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Composite scaffolds obtained by the combination of biodegradable porous scaffolds and hydroxyapatite with bone regeneration potential are feasible materials for bone tissue engineering. However, most composite scaffolds have been fabricated by complicated procedures or under thermally harsh conditions. We have previously demonstrated that hydroxyapatite coating onto various substrates under a thermally mild condition was achieved by erbium-doped yttrium aluminum garnet (Er: YAG) pulsed laser deposition (PLD). The purpose of this study was to prepare a polycaprolactone (PCL) porous scaffold coated with the hydroxyapatite by the Er: YAG-PLD method. Hydroxyapatite coating by the Er: YAG-PLD method was confirmed by morphology, crystallographic analysis, and surface chemical characterization studies. When cultured on PCL porous scaffold coated with hydroxyapatite, rat bone marrow-derived mesenchymal stem cells adhered, spread, and proliferated well. The micro-CT and staining analyses after the implantation of scaffold into the critical-sized calvaria bone defect in rats indicate that PCL porous scaffold coated with hydroxyapatite demonstrates accelerated and widespread bone formation. In conclusion, PCL porous scaffold coated with hydroxyapatite obtained by the Er: YAG-PLD method is a promising material in bone tissue engineering.

OBJECTIVES Excessive and chronic inflammation after a myocardial infarction (MI) is associated with left ventricular remodelling and impaired cardiac function. Among inflammatory cells, macrophages play a critical role in polarizing proinflammatory M1 or the reparative M2 subtype. Pioglitazone (PGZ) is reported to regulate macrophage polarization to the M2 subtype. Our goal was to validate the therapeutic effects and the mechanisms of PGZ utilizing a drug delivery system. METHODS Poly L-lactic-co-glycolic acid microspheres (MS) incorporating PGZ were prepared. To validate the therapeutic potential of PGZ-MS, Sprague-Dawley rats were subjected to permanent left coronary artery ligation to induce an MI. Placebo-MS (100 μg) or PGZ-MS (100 μg) was injected to the infarct region just after induction. Cardiac function and size were assessed by echocardiography. At 28 days after surgery, the rats were sacrificed, and the excised hearts were evaluated histologically. RESULTS Sustained release of PGZ from the PGZ-MS was confirmed in vitro. PGZ-MS significantly rehabilitated cardiac dysfunction after an MI (fractional shortening: MI vs MI+placebo-MS vs MI+PGZ-MS, 24.4 ± 1.1 vs 24.3 ± 1.6 vs 32.2 ± 1.4%; P = 0.0035) with reverse remodelling. Immunohistochemical analyses revealed that PGZ-MS enhanced macrophage polarization (ratio of M2 subtype: 0.39 ± 0.03 vs 0.42 ± 0.02 vs 0.54 ± 0.02; P = 0.0004) and attenuated apoptosis of cardiomyocytes in the ischaemic border zone. CONCLUSIONS We confirmed macrophage polarization by sustained release of PGZ, which resulted in amelioration of adverse left ventricular remodelling and cardiac dysfunction. Drug delivery system-based macrophage polarization might serve as a promising strategy in cardiac regenerative therapy for ischaemic heart disease. (241 words)

Irisin is a proteolytic product of fibronectin type II domain-containing 5, which is related to the improvement in glucose metabolism. Numerous studies have suggested that irisin is a crucial myokine linking muscle to bone in physiological and pathophysiological states.We examined the effects of local irisin administration with gelatin hydrogel sheets and intraperitoneal injection of irisin on the delayed femoral bone repair caused by streptozotocin (STZ)-induced diabetes in female mice. We analyzed the femurs of mice using quantitative computed tomography and histological analyses and then measured the mRNA levels in the damaged mouse tissues.Local irisin administration significantly blunted the delayed bone repair induced by STZ 10 days after a femoral bone defect was generated. Local irisin administration significantly blunted the number of Osterix-positive cells that were suppressed by STZ at the damaged site 4 days after a femoral bone defect was generated, although it did not affect the mRNA levels of chondrogenic and adipogenic genes 4 days after bone injury in the presence or absence of diabetes. On the other hand, intraperitoneal injection of irisin did not affect delayed bone repair induced by STZ 10 days after bone injury. Irisin significantly blunted the decrease in Osterix mRNA levels induced by advanced glycation end products or high-glucose conditions in ST2 cells in the presence of bone morphogenetic protein-2.We first showed that local irisin administration with gelatin hydrogel sheets improves the delayed bone repair induced by diabetic state partially by enhancing osteoblastic differentiation.

The diagnosis of the dynamics, accumulation, and engraftment of transplanted stem cells in vivo is essential for ensuring the safety and the maximum therapeutic effect of regenerative medicine. However, in vivo imaging technologies for detecting transplanted stem cells are not sufficient at present. We developed nanohybrid particles composed of dendron-baring lipids having two unsaturated bonds (DLU2) molecules, quantum dots (QDs), and magnetic nanoparticles in order to diagnose the dynamics, accumulation, and engraftment of transplanted stem cells, and then addressed the labeling and in vivo fluorescence and magnetic resonance (MR) imaging of stem cells using the nanohybrid particles (DLU2-NPs). Five kinds of DLU2-NPs (DLU2-NPs-1-5) composed of different concentrations of DLU2 molecules, QDs525, QDs605, QDs705, and ATDM were prepared. Adipose tissue-derived stem cells (ASCs) were labeled with DLU2-NPs for 4 h incubation, no cytotoxicity or marked effect on the proliferation ability was observed in ASCs labeled with DLU2-NPs (640- or 320-fold diluted). ASCs labeled with DLU2-NPs (640-fold diluted) were transplanted subcutaneously onto the backs of mice, and the labeled ASCs could be imaged with good contrast using in vivo fluorescence and an MR imaging system. DLU2-NPs may be useful for in vivo multimodal imaging of transplanted stem cells.

The objective of this study is to visualize cell apoptosis in three-dimensional (3D) cell aggregates based on molecular beacons (MB). Two types of MB for messenger RNA were used: caspase-3 MB as a target for apoptosis and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) MB as a control of stable fluorescence in cells. To enhance the MB internalization into cells, caspase-3 and GAPDH MB were incorporated in cationized gelatin nanospheres (cGNS), respectively (cGNScasp3 MB and cGNSGAP MB). In addition, cGNS co-incorporating caspase-3 and GAPDH MB (cGNSdual MB) were prepared to perform the dual-color imaging for the same cell aggregate. The cGNSMB were incubated with mouse mesenchymal stem cells to label with MB in the two-dimensional culture. The cell apoptosis mediated by the addition of antibody for a death receptor Fas was ratiometrically detected by the cGNSdual MB to the same extent as single MB. The cell aggregates were prepared from MB-labeled cells, and the MB fluorescence was detected from almost all the cells even in the 3D aggregates to show the homogenous distribution. In addition to the Fas-mediated apoptosis, the aggregates were treated with camptothecin of a low-molecular weight apoptosis inducer. The fluorescence of caspase-3 MB was mainly distributed at the surface surrounding site of Fas-mediated apoptotic aggregates rather than the center site, while that of GAPDH MB was detected even in the interior site. On the other hand, in the camptothecin-induced apoptotic aggregates, both caspae-3 and GAPDH MB fluorescence were detected from the interior site of aggregates as well as the surrounding site. It is likely that the MB fluorescence reflected the localization of apoptotic position caused by the different molecular sizes of apoptosis inducer and the consequent penetration into the aggregates. It is concluded that the cGMSMB are a promising system to visualize cell apoptosis in 3D cell aggregates without the destruction of aggregates.

Runt-related transcription factor 2 (Runx2)-deficient mice can be used to model congenital tooth agenesis in humans. Conversely, uterine sensitization-associated gene-1 (Usag-1)-deficient mice exhibit supernumerary tooth formation. Arrested tooth formation can be restored by crossing both knockout-mouse strains; however, it remains unclear whether topical inhibition of Usag-1 expression can enable the recovery of tooth formation in Runx2-deficient mice. Here, we tested whether inhibiting the topical expression of Usag-1 can reverse arrested tooth formation after Runx2 abrogation. The results showed that local application of Usag-1 Stealth small interfering RNA (siRNA) promoted tooth development following Runx2 siRNA-induced agenesis. Additionally, renal capsule transplantation of siRNA-loaded cationized, gelatin-treated mouse mandibles confirmed that cationized gelatin can serve as an effective drug-delivery system. We then performed renal capsule transplantation of wild-type and Runx2-knockout (KO) mouse mandibles, treated with Usag-1 siRNA, revealing that hindered tooth formation was rescued by Usag-1 knockdown. Furthermore, topically applied Usag-1 siRNA partially rescued arrested tooth development in Runx2-KO mice, demonstrating its potential for regenerating teeth in Runx2-deficient mice. Our findings have implications for developing topical treatments for congenital tooth agenesis.

The objective of this study is to prepare cationized gelatin-molecular beacon (MB) complexes for the visualization of intracellular messenger RNA (mRNA). The complexes were prepared from cationized gelatins with different extents of cationization and different mixing ratios of MB to cationized gelatin. The apparent size of complexes was almost similar, while the zeta potential was different among the complexes. Irrespective of the preparation conditions, the complexes had a sequence specificity against the target oligonucleotides in hybridization. The cytotoxicity and the amount of complexes internalized into cells increased with an increase in the cationization extent and the concentration of cationized gelatin. After the incubation with complexes prepared from cationized gelatin with the highest extent of cationization and at mixing ratios of 10 and 20 pmole MB/μg cationized gelatin, a high fluorescent intensity was detected. On the other hand, the complex prepared with the mixing ratio at 20 pmole/μg did not show any cytotoxicity. The complex was the most effective to visualize the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA endogenously present. In addition, even for enhanced green fluorescent protein (EGFP) mRNA exogenously transfected, the complex permitted to effectively detect it as well. It is concluded that both the endogenous and exogenous mRNA can be visualized in living cells by use of cationized gelatin-MB complexes designed.

Magnetic Resonance Imaging (MRI) contrast agents with rapid renal excretion that do not penetrate the blood brain barrier (BBB) and blood cerebrospinal fluid barrier (BCFB) are preferred for safer and low-risk diagnosis. Gadolinium (Gd)-conjugated nanoparticles have been proposed for use as contrast agents; however, the particle size must range between 1 to 7 nm to ensure rapid renal excretion. In this study, three types of gelatin, dissolved in water at varying concentrations of 0.1-2 wt.%, were irradiated with 5 kGy γ-rays at 25°C under aerated conditions to produce ultra-small gelatin nanogels having an average particle size ranging between 6 ± 2 to 21 ± 4 nm. Ultra-small Gd-coordinated gelatin nanogels (GdGN) suitable for use as MRI contrast agents were produced using 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS) and DOTA-butylamine as Gd ligand derivatives. Non-cytotoxicity and effective relaxivity of GdGN as a positive MRI contrast agent were verified using in vivo experiments. Rapid renal excretion of GdGN was observed in mice within 1 h with no accumulation in the liver. GdGN did not migrate across the BCFB in normal mice, thus emphasizing its safety as an MRI contrast agent. STATEMENT OF SIGNIFICANCE: The authors developed ultra-small sized gelatin nanogels as blood-brain-barrier impermeable contrast agents for magnetic resonance imaging (MRI). The authors used radiation crosslinking technique to ensure better integrity of the amino acids present in the gelatin nanogels while conjugating with gadolinium (Gd) to form gadolinium-coordinated gelatin nanogels (GdGN). The safety and efficacy of GdGN, as MRI contrast agents, were verified by in vivo studies. GdGN exhibited rapid renal excretion within 90 minutes and no passage across the barriers in the brain.

The objective of this study is to visualize the ability of cell proliferation based on molecular beacons (MB). Two types of MB to detect messenger RNA (mRNA) were used. One is a Ki67 MB of a target for cell proliferation ability. The other one is a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) MB as a control of stable fluorescence in cells. To enhance the MB internalization into cells, the MB were incorporated into cationized gelatin nanospheres (cGNS). There was no difference in the physicochemical properties and the cell internalization between the cGNS

Background Nanoparticle-loaded delivery systems have attracted much attention recently. Poly(lactic-co-glycolic acid) (PLGA) is one of the most successful biodegradable polymers for biomedical applications. There are only a few studies on the treatment of dermal fibrosis with sustained-release drugs. Peroxisome proliferator-activated receptor-γ (PPAR-γ) plays an important role in endogenous anti-fibrotic defense mechanisms. Recent studies have suggested that pioglitazone, a synthetic PPAR-γ activator, has effects beyond reducing blood sugar and it can reduce fibrosis and inflammation when used systemically. Objective We aimed to assess the effects of local injections of pioglitazone-loaded PLGA nanoparticles (PGN-NP) on an experimental sclerosis and to demonstrate the in vivo pharmacokinetics of subcutaneously administered PLGA nanoparticles. Methods Locally injectable PGN-NP were prepared and subcutaneously administered to bleomycin (BLM)-induced scleroderma model mice. The effect of pioglitazone was also evaluated with cultured fibroblasts. Coumarin-6-loaded fluorescent PLGA nanoparticles (FL-NP) and silicon naphthalocyanine-loaded near-infrared PLGA nanoparticles (NIR-NP) were used to demonstrate in vitro cellular uptake by cultured fibroblasts and the in vivo pharmacokinetics of subcutaneously administered nanoparticles. Results Weekly subcutaneous injections of PGN-NP attenuated skin fibrosis in BLM-induced scleroderma model mice. Pioglitazone significantly suppressed migration ability and TGF-β-mediated myofibroblast differentiation in cultured fibroblasts. FL-NP were internalized into cultured fibroblasts within 60 min, and PGN-NP-primed fibroblasts expressed anti-fibrotic phenotypes. Subcutaneously injected NIR-NP remained in the vicinity of the injection site more than non-particulate silicon naphthalocyanine. Conclusion These results provide a basis for the development of new treatments for dermal fibrosis and a better understanding of the potential of PLGA nanoparticles in dermatology.

A promising strategy for augmenting bone formation involves the local delivery of multiple osteoinductive and vasculogenic growth factors. However, success depends on sustained growth factor release and its appropriate combination to induce stem cells and osteogenic cells at the bony site. Herein, we have developed a nanocomposite fibrous scaffold loaded with fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2) and its ability to promote vascularisation and bone regeneration in critical sized calvarial defect was compared to the scaffold with VEGF + BMP2 and FGF2 + BMP2. Simple loading of growth factors on the scaffold could provide a differential release pattern, both in vitro and in vivo (VEGF release for 1 week where as BMP2 and FGF2 release for 3 weeks). Among all the groups, dual growth factor loaded scaffold (VEGF + BMP2 & FGF2 + BMP2) enhanced vascularisation and new bone formation, but there was no difference between FGF2 and VEGF loaded scaffolds although its release pattern was different. FGF2 mainly promoted stem cell migration, whereas VEGF augmented new blood vessel formation at the defect site. This study suggests that biomimetic nanocomposite scaffold is a promising growth factor delivery vehicle to improve bone regeneration in critical sized bone defects. Statement of Significance Many studies have shown the effect of growth factors like VEGF-BMP2 or FGF2-BMP2 in enhancing bone formation in critical sized defects, but there are no reports that demonstrate the direct comparison of VEGF-BMP2 and FGF2-BMP2. In this study, we have developed a nanocomposite fibrous scaffold that could differentially release growth factors like VEGF, BMP2 and FGF2 (VEGF release for 1 week where as BMP2 and FGF2 release for 3 weeks), which in turn promoted neovascularisation and new bone formation in critical sized defects. There was no difference in vascularisation and bone formation induced by VEGF + BMP2 or FGF2 + BMP2. The growth factor was loaded in a simple manner, which would ensure ease of use for the end-user, especially for the surgeons treating a patient in an operating room.

Chronic myocardial infarction (CMI) tends to be resistant to treatments possibly due to extensive solid fibrotic scar, hypoxia mediated by poorly vascularized environment, and/or inflammation and apoptosis. Here we aimed to testify the therapeutic effects of sustained release of basic fibroblast growth factor (bFGF) using gelatin hydrogel (GH) in a rat chronic MI model and to elucidate the therapeutic mechanism including the alteration of extracellular matrix component. CMI model rats are prepared by the permanent ligation of proximal left anterior descending coronary artery. After 4 weeks, GH sheets (GHSs) with bFGF (100 µg) (bFGF group) or with phosphate-buffered saline (Vehicle group) were implanted to the CMI models to evaluate the effect of bFGF–GHS on chronic scar tissue. Sham operation group was also prepared (n = 5 for each). 4 weeks after implantation, bFGF–GHS significantly improved cardiac contractile function (fractional shortening: 21.8 ± 1.1 vs 21.5 ± 1.3 vs 29.7 ± 1.8%; P

Summary Both glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are bona fide self-renewal factors for spermatogonial stem cells, whereas retinoic acid (RA) induces spermatogonial differentiation. In this study, we investigated the functional differences between FGF2 and GDNF in the germline niche by providing these factors using a drug delivery system in vivo. Although both factors expanded the GFRA1+ subset of undifferentiated spermatogonia, the FGF2-expanded subset expressed RARG, which is indispensable for proper differentiation, 1.9-fold more frequently than the GDNF-expanded subset, demonstrating that FGF2 expands a differentiation-prone subset in the testis. Moreover, FGF2 acted on the germline niche to suppress RA metabolism and GDNF production, suggesting that FGF2 modifies germline niche functions to be more appropriate for spermatogonial differentiation. These results suggest that FGF2 contributes to induction of differentiation rather than maintenance of undifferentiated spermatogonia, indicating reconsideration of the role of FGF2 in the germline niche., Graphical Abstract, Highlights • FGF2 and GDNF induce morphologically distinct spermatogonial clusters in vivo • FGF2-expanded undifferentiated spermatogonia are prone to be RARG+ in vivo • FGF2 is more permissive than GDNF for Rarg expression in cultured spermatogonia • FGF2 suppresses GDNF production and retinoic acid metabolism in the germline niche, Takashima and colleagues demonstrate the novel functions of FGF2 in the germline niche. Although FGF2 induces self-renewal of spermatogonial stem cells in vitro, this molecule expands “differentiation-prone” GFRA1+RARG+ spermatogonia and facilitates retinoic acid actions required for differentiation in vivo. The present study suggests that FGF2 contributes to spermatogonial differentiation, indicating reconsideration of the role of FGF2 in the germline niche.

Recent studies on bone regeneration demonstrate the use of low cost and stable small molecules, which avoid the adverse effect and high cost of growth factors. Herein, we investigate the chemotactic, angiogenic and osteoinductive potential of a prostacyclin analogue, ONO-1301, when delivered through a biomimetic nanocomposite scaffold (nanohydroxyapatite-gelatin matrix reinforced with fibers) for bone tissue regeneration. The small molecule was loaded onto the scaffold in three different concentrations. There was burst release from all the groups of scaffolds within 24 h followed by a sustained release up to 14 days, but the concentration was dependent on loading percentage. ONO-1301 loaded scaffolds augmented the migration, proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs), but increasing the concentration beyond a certain dose did not show any effect. The osteoinduction was mediated through the prostaglandin I2 receptor and cyclic AMP (cAMP) signaling pathway. They also promoted new bone formation in large sized calvarial defects in rats compared to the scaffold alone, but did not show any impact on angiogenesis. Hence, this study suggests the chemotactic and osteoinductive capability of ONO-1301 for the repair and regeneration of critical sized bone defects.

Acute myocardial infarction is a leading cause of death among single organ diseases. Despite successful reperfusion therapy, ischaemia reperfusion injury (IRI) can induce oxidative stress (OS), cardiomyocyte apoptosis, autophagy and release of inflammatory cytokines, resulting in increased infarct size. In IRI, mitochondrial dysfunction is a key factor, which involves the production of reactive oxygen species, activation of inflammatory signalling cascades or innate immune responses, and apoptosis. Therefore, intercellular mitochondrial transfer could be considered as a promising treatment strategy for ischaemic heart disease. However, low transfer efficiency is a challenge in clinical settings. We previously reported uptake of isolated exogenous mitochondria into cultured cells through co‐incubation, mediated by macropinocytosis. Here, we report the use of transactivator of transcription dextran complexes (TAT‐dextran) to enhance cellular uptake of exogenous mitochondria and improve the protective effect of mitochondrial replenishment in neonatal rat cardiomyocytes (NRCMs) against OS. TAT‐dextran–modified mitochondria (TAT‐Mito) showed a significantly higher level of cellular uptake. Mitochondrial transfer into NRCMs resulted in anti‐apoptotic capability and prevented the suppression of oxidative phosphorylation in mitochondria after OS. Furthermore, TAT‐Mito significantly reduced the apoptotic rates of cardiomyocytes after OS, compared to simple mitochondrial transfer. These results indicate the potential of mitochondrial replenishment therapy in OS‐induced myocardial IRI.

The objective of this study is to prepare cationized gelatin nanospheres incorporating molecular beacon (MB) (cGNSMB) and a cationized gelatin-molecular beacon complex, and evaluate the time period of messenger RNA (mRNA) visualization. The cGNS with different degradabilities were prepared to incorporate MB. There was no difference in the apparent size and zeta potential between the cGNSMB and the complex, while the MB release from the complex was faster than that from the cGNSMB. When mouse mesenchymal stem cells were incubated with the complex and cGNSMB, the amount of MB internalized into cells and cytotoxicity of complex were higher compared with cGNSMB. However, in the amount range of noncytotoxicity, the amount of MB internalized was at a similar level among them. The intracellular fluorescence of cGNSMB was observed over 14 days, whereas that of complex disappeared within 5 days. Moreover, the time period of cGNSMB remaining in the cells prolonged with the increase of glutaraldehyde amount used in cGNSMB preparation. As the result, it is likely that the intracellular fluorescence was retained at a high level for longer time periods. It is concluded that the intracellular controlled release of MB is promising, to achieve long-term and continuous visualization of mRNA. Impact Statement Molecular beacon (MB) is a versatile activatable imaging probe to detect messenger RNA (mRNA). Cationized gelatin nanospheres incorporating MB (cGNSMB) with different degradabilities and a cationized gelatin-MB complex were prepared. The intracellular fluorescence of cGNSMB was observed over 14 days, whereas that of complex disappeared within 5 days. This is because the intracellular decrease of cGNSMB was slower than that of complex. It is concluded that the intracellular controlled release of MB is promising, to achieve long-term and continuous visualization of mRNA.

Osteonecrosis of the jaw induced by administration of bisphosphonates (BPs), BP-related osteonecrosis (BRONJ), typically develops after tooth extraction and is medically challenging. As BPs inhibit oral mucosal cell growth, we hypothesized that suppression of the wound healing-inhibiting effects could prevent BRONJ onset after tooth extraction. Since basic fibroblast growth factor (bFGF) promotes wound healing, but has a short half-life, we examined whether the initiation of BRONJ could be prevented by applying a bFGF-containing gelatin hydrogel over the extraction sockets of BRONJ model rats. Forty-three rats, received two intravenous injections of zoledronic acid 60 μg/kg, once per week for a period of 2 weeks, underwent extraction of a unilateral lower first molar. The rats here were randomly assigned to the bFGF group (n = 15 rats, gelatin hydrogel sheets with incorporated bFGF applied over the sockets); the phosphate-buffered saline (PBS) group (n = 14 rats, gelatin hydrogel sheets without bFGF applied over the sockets); or the control group (n = 14 rats, nothing applied over the sockets). One rat in the bFGF group was sacrificed immediately after tooth extraction. Twenty-one rats were sacrificed at 3 weeks, and the remaining 21 rats were sacrificed at 8 weeks after tooth extractions. The harvested mandibles were analyzed using micro-computed tomography and sections were evaluated qualitatively for mucosal disruption and osteonecrosis. The incidence of osteonecrosis at 8 weeks after tooth extraction was 0% in the bFGF group, 100% in the PBS group, and 85.7% in the control group. The frequency of complete coverage of the extraction socket by mucosal tissue was significantly greater in the bFGF group than in the other groups. These results suggest that application of bFGF in the extraction socket promoted socket healing, which prevented BRONJ development. The growth-stimulating effects of bFGF may have offset the inhibition of wound healing by BP.

Macrophages play an important role in regulating inflammation and tissue regeneration. In the present study, uniform fibrin hydrogel scaffolds were engineered in millimeters. These scaffolds induced anti-inflammatory macrophages to digest and infiltrate the scaffold. The culture conditions of the fibrin hydrogels decreased the secretion of tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine, and increased the secretion of interleukin-10 (IL-10), an anti-inflammatory cytokine, in mouse bone marrow-derived macrophages. Similar results were also observed in human monocyte-derived macrophages (HMDMs). In addition, most of cells that infiltrated the fibrin hydrogels were macrophages expressing CD163, CD204, and CD206, which are anti-inflammatory macrophages markers, both in mice and in human cells. Therefore, to induce increased macrophage infiltration, we attempted to combine fibrin hydrogels with SEW2871, a monocyte/macrophage recruitment agent that is known to be a sphingosine-1 phosphate receptor 1 agonist, solubilized in water by micelle formation with a cholesterol-grafted gelatin. However, the fibrin hydrogels alone retained the same monocyte migration activity as the hydrogels with SEW2871-incorporated micelles in the hydrogel-bearing mouse model. These findings indicate that fibrin hydrogels have a strong promoting effect on the recruitment of anti-inflammatory macrophages. Therefore, fibrin hydrogels may be an optimal biomaterial in the design of medicines for macrophage-induced regenerative therapies. STATEMENT OF SIGNIFICANCE: The immune response to tissue injury is important for determining the speed and the result of the regeneration. Alternatively activated macrophages (M2 macrophages) resolve inflammatory response and promote tissue repair by producing anti-inflammatory factors. Promoting the recruitment of macrophages is a hopeful strategy in the design of biomaterials for tissue regeneration. In the present study, we combined the fibrin hydrogel, which promotes anti-inflammatory polarization, with a macrophage recruitment agent. We revealed that the fibrin hydrogel significantly promoted anti-inflammatory polarization in mouse in vivo and human in vitro. Moreover, macrophages significantly infiltrated into the fibrin hydrogel regardless of the agent combination. Fibrin hydrogels may become a reliable biomaterial for tissue regeneration, and the present study is believed to provide information for many researchers.

We previously have developed the photoresponsive tetra-gel and nanoparticles for controlling the function of the encapsulated substance by UV irradiation. However, the penetration ability of the UV is not high enough. Here, we developed a radiation-responsive tetra-gel and nanoparticle based on γ-ray-responsive X-shaped polyethylene glycol (PEG) linker with a disulfide bond. The nanoparticle could retain small molecules and biomacromolecules. γ-Rays were used as a trigger signal because of their higher penetrating ability. This allowed a spatiotemporal release and control of the encapsulated substances from the nanoparticle in the deeper region, which is impossible by using light exposure (ultraviolet, visible, and near-infrared).

Many types of gene delivery systems have been developed to enhance the level of gene expression. Controlled release technology is a feasible gene delivery system which enables genes to extend the expression duration by maintaining and releasing them at the injection site in a controlled manner. This technology can reduce the adverse effects by the bolus dose administration and avoid the repeated administration. Biodegradable biomaterials are useful as materials for the controlled release-based gene delivery technology and various biodegradable biomaterials have been developed.Controlled release-based gene delivery plays a critical role in a conventional gene therapy and genetic engineering. In the gene therapy, the therapeutic gene is released from biodegradable biomaterial matrices around the tissue to be treated. On the other hand, the intracellular controlled release of gene from the sub-micro-sized matrices is required for genetic engineering. Genetic engineering is feasible for cell transplantation as well as research of stem cells biology and medicine.DNA hydrogel containing a sequence of therapeutic gene and the exosome including the individual specific nucleic acids may become candidates for controlled release carriers. Technologies to deliver genes to cell aggregates will play an important role in the promotion of regenerative research and therapy.

The advent of nanoparticle DDSs (drug delivery systems, nano-DDSs) is opening new pathways to understanding physiology and pathophysiology at the nanometer scale. A nano-DDS can be used to deliver higher local concentrations of drugs to a target region and magnify therapeutic effects. However, interstitial cells or fibrosis in intractable tumors, as occurs in pancreatic or scirrhous stomach cancer, tend to impede nanoparticle delivery. Thus, it is critical to optimize the type and size of nanoparticles to reach the target. High-resolution 3D imaging provides a means of “seeing” the nanoparticle distribution and therapeutic effects. We introduce the concept of “nano-pathophysiological imaging” as a strategy for theranostics. The strategy consists of selecting an appropriate nano-DDS and rapidly evaluating drug effects in vivo to guide the next round of therapy. In this article we classify nano-DDSs by component carrier materials and present an overview of the significance of nano-pathophysiological MRI.

Gene engineered mesenchymal stem cells (MSCs) have been proposed as promising tools for their various applications in biomedicine. Nevertheless, the lack of an effective and safe way to genetically modify these stem cells is still a major obstacle in the current studies. Herein, we designed novel magnetic complexes by assembling cationized pullulan derivatives with magnetic iron oxide nanoparticles for delivering target genes to MSCs. Results showed that this complexes achieved effective gene expression with the assistance of external magnetic field, and resisted the adverse effect induced by serum proteins on the gene delivery. Moreover, neither significant cytotoxicity nor the interference on the osteogenic differentiation to MSCs were observed after magnetofection. Further studies revealed that this effective and serum resistant gene transfection was partly due to the accelerated and enhanced intracellular uptake process driven by external magnetic field. To conclude, the current study presented a novel option for genetic modification of MSCs in an effective, relatively safe and serum compatible way.

The objective of this study was to design and prepare a new contrast agent of magnetic resonance (MR) imaging for the evaluation of therapeutic angiogenesis. Diethylenetriaminepentaacetic acid (DTPA) residue of a chelator was chemically introduced to dextran with a molecular weight of 74,000 (dextran-DTPA). Cyclic peptide containing an arginine-glycine-aspartic acid (RGD) sequence (cyclic RGD) with an inherent affinity for the α(v)β(3) integrin was then introduced to dextran-DTPA (Cyclic RGD-dextran-DTPA). Gd(3+) was added to cyclic RGD-dextran-DTPA to prepare a dextran-based MR contrast agent (Cyclic RGD-dextran-DTPA-Gd). Cyclic RGD-dextran-DTPA-Gd had affinity for cells expressing the α(v)β(3) integrin and showed a higher longitudinal relaxivity compared with DTPA-Gd of an MR contrast agent clinically used. Right femoral, external iliac, and deep femoral and circumflex arteries and veins were surgically ligated to prepare a mouse model of hindlimb ischemia. A laser Doppler analysis and histological evaluation confirmed that hindlimb ischemia healed naturally and was accompanied by angiogenesis, while α(v)β(3) integrin was expressed in the ischemic-angiogenic region without any treatment. Mice at 7 days after vascular ligation were used as an angiogenesis model. When intravenously injected into mice with hindlimb ischemia, cyclic RGD-dextran-DTPA accumulated in the ischemic-angiogenic region and showed the MR ability to detect the ischemic-angiogenic region. It is concluded that cyclic RGD-dextran-DTPA-Gd is a promising material for evaluation of therapeutic angiogenesis.

The objective of this study is to investigate the anti-fibrotic effect of combined mesencymal stem cells (MSCs) and gene therapy on liver fibrosis. When transfected by the complex with a plasmid DNA of hepatocyte growth factor (HGF) and the spermine-introduced pullulan of gene carrier, MSCs secreted HGF protein over 1 week. The HGF secreted from transfected MSC had the biological activity to promote the albumin production of hepatocytes. After intravenous injection, the HGF-secreting MSCs (HGF-MSC) accumulated in the liver. The injection of HGF-MSC decreased the fibrosis area in a rat model of liver fibrosis to a significantly great extent compared with that of original MSC. In the in vitro experiment, the higher number of HGF-transfected MSCs was migrated by stromal cell-derived factor (SDF)-1α more strongly than the original MSC. Considering the promotion of SDF-1α secretion in the liver fibrosis, it is possible that, when transplanted, genetically-engineered MSCs are accumulated in the liver due to their higher response to SDF-1α. It is concluded that the intravenous injection of genetically-engineered MSCs is a promising therapy for liver fibrosis.

Miho Watanabe, Hiaying Li, Jessica Roybal, Matthew Santore, Antonetta Radu, Jun-Ichiro Jo, Michio Kaneko, Yasuhiko Tabata, and Alan Flake. Tissue Engineering Part A.Apr 2011.1099-1110. http://doi.org/10.1089/ten.tea.2010.0390, Myelomeningocele (MMC) is a common and devastating malformation. As an alternative to fetal surgical repair, tissue engineering has the potential to provide a less invasive approach for tissue coverage applicable at an earlier stage of gestation. We have previously evaluated the use of gelatin hydrogel composites composed of gelatin sponges and sheets as a platform for tissue coverage of the MMC defect in the retinoic acid induced fetal rat model of MMC. In the current study, we compare our previous composite with gelatin microspheres as a scaffold for tissue ingrowth and cellular adhesion within the amniotic fluid environment. We also examine the relative efficacy of various bioactive protein coatings on the adhesion of amniotic fluid cells to the construct within the amniotic cavity. We conclude from this study that gelatin microspheres are as effective as gelatin sponges as a scaffold for cellular ingrowth and amniotic fluid cell adhesion and that collagen type I and fibronectin coatings enhance amniotic fluid cell adhesion to the gelatin-based scaffolds. These findings support the potential for the development of a tissue-engineered injectable scaffold that could be applied by ultrasound-guided injection, much earlier and less invasively than sponge or sheet-based composites. © 2011 Mary Ann Liebert, Inc.

The objective of this study was to design three-dimensional scaffolds of bone marrow-derived stem cells capable of their gene transfection and evaluate the transfection extent. Three-dimensional scaffolds of gelatin and β-tricalcium phosphate (β-TCP) were prepared. Spermine-introduced pullulan (spermine-pullulan) was prepared as the non-viral carrier of gene transfection. The spermine-pullulan was mixed with a luciferase plasmid DNA to prepare their polyion complex. The scaffolds were treated with succinylated gelatin (suc-gel) at different concentrations, treated in different methods of freeze-drying and dehydrothermal treatment, and placed in the aqueous of complexes to prepare various scaffolds containing the complex. When the stem cells were seeded into the scaffolds to evaluate the gene transfection of cells, the level of plasmid DNA transfection depended on the method of complex containing scaffolds preparation. The complexes were released with time from the scaffolds although the release profile depended on the type of scaffolds. The order of gene transfection for the stem cells in the scaffolds was in good accordance with that of plasmid DNA released. It is possible that cells were transfected with the complexes released from the scaffolds.

We report a microvascular-interstitium model on microfluidic devices to study leakage of drugs from blood vessels under in vivo-like flow conditions. We employed magnetic resonance imaging to demonstrate the compatibility of the model for experimental animals and humans. We observed transport of two types of different molecular-weight contrast agents into the model interstitium. The ratio of the transport rates of agents agreed with the ratio calculated from diffusion coefficients of the agents. We expect that the model will be useful for the estimation and evaluation of leakage of many kinds of agents in vivo.

Miho Watanabe, Jun-ichiro Jo, Antonetta Radu, Michio Kaneko, Yasuhiko Tabata, and Alan W. Flake. Tissue Engineering Part A.May 2010.1645-1655. http://doi.org/10.1089/ten.tea.2009.0532., Myelomeningocele (MMC) is a common and devastating malformation. Although fetal surgical closure may improve outcome, a less invasive approach that can be applied earlier in gestation is desirable. The objective of this study was to evaluate the therapeutic feasibility of a tissue engineering approach for prenatal coverage of MMC. A gelatin hydrogel composite combining a gelatin sheet and gelatin sponge was prepared with or without basic fibroblast growth factor incorporation, and applied prenatally to retinoic-acid-induced fetal MMC in the rat model. Most of the composites were adherent to the MMC within the amniotic fluid environment with the help of cyanoacrylate adhesive. Histological examination revealed cells layered over the composites with associated extracellular matrix as well as cellular ingrowth into the sponges. The layer over the composite was composed of mixed nonepithelial and epithelial cells with the extracellular matrix consisting of collagen type I and hyaluronic acid. The tissue inside the sponge consisted of nonepithelial cells and hyaluronic acid. Epidermal ingrowth underneath the sponges and neovascularization into the sponges occurred and were significantly increased by the incorporation of basic fibroblast growth factor. Although further development is needed, this study supports the therapeutic potential of a tissue engineering approach for prenatal coverage of MMC. © 2010 Mary Ann Liebert, Inc.

The objective of this study is to prepare iron oxide nanoparticles with different sizes and surface potentials and evaluate the labeling efficiency of bone marrow-derived mesenchymal stem cells (MSC). The ferric and ferrous ions were co-precipitated in the presence of pullulan or the cationized and anionized derivatives to prepare iron oxide-pullulan nanoparticles of a contrast agent of magnetic resonance imaging (MRI). The size and surface potential of iron oxide-pullulan nanoparticles were changed by altering the mixing molar ratio of pullulan OH groups to ferric ions and the mixing percentage of pullulan derivatives, respectively. When MSC were labeled with the iron oxide-pullulan nanoparticles by the co-culture for 1 h, the labeling efficiency and 1H MRI relaxivity were greatly influenced by the particle size and surface potential, while the labeling procedure did not affect the viability and differentiation ability of MSC. These findings indicate that iron oxide-pullulan nanoparticle is a promising tool for the MRI labeling of MSC.

The objective of this study is to prepare a non-viral carrier of gene transfection from various polysaccharides and evaluate the feasibility in gene expression for mesenchymal stem cells (MSCs). Various amounts of spermine were chemically introduced into pullulan, dextran and mannan with a molecular weight of around 40 000 or pullulan with different molecular weights to prepare cationized polysaccharides with different extents of spermine introduced (spermine-polysaccharide). Each cationized polysaccharide was complexed with a plasmid DNA at various ratios and in vitro gene transfection was investigated for rat bone marrow-derived MSCs. The level of gene expression depended on the type of cationized polysaccharide. The highest level was observed for the complex of spermine-pullulan and plasmid DNA. Additionally, the level also depended on the molecular weight of pullulan and the extent of spermine introduced to pullulan. Suppression of gene expression with chlorpromazine and methyl-beta-cyclodextrin of endocytosis inhibitors demonstrated that the cellular uptake of spermine-pullulan-plasmid DNA complexes was mediated by clathrin- and raft/caveolae-dependent endocytic pathways. The cationized pullulan is a promising non-viral carrier of plasmid DNA for MSCs.

Purpose To assess whether Gd-DTPA-Gel-Cis, a conjugate of gadolinium (Gd), cis diamminedichloroplatinum (Cis), diethylenetriaminepentaacetic acid (DTPA)-dianhydride, and bovine gelatin (Gel) can be used as an intravascular contrast agent at MRI and as an antitumor cell proliferation agent in vitro. Materials and Methods We injected Gd-DTPA-Gel-Cis (200 mg/mL) into the caudal vein of female HER-2/neu transgenic mice with spontaneous mammary tumors. The tumor signal intensity was measured with a 0.3 Tesla MRI scanner. HER-2/neu-expressing NT cells were treated with Gd-DTPA-Gel-Cis (5 μM cisplatin, 200 mg/mL Gel), Cis alone (5 μM cisplatin), or Gel alone (200 mg/mL gelatin). Differences of P < 0.05 were considered to be statistically significant. Results On T1-weighted MRI scans of mice injected with Gd-DTPA-Gel-Cis we observed a 23% increase in signal intensity. The survival rates of cells exposed to Gd-DTPA-Gel-Cis or Cis were 70.9% and 58.3%, respectively, of the survival rates observed after treatment with Gel alone. Gd-DTPA-Gel-Cis showed significant toxicity (P < 0.05). Conclusion Gd-DTPA-Gel-Cis shows promise for use as an MRI contrast medium and as an antitumor agent. J. Magn. Reson. Imaging 2009;30:907–910. © 2009 Wiley-Liss, Inc.

Background— In-stent thrombosis is mainly triggered by adenosine diphosphate (ADP)-dependent platelet aggregation after percutanous coronary stent implantation. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) rapidly hydrolyzes ADP to adenosine monophosphate, inhibiting platelet aggregation. We tested the hypothesis that local delivery of human placental E-NTPDase (pE-NTPDase) gene into injured arteries via gene-eluting stent could prevent subacute in-stent thrombosis. Methods and Results— We generated gene-eluting stents by coating bare metal stents with cationic gelatin hydrogel containing pE-NTPDase cDNA (pE-NTPDase stent), and implanted the stents into rabbit femoral arteries (FA) prone to production of platelet-rich thrombi due to repeated balloon injury at 4-week intervals. After the second injury, E-NTPDase gene expression was severely decreased; however, the implantation of pE-NTPDase stent increased E-NTPDase mRNA levels and NTPDase activity to higher level than normal FA. The FAs with pE-NTPDase stents maintained patency in all rabbits ( P Conclusions— Human pE-NTPDase gene transfer via cationic gelatin-coated stents inhibited subacute in-stent thrombosis and suppressed neointimal hyperplasia and inflammation without antiplatelet drugs.

Autoimmune alopecia (alopecia areata) is considered to be triggered by a collapse of immune privilege in hair follicles. Here we confirmed that infiltrating CD4 T lymphocytes around hair follicles of patients with alopecia areata were primarily CCR5-positive with few CCR4-positive cells, suggesting a dominant role of Th1 cells in the alopecic lesion. Given this finding, we sought to elucidate the effect of cytokine therapy in C3H/HeJ mice, a mouse model of alopecia areata, by applying recombinant interleukin-4 and neutralizing anti-interferon-gamma antibody. We found that local injections of both interleukin-4 and neutralizing anti-interferon-gamma antibody effectively treated alopecia in C3H/HeJ mice. Results from immunohistochemistry and semiquantitative reverse transcription-polymerase chain reaction demonstrated that intralesional injection of interleukin-4 suppressed CD8 T cell infiltrates around the hair follicles and repressed enhanced interferon-gamma mRNA expression in the affected alopecic skin. Furthermore, Th1 transcription factor T-box21 small interfering RNAs conjugated to cationized gelatin showed mitigating effects on alopecia in C3H/HeJ mice, resulting in the restoration of hair shaft elongation. Taken together, the use of gelatin-small interfering RNA conjugates promises to be a novel, efficient, and safe tool as an alternative gene therapy for the treatment of various human diseases. To our knowledge, this is the first report of effective controlled delivery of small interfering RNA using biodegradable cationized gelatin microspheres in an animal model of disease.

It is expected that mesenchymal stem cells (MSCs) will be a cell source for cardiac reconstruction because of their differentiation potential and ability to supply growth factors. However, poor viability at the transplanted site often hinders the therapeutic potential of MSCs. Here, in a trial designed to address this problem, a non-viral carrier of cationized polysaccharide is introduced for genetic engineering of MSCs. Spermine-introduced dextran of cationized polysaccharide (spermine-dextran) was internalized into MSCs by way of a sugar-recognizable receptor to enhance the expression level of plasmid deoxyribonucleic acid (DNA). When genetically engineered by the spermine-dextran complex with plasmid DNA of adrenomedullin (AM), MSCs secreted a large amount of AM, an anti-apoptotic and angiogenic peptide. Transplantation of AM gene-engineered MSCs improved cardiac function after myocardial infarction significantly more than MSCs alone. Thus, this genetic engineering technology using the non-viral spermine-dextran is a promising strategy to improve MSC therapy for ischemic heart disease.

The human noncoding RNA gene RGM249 has been shown to regulate the degree of cancer cell differentiation. In this study, we investigated the effects of 3 microRNA-like molecules digested from RGM249 on the loss of malignant properties in cancer cells in immunodeficient KSN/Slc mice. We utilized small interfering RNAs (siRNAs) alone or in combination with a cationized drug delivery system (DDS) consisting of atelocollagen or gelatin hydrogel microspheres. The results demonstrated growth inhibition and apoptosis and the inhibition of both neovascularization and metastasis, indicating that the DDSs effectively infiltrated the majority of tumor cells in vivo. Systemic administration of the 3 siRNAs inhibited the metastatic ability of malignant cells. Cotransfection of these siRNAs exerted a regulatory effect upon the genes involved in differentiation, pluripotency, and proliferation in cancer cells. These results suggest that RGM249-derived oligonucleotides may be involved in the regulation of metastasis, proliferation, and differentiation in vivo, and that the tested siRNAs may therefore represent a new anticancer therapeutic approach.

The objective of this study is to develop a sustained release system of small interfering RNA (siRNA) inside cells aiming at a prolonged time period of gene suppression. Gelatin aqueous solution containing luciferase siRNA was coacelvated by acetone addition, followed by the glutaraldehyde (GA) crosslinking of gelatin to prepare gelatin nanospheres incorporating siRNA. The nanospheres were degraded with time in phosphate-buffered saline solution containing collagenase to release siRNA incorporated. The nanospheres were degraded more slowly as the GA concentration become higher, and consequently the rate of siRNA become lower. siRNA was released from the nanospheres as a result of nanospheres degradation. The nanospheres were internalized into colon 26 cells luciferase stably expressed, irrespective of the GA concentration. The gene expression was suppressed by the nanospheres incorporating siRNA capable for the longer-term release, and subsequently the time period of gene suppression was prolonged. The siRNA release inside the cell was observed, while the release period became longer for the slow-degraded nanospheres. It is possible that the intracellular siRNA release for a longer time period contributes to the prolonged time period of gene suppression.

Theranostics means a therapy conducted in a diagnosis-guided manner. For theranostics of solid tumors by means of ultrasound, we designed a nano-sized emulsion containing perfluoropentane (PFC5). This emulsion can be delivered into tumor tissues through the tumor vasculatures owing to its nano-size, and the emulsion is transformed into a micron-sized bubble upon sonication through phase transition of PFC5. The micron-sized bubbles can more efficiently absorb ultrasonic energy for better diagnostic images and can exhibit more efficient ultrasound-driven therapeutic effects than nano-sized bubbles. For more efficient tumor delivery, smaller size is preferable, yet the preparation of a smaller emulsion is technically more difficult. In this paper, we used a bath-type sonicator to successfully obtain small PFC5-containing emulsions in a diameter of ca. 200nm. Additionally, we prepared these small emulsions at 40°C, which is above the boiling temperature of PFC5. Accordingly, we succeeded in obtaining very small nano-emulsions for theranostics through a very facile method.

The objective of this study is to design biodegradable nanospheres of cationized gelatin as a carrier of cellular internalization of plasmid DNA. Ethylenediamine was chemically introduced into the carboxyl groups of gelatin to obtain cationized gelatin. The gelatin solution was filtered through a glass membrane under high pressure and dropped into 2-butanol, acetone or a mixture of the two to form nanospheres of cationized gelatin. The microspheres of cationized gelatin were prepared by the conventional water-in-oil emulsion method. The resulting nano- and microspheres of cationized gelatin were dehydrothermally treated at 160°C for different time periods to allow them to cross-link chemically. The size of nanospheres, prepared by the filtration method and changed by the type of solvents, was 1.86, 0.83 or 0.24 μm. The in vitro degradation of spheres became faster as the time period of dehydrothermal treatment was shorter. The degradation time of spheres in HCl solution linearly increased with an increase in the cross-linking time, irrespective of the sphere size. However, in the collagenase solution, when compared at the similar cross-linking density, the smaller spheres were degraded more slowly than the larger ones. The plasmid DNA incorporated in the nanospheres was released from the nanospheres with their degradation. The nanospheres incorporating plasmid DNA were internalized into cells, and intracellularly degraded with time to release plasmid DNA. The time period of plasmid DNA release was prolonged by increasing the nanosphere degradation time.

Akinaga Sonoda1, Norihisa Nitta1, Ayumi Nitta-Seko1, Shinich Ohta1, Shigeyuki Takamatsu2, Yoshio Ikehata3, Isamu Nagano3, Jun-ichiro Jo4, Yasuhiko Tabata4, Masashi Takahashi1, Osamu Matsui3, Kiyoshi Murata11Department of Radiology, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga, 520-2192, Japan; 2Department of Radiology, Graduate School of Medical Science, Kanazawa University, Takara-machi 13-1, Kanazawa Ishikawa, 920-8641, Japan; 3Department of Natural Science and Technology, Graduate School of Engineering, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan; 4Department of Biomaterials, Institute for Frontier Medical Sciences, Kyoto University, Shogoin kawara-machi 53, Sakyo-ku 606-8507, Kyoto, JapanAbstract: We developed a dextran-magnetite conjugated cisplatin (DM-Cis) complex for use in thermal ablation and as a chemotherapeutic drug. To produce DM-Cis we reacted Cis with 1 mL DM (56 mg/mL iron). The temperature rise of DM-Cis was measured in vitro and in vivo under a portable induction-heating (IH) device. Platinum desorption from DM-Cis over 24 hours was measured in bovine serum. In in vivo accumulation and magnet and exothermic experiments we used four rat groups. In group 1 we delivered DM-Cis intraperitoneally (ip) and placed magnets subcutaneously (sc). In group 2 we injected saline (ip) and placed magnets (sc). In group 3 we injected DM-Cis (ip) and placed a sc incision (sham). The control (group 4) received an ip injection of saline. Rectus abdominis muscle tissue was stained with hematoxylin-eosin and iron-stained tissue areas (µm2) were calculated. The maximum platinum concentration in DM-Cis was approximately 105.6 µg/mL. Over 24 hours, 33.48% of platinum from DM-Cis was released. There was a significant difference (P < 0.05) in the iron-stained area between group 1 and the other groups. The temperature in muscle tissue registered a maximum of 56°C after about 4 min. DM-Cis may represent a magnetically-accumulated anticancer drug with hyperthermic effects.Keywords: magnetic nanoparticle-conjugated anticancer agents, DM, portable induction ­heating device, carboxyl group, rat

A simple modification of a DNA aptamer with poly(ethylene glycol) (PEG) based on metal coordination was developed. N,N-bis(carboxymethyl)-L-lysine (NTA) of a metal chelate residue was chemically introduced to one terminus of PEG. The NTA-introduced PEG (PEG-NTA) chelated Cu(2+) ions form a Cu(2+)-chelated PEG (PEG-Cu). When PEG-Cu was mixed with a DNA aptamer of anti-tumor activity (AS1411) in aqueous solution, a complex of PEG-Cu and AS1411 based on metal coordination was formed. The complex inhibited in vitro tumor growth in a dose-dependent manner. A body distribution study with tumor-bearing mice revealed that PEG-Cu-AS1411 complexes injected intravenously had a significant longer lifetime in the blood circulation and 1.5-2.0-fold higher accumulation in the tumor tissue than free AS1411. Intravenous injection of complexes suppressed the in vivo growth of tumor mass to a significantly greater extent compared with that of free AS1411. The Cu(2+)-coordinated PEG modification is a simple and promising method to enhance accumulation of the aptamer in the tumor, resulting in the augmented anti-tumor effect.

This study is undertaken to design a novel nano-sized delivery system of tissue-type plasminogen activator (t-PA) which has a suppressed thrombolytic activity of t-PA, but recovered the activity only when exposed to ultrasound. Various amounts of ethylenediamine were chemically introduced into gelatin (cationized gelatins) to complex with t-PA. To modify the surface of complexes with polyethylene glycol (PEG), PEG was chemically grafted to the anionic gelatin (PEG-gelatin). The simple mixing with the PEG-gelatin enabled the t-PA-cationized gelatin complex to form a nano-sized delivery complex with PEG chains on the surface. The t-PA activity of PEG-modified complexes was significantly suppressed to be 45% of original t-PA. However, when exposed to ultrasound in vitro, the t-PA activity was fully recovered. A cell culture experiment demonstrated no cytotoxicity of PEG-modified complexes. The body distribution study indicated that the half-life of t-PA in the blood circulation was prolonged about 3 times. In a rabbit thrombosis model, the intravenous administration of PEG-modified complexes followed by ultrasound irradiation resulted in complete recanalization, in remarked contrast to the complex administration alone. It is concluded that the PEG-modified complex is a promising t-PA delivery system to enhance the biological activity at the site necessary only by a local ultrasound irradiation.

The objective of this study is to design a new multimodal imaging system for the evaluation of bone regeneration process. Pamidronate (PA) of bisphosphonates with a high affinity for hydroxyapatite, was introduced to pullulan with different molecular weights (PA–pullulan). Then, two probes for fluorescence and magnetic resonance (MR) imagings were introduced into the PA–pullulan to prepare the PA–pullulan conjugates containing both the imaging probes (PA–pullulan–F/M). The PA–pullulan–F/M conjugates had an affinity for hydroxyapatite. A gelatin hydrogel incorporating bone morphogentic protein (BMP)-2 was prepared and implanted subcutaneously into mice to obtain an animal model of bone regeneration. When intravenously injected into mice with the bone tissue ectopically formed by the BMP-2-incorporated hydrogel to fluorescently evaluate their body distribution, the PA–pullulan–F/M conjugates were accumulated in the bone tissue regenerated. The time profile of fluorescent intensity well corresponded with that of calcium amount in the bone tissue newly formed. In addition, the PA–pullulan–F/M conjugates showed an MR ability similar to Gd-DTPA (gadopentetate dimeglumine) of a MR imaging agent clinically used. The MR signal around the bone tissue newly formed was enhanced for mice injected with PA–pullulan–F/M prepared from pullulan with the molecular weight of 6000. It is concluded that the PA–pullulan–F/M conjugate is a useful multimodal agent of polymeric delivery system to evaluate the process of bone regeneration.

The objective of this study was to genetically engineer dendritic cells (DC) for biological activation and evaluate their anti-tumor activity in a tumor-bearing mouse model. Mouse DC were incubated on the surface of culture dishes which had been coated with the complexes of a cationized dextran and luciferase plasmid DNA complexes plus a cell adhesion protein, Pronectin®, for gene transfection (reverse transfection). When compared with the conventional transfection where DC were transfected in the medium containing the complexes, the level of gene expression by the reverse method was significantly higher and the time period of gene expression was prolonged. Following the reverse transfection of DC by a plasmid DNA of mouse interleukin-12 (mIL-12) complexed with the cationized dextran, the mIL-12 protein was secreted at higher amounts for a longer time period. When injected intratumorally into mice carrying a mass of B16 tumor cells, the DC genetically activated showed significant anti-tumor activity.

The objective of this study is to investigate the possibility of small interfering RNA (siRNA) complexed with a cationized dextran of nonviral carrier to biologically modify the adipogenesis extent of bone marrow-derived mesenchymal stem cells (MSC). Spermine was chemically introduced to the hydroxyl groups of dextran to prepare the cationized dextran (spermine-dextran). The spermine-dextran could form a complex with siRNA, and the physicochemical properties were changed by the molecular weight of dextran, the molar percentage of spermine introduced to dextran, and the molar ratio of nitrogen molecule of spermine-dextran to the phosphorous ones of siRNA (N/P ratio). The gene expression level of luciferase or green fluorescence protein was significantly suppressed by transfection with the complex of spermine-dextran and siRNA. The gene expression level by the complex decreased with an increase in the extent of complex internalized. Biochemical experiments revealed that culture in an adipogenic differentiation medium allowed MSC to differentiate into adipogenic cells. However, upon culturing with siRNA of anti-transcription coactivator containing PDZ-binding motif (TAZ) for osteogenic differentiation complexed with the spermine-dextran, the adipogenesis of MSC was further promoted. It is concluded that the spemine-dextran was a promising nonviral carrier to suppress the expression level of differentiation gene, resulting in the modification of cell differentiation direction.

The objective of this study is to prepare a novel gene carrier from pullulan, a polysaccharide with an inherent affinity for the liver, and evaluate the feasibility in gene transfection. Various amounts of spermine were chemically introduced into pullulan with molecular weights of 22,800, 47,300, and 112,000 to prepare cationized pullulan derivatives with different percentages of spermine introduced. Each cationized pullulan derivative was complexed with a plasmid DNA at various ratios and applied to HepG2 cells for in vitro gene transfection. The level of gene expression depended on the percent spermine introduced of cationized pullulan derivatives and the molecular weight of pullulan. However, when compared at the complexation molar ratio of pullulan derivative to the plasmid DNA, the expression level became maximum around the ratio of 10(2), irrespective of the pullulan molecular weight. Pre-treatment of cells with asialofetuin of asialoglycoprotein receptor ligand decreased the level of gene expression by the complexes. The cationized pullulan derivative with an appropriate physicochemical character is a promising non-viral carrier which promotes the receptor-mediated internalization of plasmid DNA and consequently enhances the expression level.

A new non-viral method of gene transfection was designed to enhance the level of gene expression for rat mesenchymal stem cells (MSCs). Pullulan was cationized using chemical introduction of spermine to prepare cationized pullulan of non-viral carrier (spermine-pullulan). The spermine-pullulan was complexed with a plasmid deoxyribonucleic acid (DNA) of luciferase and coated on the surface of culture substrate together with Pronectin of artificial cell adhesion protein. MSCs were cultured and transfected on the complex-coated substrate (reverse transfection), and the level and duration of gene expression were compared with those of MSCs transfected by culturing in the medium containing the plasmid DNA-spermine-pullulan complex (conventional method). The reverse transfection method enhanced and prolonged gene expression significantly more than did the conventional method. The reverse method permitted the transfection culture of MSCs in the presence of serum, in contrast to the conventional method, which gave cells a good culture condition to lower cytotoxicity. The reverse transfection was carried out for a non-woven fabric of polyethylene terephthalate (PET) coated with the complex and Pronectin using agitation and stirring culture methods. The two methods enhanced the level and duration of gene expression for MSCs significantly more than did the static method. It is possible that medium circulation improves the culture conditions of cells in terms of oxygen and nutrition supply and waste excretion, resulting in enhanced gene expression.

The objective of this study is to prepare a novel gene carrier from pullulan, a polysaccharide with an inherent affinity for the liver and evaluate the feasibility in gene transfection in the mice liver. Pullulan with a weight-average molecular weight of 22,800 was cationized by the chemical introduction of spermine (spermine-pullulan). The cationized pullulan derivative was complexed with a plasmid DNA and intravenously injected for in vivo gene transfection. The level of gene expression by the spermine-pullulan in the liver depended on the extent of spermine introduced and the highest level was observed for the spermine-pullulan with an introduction extent of 5.60. When a plasmid DNA coding NK4 of a hepatocyte growth factor (HGF)/scatter factor antagonist complexed with the spermine-pullulan was intravenously injected to mice 1 day before the inoculation of RLmale1 tumor cells, the tumor-bearing mice survived for a longer time period, while the GPT level and the number of tumor cells grown in the liver were low compared with those of free plasmid DNA injection. These findings indicate that the liver targeting of NK4 plasmid DNA by complexation with the spermine- pullulan specifically enhanced the liver expression level, resulting in augmented suppression effect on tumor growth therein.