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Smart Nanoparticles Based on Hyaluronic Acid for Redox-Responsive and CD44 Receptor-Mediated Targeting of Tumor
- Job Stanley Brown
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1 Park et al. Nanoscale Research Letters (2015) 10:288 DOI /s NANO EXPRESS Open Access Smart Nanoparticles Based on Hyaluronic Acid for Redox-Responsive and CD44 Receptor-Mediated Targeting of Tumor Hyung-Kyu Park 1, Sang Joon Lee 2, Jong-Suk Oh 1, Sam-Gyu Lee 3, Young-IL Jeong 4 and Hyun Chul Lee 1* Abstract Background: Since aggressive cancer cells highly express the CD44 receptor compared to normal cells, hyaluronic acid (HA) can be used for CD44 targeting molecule. Since glutathione (GSH) level is normally elevated in the intracellular compartment and in the tumor cell, the fact that disulfide bond can be cleaved by GSH is widely used for intracellular drug delivery. Methods: HA was connected with poly(dl-lactide-co-glycolide) (PLGA) using disulfide linkage, and then a diblock copolymer (HAssLG) was prepared. Doxorubicin (DOX)-loaded HAssLG nanoparticles were prepared by dialysis procedures. Results and Discussion: DOX-loaded HAssLG nanoparticles have spherical shapes with small particle size of less than 300 nm. In fluorescence measurement, DOX was dose-dependently liberated from nanoparticles by the addition of GSH. DOX release rate from HAssLG nanoparticles was increased by the addition of GSH. To confirm CD44 receptor-mediated endocytosis of nanoparticles, CD44-positive MDA-MB231 cells were employed and fluorescence intensity was strong when nanoparticles were treated to tumor cells. However, fluorescence intensity was significantly decreased through blocking of the CD44 receptor by pretreatment of cells with free HA. Fluorescence intensity of cells was increased again when GSH was added, indicating that HAssLG nanoparticles have CD44 receptor targetability and potential of redox-responsive drug delivery. For animal imaging study, CD44-positive MDA-MB231 cells and CD44-negative NIH3T3 cells were simultaneously implanted into the right flank and left flank of mice, respectively. Fluorescence intensity was significantly stronger at tumor mass of MDA-MB231 cells than solid mass of NIH3T3 cells, indicating that HAssLG nanoparticles were specifically delivered to tumor cells. Conclusions: The results indicated that HAssLG nanoparticles have specificity against the CD44 receptor and can be used for anticancer drug targeting. We recommend HAssLG nanoparticles as a promising vehicle for cancer drug targeting. Keywords: Hyaluronic acid; CD44 receptor-mediated endocytosis; Redox-responsive; Block copolymer; Nanoparticles * Correspondence: 1 Department of Microbiology, Chonnam National University Medical School, 160, Baekseo-ro, Dong-gu, Gwangju , Republic of Korea Full list of author information is available at the end of the article 2015 Park et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.
2 Park et al. Nanoscale Research Letters (2015) 10:288 Page 2 of 10 Background Nanomedicines based on nanotechnology have been extensively investigated for drug delivery issues, and they can be considered as appropriate vehicles for tumor targeting due to their small sizes [1 4]. Especially, nanosized vehicles such as nanoparticles or polymeric micelles have superior potentials for solid tumor targeting because the enhanced permeation and retention (EPR) effect against tumor blood vessels is regarded as a preferential advantage of nanovehicles for solid tumor targeting [5, 6]. Furthermore, nanoparticles can be decorated with various targeting moieties for specific targeting of cancer cells since they have a big surface area [7 10]. Due to these intrinsic properties, nanoparticles can concentrate cytotoxic anticancer drug to the solid tumor with minimal unwanted side effects for normal tissues/cells. From these points of view, nanoparticles decorated with targeting moieties have been also extensively investigated [7 10]. Aggressive tumor cells frequently overexpress the CD44 receptor in the cell membrane [11 18]. This is a specific receptor for hyaluronic acid (HA), and HA is known to stimulate spreading, migration, invasion, and metastasis of cancer cells [11 14, 17]. From these reasons, hyaluronic acid has been extensively used as a vehicle for specific targeting of the CD44 receptor of tumor cells [8, 18 22]. Especially, Prestwich s group extensively investigated the potential of CD44 receptor delivery of HA-anticancer agent conjugates against cancer cells [19 22]. Furthermore, HA was also investigated for tissue engineering, gene delivery, and peptide delivery due to the biodegradability and biocompatibility [23 25]. Nanoparticles based on redox-responsive drug targeting for cancer cells also have been extensively investigated in the last decades [26 29]. Redox-responsive drug delivery is based on the fact that disulfide bond can be reduced by glutathione and intracellular glutathione (GSH) level is significantly higher than the extracellular fluid level [30]. This peculiar property between GSH and disulfide bond stimulated the redox-responsive drug delivery concept. For example, Sun et al. synthesized a poly(ethylene glycol)/poly (ε-caprolactone) block copolymer using disulfide bond [27]. They showed that drug release rate was increased by the addition of dithiothreitol (DTT) and enhanced tumor cell uptake of anticancer drugs was observed. Furthermore, redox-responsive delivery of DNA drugs using disulfidelinked polymer nanovehicles was also investigated by several authors [26 29]. In spite of successful development of the redox-responsive drug delivery system, GSH levels in normal tissues and low specificity of nanoparticles are still obstacles for efficient drug targeting. For example, GSH levels can be increased in the tissues of a tumor-bearing mice compared to a normal mice [30]. Therefore, multiple targeting strategies are required to attain efficient targeting of tumors. In this study, we synthesized a HA-b-PLGA (HAssLG) copolymer having disulfide linkage between HA and PLGA for targeting of tumor cells through the CD44 receptor and redox-responsive process. Since PLGA is a biodegradable and lipophilic polymer, PLGA must form the core of the nanoparticles while HA would form the outer shell of the nanoparticles. We studied the potential of CD44 receptor-mediated and redox-responsive targeting of tumor cells by HAssLG nanoparticles using tumor cells and fibroblast cells in vitro and in vivo. Methods Materials HA (molecular weight from the manufacturer s data, 7460 g/mol) was purchased from Lifecore Biomedical (Chaska, MN, USA). Triethylamine (TEA), L-glutathione (GSH), sodium cyanoborohydride, N-(3-dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride, and cystamine hydrochloride were purchased from Sigma Chem. Co. (St. Louis, USA). N,N -dicyclohexyl carbodiimide (DCC) and N-hydroxysuccimide (NHS) were purchased from Sigma- Aldrich Chemical Co, USA. The dialysis membranes (Spectra/Pro membranes) with molecular weight cutoffs (MWCO) of 2000 and 12,000 g/mol were purchased from Spectrum Labs. Doxorubicin (DOX) HCl was purchased from LC Labs Co. Ltd. (Woburn, MA, USA). Dichloromethane (DCM) and dimethyl sulfoxide (DMSO) were of HPLC grade or extra-pure grade. PLGA (PLGA-5005, MW = 5000 g/mol) was purchased from Wako Pure Chemicals Co. (Osaka, Japan). The molecular weight (MW) of PLGA was measured by GPC, as described previously [8]. The weight average MW, number average MW, and polydispersity of PLGA were 4920, 4780, and 1.029, respectively. Synthesis of HAssLG Block Copolymer HA having disulfide linkage in the end of the chain was synthesized as described by Maruyama et al. [25] with brief modification. HA (400 mg) was dissolved in 10 ml of H 2 O/DMSO (3/7, v/v) solution, and then excess amount of sodium cyanoborohydride was added to this solution followed by magnetic stirring for 12 h at room temperature. After that, 10 equivalents of cystamine were added and the mixtures were further stirred for 24 h. The resulting solution was put into a dialysis membrane (MWCO, 2000 g/mol), and by-products were removed by dialysis procedure for 2 days followed by lyophilization for 3days. NHS-activated PLGA (PLGA-NHS) was separately prepared. Five hundred milligrams of PLGA was dissolved in DCM with 1.2 equivalents of DCC and NHS. This solution was stirred for 12 h, and then reactants were precipitated into the excess amount of methanol followed by filtration under reduced pressure. The white solid was washed with methanol three times followed by vacuum drying for a day.
3 Park et al. Nanoscale Research Letters (2015) 10:288 Page 3 of 10 The HAssLG block copolymer was synthesized as follows: 400 mg HA-cystamine conjugates and 250 mg of PLGA-NHS were simultaneously dissolved in dry DMSO and stirred in nitrogen atmosphere for 2 days. After that, this solution was then introduced into a dialysis membrane (MWCO, 12,000 g/mol) to remove the unreacted HA and organic solvent. During the dialysis procedure, water was exchanged at 3-h intervals for 2 days and then the resulting solution was lyophilized for 3 days. The lyophilized solid was precipitated into DCM to remove unreacted PLGA for three times, and then filtered precipitates were dried under a vacuum for 3 days. The yield of the final product was 81 % (w/w). Yield of product (%, w/w) = [(weight of final product)/(weight of aminated HA + PLGA-NHS)] H Nuclear Magnetic Resonance Spectroscopy 1 H nuclear magnetic resonance (NMR) spectroscopy (500 MHz superconducting FT-NMR spectrometer, Unity-Inova 500, Varian Inc., Agilent Tech., CA, USA) was employed to confirm synthesis of polymers. Preparation of HAssLG Nanoparticles Empty or DOX-incorporated nanoparticles were fabricated as follows (Table 1): 50 mg of HAssLG block copolymer was dispersed in 1 ml of deionized water and then mixed with 3 ml DMSO. DOX HCl was separately dissolved in 1 ml DMSO with trace amounts of TEA, and then this solution was added to the polymer/dmso solution. Then, the mixed solution was dialyzed against deionized water using a dialysis membrane (2000 g/mol) with exchange of water (2-h intervals) for 1 day. After that, the resulting solution was analyzed or lyophilized. Empty nanoparticles were fabricated except DOX. To measure DOX contents in the HAssLG nanoparticles, 5 mg of DOX-loaded nanoparticles was dissolved in 10 ml of DMSO/water mixtures (8/2, v/v) and then appropriately diluted with DMSO. The DOX contents in the nanoparticles were evaluated with a UV spectrophotometer (UV spectrophotometer 1601, Shimadzu Co., Japan) at 486 nm. To avoid interference of the polymer, empty HAssLG nanoparticles were used as blank. Drug contents were calculated with the following equation: [(drug weight in the nanoparticle)/(total weight of the nanoparticle)] 100. Table 1 Characterization of HAssLG nanoparticles Polymer/ drug (mg/mg) Drug contents (%, w/w) Theoretical Experimental 50/0 50/ / Analysis of Nanoparticles Transmission electron microscopy (TEM, JEOL JEM-2000 FX II, Japan) was used to observe the morphology of nanoparticles. One drop of nanoparticle solution (0.1 mg/ml) was dropped onto a carbon film coated on a copper grid and then dried in room temperature. Three hours later, phosphotungstic acid (0.05 %, w/w) was then dropped onto nanoparticles for negative staining and dried in room temperature for more than 6 h. Observation of nanoparticles was performed at 80 kv. Particle size was measured with dynamic laser scattering (DLS-7000, Otsuka Electronics Co., Japan) (nanoparticle concentration, 0.1~1 mg/ml). Drug Release Study DOX release from HAssLG nanoparticles was performed as follows: 10 mg of DOX-incorporated nanoparticles was dispersed in 5 ml of phosphate-buffered saline (PBS, 0.1 M, ph 7.4), and this solution was put into a dialysis membrane (MWCO, 12,000 g/mol). After that, the dialysis membrane was introduced into 95 ml PBS in a bottle. Drug release was performed in a shaking incubator with 100 rpm of stirring speed at 37 C. Whole media were replaced with fresh media at specific time intervals. The released DOX was measured with a UV spectrophotometer (UV spectrophotometer 1601, Shimadzu Co., Japan) at 486 nm. Cell Culture MDA-MB231 human breast carcinoma cells and NIH3T3 mouse fibroblast cells were obtained from Korea Cell Line Bank Co. (KCLB, Seoul, Korea). MDA-MB231 human breast carcinoma cells and NIH3T3 mouse fibroblast cells were maintained in RPMI1640 and DMEM supplemented with 10 % fetal bovine serum (5 % CO 2 at 37 C), respectively. Cell viability test was performed as follows: MDA-MB231 ( cell/well) cells were seeded into 96-well plates and then incubated overnight (5 % CO 2 at 37 C). DOX or nanoparticles were treated to these cells. The CD44 receptor of the cells was blocked with 10 equivalents of free HA 1 h before nanoparticle treatment, and then the cells were washed with PBS. After that, DOX or nanoparticles were added to the cells in the 96-well plates and incubated in a CO 2 incubator (5 % CO 2 at37 C)for4or24h.Afterthat, media were discarded and then replaced with 100 μl of fresh media. To this solution, 30 μl of MTT (5 mg/ml) was added, and then the solution was further incubated in a CO 2 incubator (5 % CO 2 at 37 C) for 4 h. Since the contents of the generated formazan were proportional to the number of viable cells, the generated formazan was solubilized with DMSO and determined with an automated computer-linked microplate reader (Molecular Device Co., USA) (560 nm test/630 nm reference).
4 Park et al. Nanoscale Research Letters (2015) 10:288 Page 4 of 10 Receptor-Mediated Delivery of Nanoparticles to Tumor Cells Cellular uptake of DOX-loaded HAssLG nanoparticles was studied with fluorescence microscopy observation and flow cytometry analysis. MDA-MB231 cells were seeded onto a cover glass and incubated overnight in a CO 2 incubator (5 % CO 2 at 37 C). Then, nanoparticles were treated to cells for 1 h in a CO 2 incubator. One hour before the addition of nanoparticles, the CD44 receptor was blocked by pretreatment of free HA (10 equivalents of nanoparticle contents). Following this, cells were washed with PBS (ph 7.4, 0.1 M) and fixed with 4 % paraformaldehyde. This was fixed with immobilization solution (IMMU-MOUNT, Thermo Electron Corporation, Pittsburgh, PA, USA). Cells were then observed with a confocal laser scanning microscope (CLSM, TCS-SP2; Leica, Wetzlar, Germany). For flow cytometry analysis of cells, cells were seeded in each well of six-well plates. Nanoparticles were treated to the cells and incubated for 1 h in a CO 2 incubator. One hour before the addition of nanoparticles, the CD44 receptor was blocked by pretreatment of free HA (10 equivalents of nanoparticle contents). Cells were washed with PBS (ph 7.4, 0.1 M) and trypsinized to harvest cells. The cells were analyzed with flow cytometery (FACScan) at an excitation wavelength of 488 nm and emission wavelength of 522 nm for DOX fluorescence intensity. Near-Infrared Absorption Dye Conjugation A near-infrared (NIR) dye was conjugated with HAssLG for in vivo animal imaging studies. Fifty milligrams of HAssLG in 5 ml DMSO/water mixtures (4/1, v/v) was mixed with 3 mg of sulfo-cyanine7 amine (Lumiprobe Co., Florida, USA) with 1.2 equivalent moles of EDAC and NHS. This solution was then stirred for 24 h in room temperature. After that, the solution was dialyzed to remove unreacted dye with exchange of water at intervals of 2 h until the dye was not detected in the water. The detection of the dye and the contents in the polymer were measured with a UV spectrophotometer (UV-1601 UV-VIS spectrophotometer, Shimadzu, Kyoto, Japan) at 750 nm. The content of the NIR dye was 2.8 %, w/w. (Dye content (%, w/w) = (dye amount in the nanoparticles/total weight of the nanoparticles) 100). In Vivo Fluorescence Imaging For fluorescence imaging of the biodistribution of nanoparticles, MDA-MB231 cells and NIH3T3 cells were implanted into the right flank and left flank of nude BALb/C mice (5 weeks, 20 g), respectively. After 20 days, NIR-dye-conjugated nanoparticles were administered intravenously (i.v.) into the tail vein of mice at a dose of 20 mg/kg (injection volume was 100 μl). Mice were observed with a Maestro 2 small-animal imaging instrument (Cambridge Research and Instruments, Inc., Woburn, MA, USA). Twenty-four hours later, mice were sacrificed to evaluate organ distribution of nanoparticles with the Maestro 2 small-animal imaging instrument. All animal studies were performed under the guidelines of the Committee of Care and Use of Laboratory Animals of Chonnam National University. Results Characterization of HAssLG Block Copolymer HA and PLGA were linked with cystamine linkage, and the block copolymer was synthesized as shown in Fig. 1. Prior to PLGA conjugation, cystamine was conjugated in the end of the HA chain by treatment with sodium cyanoborohydride because polysaccharides have one reductive end. To this, excess amount of cystamine was added to make aminated end of HA having disulfide linkage. Intrinsic peaks of HA were confirmed at 1.6~4.6 ppm at 1 H NMR spectra. The ethylene proton of cystamine linkage was observed at 2.9 ppm as shown in Fig. 1. To the terminal amine of HA, PLGA was conjugated to construct the block copolymer having disulfide linkage. The specific proton peaks of PLGA were observed at 1.2~1.6 ppm and 4.6~5.2 ppm. The final yield of the HAssLG block copolymer was approximately 81 % (w/w). Characterization of Nanoparticles Generally, diblock copolymers composed of a hydrophilic domain and hydrophobic domain can form coreshell-type nanoparticles in the aqueous environment. Since HA is the hydrophilic one, it would form the outer shell of the nanoparticles while PLGA would form the core of the nanoparticles. Nanoparticles of the HAssLG block copolymer were fabricated in water by dialysis procedure. The morphology of empty HAssLG nanoparticles was a spherical shape as shown in Fig. 2a, and their sizes were less than 200 nm. Particle sizes were increased when DOX was incorporated into the nanoparticles as shown in Fig. 2b, d. DOX-loaded nanoparticles also have spherical shapes (Fig. 2b) and unimodal size distribution (Fig. 2c). To study redox-response properties of nanoparticles, DOX-loaded nanoparticles were incubated at 37 C with and without GSH as shown in Fig. 3a. DOX-loaded nanoparticles were treated with GSH for 4 h, and then fluorescence intensity of DOX was measured using a microplate reader and observed with imaging equipment as shown in Fig. 3a. As shown in Fig. 3a, fluorescence intensity rapidly increased according to the increase of GSH concentration. Furthermore, fluorescence emission spectra of DOXloaded nanoparticles were recorded at 520~700 nm as shown in Fig. 3b. Fluorescence intensity was gradually increased according to the GSH concentration, indicating that DOX-incorporated HAssLG nanoparticles responded
5 Park et al. Nanoscale Research Letters (2015) 10:288 Page 5 of 10 Fig. 1 Synthesis scheme of HAssLG block copolymer to GSH addition and then liberated DOX was controlled by GSH content. Furthermore, DOX release rate was increased when GSH was added to release media as shown in Fig. 3c. These results indicated that HAssLG nanoparticles have redox-responsive characters and delivery of anticancer drugs would be prompted according to the concentration of GSH. CD44 Receptor-Mediated Targeting of HAssLG Nanoparticles To investigate CD44-mediated delivery of HAssLG nanoparticles, CD44 receptor-overexpressed MDA-MB231 cells were employed. Since DOX itself expresses strong fluorescence intensity, DOX-loaded HAssLG nanoparticles were treated to MDA-MB231 cells as shown in Fig. 4a. As shown in Fig. 4a, red fluorescence of MDA-MB231 cells was strongly expressed when DOX-loaded nanoparticles were treated. However, fluorescence intensity was significantly decreased when the CD44 receptor was blocked by pretreatment with free HA as shown in Fig. 3a, indicating that HAssLG nanoparticles can target tumor cells through receptor-mediated endocytosis using the CD44 receptor. However, fluorescence intensity was slightly increased when GSH was added to the cell culture even though the CD44 receptor was blocked. These results indicate that HAssLG nanoparticles are responsive to both the CD44 receptor and redox environment. Furthermore, flow cytometry analysis of cells also supported these results, indicating that treatment of DOX-loaded HAssLG nanoparticles significantly increased fluorescence intensity
6 Park et al. Nanoscale Research Letters (2015) 10:288 Page 6 of 10 Fig. 2 TEM photographs (a, b), particle size (c, d). TEM photographs of a empty HAssLG nanoparticles and b DOX-incorporated HAssLG nanoparticles (DOX contents, 7.8 % (w/w)). c Particle size distribution of DOX-incorporated HAssLG nanoparticles (DOX contents, 7.8 % (w/w)). d Particle size changes of HAssLG nanoparticles according to the increase of DOX contents Fig. 3 Fluorescence intensities (a) and fluorescence emission scan (b) of DOX-incorporated HAssLG nanoparticles according to the GSH concentration. DOX-incorporated nanoparticles were incubated 4 h at 37 C in the presence of GSH. Fluorescence emission spectra were recorded in the range of 520~700 nm (excitation wavelength, 486 nm). Nanoparticle concentration was 1 mg/ml (DOX contents, 7.8 % (w/w)). c DOX release from HAssLG nanoparticles in the presence (GSH(+), 10 mm) or absence (GSH( ), 0 mm) of GSH
7 Park et al. Nanoscale Research Letters (2015) 10:288 Page 7 of 10 Fig. 4 a Fluorescence images of MDA-MB231 cells. MDA-MB231 cells were exposed to DOX-incorporated HAssLG nanoparticles (DOX concentration, 5 μg/ml) for 1 h. Fluorescence images of cells were observed with CLSM. The CD44 receptor of MDA-MB231 cells was blocked with free HA (0.5 mg/ml) 1 h before treatment of DOX-incorporated nanoparticles. GSH (10mM) was added to study the effect of redox-responsive delivery of nanoparticles. b Flow cytometry analysis of MDA-MB231 cells as shown in Fig. 4b. Blocking of the CD44 receptor decreased fluorescence intensity of cells, and GSH addition increased fluorescence intensity of cells again. These results clearly supported Fig. 3a. Figure 5 shows the anticancer activity of nanoparticles against MDA-MB231 cells. As shown in Fig. 5, viability of MDA-MB231 cells was gradually decreased according to the increase of DOX concentration. Interestingly, HAssLG nanoparticles showed higher anticancer activity against MDA-MB231 cells, i.e., viability of cells with treatment of HAssLG nanoparticles was lower than that of DOX alone. Furthermore, cell viability with treatment of HAssLG nanoparticles was increased when free HA was pretreated as shown in Fig. 5b. These results indicated that HAssLG nanoparticles have enhanced anticancer activity with CD44 receptor targetability. In Vivo Animal Imaging Study To prove the CD44 receptor-mediated delivery capacity of HAssLG nanoparticles in vivo, CD44-positive MDA-MB231 cells and CD44-negative NIH3T3 cells were implanted into the right flank and left flank of the mice, respectively. NIRdye-conjugated HAssLG nanoparticles were administered intravenously via the tail vein of mice, and distribution of nanoparticles in mice was observed with NIR imaging equipment as shown in Fig. 6. As shown in Fig. 6, fluorescence intensity of the MDA-MB231 tumor mass was significantly higher than that of the solid mass of NIH3T3. And then, these differences were increased 24 h after nanoparticle administration. As shown in Fig. 6b, fluorescence observation of each organ showed that the solid tumor of MDAMB231 cells expressed stronger fluorescence intensity than that of NIH3T3 cells even though the liver also expressed strong fluorescence intensity. These results indicated that HAssLG nanoparticles have drug targeting potential against solid tumor via a specific pathway. Discussion HA is a linear polysaccharide composed of D-glucuronic acid/n-acetyl-d-glucosamine units and has a vital role in the physiological phenomenon of cells such as adhesion, growth, migration, cell motility, inflammation, and invasion [31 36]. Especially, specific receptors for HA such as CD44 and RHAMM are frequently overexpressed in cancer cells rather than normal cells [11 18]. In addition to these findings, the fact that HA has biocompatibility, biodegradability, and functional moiety such as carboxylic acid stimulates application of HA in the biomedical field and drug targeting research [8, 19 25]. Hyung et al. reported that HAdecorated nanoparticles on the surface effectively targeted the CD44 receptor of CD44-overexpressed cells rather than lower CD44-expressed cells, while pure PLGA nanoparticles did not show targeting differences in both cells [7]. Since invasive cancer cells generally plentifully secreted hyaluronidase, anticancer agent release and delivery can be stimulated at invasive cancer cells and then specifically attack these cells through accelerated drug release by hyaluronidase [8]. Meanwhile, redox-responsive drug delivery has been also widely investigated for targeted drug delivery to tumor [26, 28, 29, 37]. Disulfide bond, which is abundant in the peptide and protein, can be cleaved by an intracellular reducing molecule, GSH, while it is stable at the extracellular environment [30]. Furthermore, the GSH level in the intracellular compartment (~10 mm) is known to be elevated compared to that in the extracellular environment (~10 μm). Especially, GSH level is elevated in the tumor cells [38]. These findings facilitated intracellular
8 Park et al. Nanoscale Research Letters (2015) 10:288 Page 8 of 10 Fig. 5 a Cytotoxicity of free DOX or DOX-incorporated HAssLG nanoparticles against MDA-MB231 cells. DOX or DOX-incorporated nanoparticles were exposed to cells 24h. b. Cytotoxicity of DOX-incorporated nanoparticles in the presence (+) or absence (-) of free HA against MDA-MB231 cells (*p < 0.05). MDA-MB231 cells were pretreated with free HA 1 h before drug treatment, and then cells were exposed to 50 μg/ml of DOX or nanoparticles for 4 h. Twenty-four hours later, viable cells were determined using the MTT assay drug targeting strategy by the introduction of disulfide bond in the polymer chain [27, 37]. Sun et al. synthesized a diblock copolymer having a disulfide bond between dextran and poly(ε-caprolactone), and then drug release rate was increased through cleavage of disulfide bond by GSH [37]. These phenomena enabled nanovehicles to target the intracellular compartment. Our concept is to attain sequential targeting of tumor cells as shown in Fig. 7, i.e., nanoparticles are accumulated in the tumor tissue by the EPR effect and then they target tumor cells by specific interaction with the CD44 receptor on the tumor cell membrane followed by receptor-mediated endocytosis of them. Then, GSH in the cytosol cleaved the HA outer shell of HAssLG nanoparticles, and DOX release would be facilitated in the intracellular environment. This hypothesis was proved by fluorescence intensity measurement of DOX-loaded HAssLG nanoparticles in the presence of GSH as shown in Fig. 3a, b. Fluorescence intensity of DOX was dosedependently increased by GSH, indicating that GSH cleaved the HA outer shell of the nanoparticles and then drug release rate was promoted as shown in Fig. 3c. Furthermore, CD44 receptor-specific endocytosis of nanoparticles was observed and, otherwise, CD44 receptor blocking is to interrupt cellular uptake of nanoparticles as shown in Fig. 4a. Furthermore, DOX delivery into the
9 Park et al. Nanoscale Research Letters (2015) 10:288 Page 9 of 10 Fig. 6 a Fluorescence imaging of MDA-MB231 cell-bearing (right flank) and NIH3T3 cell-bearing (left flank) mice. b. Organ-distribution of nanoparticles 24h later MDA-MB231 cells and NIH3T3 cells were implanted into the right flank and left flank of BALb/C nude mice (5 weeks, 20~25 g), respectively. Three weeks later, NIR-dye-conjugated HAssLG nanoparticles (10 mg/kg) were intravenously injected via the tail vein. The major organs of the mice were taken 24 h after nanoparticle administration tumor cells was increased again by the addition of GSH even though the CD44 receptor of tumor cells was blocked and nanoparticle uptake was inhibited, indicating that DOX release was facilitated by GSH addition and subsequent cleavage of the HA outer shell. CD44 receptor specificity of HAssLG nanoparticles was also proved by an in vivo animal NIR fluorescence imaging study as shown in Fig. 6. To prove the specificity of HAssLG nanoparticles, CD44positive MDA-MB231 cells and CD44-negative NIH3T3 mouse fibroblast cells were simultaneously implanted into the right flank and left flank of the mice, respectively, and then the solid tumor was prepared. As shown in Fig. 6, NIR fluorescence intensity in the back of mice was significantly higher at tumor mass of MDA-MB231 than at solid mass of NIH3T3, indicating that HAssLG nanoparticles specifically targeted MDA-MB231 cells in vivo and they would deliver the drug to tumor cells in a specific manner. Conclusions A HAssLG diblock copolymer was synthesized for application in the sequential targeting concept of nanoparticles by CD44 receptor-mediated and redox-responsive drug delivery. HAssLG nanoparticles have spherical shapes with nanosized diameter. DOX-loaded HAssLG nanoparticles were Fig. 7 Schematic illustration of tumor cell targeting using HAssLG nanoparticles
10 Park et al. Nanoscale Research Letters (2015) 10:288 Page 10 of 10 prepared by dialysis procedure. DOX release was facilitated by addition of GSH, indicating that the disulfide bond of HAssLG nanoparticles was cleaved and then DOX release rate was increased. In an in vitro cell culture study, DOX-loaded HAssLG nanoparticles were endocytosed by CD44 receptor-mediated pathway. An animal NIRF imaging study also proved that HAssLG nanoparticles were delivered to tumor tissues through a targeting mechanism. We suggest that HAssLG nanoparticles are a promising candidate for tumor targeting. Competing Interest The authors declare that they have no competing interest. Authors contributions HKP carried out fabrication of nanoparticles, drug release experiment, fluorescence study in vitro and conceived the study. SJL participated in the NIRF imaging of animals and in vivo study. JSO and SGL participated in the cell culture study, fluorescence observation of cells, and performed the statistical analysis. YIJ participated in the analysis of polymers using NMR spectra. HCL participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript. Author details 1 Department of Microbiology, Chonnam National University Medical School, 160, Baekseo-ro, Dong-gu, Gwangju , Republic of Korea. 2 Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju , Republic of Korea. 3 Department of Physical and Rehabilitation Medicine, Chonnam National University Medical School, Gwangju , Republic of Korea. 4 Biomedical Research Institute, Pusan National University Hospital, Pusan , Republic of Korea. Received: 16 March 2015 Accepted: 14 June 2015 References 1. Greish K. Enhanced permeability and retention of macromolecular drugs in solid tumors: a royal gate for targeted anticancer nanomedicines. 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Biomacromolecules. 2010;11: Balendiran GK, Dabur R, Fraser D. The role of glutathione in cancer. Cell Biochem Funct. 2004;22:
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Beautiful family home situation in Warwick, Qld. Only two hours to Brisbane, two hours to the Gold Coast and one hour to Toowoomba making Warwick the perfect location.
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4 bedrooms - 3 with built-ins, 2 with ceiling fans. Recently renovated eat-in kitchen, floor coverings, interior paintwork, new HW system, completly re-wired throughout with new electrical fittings, new water tank. House is air-conditioned plus has oil heater which is in original fireplace and could be converted to wood/gas or back to open fire. French doors lead from two bedrooms onto sleepout which would make a perfect play area/office/sewing room. Front and back sunrooms make the house cosy in any season. Original fretwork divides lounge/dining rooms with built-in glass display cabinets. Nice views over farmland from front sunroom. Downstairs is internal garden, second toilet, 8m x 6m rumpus room with brick bar and another fireplace. This could easily be converted to a flat or teenager retreat and has been used as home business. Downstairs also is a storage room and 4 car accommodation. In fact you could say, with a little work that you had two houses in one. Large covered greenhouse/BBQ area. To find out more or to arrange an inspection, call today. ABOUT WARWICK:- Warwick is situated 160 kilometres south west of Brisbane and borders on fertile farmlands and the rolling hills of the Great Dividing Range. The town is strategically located at the junction of major highways from Melbourne, Sydney and Brisbane. Warwick has excellent shopping facilities and the Indoor Recreation and Aquatic Centre provides a first class gymnasium with swimming pools which are heated during winter allowing for use all year round. Other attractions include the Historical Museum, Regional Art Gallery, and an 18 hole golf course. Leslie Dam is located 13 kilometres west of Warwick and provides for fishing, swimming, and boating, and also has picnic areas for day visitors and camping areas for longer stays.
Warwick QLD
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text:
DEADLY ADDICTION is the second book in my Deadly Vices series. It's the story of a half-white native man who teams up with a white policewoman to restore policing to his native community. Here's a short description:
Rife with crime and drug abuse, the Blackriver Reservation is a powder keg ready to blow. Fed up with white interference, a radical sovereigntist sets off a chain of events that can only end in catastrophe. Two people, Rémi Whitedeer, a proud warrior, and Alyssa Morgan, a maverick cop, join forces to save a nation.
2) What inspired you to write this book?
I grew up in the Montréal area, where there are several native reserves. About twenty years ago, there was a huge altercation between the native populations and the provincial police that resulted in the Canadian Armed Forces being called in. Hard feelings still exist on both sides. The other aspect that drew me to this story is that although I had grown up within a few miles of one of the reserves, I knew virtually nothing about these amazing people.
Regarding the unpoliced reserve aspect of the story, one of the reserves in the area did disband their police services several years ago and has remained without a force ever since. This has led to even more difficult relations with the provincial police, because reserves fall under their jurisdiction if the reserve does not have a police service of its own. I thought all of this made for a fabulously intriguing story with opportunities to learn about a little-known people and explore motivations on both sides of the issue.
3) Tell us about your leads, Rémi and Alyssa?
Rémi Whitedeer is a police officer turned substance-abuse counselor who dreams of restoring order to his tribe. The tribe has been without law enforcement since their previous police force was disbanded four years earlier on charges of corruption. Matters are coming to a head though, as crime is escalating, and tensions are rising between the various factions on the reserve. As the mixed-race cousin of Chaz Whitedeer, the Guardians' leader, Rémi is caught in a no-man's land—several groups lay claim to him, but all want him to deny his white blood.
Alyssa Morgan is a sergeant with the provincial police (Sûreté du Québec). In her previous assignment, she infiltrated the Vipers to take down the leader of the outlaw biker gang responsible for her brother's death. She got her man, but her superiors think she went too far. Her disregard for protocol and her ends-justify-the-means ethics have branded her an unreliable maverick. To salvage her career, she accepts an assignment to set up a squad of native provincial officers on a reserve.
Together, Rémi and Alyssa must find a way to bring policing back to the reserve. But fed up with white interference and his cousin Rémi's growing affection for Alyssa, radical sovereigntist Chaz Whitedeer sets off a catastrophic chain of events.
4) Your book deals quite heavily with both Native American substance abuse and radical politics. Did you do a lot of research on these issues?
Substance abuse does have a role in my book. It is a big problem on most reserves and reservations, especially those with high poverty rates. However, many native communities are taking an active role in dealing with these issues and offering services to help those affected.
The political issues, I researched in-depth. I read reports and studies on the Akwesasne and Oka Crises (1990) both from the native and the military perspective. I watched documentaries, read fictional and non-fictional accounts. But most importantly, to understand the current political situation within the Iroquois, I went to Kahnesatake and spoke with various tribe members. I was fascinated to learn how divided the Iroquois are. And most of those divisions stem from the past. For example, there are tensions between people who believe in the traditional political system—the Longhouse—and those who support the government-imposed political system—the band council. However, the Canadian government only recognizes band leaders and will not negotiate with traditional leaders. Furthermore, because of residential schools and conversions by Christian missionaries, many natives are Christian, creating another divide. Traditionalists follow the Longhouse and its code of ethics and traditions.
One interesting note to be made here: in the Montréal area, since the Crises in 1990, there has been an upsurge in conversions to the Longhouse both politically and spiritually. In addition, many communities are now offering children education in their native languages and providing language classes to adults. A clear effort is being made to return to their roots.
5) This is the second book in the Deadly Vices series. Will people who have not read the first book be able to slide into this one without too much trouble?
Yes. The books are loosely connected through the friendships of the characters. When characters from the previous book are mentioned, I'm careful to introduce them in such a way that the reader knows who they are in relation to the hero/heroine of the current book.
6) Racial and ethnic elements in stories can bring with them potential controversy. Are you concerned about anyone taking issue with any of your depictions of Native peoples in this book?
Yes and no. I've done my best to represent in as truthful a manner as I could the great diversity among First Nations people. I have characters who are band council members and supporters, moderate traditionalists, radical traditionalists, Longhouse followers, Christians, etc. Some people will feel that Chaz Whitedeer is a hero, and that's okay. Which character, Rémi or Chaz, the reader views as the protagonist and the antagonist will depend on the reader's own beliefs. Many traditionalists will find that Rémi was not a hero because as Chaz says, he put his personal needs above those of his people. Of course, Chaz says this in a much more colorful manner. Others will think Rémi is a hero because he did follow his heart. He didn't give in to the demands of those around him. He had the courage to go his own way. It's all a question of perspective.
7) What ingredients do you think make for the best thrillers?
Personally, I think the best thrillers are those that make us think. They shouldn't be so far out there that the reader is detached from what is happening. The reader needs to feel it is believable enough and plausible enough to be real.
The other aspect they need is a great villain. To me a great villain is one where I can understand his or her motives. I might even feel that in their shoes, I might experience some of the same feelings. I wouldn't go so far as to destroy a community or kill people, but I might be able to see why this person would think it necessary to achieve his goal.
8) Tell us about your other works?
I'm currently working on a short story/novella for an anthology coming out this spring. I'm also working on Deadly Betrayal, the third book in the Deadly Vices series. This book will take us to Afghanistan, where Kaden Christiansen, a character introduced in the first two books, will be forced to face some truths about his past and his present.


text:
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text:
A warning shot
Sick Hillary's people fired warning shot at me. ALL accounts (multiple banks) simultaneously frozen. Had to talk to "senior operations."
Speaking of warning shots, didn't the Alt-White just declare war on Milo yesterday? The FBI determined that a "credible threat" was made against him today.
A credible threat to MILO's event and Florida Atlantic University students has forced the cancellation of the Breitbart editor's scheduled talk today.The timing doesn't necessarily indicate that it was anyone connected with Mr. Anglin, however, given that Milo has been dealing with bomb threats since the first one was telephoned into the venue of GGinDC more than a year ago.
According to the FAU Police Department, student organizers received a communication threatening to bring firearms to the talk or plant explosives at the venue.
Threats were also made to FAU students.
The threat was relayed to the F.B.I, which after investigation deemed it to be credible. The F.B.I. contacted Florida Atlantic University this morning recommending cancellation of the event and the university took the decision to pull MILO's event.
MILO was due to give a lecture this evening entitled, "How Feminism Hurts Women."
It is not clear who is responsible for the threat.
Labels: conspiracy, politics


text:
since i have nothing planned for today here on The Chevron Rose, i decided to share some Instagram photos. i don't even know if y'all find these interesting at all. but i'll go in faith and share them anyway.
|Hannah and Robert came through our neck of the woods, and we met them for breakfast at The Chef. delicious.
|I was mushroom hunting in my grandparents yards, and there were a bunch of nasty looking ones, and them i came upon this cute little perfect one.
|There is no caption. i don't know... haha.
|My beautiful, cute, sweet and hilarious momma. i'm glad i have her.
|FINALLY!!!!!!!!! After years and years of being taught chords, and then forgetting a few days later, i finally picked up on it! i finally can remember where to put my fingers. finally.
|He died for our sins. we don't have to go back to him everyday to ask for forgiveness, that's not freedom, he already forgave us. we're clean.
|I got stuck in the boy van. it turned out really fun, once i got passed the loud music and the smell of beef jerky and feet.
|Christy... this is for you.
|The Royals!!!! :( we didn't win. therefore, no flipping fireworks!
|A very late night game of Apples to Apples.
|The early morning talks with my dad are the best.
|Miss Brittany C. inspired me to wear my boots in the summer. i've missed these navy blue babies!
|I have cute siblings! Finny gets VERY into his Dinosaur Documentaries, have no clue how he sits through those!!
|Radina's Gelato! ooooooh yum!
|Radina's Iced Coffee, also super yummy!
|Aaaah! I can't get over these shoes! i'm prepared to wear them with everything!


text:
It's time to begin thinking about spring cleaning, and since many churches contain architectural features that birds and bats find appealing, it's important to clean up their waste during this time. Oftentimes, bird and bat droppings contain fungus, bacteria or other harmful pathogens that cause human illness.
In addition to the possible health hazards presented, the droppings also are corrosive and can significantly accelerate the deterioration of building components. With spring cleanup days taking place over the next few weeks, be sure to detect areas where these droppings occur. If a large amount of waste is present, consider hiring a professional cleaning company. For smaller, more manageable amounts of feces, use the following precautions recommended by the Center for Disease Control and Prevention (CDC):
The cleanup should be done by healthy individuals;
Wear an appropriate respirator that can filter particles as small as 0.3 microns;
Wear disposable and protective gloves, hats, coveralls and boots;
During the cleanup, seal heating and cooling air ducts, or shut the system down;
Moisten the droppings with a light mist of water to keep dust and spores from becoming airborne;
Put droppings into sealed plastic garbage bags;
When finished and while still wearing a respirator, remove protective clothing and place it in a plastic bag;
Wash or shower;
Check with the local government agencies to verify that disposal of the waste is permissible through standard trash pick-up; and
Consider modifying the building structure to prevent birds or bats from reestablishing the roost.
During this same cleanup time, consider conducting your church's annual self-inspection. It's possible that a maintenance crew may already be on-site, and can correct minor concerns as they are identified. An inspection checklist detailing, window and door maintenance, proper shrub and tree length, and proper lighting fixtures can be found on SafeChurch.
As with any church activity, a spring cleaning day requires planning and coordination. To help you plan for a productive and safe event, view the following relevant material on SafeChurch.
Ladder Safety
Electrical Self-Assessment Survey
Water Damage Prevention: Self-Inspection Program


text:
January 27, 2016
This irony exists throughout the business world—when prices are low and supply is high, no one's thinking, "Uh-oh, this is trouble," but they should because underlying dynamics during such a market-favorable time can frequently turn the tide. Additionally, because these influences fly under the radar and work quietly amidst the noise made over low prices, when they hit, they can hit with major consequences.
This very dynamic was set in motion for the vanilla market five years ago. Prices reached a historic low, allowing food and beverage companies that use vanilla to enjoy a stable vanilla flavor cost of goods. While everyone was "riding the wave" of low-cost vanilla, the market forces behind the scenes for what we are seeing now were building.
Currently, vanilla prices have already quadrupled in less than 10 months. Still, many ask, "Are skyrocketing prices reason enough to elevate the situation from cause for concern to a state of crisis?"
I answer that with "it's looking more and more possible." David Michael & Co. has more than 100 years of experience in the vanilla industry, many of them accompanied by volatile vanilla prices as the ebbs and flows of the market played out, so we've seen all kinds of forces at work affecting prices. Let's take a look at the current factors at work taking vanilla prices into the high-demand, low-supply, rising-cost scenario. What is happening to vanilla is not just the rule of supply and demand. There are more forces at work keeping the supply low for the foreseeable future.
For starters, last year's poor flowering season means potentially 300–500 fewer tons available in 2016. There are also a slew of "man-driven" actions that are keeping the vanilla supply low including: farmers' switching to more lucrative crops, speculators holding back on current supplies until prices increase, a booming trend of making "green bean extract," vacuum-packing partially cured beans for future sale at higher prices (compromising the quality while bringing out an undesirable phenolic note that stays with the bean), and even a mafia-type buy-up of vanilla beans for money laundering purposes.
I'd like to tell everyone to just hang tight for a few quarters—but it doesn't seem the hand of supply-and-demand will balance things out that quickly. The good news is there are some solutions while we ride out the wave. Some suppliers, like us, saw this coming and secured quantities of vanilla during the low-cost era, offering customers the opportunity to lock in at lower prices. Additionally, technology exists that enables using a lower amount of vanilla extract while not impacting the label, and innovations in the extraction process allow for more complete extractions resulting in a more intensified product.
My family has been through three vanilla crises to date, and every time we've been able to maintain a supply for our customers. I have no doubt that as long as innovators keep innovating, and suppliers continue to work with their customers to manage costs and extend supply, the food industry will weather the crisis and emerge stronger.
Skip Rosskam
President and Chief Operating Officer
David Michael & Co.
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text:
TIPS on Applying Internet Connection.
1. AvailabilityAsk if which internet provider are available in your location, for broadband you can check your phone signal
2. Distance to their main server or Cell site.This is a very important to our internet connection speed. The shortest distance the better Bandwidth we can get and less pings. So if your to far to their main server or Cell site you can't receive the full length of your Bandwidth.
Distance you must have at least 2 to 3 kilometer away from their transmitter or server. Logic here longer cable reduce electric current.( all most Provider all ready upgrade to Fiber Optic Cables)
Wireless Broadband
Signal Strength 90 to 100% and RSSI at least you have 50% for better link quality.
3. Promo
Check if there is any promo to avail for badget conscious .


text:
|2013-09-28, 03:55
|Link #121
The Lovable SuBiTA
Join Date: Jun 2006
Location: Sleeping in Yokohama base, Section 9.
It's from Feudal East Asia culture, such as China, where colors are used to define groups of people. Japan has the colors to signify the groups of people that are close to the Emperor in terms of trust and loyalty, so its like a ranking system.
For world usage, every army wants to differentiate itself from another country, as a means of identity and pride. Far as I know, the UN blue was because of peacekeeping missions. Its probably retained in the Alterverse because BETAs don't into colors anyway, and its easier for their own human forces to tell who is who.
IRL, any soldier not wearing his or her own uniform colors, are not applicable for the POW treatment as per the Geneva Convention, aka they won't be recognized as official combatants. Any such illegal soldiers will then have no rights to human treatment. Also, not wearing the correct rank and colors when there is no reason not to do so shows a deep lack of respect and pride towards the symbolism of that rank, and lack of appreciation of what they are fighting for.
|2013-09-28, 04:21
|Link #122
Some say I'm the Reverse
Join Date: Jul 2006
Okay, so no messing around with the Takemi colors. Got it.
EDIT:...actually, waitasec (And somewhat back on-topic) I just remembered that the real-life F-5 Tiger was originally an in-house project by Northrop (EG no military funding or support). What we've seen so far in Total Eclipse are TSF projects that are partly (or fully) funded by their respective militaries. If (say for example) a private company were to test their (non-government funded) TSF in actual combat with the BETA, what sort of requirements/permits/et al woudl they need for this?
Last edited by wavehawk; 2013-09-28 at 04:35.
|2013-09-28, 11:28
|Link #124
Idar Lead
Join Date: Sep 2012
Location: World Marshall bureau
Hypothetically though...you'd need a warzone, and some kind of an excuse to be in it. Then, a CP (or a mobile equivalent) to record observation data, chaser/support craft, the resupply train, etc.
|2013-09-28, 12:30
|Link #125
Truth Martyr
Join Date: Nov 2007
Location: Doing Anzu's paperwork.
Age: 32
Should note that IRL, the F-14 started as a Grumman study internal design also... Though that was because they looked at the F-111B program, saw the writing on the wall, and decided to get a head start on design.
Interestingly, I've read that in clean configuration, Tomcats could dogfight Eagles and win, but usually they're stuck with the fuckheavy drop tanks (IRL) and so always had to dogfight with that disadvantage.
|2013-09-28, 18:04
|Link #126
Some say I'm the Reverse
Join Date: Jul 2006
It's more the legality issue I'm talking about--What do you need to have to get approval, what are the terms and conditions, etc.
|2013-09-28, 23:02
|Link #128
Truth Martyr
Join Date: Nov 2007
Location: Doing Anzu's paperwork.
Age: 32
According to Tomcat drivers in ACEVAL/AIMVAL, they had a series of unsanctioned quiet dogfights with Eagles. Tl;dr, it could really go either way in a guns duel.
Now, Muv-Luv hypotheticals: Silent Eagle probably has the advantage because stealth, mike foxtrots. You can't kill what you can't see. Strike Eagle and Super Tomcat are probably even, but I'd think Strike Eagle edges the Super Tomcat on manuverability. Slightly. On the other hand, Super Tomcat's 2-man crew allows the workload to be shared and another set of eyes in the fight.
Tl;dr: Silent has the advantage because stralth. F-14D and F-15E are close and it'll come down to the crew.
|2013-10-03, 04:15
|Link #129
Some say I'm the Reverse
Join Date: Jul 2006
Eh. Tangentially related. If the real-life Silent Eagle isn't dead it soon will be, with a daggy stake in it's heart.
I can actually see the very same thing happening to Boening in ML:A. And Heinemann going "I told you so" to everyone. Once the BETA wind down and everyone starts shifting to the more advanced and accessible F-35, if Boening doesn't develop something new and fast, it's TSF division will lose lots of jobs...
|2013-10-03, 04:29
|Link #130
Idar Lead
Join Date: Sep 2012
Location: World Marshall bureau
Although Muv-Luv 3rd Gens still take time to be developed (nearly 20 years between Raptor being designed, and entering service), so Boening can still probably squeeze a 2 decades of Strike Eagle usage and purchases, and that's without upgrade plans. Because so many people use them (U.S Army, U.N Orbital Divers, etc, etc.) and not everyone will have the funds or infrastructure to support 3rd Gens right away, Eagle TSFs most likely will still have a long future.
Not to mention that they apparently build Super Hornet TSFs, having acquired the rights for those.
Plus, F-15SE will probably due well in Alt. It's a 3rg Gen, stealthy, flies extremely well. In Japan, it takes direct intervention by Shogun for it to loose versus Type-04 in the bid for next mainstay Imperial TSF, so I imagine Boenings PR department is not slacking off either.
And someone had to make all those G-Bombs too.
|2013-10-03, 05:39
|Link #132
Truth Martyr
Join Date: Nov 2007
Location: Doing Anzu's paperwork.
Age: 32
Especially since Mitsubishi will want to make the Type-04 Shiranui locally in Japan... Boening will miss out on that contract.
Well, there's still Super Hornets.
Also, the Silent Eagle is - for those who can afford it and already operate Eagles - an easier pill to swallow than buying Raptors. Hence why Boening is probably trying it's best to pimp the Silent Eagle to Comet! US Space Force and UN. Or if the Shiranui Niigata is chosen by USSF, Boening is going to get the contract for making it in the US, since there's no way in hell Mitsubishi is going to be able to make enough Shiranuis for both the US and Japan.
|2013-10-03, 19:57
|Link #133
Lumine Passio
Join Date: Jul 2013
Location: Hanoi, Vietnam
Age: 12
Proposed version for F-15J Kai "Kagerou Kai" (not that Ayu-Mayu version):
- Using the F-15E Strike Eagle as the base, but overhauled frame with titanium joints.
- XM3 Operating System.
- Infra-red search and track targeting system.
- MIDS Fighter Data Link Terminal for better communication.
- Automatic Electronically Scanned Array.
- Customized Tactical Electronic Warfare Suite, could be use with the special paint of Silent Eagle to create limited stealth capability.
- New generator with 22% more powerful and efficiency. Complete with larger fuel tank.
- Thicker (by 1cm) armor with carbon-web structure which could reduce the damage of an attack.
Last edited by Fireminer; 2013-10-03 at 22:31.
|2013-10-03, 22:20
|Link #134
The Lovable SuBiTA
Join Date: Jun 2006
Location: Sleeping in Yokohama base, Section 9.
You might want to keep the fan based creations in the Fan-Fic threads to avoid confusion.
Also, this is supposed to be for anti-BETA or anti-human modifications? Cause basically those electronic mods are going to be worth fuck-all in a Hive.
|2013-10-04, 00:57
|Link #136
Truth Martyr
Join Date: Nov 2007
Location: Doing Anzu's paperwork.
Age: 32
Cheap? Bullshit. Your upgrades aren't going to make it any cheaper than the Silent Eagle. Also, thicker armor increases costs and has a negative effect on combat performance.
In the post-apocalyptic world of TDA, there won't be resources for making your upgraded Eagle.
|2013-10-04, 04:36
|Link #137
Idar Lead
Join Date: Sep 2012
Location: World Marshall bureau
Yeah, hahahahaha, that's not how this works.
The technology that makes stealth is expensive, and M-L stealth is probably no different. It would be important to note that Silent Eagle is not really as stealthy as Raptor or Lightning II, which is part of the reason why's it's (relatively) cheaper. Additionally while F-35 is meant as a "cheap" stealth TSF, that's really relative only to other 3rd Generation craft; it costs far more than an average 2nd Gen. Same with Silent Eagle (which is not just stealthy, it's overall a next-Gen Eagle).
Plus, you can't have extra stuff in your TSF (like titanium - which is bloody expensive by the way) and not have it cost more than base version.
If you want to make a cheap TSF, use the F-5. There's a reason why every EU force in the verse loves it.
|2013-10-04, 04:51
|Link #138
Some say I'm the Reverse
Join Date: Jul 2006
Essentially make most of the fuselage out of non-metallic or nonreflective materials.
...Hey, Stealthy and cheap, yes. Nobody ever said anything about effective against modern fighter aircraft!
|2013-10-04, 06:57
|Link #139
Lumine Passio
Join Date: Jul 2013
Location: Hanoi, Vietnam
Age: 12
When I said "cheap", I mean it's more cost-effective than the Gekkou. And if it's too expensive, then I could write only two dozen of them was produced just before the day, which Ikaruga has already forseen the world beyond it and decided to prepare himself with these machine. The F-15J Kai Kagerou Kai, together with F-16V Viper, F/A 18SA Super Hornet Advance, Su-35 and some other would be the main fighting force of human to finish BETA on Earth after the Day. F-22, F-35 and Su-47 will replace them for Operation "Zero Requiem" ten years later.
|2013-10-04, 07:08
|Link #140
Lumine Passio
Join Date: Jul 2013
Location: Hanoi, Vietnam
Age: 12
Proposed version two for F-15J Kai "Kagerou Kai":
- Using the F-15E Strike Eagle as the base, but overhauled frame with strengthern joints.
- XM3 Operating System platform.
- Automatic Infra-red Search-and-Track Targeting System.
- MIDS Fighter Data Link Terminal for better communication. Capable of establishing a local network.
- Customized Tactical Electronic Warfare Suite, could be use with the special paint of Silent Eagle to create limited stealth capability.
- Capable of bringing a larger amount of Ammunition.
- New generator with 22% more powerful and efficiency. Complete with larger fuel tank.
- Thinner (by 1cm) armor with carbon-web structure which increase durability.
Overall, the Kagerou Kai is a bit lighter and more suited for TSF v.s TSF warfare.
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