bone Graft Materials.

The development of a guided bone regeneration (GBR) membrane with non-mammalian fish collagen has the advantage of low risk for transmission of infectious diseases in tissue regeneration. In this work, a fish collagen/gellan gum and bone graft material (FC/GG-BGM) composite GBR membrane were fabricated through solution blending and casting procedures in a vacuum. The membranes were characterized using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy observation (SEM), and atomic force microscope (AFM) analyses. FT-IR results suggested that ionic interactions were formed between FC and GG both in composite powder and membranes. In vivo experiments showed that these FC/GG-BGM composite membranes could generate osteoblast minerals and promote loose bone calcification, thus accelerating bone regeneration. At 2 weeks, the defected site of rats treated with FC/GG-BGM membrane (0.377 ± 0.012 mm3) showed higher regeneration than that of rats treated with the bovine collagen membrane (0.290 ± 0.015 mm3) and control rats without membrane (0.160 ± 0.008 mm3). Compared with bovine collagen membrane, the FC/GG-BGM composite membrane displays better bone regeneration ability. Therefore, FC/GG-BGM composite membrane is suitable as a GBR membrane for bone regeneration.

Synthesis and drug release properties of melanin added functional allopurinol incorporated starch-based biomaterials.

The main objective of this study was to prepare functional allopurinol (ALP) incorporated biomaterials using mungbean starch, polyvinyl alcohol, melanin (MEL), and plasticizers. Prepared biomaterials were characterized by FE-SEM and FT-IR analysis. Photothermal conversion efficiencies and ALP release properties of biomaterials were evaluated with NIR laser irradiation. When biomaterials were irradiated with the NIR laser, temperatures increase of MEL-added biomaterials were higher than those of MEL-non-added biomaterials. After NIR laser irradiation, ALP release rates of MEL-added biomaterials were 1.62 times faster than those of MEL-non-added biomaterials. In addition, ALP release using an artificial skin was increased by NIR laser irradiation. ALP release from biomaterials followed Fickian diffusion mechanism, while ALP release using an artificial skin followed a non-Fickian diffusion mechanism. Xanthine oxidase inhibitory (%) for MEL-added biomaterials with/without the addition of GL and XL were 47.5%, 61.7%, and 65.1%, respectively.

Co-Electrospun Silk Fibroin and Gelatin Methacryloyl Sheet Seeded with Mesenchymal Stem Cells for Tendon Regeneration.

Silk fibroin (SF) is a promising biomaterial for tendon repair, but its relatively rigid mechanical properties and low cell affinity have limited its application in regenerative medicine. Meanwhile, gelatin-based polymers have advantages in cell attachment and tissue remodeling but have insufficient mechanical strength to regenerate tough tissue such as tendons. Taking these aspects into account, in this study, gelatin methacryloyl (GelMA) is combined with SF to create a mechanically strong and bioactive nanofibrous scaffold (SG). The mechanical properties of SG nanofibers can be flexibly modulated by varying the ratio of SF and GelMA. Compared to SF nanofibers, mesenchymal stem cells (MSCs) seeded on SG fibers with optimal composition (SG7) exhibit enhanced growth, proliferation, vascular endothelial growth factor production, and tenogenic gene expression behavior. Conditioned media from MSCs cultured on SG7 scaffolds can greatly promote the migration and proliferation of tenocytes. Histological analysis and tenogenesis-related immunofluorescence staining indicate SG7 scaffolds demonstrate enhanced in vivo tendon tissue regeneration compared to other groups. Therefore, rational combinations of SF and GelMA hybrid nanofibers may help to improve therapeutic outcomes and address the challenges of tissue-engineered scaffolds for tendon regeneration.

Poly (l-lactic acid) membrane crosslinked with Genipin for guided bone regeneration.

In this study, we chemically modified poly(L-lactic acid) (PLLA) with functional amine groups and fabricated a PLLA membrane crosslinked with genipin as a biomembrane for inducing guided bone regeneration (GBR). The mechanical strength of the PLLA-amine membrane was improved by crosslinking with genipin compared to pure PLLA membrane. The surface of the PLLA-amine membrane crosslinked with genipin had many more uniform pores. Attachment and proliferation of MC3T3-E1 cells were increased and improved on the PLLA-amine membrane crosslinked with genipin. In an in vitro osteogenesis study, MC3T3-E1 cells on the PLLA membrane showed higher alkaline phosphatase (ALP) activity and calcification ability evaluated by alizarin red S staining than those on the pure PLLA membrane. When a skull defect hole of a rat was covered with the PLLA-amine membrane crosslinked with genipin, vigorous new bone regeneration determined by computed tomography at 8 weeks post operation was superior to that when the skull defect was covered with the pure PLLA membrane. Taken together, these results demonstrate that the PLLA-amine membrane crosslinked with genipin has a promising therapeutic application to GBR as a barrier membrane for covering the defect site.

Development of 64Cu-loaded Perfluoropentane Nanodroplet: A Potential Tumor Theragnostic Nano-carrier and Dual-Modality PET-Ultrasound Imaging Agents.

The purpose of this study was to develop and preliminarily evaluate phospholipid-shelled nanodroplets (NDs) encapsulating perfluoropentane (PFP) and radioactive 64Cu as a hybrid positron emission tomography (PET)-ultrasound (US) probe. PFP NDs were fabricated by mixing liquid-phase PFP with a phospholipid solution. The 64Cu was encapsulated into the NDs in a size-controlled manner by exploiting the hydrophobicity of 64Cu-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM) using a vial mixer and an extruder. The fabricated 64Cu-loaded PFP NDs (64Cu-PFP NDs) were evaluated using in vitro/in vivo PET-computed tomography (PET-CT), US imaging and transmission electron microscopy. In the in vitro PET images, the 64Cu-PFP NDs were observed as a hot spot in the lower section of the test tube. In the acquired US images, the mean region of interest brightness values of 64Cu-PFP NDs were revealed by their strong echo image. In a tumor-bearing mouse animal model, tumor uptake of the 64Cu-PFP NDs was low, that is, approximately 65%, compared with that of only free 64Cu, as determined by PET-delayed imaging analysis. The dual-function concept of the NDs is expected to contribute to the prognosis and effectiveness of therapy by fusing the science and technology of nuclear medicine and US.

Inulin/PVA biomaterials using thiamine as an alternative plasticizer.

In this study, biodegradable biomaterials were prepared by using inulin (INL), PVA and plasticizers (citric acid (CA), glycerol (GL) and thiamine (TH)) with UV curing process. INL was extracted from Jerusalem artichoke flour using hot water extraction method. Extracted INL and INL/PVA biomaterials were characterized by TLC, FT-IR, and SEM analysis. Physical properties such as mechanical and water resistance properties of biomaterials prepared with UV curing time from 0 to 20 min and types of plasticizers were investigated. Their antimicrobial activities, biodegradability, and application of coating materials for foods were also determined. Results indicated that their physical properties were improved by the UV curing process. In addition, physical properties of TH-added biomaterials were 1.5 to 2 times higher than those of GL-added and CA-added biomaterials. Biodegradability in soil revealed that biomaterials were degraded by about 20-40% after 140 days.

Sulindac imprinted mungbean starch/PVA biomaterial films as a transdermal drug delivery patch.

In this work, biodegradable biomaterial films for sulindac (SLD) recognition are synthesized from mungbean starch (MBS), PVA, and plasticizers by using UV irradiation process and casting methods. The optimal UV irradiation time for the preparation of SLD imprinted biomaterials films was about 30 min. Mechanical properties, recognition ability, and SLD release property for prepared films were investigated. From the results of recognition ability, we verified that these SLD imprinted biomaterial films have the binding site for SLD. The release properties of SLD was examined with the change of pH and temperature. The results indicate that the SLD release in pH 10.0 was higher than in pH 4.0. SLD release was also evaluated using an artificial skin. Results of the artificial skin test verified that SLD was released constantly for 20 days.

Natural melanin-loaded nanovesicles for near-infrared mediated tumor ablation by photothermal conversion.

Photothermal therapy requires a biocompatible material to absorb near-infrared (NIR) light and generate sufficient heat. Herein, we suggest natural melanin-loaded nanovesicles (melasicles) as photothermal therapeutic agents (PTA) for NIR mediated cancer therapy in vivo. The mean size of these melasicles was 140 ± 15 nm. They showed excellent colloidal stability. After irradiation from 808 nm NIR laser at 1.5 W cm-2, the melasicles showed good photothermal conversion efficiencies both in vitro and in vivo. In drug release study, laser irradiation increased fluidity of vesicle membrane due to photothermal generation from melanin. Initial drug release in the laser irradiation group was higher than that in the no laser irradiation group. After injecting the melasicles into tail veins of CT-26 bearing mice, tumors were suppressed or eliminated after irradiation at 1.5 W cm-2 for 5 min once or twice. These results suggest that melasicles could be used as attractive PTA for cancer therapy and localized drug release.

Preparation of chitosan/polyvinyl alcohol blended films containing sulfosuccinic acid as the crosslinking agent using UV curing process.

This paper reports on a method of preparing chitosan-based films to which sulfosuccinic acid (SSA) is added for crosslinking agent with/without UV curing treatment and applications of a coating materials for foods. The physical, thermal, and optical properties of the UV cured chitosan-based films are investigated including their tensile strength (TS), elongation at break (%E), degree of swelling (DS), solubility (S), and water vapor absorption as well as their biodegradability in soil and applicability of the coating on a fruit. We also evaluated the physical properties of the prepared films to which glycerol (GL), xylitol (XL), and sorbitol (SO) are added to be used as plasticizers. The surface and topography of the prepared films are investigated by scanning electronic microscopy (SEM) and atomic force microscopy analysis (AFM). The results indicate that the films UV cured for 20min possess optimal physical and thermal properties compared to that of non-cured films. The mechanical, thermal, and water barrier properties of SO-added film are also found to be superior to other films with added GL and XL. The degree of biodegradability revealed that the films are degraded by about 40-65% after 220days.

A drug release system induced by near infrared laser using alginate microparticles containing melanin.

The photothermal effect is used in a new drug release system to control drug delivery in a specific region. Melanin absorbs near-infrared (NIR) light with a high photothermal conversion efficiency, and as a result, an NIR laser can be used to induced drug release from alginate microparticles containing melanin (ALG-Mel microparticles). The temperature of the ALG-Mel microparticle solution at a concentration of 5mg/mL increased to 38.1°C from 26.0°C after irradiation with 808nm NIR at 1.5W/cm2 for 5min, and this increase in temperature was found to be independent of the ALG-Mel microparticle concentration. After the NIR laser irradiation, 5-fluorouracil (5-FU) was released from the ALG-Mel microparticles to 87.4±0.5% of the total loaded drug for 24h. Without NIR laser irradiation, 5-FU was released from the ALG-Mel microparticles to 60.8±1.5% of the total loaded drug for 24h. These results indicate that NIR laser irradiation can be used with ALG-Mel microparticles as a drug delivery system for release within a target region.

Wound healing potential of a polyvinyl alcohol-blended pectin hydrogel containing Hippophae rahmnoides L. extract in a rat model.

In this study, we investigated the effect of a polyvinyl alcohol-blended pectin hydrogel (PVA-PT HG) containing the extracts of Hippophae rhamnoides L. (H. rhamnoides L.) leaves on wound healing in a rat model. The total phenolic content in the extract solution was 40.64±2.7 GAE mg/g and that of flavonoids was 13.15±1.8 QE mg/g. Of the total flavonoids in HGs, 61.6 and 50.0% were released at pH 5.5 and 7.4 after 60min. In rat acute wound models, the wound size was reduced significantly and the recovery rate was significantly higher after treatment with HG containing the extracts, compared with treatment with the control and HG only. The wound healing effects of the HG containing the extracts were confirmed by histological evaluation of the wound tissue. Therefore, HG containing extracts from H. rhamnoides L. leaves enhanced wound healing effectively, and so may be developed as a cover to promote wound healing.

Thermohydrogel Containing Melanin for Photothermal Cancer Therapy.

Melanin is an effective absorber of light and can extend to near infrared (NIR) regions. In this study, a natural melanin is presented as a photothermal therapeutic agent (PTA) because it provides a good photothermal conversion efficiency, shows biodegradability, and does not induce long-term toxicity during retention in vivo. Poloxamer solution containing melanin (Pol-Mel) does not show any precipitation and shows sol-gel transition at body temperature. After irradiation from 808 nm NIR laser at 1.5 W cm-2 for 3 min, the photothermal conversion efficiency of Pol-Mel is enough to kill cancer cells in vitro and in vivo. The tumor growth of mice bearing CT26 tumors treated with Pol-Mel injection and laser irradiation is suppressed completely without recurrence postirradiation. All these results indicate that Pol-Mel can become an attractive PTA for photothermal cancer therapy.

Selenium Nanoparticles Formed by Modulation of Carrageenan Enhance Osteogenic Differentiation of Mesenchymal Stem Cells.

Fabrication of nano-sized selenium (Se) particles may help to expend the applications of Se. In this study, we focused on the preparation and characterization of Se nanoparticles (Se NPs) modulated with carrageenan (CA). Furthermore, their influence on osteoblast cell growth was investigated in vitro. Spherical Se-NPs, of 100-200 nm diameter, were prepared simply by adding κ-, ι-, and λ-CA, which has sulfate groups, hydroxyl groups, and carboxyl groups. CA-modulated Se NPs (CA-Se NPs) were readily suspended in liquid medium with no precipitation over long time periods. In particular, it was found through Alizarin Red S staining that the growth of osteoblast D1 cells treated with λ-CA-Se NPs was improved significantly. These results suggest that Se NPs can be prepared simply, using CA, have good suspension stability in liquid medium, and λ-CA-Se NPs may induce the growth of osteoblast cells.

The Alginate Layer for Improving Doxorubicin Release and Radiolabeling Stability of Chitosan Hydrogels.

PURPOSE: Chitosan hydrogels (CSH) formed through ionic interaction with an anionic molecule are suitable as a drug carrier and a tissue engineering scaffold. However, the initial burst release of drugs from the CSH due to rapid swelling after immersing in a biofluid limits their wide application as a drug delivery carrier. In this study, alginate layering on the surface of the doxorubicin (Dox)-loaded and I-131-labeled CSH (DI-CSH) was performed. The effect of the alginate layering on drug release behavior and radiolabeling stability was investigated. METHODS: Chitosan was chemically modified using a chelator for I-131 labeling. After labeling of I-131 and mixing of Dox, the chitosan solution was dropped into tripolyphosphate (TPP) solution using an electrospinning system to prepare spherical microhydrogels. The DI-CSH were immersed into alginate solution for 30 min to form the crosslinking layer on their surface. The formation of alginate layer on the DI-CSH was confirmed by Fourier transform infrared spectroscopy (FT-IR) and zeta potential analysis. In order to investigate the effect of alginate layer, studies of in vitro Dox release from the hydrogels were performed in phosphate buffered in saline (PBS, pH 7.4) at 37 °C for 12 days. The radiolabeling stability of the hydrogels was evaluated using ITLC under different experimental condition (human serum, normal saline, and PBS) at 37 °C for 12 days. RESULTS: Formatting the alginate-crosslinked layer on the CSH surface did not change the spherical morphology and the mean diameter (150 ± 10 µm). FT-IR spectra and zeta potential values indicate that alginate layer was formed successfully on the surface of the DI-CSH. In in vitro Dox release studies, the total percentage of the released Dox from the DI-CSH for 12 days were 60.9 ± 0.8, 67.3 ± 1.4, and 71.8 ± 2.5 % for 0.25, 0.50, and 1.00 mg Dox used to load into the hydrogels, respectively. On the other hand, after formatting alginate layer, the percentage of the released Dox for 12 days was decreased to 47.6 ± 1.4, 51.1 ± 1.4, and 57.5 ± 1.6 % for 0.25, 0.50, and 1.00 mg Dox used, respectively. The radiolabeling stability of DI-CSH in human serum was improved by alginate layer. CONCLUSIONS: The formation of alginate layer on the surface of the DI-CSH is useful for improving the drug release behavior and radiolabeling stability.

The effect of PPAR-γ agonist on (18)F-FDG PET imaging for differentiating tumors and inflammation lesions.

INTRODUCTION: (18)F-2-deoxy-2-fluoro-d-glucose ((18)F-FDG) positron emission tomography (PET) has been used for imaging human cancers for several decades. Despite its extensive use, (18)F-FDG PET imaging has limitations in the tumor findings. The goal of this study was to investigate the potential of a PPAR-γ agonist pioglitazone (PIO) to distinguish tumors and inflammatory lesions in (18)F-FDG PET imaging. METHODS: Studies of cellular uptake of (18)F-FDG and Western blot were performed in macrophage (RAW264.7) and three tumor cell lines (A549, KB, and MDA-MB-231) after treatment with PIO. In vivo microPET/CT imaging and biodistribution were performed in animal models. RESULTS: The uptake of (18)F-FDG in the macrophages was decreased and uptake of (18)F-FDG in the tumor cells was increased when these cells were treated with PIO. Western blot showed that the expression of Glut1 was reduced by treatment of PIO in the macrophage cells, whereas the expression of Glut1 in the tumor cells was increased. In vivo PET/CT imaging revealed that (18)F-FDG uptake (%ID/g) in the tumors was enhanced from 4.05±1.46 to 5.28±1.92 for A549, from 3.9±0.5 to 4.9±0.2 for KB, and from 9.14±0.86 to 13.48±2.07 for MDA-MB-231 tumors after treatment with PIO. Unlike tumors, the RAW264.7 xenograft model showed the reduced (18)F-FDG uptake in the inflammatory lesion from 11.74±1.19 to 6.50±1.47. The results of biodistribution also showed that (18)F-FDG uptake in the tumors were increased after treatment of PIO. However, the uptake of inflammation lesions was reduced. CONCLUSIONS: In this study, we demonstrated the effect of a PPAR-γ agonist PIO on (18)F-FDG uptake in tumors and inflammation in vitro and in vivo. PIO has potential to differentiate tumors and inflammatory lesions on (18)F-FDG PET imaging.

Effect of angiogenesis induced by consecutive intramuscular injections of vascular endothelial growth factor in a hindlimb ischemic mouse model.

PURPOSE: Angiogenesis plays a major role in various physiological and pathological situations. Thus, an angiogenic therapy with vascular endothelial growth factor (VEGF) has been commonly recommended as a representative therapeutic solution to recover the insufficient blood supply of collateral vessels in an ischemic lesion. In this study, the injection method and injection time point of VEGF proteins were focused to discover how to enhance the angiogenic effect with VEGF. METHODS: Mouse models (n = 15) were divided into control, VEGF treatment by intra-venous injection (VEGF-IV) and VEGF treatment by intra-muscular injection (VEGF-IM). Right proximal femoral arteries of mice were firmly sutured to obstruct arterial blood-flow. In the VEGF-IV treatment group, VEGF proteins were injected into the tail vein and, in the VEGF-IM treatment group, VEGF proteins were directly injected into the ischemic site of the right thigh after postoperative day 5, 10, 15, 20 and 25 follow-ups. Blood-flow images were acquired by (99m)Tc Gamma Image Acquisition System to compare the ischemic-to-non-ischemic bloodstream ratio at postoperative days 5, 15, and 30. RESULTS: VEGF-IM treatment significantly induced higher an angiogenic effect rather than both the control group (P = 0.008) and VEGF-IV treatment group (P = 0.039) at the 30th day. CONCLUSION: During all experiments, angiogenesis of VEGF-IM treatment represented the most evident effect compared with control and VEGF-IV group in a mouse model of hindlimb ischemia.