Becoming the Bimboy (Bimbo Transformation Story, Gay Alpha Male M/M Romance)

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Fig. 4 Cell viability of intestinal HT-29 cancer cells after 3 h incubation with different concentrations (mg mL −1) of the bare THCPSi and THCPSi–lipid vesicle microparticles assessed by a luminescence-based assay. All experiments were conducted at 37 °C. Errors bars represent the mean ± SD ( n = 4). After removing the medium and washing the wells twice with fresh 1× Hanks Balanced Salt Solution pH 7.4 (HBSS), the samples were added to the wells and incubated for 3 h. For positive and negative controls, HBSS (pH 7.4; 100% cell viability) and triton X-100 (<1% cell viability) were used. Statistical analysis was made by a one-way analysis of variance (ANOVA), followed by a Dunnett's multiple comparison test to analyze the data using GraphPad Prism v. 5.01 (GraphPad Software). The level of significance was set at probability of * p< 0.05. E. Tasciotti, X. Liu, R. Bhavane, K. Plant, A. D. Leonard, B. K. Price, M. M. Cheng, P. Decuzzi, J. M. Tour, F. Robertson and M. Ferrari, Nat. Nanotechnol., 2008, 3, 151–157 CrossRef CAS PubMed. Scheme 1 (A) Schematic illustration of the synthesis of HA +, fabrication of the UnTHCPSi nanoparticles from boron doped p+ Si <100> wafers, conjugation of HA + onto the surface of the UnTHCPSi nanoparticles via EDC/NHS coupling chemistry. (B) Hypothesis of the application of the resulting UnTHCPSi–HA + nanoparticles for the targeting of CD44-overexpressing breast cancer cells.

The size and morphology of the UnTHCPSi and UnTHCPSi–HA + nanoparticles were analyzed by transmission electron microscopy (TEM). The TEM pictures were captured using a Jeol JEM-1400 microscope (Jeol Ltd., Tokyo, Japan) at an 80 kV voltage. The nanoparticle suspensions were centrifuged, re-dispersed in ethanol at a concentration of 10 μg mL −1, and dropped on a carbon-coated copper TEM grid, followed by drying for 48 h at room temperature. Stability studies in human plasma For performing these experiments, 300 μg of UnTHCPSi and UnTHCPSi–HA + were dispersed in 200 μL of 1× PBS (pH 7.4), exposed to 1500 μL of human plasma, and left under stirring at 800 rpm and 37 °C for 2 h. Sample aliquots of 200 μL were withdrawn at pre-determined time points (1, 5, 10, 15, 30, 60, 90, and 120 min), and the corresponding size, ζ-potential, and PdI were subsequently determined by DLS and ELS. The results are shown as the average of at least three independent measurements. Anonymous human plasma donors were obtained from the Finnish Red Cross Blood Service, with the permission from the respective institutional ethical committee. Cell lines and culturing conditions For the in vitro studies, MCF-7 and MDA-MB-231 breast cancer cells were cultured according to the protocols described in detail in the ESI. † In vitro cytotoxicity studies The in vitro cytotoxicity of both UnTHCPSi and UnTHCPSi–HA + was evaluated by a CellTiter-Glo ® Luminescent Cell Viability assay, as previously described elsewhere. 26,29 MCF-7 and MDA-MB-231 breast cancer cells were suspended in the corresponding cell culture media at a concentration of 2 × 10 5 cells per mL, and approximately 2 × 10 4 cells per well were seeded in 96-well plates (Corning Inc. Life Sciences, USA). The cells were allowed to attach overnight at 37 °C, after which the cell culture medium was removed and replaced with 100 μL of UnTHCPSi and UnTHCPSi–HA + nanoparticle suspensions at concentrations of 25, 50, and 100 μg mL −1, with 1× HBSS (pH 7.4) and 1% Triton X-100 as positive and negative controls, respectively. After incubating for 6 and 24 h at 37 °C, 100 μL of the assay reagent was added to each well and the number of viable cells was determined by measuring the luminescence from the living cells using a Varioskan Flash fluorometer (Thermo Fisher Scientific Inc., USA). The results presented correspond to the average of at least three independent measurements. Cellular internalization The cellular uptake and intracellular localization of the UnTHCPSi and UnTHCPSi–HA + nanoparticles was assessed by TEM. Approximately 10 5 cells per well of MCF-7 and MDA-MB-231 breast cancer cells were seeded in 24-well plates (Corning Inc. Life Sciences, USA) with each well containing a 13 mm round shaped coverslip and allowed to attach overnight at 37 °C. After removing the cell culture media, 500 μL per well of the nanoparticle suspensions at a concentration of 50 μg mL −1 were added to the wells. After an incubation period of 6 h at 37 °C, the nanoparticle suspensions were carefully removed and the samples were rinsed twice with HBSS–HEPES (pH 7.4). Then, the cells were fixed with 2.5% glutaraldehyde in 0.1 M PBS buffer (pH 7.4) for 1 h at room temperature, and subsequently washed twice with HBSS–HEPES (pH 7.4) and sodium cacodylate buffer (NaCac) for 3 min, prior to post-fixation with 1% osmium tetroxide in 0.1 M NaCac buffer (pH 7.4). Thereafter, the cells were dehydrated with 30–100% ethanol for 10 min and embedded in epoxy resin. Ultrathin sections with approximately 60 nm were sliced parallel to the coverslips, post-stained with uranyl acetate and lead citrate, and finally analyzed by TEM as described above. Flow cytometric analysis of CD44 expression and cellular association The expression of CD44 receptor in MCF-7 and MDA-MB-231 breast cancer cell lines was evaluated by flow cytometry according to a protocol adapted from the manufacturer's instructions, as described in the ESI. † M.-A. Shahbazi, M. Hamidi, E. M. Mäkilä, H. Zhang, P. V. Almeida, M. Kaasalainen, J. J. Salonen, J. T. Hirvonen and H. A. Santos, Biomaterials, 2013, 34, 7776–7789 CrossRef CAS PubMed.

Players were responsible for feeding their bimbo, maintaining her mood, and keeping her clean and healthy. They could earn Bimbo Attitude (BA) and IQ points by playing mini-games or spending time at locations in the city. For instance, bimbos could earn IQ by spending an hour in the library or earn BA by paying for plastic surgery at the clinic or changing their style at the salon. Bimbos could also accumulate BA regularly from their boyfriends and homes. W. Wang, M.-J. Zhang, R. Xie, X.-J. Ju, C. Yang, C.-L. Mou, D. A. Weitz and L.-Y. Chu, Angew. Chem., Int. Ed., 2013, 52, 8084–8087 CrossRef CAS PubMed. R. E. Serda, B. Godin, E. Blanco, C. Chiappini and M. Ferrari, Biochim. Biophys. Acta, 2011, 1810, 317–329 CrossRef CAS PubMed. Fig. 5 Flow cytometric analysis of the cellular association of UnTHCPSi and UnTHCPSi–HA + nanoparticles with MDA-MB-231 and MCF-7 breast cancer cells. The cells were incubated with UnTHCPSi and UnTHCPSi–HA + nanoparticles at the concentrations of 50 and 100 μg mL −1 for 6 h at 37 °C. An enhanced association of the UnTHCPSi nanoparticles with both cell lines was observed after the biofunctionalization with HA +.

P. J. Kinnari, M. L. K. Hyvönen, E. M. Mäkilä, M. H. Kaasalainen, A. Rivinoja, J. J. Salonen, J. T. Hirvonen, P. M. Laakkonen and H. A. Santos, Biomaterials, 2013, 34, 9134–9141 CrossRef CAS PubMed. Kogal or, more correctly, kogyaru and ganguro carry similar connotations as a Japanese version of a "valley girl" or bimbo.H. A. Santos, L. M. Bimbo, V. P. Lehto, A. J. Airaksinen, J. Salonen and J. Hirvonen, Curr. Drug Discovery Technol., 2011, 8, 228–249 CrossRef CAS. Oxford dictionary of word origins. Cresswell, Julia, 1950-, Oxford University Press. (Seconded.). New York. 9 September 2010. ISBN 978-0199547937. OCLC 663824301. {{ cite book}}: CS1 maint: location missing publisher ( link) CS1 maint: others ( link) L. M. Bimbo, M. Sarparanta, H. A. Santos, A. J. Airaksinen, E. Mäkilä, T. Laaksonen, L. Peltonen, V.-P. Lehto, J. Hirvonen and J. Salonen, ACS Nano, 2010, 4, 3023–3032 CrossRef CAS PubMed. K. K. Upadhyay, A. K. Mishra, K. Chuttani, A. Kaul, C. Schatz, J.-F. Le Meins, A. Misra and S. Lecommandoux, Nanomedicine, 2012, 8, 71–80 CrossRef CAS PubMed. Like all LBOs, there are still risks of disruption, conflicts, and reduced performance - but these may be minimized as the new managers have bought in as owners as well.

Because of this U-turn, older fans felt rejected and cringed at the staff's attempt at communicating with young players as they exited the community. L. M. Bimbo, M. Sarparanta, E. Mäkilä, T. Laaksonen, P. Laaksonen, J. Salonen, M. B. Linder, J. Hirvonen, A. J. Airaksinen and H. A. Santos, Nanoscale, 2012, 4, 3184–3192 RSC.T. Tanaka, B. Godin, R. Bhavane, R. Nieves-Alicea, J. Gu, X. Liu, C. Chiappini, J. R. Fakhoury, S. Amra, A. Ewing, Q. Li, I. J. Fidler and M. Ferrari, Int. J. Pharm., 2010, 402, 190–197 CrossRef CAS PubMed. S. H. Kim, J. W. Kim, D. H. Kim, S. H. Han and D. A. Weitz, Small, 2013, 9, 124–131 CrossRef CAS PubMed.