Anisotropic NMR data acquisition with a prototype 400 MHz cryogen-free NMR spectrometer.

High-temperature superconducting (HTS) materials have recently been incorporated into the construction of HTS cryogen-free magnets for nuclear magnetic resonance (NMR) spectroscopy. These HTS NMR spectrometers do not require liquid cryogens, thereby providing significant cost savings and facilitating easy integration into chemistry laboratories. However, the optimal performance of these HTS magnets against standard cryogen NMR magnets must be evaluated, especially with demanding modern NMR applications such as NMR in anisotropic media. The stability of the HTS magnets over time and their performance with complex pulse sequence experiments are the main unknown factors of this new technology. In this study, we evaluate the utility of our prototype 400 MHz cryogen-free power-driven HTS NMR spectrometer, installed in the fumehood of a chemistry laboratory, for stereochemical analysis of three commercial natural products (artemisinin, artemether, and dihydroartemisinin) via measurement of anisotropic NMR data, in particular, residual dipolar couplings. The accuracy of measurement of the anisotropic NMR data with the HTS magnet spectrometer is evaluated through the CASE-3D fitting protocol, as implemented in the Mestrenova-StereoFitter software program.

Transcriptome analysis of buffalo granulosa cells in three dimensional culture systems.

Hanging drop (HD) three-dimensional (3D) culture model for buffalo granulosa cells (GC) was reported to mimic the preovulatory stage of ovarian follicles in our previous study. To further verify its reliability, the present study attempted a comparative transcriptome profile of buffalo GC freshly isolated from ovarian follicles (<8 mm diameter) (FC) and their cultures in normal culture dish (ND or 2D), polyHEMA coated dish (PH) and HD culture systems (3D). Out of 223 significantly (-log2 fold change: >3; p < .0005; false discovery rate [FDR]: <0.1) differentially expressed genes (SDEGs) among different culture systems, 137 were found unannotated, and 94, 29, and 66 were exclusively expressed in FC, PH, and HD, respectively. However, on eliminating the fixed points of p values and FDR from the entire raw data, only 11 genes related to long noncoding RNA, 12 genes related to luteinization, and 3 genes related to follicular maturation were exclusively expressed in FC, PH, and HD culture systems, respectively. The quantitative real time-PCR validation and the next generation sequencing data had more than 90% correlation. Bioinformatics analyses of the exclusively expressed SDEG revealed that the freshly aspirated GCs were a true representative of GCs from small follicles (<8 mm diameter), the GC spheroids under PH maintained mitochondrial function, and those cultured in HD system for 6 days simulated the inflammatory milieu required for ovulation. Therefore, the comparative transcriptome profile also reinforced that HD culture system is better in vitro culture method than the other methods analyzed in this study for buffalo GC.

Integrin-EGFR interaction regulates anoikis resistance in colon cancer cells.

Anoikis resistance is an essential property of cancer cells that allow the extra-cellular matrix-detached cells to survive in a suspended state in body fluid in order to metastasize and invade to distant organs. It is known that integrins play an important role in anoikis resistance, but detailed mechanisms are not well understood. Here we report that highly metastatic colon cancer cells showed a higher degree of anoikis resistance than the normal intestinal epithelial cells. These anoikis-resistant cancer cells express high-levels of integrin-α2, ß1, and activated EGFR in the anchorage-independent state than the anchorage-dependent state. In contrast, normal intestinal epithelial cells failed to elevate these proteins. Interestingly, a higher co-association of EGFR with integrin-α2ß1/-α5ß1 was observed on the surface of anoikis-resistant cells. Thus, in the absence of extra-cellular matrix, integrins in association with EGFR activates downstream effectors ERK and AKT and suppress Caspase-3 activation to induce anoikis resistance as was confirmed from the gene-ablation and pharmacological inhibitor studies. Interestingly, these anoikis-resistant cancer cells express high-level of cancer stem cell signatures (CD24, CD44, CD133, EpCAM) and pluripotent stem cell markers (OCT-4, SOX-2, Nanog) as well as drug-resistant pumps (ABCG2, MDR1, MRP1). Altogether, our findings unravel the interplay between integrin-α2ß1/-α5ß1 and EGFR in anoikis resistance and suggest that the resistant cells are cancer initiating or cancer stem cells, which may serve as a promising target to combat metastasis of cancer.

Three-dimensional culture of buffalo granulosa cells in hanging drop mimics the preovulatory follicle stage.

Granulosa cell (GC) culture models mimicking the intrafollicular environment are limited. Such models have a great potential in reproductive toxicity studies. The buffalo, a monovulatory species like humans, could be a better model than polyovulatory rodents. Therefore, we targeted the development and characterization of three-dimensional (3D) culture systems for buffalo GCs. The GCs from small ovarian follicles (SF) maintained the CYP19 gene expression for 144 hr in a 2D culture system. Hence, GCs from SF were cultured directly in 3D using hanging drop and Poly-([2-hydroxyethyl methacrylate]) (polyHEMA) methods in the DMEM media containing 1 ng/ml FSH and 10 ng/ml IGF-1 for 144 hr. The expression profile of nine GC-specific transcripts; CYP19, TNFAIP6, AMH, PTI, NR4A1, FSHR, RUNX, LHR, and COX2/PTGS2; revealed that 3D-spheroids developed in hanging drop method maintained the GC phenotype of preovulatory follicles. Therefore, hanging drop method is a best method for culturing GCs to mimic the intrafollicular environment.

Induced Retro-Differentiation of Human Retinal Pigment Epithelial Cells on PolyHEMA.

Retinal pigment epithelium (RPE) cells represent a great potential to rescue degenerated cells of the damaged retina. Activation of the virtually plastic properties of RPE cells may aid in recovery of retinal degenerative disorders without the need for entire RPE sheet transplantation. Poly (2-hydroxyethyl methacrylate)(PolyHEMA) is one of the most important hydrogels in the biomaterials world. This hydrophobic polymer does not normally support attachment of mammalian cells. In the current study we investigated the effect of PolyHEMA as a cell culture substrate on the growth, differentiation, and plasticity of hRPE cells. hRPE cells were isolated from neonatal human globes and cultured on PolyHEMA and polystyrene substrates (as controls) in 24-well culture plates. DMEM/F12 was supplemented with 10% fetal bovine serum (FBS) and/or 30% human amniotic fluid (HAF) for cultured cells on polystyrene and PolyHEMA coated vessels. Morphology, rate of cell proliferation and cell death, MTT assay, immunocytochemistry and Real-Time RT-PCR were performed to investigate the effects of PolyHEMA on the growth and differentiation of cultured hRPE cells. Proliferation rate of the cells that had been cultured on PolyHEMA was reduced; PolyHEMA did not induce cell death in the hRPE cultures. hRPE cells cultured on PolyHEMA formed many giant spheroid colonies. The giant colonies were re-cultured and the presence of retinal progenitor markers and markers of hRPE cells were detected in cell cultures on PolyHEMA. PolyHEMA seems to be promising for both maintenance and de-differentiation of hRPE cells and expansion of the retinal progenitor cells from the cultures that are originated from hRPE cells. J. Cell. Biochem. 118: 3080-3089, 2017. © 2017 Wiley Periodicals, Inc.

Radiolabeling of new generation magnetic poly(HEMA-MAPA) nanoparticles with (131) I and preliminary investigation of its radiopharmaceutical potential using albino Wistar rats.

In this study, N-methacryloyl-l-phenylalanine (MAPA) containing poly(2-hydroxyethylmethacrylate) (HEMA)-based magnetic poly(HEMA-MAPA) nanobeads [mag-poly(HEMA-MAPA)] were radiolabeled with (131) I [(131) I-mag-poly(HEMA-MAPA)], and the radiopharmaceutical potential of (131) I-mag-poly(HEMA-MAPA) was investigated. Quality control studies were carried out by radiochromatographic method to be sure that (131) I binded to mag-poly(HEMA-MAPA) efficiently. In this sense, binding yield of (131) I-mag-poly(HEMA-MAPA) was found to be about 95-100%. In addition to this, optimum radiodination conditions for (131) I-mag-poly(HEMA-MAPA) were determined by thin-layer radiochromatography studies. In addition to thin-layer radiochromatography studies, lipophilicity (partition coefficient) and stability studies for (131) I-mag-poly(HEMA-MAPA) were realized. It was determined that lipophilicities of mag-poly(HEMA-MAPA) and (131) I-mag-poly(HEMA-MAPA) were 0.12 ± 0.01 and 1.79 ± 0.76 according to ACD/logP algorithm program, respectively. Stability of the radiolabeled compound was investigated in time intervals given as 0, 30, 60, 180, and 1440 min. It was found that (131) I-mag-poly(HEMA-MAPA) existed as a stable complex in rat serum within 60 min. After that, biodistribution and scintigraphy studies were carried out by using albino Wistar rats. It was determined that the most important (131) I activity uptake was observed in the breast, the ovary, and the pancreas. Scintigraphy studies well supported biodistribution results.

Cell Viability Assay with 3D Prostate Tumor Spheroids.

WST-8 (Cell Counting Kit 8; CCK-8) is the last generation tetrazolium-based cell viability assay and has recently been accepted as a validated method for measuring the cell viability of 3D in vitro models. Here, we describe how to form 3D prostate tumor spheroids using the polyHEMA technique, apply drug treatments and WST-8 assay to these spheroids, and calculate their cell viability. The advantages of our protocol are the formation of spheroids without adding extracellular matrix components, and the elimination of the critique handling process needed for transferring spheroids. Although this protocol exemplifies the determination of percentage cell viability in PC-3 prostate tumor spheroids, it can be adapted and optimized for other prostate cell lines and other types of cancers.

Development of endodontic sealers containing antimicrobial-loaded polymer particles with long-term antibacterial effects.

OBJECTIVE: The objective of this study is to prepare new dental resins with a long-lasting antimicrobial activity. Specifically, this study evaluates an approach for controlling infection in root canals using sealers containing polyhydroxyethyl methacrylate trimethylolpropane trimethacrylate (polyHEMA/TMPT) particles loaded with cetylpyridinium chloride (CPC). In addition, the physical properties of sealers containing CPC-loaded polyHEMA/TMPT particles (CLP) are determined. METHODS: PolyHEMA/TMPT particles with 10 (10%-CLP) and 25wt.% CPC (25%-CLP) with different particle sizes were fabricated and incorporated in HEMA-based sealers. CPC-release profiles were evaluated over 14 days of immersion in water, followed by 14 days of storage and 14 days of water immersion. The antibacterial activity of these sealers against Enterococcus faecalis in dentinal tubules was assessed using a root-canal-infection model. Their sealing abilities were evaluated by fluid filtration and physical properties were tested according to the ISO 6876 standard. The long-term antibacterial activity of the cured sealer containing 25%-CLP (∼21µm particle diameter) was re-assessed after 1 year of storage. RESULTS: After 28 days of immersion, 25%-CLP exhibited a higher and sustained CPC release unlike 10%-CLP. Residual bacteria in root dentinal tubules were eradicated by obturation with 25%-CLP-containing sealers. The incorporation of 25%-CLP (∼21µm) had no adverse effects on the sealing ability and physical properties of the sealer and resulted in long-term antibacterial activity. SIGNIFICANCE: The incorporation of CPC-loaded particles in HEMA resins yielded endodontic sealers with long-term bactericidal activity against E. faecalis in root canals. These sealers can potentially be used to prevent recurrent apical periodontitis.

Recombinant human Erythropoietin enhanced the cytotoxic effects of tamoxifen toward the spheroid MCF-7 breast cancer cells.

Erythropoietin (EPO) is widely used to treat anemia in patients undergoing chemotherapy for cancers. The main objective of this study was to investigate the effect of rHuEPO on the response of spheroid breast cancer, MCF-7, cells to tamoxifen treatment. The MCF-7 spheroids were treated with 10 mg/mL tamoxifen in combination with either 0, 10, 100 or 200 IU/mL rHuEPO for 24, 48 or 72 h. The viability of the MCF-7 cells was determined using the annexin-V, cell cycle, caspases activation and acridine orange/propidium iodide staining. rHuEPO-tamoxifen combination significantly (p greater than 0.05) increased the number of spheroid MCF-7 cells entering early apoptotic phase after 12 h and late apoptotic phase after 24 h of treatment; primarily the result of the antiproliferative effect tamoxifen. Tamoxifen alone significantly (p < 0.05) increased the caspase-3 and -9 activities in the spheroid MCF-7 cells by 200 to 550% of the control. Combination rHuEPO and tamoxifen produced much lesser effect on the caspase-8 activity. The rHuEPO in the combination treatment had concentration-dependently caused decrease in the caspase activities. rHuEPO-tamoxifen combination markedly increased MCF-7 cells entering the SubG0/G1 phase of the cell cycle by more than 500% of the control, while decreasing those entering the G2 + M and S phases by 50%. After 72 h, the combination treatment produced greater (p < 0.05) change in the SubG0/G1 phase than tamoxifen treatment alone. Morphologically, spheroid MCF-7 cells subjected to combination rHuEPO-tamoxifen treatment showed nuclear condensation and margination, cytoplasmic blebbing, necrosis, and early and late apoptosis. Thus, the study showed that rHuEPO-tamoxifen combination induced apoptosis in the spheroid MCF-7 cells. The apoptotic effect of the rHuEPO-tamoxifen combination treatment on the MCF-7 cells was greater than that produced by tamoxifen alone. The rHuEPO-tamoxifen treatment enhanced the caspase-independent apoptotic effects of tamoxifen on the spheroid MCF-7 cells.

FGF-2 release and bonding/physical properties of 4-META/MMA-based adhesive resins incorporating small FGF-2-loaded polymer particles.

OBJECTIVES: Non-biodegradable particles comprising hydroxyethyl methacrylate (HEMA) and trimethylolpropane trimethacrylate (TMPT) have been reported as useful carriers for fibroblast growth factor-2 (FGF-2). They have also been successfully incorporated into the 4-[2-(methacryloyloxy)ethoxycarbonyl]phthalic anhydride/methyl methacrylate-tri-n-butyl borane (4-META/MMA-TBB) resin to promote tissue regeneration. However, smaller particles are required to obtain restorative materials acceptable for clinical use. The aim of this study was to investigate the ability of the 4-META/MMA-TBB resin that comprises small FGF-2-loaded particles to release FGF-2 and promote cell proliferation. In addition, the bonding and physical properties of the experimental resin were evaluated. METHODS: The small particles loaded with FGF-2 were newly fabricated and incorporated into the commercial 4-META/MMA-TBB resin. Release profiles of FGF-2 from the experimental resins were assessed, and the cell proliferation cultured with the eluate was evaluated. The bonding and physical properties of the resins were evaluated using shear bond strength and three-point bending tests, and by measuring the curing time, water absorption, and water dissolution. RESULTS: Sustained release of FGF-2 from the experimental resins for two weeks was observed, and the released FGF-2 was demonstrated to promote cell proliferation. All bonding and physical properties of the 4-META/MMA-TBB resins were found acceptable for clinical use. SIGNIFICANCE: The small FGF-2-loaded particles incorporated into the 4-META/MMA-TBB resin had the same abilities to release FGF-2 and proliferate cells, as those exhibited by the conventionally sized particles. In addition, there were no adverse influences on bonding and physical properties, suggesting that the bioactive adhesive resin was acceptable for clinical use.

Biodegradable and Drug-Eluting Inorganic Composites Based on Mesoporous Zinc Oxide for Urinary Stent Applications.

Conventional technologies for ureteral stent fabrication suffer from major inconveniences such as the development of encrustations and bacteria biofilm formation. These drawbacks typically lead to the failure of the device, significant patient discomfort and an additional surgery to remove and replace the stent in the worst cases. This work focuses on the preparation of a new nanocomposite material able to show drug elution properties, biodegradation and eventually potential antibacterial activity. Poly(2-hydroxyethyl methacrylate) or the crosslinked poly(2-hydroxyethyl methacrylate)-co-poly(acrylic acid) hydrogels were prepared by the radical polymerization method and combined with a biodegradable and antibacterial filling agent, i.e., flower-like Zinc Oxide (ZnO) micropowders obtained via the hydrothermal route. The physico-chemical analyses revealed the correct incorporation of ZnO within the hydrogel matrix and its highly mesoporous structure and surface area, ideal for drug incorporation. Two different anti-inflammatory drugs (Ibuprofen and Diclofenac) were loaded within each composite and the release profile was monitored up to two weeks in artificial urine (AU) and even at different pH values in AU to simulate pathological conditions. The addition of mesoporous ZnO micropowders to the hydrogel did not negatively affect the drug loading properties of the hydrogel and it was successfully allowed to mitigate undesirable burst-release effects. Furthermore, the sustained release of the drugs over time was observed at neutral pH, with kinetic constants (k) as low as 0.05 h-1. By exploiting the pH-tunable swelling properties of the hydrogel, an even more sustained release was achieved in acidic and alkaline conditions especially at short release times, with a further reduction of burst effects (k ≈ 0.01-0.02 h-1). The nanocomposite system herein proposed represents a new material formulation for preparing innovative drug eluting stents with intrinsic antibacterial properties.

Spermine synthesis inhibitor blocks 25-hydroxycholesterol-induced- apoptosis via SREBP2 upregulation in DLD-1 cell spheroids.

The oxysterol 25-hydroxycholesterol (25-HC) has diverse physiological activities, including the ability to inhibit anchorage-independent growth of colorectal cancer cells. Here, we found that a polyamine synthesis inhibitor, DFMO, prevented 25-HC-induced apoptosis in non-anchored colorectal cancer DLD-1 cells. Additionally, we found that the spermine synthesis inhibitor APCHA also inhibited 25-HC-induced apoptosis in DLD-1 spheroids. Inhibiting the maturation of SREBP2, a critical regulator of cholesterol synthesis, reversed the effects of APCHA. SREBP2 knockdown also abolished the ability of APCHA to counteract 25-HC activity. Furthermore, APCHA induced SREBP2 maturation and upregulated its transcriptional activity, indicating that altered polyamine metabolism can increase SREBP2 activity and block 25-HC-induced apoptosis in spheroids. These results suggest that crosstalk between polyamine metabolism and cholesterol synthetic pathways via SREBP2 governs the proliferative and malignant properties of colorectal cancer cells.

Formation of Stem Cell Aggregates and Their Differentiation on Surface-Patterned Hydrogels Based on Poly(2-hydroxyethyl Methacrylate).

Micropillar patterns were fabricated and used to study cell adhesion, morphology, and function. Micropillars were produced in poly(2-hydroxyethyl methacrylate (HEMA)/N,N-(dimethylaminoethyl)methacrylate (DMAEMA)/tetraethylene glycol dimethacrylate (TEGDMA)) hydrogels using soft lithography, had dimensions of 1 µm diameter, and were either 2.05 or 4.91 µm tall. The patterned hydrogel substrates increased adhesion and induced the formation of cellular aggregates. Digital micrographs were used to quantify aggregate size and number. Differentiation of hMSCs toward adipocytes and chondrocytes was performed using the respective complete culture and differentiation medium for 2 weeks. Cells were stained for Oil red O, Alcian blue, and Type II collagen. Hydrogel substrates supported the differentiation of hMSCs to adipocytes and chondrocytes. The taller micropillar patterns supported the attachment and growth of larger aggregates and were more amenable to aid chondrogenic differentiation.

PEP06 polypeptide 30 is a novel cluster-dissociating agent inhibiting α v integrin/FAK/Src signaling in oral squamous cell carcinoma cells.

Collectively migrating tumor cells have been recently implicated in enhanced metastasis of epithelial malignancies. In oral squamous cell carcinoma (OSCC), αv integrin is a crucial mediator of multicellular clustering and collective movement in vitro; however, its contribution to metastatic spread remains to be addressed. According to the emerging therapeutic concept, dissociation of tumor clusters into single cells could significantly suppress metastasis-seeding ability of carcinomas. This study aimed to investigate the anti-OSCC potential of novel endostatin-derived polypeptide PEP06 as a cluster-dissociating therapeutic agent in vitro. Firstly, we found marked enrichment of αv integrin in collectively invading multicellular clusters in human OSCCs. Our study revealed that metastatic progression of OSCC was associated with augmented immunostaining of αv integrin in cancerous lesions. Following PEP06 treatment, cell clustering on fibronectin, migration, multicellular aggregation, anchorage-independent survival and colony formation of OSCC were significantly inhibited. Moreover, PEP06 suppressed αv integrin/FAK/Src signaling in OSCC cells. PEP06-induced loss of active Src and E-cadherin from cell-cell contacts contributed to diminished collective migration of OSCC in vitro. Overall, these results suggest that PEP06 polypeptide 30 inhibiting αv integrin/FAK/Src signaling and disrupting E-cadherin-based intercellular junctions possesses anti-metastatic potential in OSCC by acting as a cluster-dissociating therapeutic agent.

Three-Dimensional Spheroid Culture Increases Exosome Secretion from Mesenchymal Stem Cells.

BACKGROUND: Mass production of exosomes is a prerequisite for their commercial utilization. This study investigated whether three-dimensional (3D) spheroid culture of mesenchymal stem cells (MSCs) could improve the production efficiency of exosomes and if so, what was the mechanism involved. METHODS: We adopted two models of 3D spheroid culture using the hanging-drop (3D-HD) and poly(2-hydroxyethyl methacrylate) (poly-HEMA) coating methods (3D-PH). The efficiency of exosome production from MSCs in the 3D spheroids was compared with that of monolayer culture in various conditions. We then investigated the mechanism of the 3D spheroid culture-induced increase in exosome production. RESULTS: The 3D-HD formed a single larger spheroid, while the 3D-PH formed multiple smaller ones. However, MSCs cultured on both types of spheroids produced significantly more exosomes than those cultured in conventional monolayer culture (2D). We then investigated the cause of the increased exosome production in terms of hypoxia within the 3D spheroids, high cell density, and non-adherent cell morphology. With increasing spheroid size, the efficiency of exosome production was the largest with the least amount of cells in both 3D-HD and 3D-PH. An increase in cell density in 2D culture (2D-H) was less efficient in exosome production than the conventional, lower cell density, 2D culture. Finally, when cells were plated at normal density on the poly-HEMA coated spheroids (3D-N-PH); they formed small aggregates of less than 10 cells and still produced more exosomes than those in the 2D culture when plated at the same density. We also found that the expression of F-actin was markedly reduced in the 3D-N-PH culture. CONCLUSION: These results suggested that 3D spheroid culture produces more exosomes than 2D culture and the non-adherent round cell morphology itself might be a causative factor. The result of the present study could provide useful information to develop an optimal process for the mass production of exosomes.

Development of a novel dental resin cement incorporating FGF-2-loaded polymer particles with the ability to promote tissue regeneration.

OBJECTIVE: Aiming to achieve bioactive dental resins that promote healing of surrounding tissues, we developed novel poly(2-hydroxyethyl methacrylate/trimethylolpropane trimethacrylate) (polyHEMA/TMPT) particles. These particles have been reported to be useful as a non-biodegradable carrier for fibroblast growth factor-2 (FGF-2) in vitro. The aim of this study was to evaluate the ability of an adhesive resin incorporating FGF-2-loaded polymer particles to promote tissue regeneration in vitro and in vivo. METHODS: Experimental adhesive resins were prepared by incorporating FGF-2-loaded polyHEMA/TMPT particles into a 4-META/MMA-based adhesive resin, and the release profiles of FGF-2 were evaluated. The proliferation of osteoblast-like cells in the eluate from cured experimental resin was assessed. When the experimental resin was implanted into rat calvaria defects, bone regeneration was evaluated by microcomputed tomography and histological observations. RESULTS: Sustained release of FGF-2 from the experimental resin was observed for 14 days. Eluate from the cured experimental resin significantly promoted the proliferation of osteoblast-like cells. Significantly greater bone regeneration was observed using the experimental resin compared with the control resin without FGF-2. SIGNIFICANCE: 4-META/MMA-based adhesive resin incorporating FGF-2-loaded polymer particles is useful to promote tissue regeneration, suggesting that its application would be beneficial for root-end filling or the repair of fractured roots in cases with severely damaged periodontal tissue.

Loading studies of the anticancer drug camptothecin into dual stimuli-sensitive nanoparticles. Stability scrutiny.

In recent years, the preparation of valuable drug delivery systems (DDS) from self-assembled amphiphilic copolymers has attracted much attention since these nanomaterials provide new opportunities to solve problems such as the lack of solubility in water of lipophilic drugs, improve their bioavailability, prolong their circulation time and decrease the side effects associated with their administration. In the current study two types of biocompatible pH-responsive nanoparticles derived from poly(2-hydroxyethyl methacrylate) (pHEMA) have been used as drug nano-carriers, being one of them core cross-linked to circumvent their instability upon dilution in human fluids. The present paper deals with the optimization of the loading process of the labile, hydrophobic and highly active anticancer drug, Camptothecin (CPT) into the nanoparticles with regard to four independent variables: CPT/polymer ratio, sonication, temperature and loading time. Forty experiments were carried out and a Box-Behnken experimental design was used to evaluate the significance of the independent variables related to encapsulation efficiency and drug retention capacity. The enhanced drug loading and encapsulation efficiency values (58% and >92%, respectively) of CPT were achieved by the core cross-linked NPs in 2 h at 32 °C at CPT/polymer ratio 1.5:1 w/w and 14 min of sonication. The optimized CPT-loaded NPs were studied by dynamic light scattering and scanning electron microscopy, and an increase in size of the loaded-NP compared to the unloaded counterparts was found. Other twenty experiments were conducted to study the enability to retain CPT into the conjugates at different ionic strength values and times. The stability studies demonstrated that the core cross-linked nanocarriers displayed an excellent drug retention capacity (>90%) at 25 °C for 15 days in every ionic-strength environments whereas the non-cross-linked ones were more stable at physiological ionic strength. The optimized systems proved to be a major step forward to encapsulate and retain CPT in the NP nuclei, what makes them ideal devices to control the delivery of CPT upon the triggered acidic conditions of solid tumors.

A poly(2-hydroxyethyl methacrylate)-based resin improves the dentin remineralizing ability of calcium silicates.

Bioactive polymeric composites have received great attention for their capability to remineralize the dentin tissue. This study was aimed at evaluating if a poly(HEMA-co-TEGDMA) resin (HEMA: 2-hydroxyethyl methacrylate; TEGDMA: triethyleneglycol dimethacrylate) may increase the in vitro apatite forming ability of a calcium silicate cement (CaSi), in view of developing a hydrophilic light-curable composite bio-remineralizing restorative material (R-CaSi). To this purpose, the following experiments were carried out: (1) In vitro apatite forming ability of R-CaSi and CaSi was comparatively assessed by micro-Raman spectroscopy after immersion of the cement disks in Dulbecco's Phosphate Buffered Saline (DPBS) at 37°C for 1-28days; (2) Previously demineralized human dentin slices were soaked for 7days in close contact with the CaSi and R-CaSi cements as well as poly(HEMA), poly(TEGDMA) and poly(HEMA-co-TEGDMA), and then were comparatively analyzed by IR spectroscopy. Micro-Raman spectroscopy showed that in calcium phosphate nucleation tests, the B-type carbonated apatite deposit formed on R-CaSi was thicker than that on CaSi; therefore, the poly(HEMA-co-TEGDMA) resin proved able to increase the in vitro apatite forming ability of the calcium silicate-based cement. Both cements were found to induce dentin remineralization, R-CaSi to a higher extent, in agreement with the calcium phosphate nucleation tests. This result may be ascribed to the positive role played by the polymeric component, which was found to interact with collagen and to chelate calcium ions. Upon remineralization, collagen underwent conformational rearrangements and the formed apatite phase, rather than a simple deposit, was intimately bound to the collagen matrix, thanks to the calcium ions chelated by it.

Preparation of three-dimensional macroporous chitosan-gelatin B microspheres and HepG2-cell culture.

Chitosan-gelatin B microspheres with an open, interconnected, highly macroporous (100-200 µm) structure were prepared via a three-step protocol combining freeze-drying with an electrostatic and ionic cross-linking method. Saturated tripolyphosphate ethanol solution (85% ethanol) was chosen as the crosslinking agent to prevent destruction of the porous structure and to improve the biostability of the chitosan-gelatin B microspheres, with N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide/N-hydroxysuccinimide as a second crosslinking agent to react with gelatin A and fixed chitosan-gelatin B microspheres to attain improved biocompatibility. Water absorption of the three-dimensional macroporous chitosan-gelatin B microspheres (3D-P-CGMs) was 12.84, with a porosity of 85.45%. In vitro lysozyme degradation after 1, 3, 5, 7, 10, 14, and 21 days showed improved biodegradation in the 3D-P-CGMs. The morphology of human hepatoma cell lines (HepG2 cells) cultured on the 3D-P-CGMs was spherical, unlike that of cells cultured under traditional two-dimensional conditions. Scanning electron microscopy and paraffin sections were used to confirm the porous structure of the 3D-P-CGMs. HepG2 cells were able to migrate inside through the pore. Cell proliferation and levels of albumin and lactate dehydrogenase suggested that the 3D-P-CGMs could provide a larger specific surface area and an appropriate microenvironment for cell growth and survival. Hence, the 3D-P-CGMs are eminently suitable as macroporous scaffolds for cell cultures in tissue engineering and cell carrier studies. Copyright © 2014 John Wiley & Sons, Ltd.

Fibroblast adhesion on ECM-derived peptide modified poly(2-hydroxyethyl methacrylate) brushes: ligand co-presentation and 3D-localization.

Polymer brushes prepared via surface-initiated polymerization of 2-hydroxyethyl methacrylate are powerful platforms for the fabrication of model biointerfaces to study cell-substrate interactions. In this manuscript, the versatility of surface-initiated polymerization and the poly(2-hydroxyethyl methacrylate) (PHEMA) polymer brush platform are used to address two fundamental questions, viz. the effects of ligand co-presentation and of the 3D localization of biochemical cues on cell behavior. Using a series of PHEMA brushes that present RGD and PHSRN ligands in various relative surface concentrations, the present study unequivocally demonstrates that: (i) co-presentation of PHSRN cues on an RGD functionalized substrate enhances cell adhesion and (ii) this synergetic effect is highest when the two ligands are presented at equal surface concentrations. In the second part of this study, adhesion of 3T3 fibroblasts on a series of PHEMA brushes that present the RGD ligand at a distance of 12, 23 or 42 nm away from the cell substrate interface is investigated. While cells were found to adhere to surfaces that presented the cell adhesive peptides at distances up to 23 nm from the interface, polymer brushes that contained the RGD ligands 42 nm away from the interface did not support cell adhesion.