fsi cf  (MatTek)

Journal: Bioconjugate chemistry

Article Title: Bifunctional Elastin-like Polypeptide Nanoparticles Bind Rapamycin and Integrins and Suppress Tumor Growth in Vivo

doi: 10.1021/acs.bioconjchem.7b00469

Figure Lengend Snippet: Coassembly of bifunctional ELP nanoparticles with specificity of drug and receptor binding. Obtained from recombinant cellular expression, ELPs are a useful platform with which to engineer precision macromolecules for drug delivery. For example, the ELP diblock copolymer known as FSI assembles nanoparticles when heated above its critical micelle temperature (CMT); furthermore, these nanoparticles are decorated with a small protein (FKBP) that binds a potent cytostatic small molecule (Rapa). To examine the hypothesis that mixtures of ELP diblock copolymers can coassemble bifunctional nanoparticles, this manuscript explores triggered assembly of FSI with a second ELP called ISR. ISR contains the RGD ligand, which binds cells expressing heterodimeric integrins in tumors. When mixed and heated to physiological temperature, ISR and FSI assemble bifunctional nanoparticles with both drug-binding and receptor-binding capacity. In comparison to FSI nanoparticles alone, the bifunctional nanoparticles demonstrate superior tumor binding in vitro and enhanced tumor suppression in vivo.

Article Snippet: FSI–CF and ISR–Rho were mixed (100 μ M each) on a 35 mm glass-bottom dish (MatTek, MA) and imaged at 30 °C using a Zeiss LSM 510 Meta NLO confocal microscopy (Thornwood, NY) with an Instec HCS60 temperature control stage (Denver, CO; ).

Techniques: Binding Assay, Recombinant, Expressing, In Vitro, In Vivo

Journal: Bioconjugate chemistry

Article Title: Bifunctional Elastin-like Polypeptide Nanoparticles Bind Rapamycin and Integrins and Suppress Tumor Growth in Vivo

doi: 10.1021/acs.bioconjchem.7b00469

Figure Lengend Snippet: ISR and FSI spatial co-localization into bifunctional nanoparticles. (a) To explore the coassembly of ISR and FSI, the two diblock copolymers were fluorescently labeled, mixed in a 1:1 ratio, and imaged above the CMT (30 °C) using confocal laser scanning microscopy. When merged, FSI–CF (green), and ISR–Rho (red) show a high degree of spatial co-localization (yellow). (b) ImageJ and JACoP analysis confirmed a Pearson’s coefficient (PC) of 0.976, indicating the strong co-localization of two nanoparticles. (c) Cryo-TEM was used to observe ISR nanoparticles alone (left) or the 50% ISR and 50% FSI bifunctional nanoparticles (right). Both samples have a similar distribution of particle shape and have particles sizes consistent with DLS (Table 1). ISR nanoparticles have an average diameter of 33.8 ± 3.4 nm (n = 6, mean ± SD), and 50% ISR and 50% FSI nanoparticles have an average diameter of 33.7 ± 3.7 nm (n = 6, mean ± SD). Scale bar length = 100 nm.

Article Snippet: FSI–CF and ISR–Rho were mixed (100 μ M each) on a 35 mm glass-bottom dish (MatTek, MA) and imaged at 30 °C using a Zeiss LSM 510 Meta NLO confocal microscopy (Thornwood, NY) with an Instec HCS60 temperature control stage (Denver, CO; ).

Techniques: Labeling, Confocal Laser Scanning Microscopy

Journal: Bioconjugate chemistry

Article Title: Bifunctional Elastin-like Polypeptide Nanoparticles Bind Rapamycin and Integrins and Suppress Tumor Growth in Vivo

doi: 10.1021/acs.bioconjchem.7b00469

Figure Lengend Snippet: Bifunctional ISR/FSI nanoparticle recognition and binding of integrin receptors similar to ISR nanoparticles. Confocal microscopy was used to study the binding of 200 μM rhodamine-labeled nanoparticles (red) with or without coassembly for 1 h at 37 °C. (a) MDA-MB-468 cells were incubated with ISR–Rho, FSI–Rho, and FSI–Rho mixed with unlabeled ISR (50% ISR and 50% FSI). Panels i–iii represent integrin binding with ISR–Rho. Panels iv–vi represents integrin binding with FSI–Rho and panels vii–ix represents integrin binding with 50% ISR and 50% FSI. Nuclei were stained with DAPI (blue). (b) Normalized fluorescence (n = 3, mean ± SD) showed statistically significant differences between ISR–Rho and FSI–Rho, and between ISR–Rho and 50% ISR and 50% FSI (Tukey’s post-hoc test; α = 0.05; ***, p < 0.0001; ○○○, p < 0.0001) and between 50% ISR and 50% FSI and FSI–Rho (Tukey’s post-hoc test; α = 0.05; •••, p < 0.0001). (c) To further characterize binding, flow cytometry was used to identify the percent of cells positive for FSI–Rho assembled with increasing proportions of unlabeled ISR. For 50% ISR and 50% FSI, more than 90% of cells were positive, which was equivalent to that observed for cells treated with a positive ISR–Rho control.

Article Snippet: FSI–CF and ISR–Rho were mixed (100 μ M each) on a 35 mm glass-bottom dish (MatTek, MA) and imaged at 30 °C using a Zeiss LSM 510 Meta NLO confocal microscopy (Thornwood, NY) with an Instec HCS60 temperature control stage (Denver, CO; ).

Techniques: Binding Assay, Confocal Microscopy, Labeling, Incubation, Staining, Fluorescence, Flow Cytometry

Journal: Bioconjugate chemistry

Article Title: Bifunctional Elastin-like Polypeptide Nanoparticles Bind Rapamycin and Integrins and Suppress Tumor Growth in Vivo

doi: 10.1021/acs.bioconjchem.7b00469

Figure Lengend Snippet: Bifunctional nanoparticles retaining of Rapa similarly to FSI nanoparticles. (a) Rapa loading of coassembled ISR/FSI nanoparticles is lower compared to FSI alone due to the reduced number of FKBP domains, whereas ISR alone failed to demonstrate high Rapa loading (n = 3, mean ± SD). (b) The coassembled nanoparticles remained colloidally stable with and without Rapa loading as assessed by DLS over 24 h at 37 °C (n = 3, mean ± SD). (c) Rapa-loaded formulations were then dialyzed to assess drug retention under sink conditions. Bifunctional ISR/FSI nanoparticles retained drug similarly to FSI nanoparticles. Even though the ISR alone can solubilize low levels of Rapa, it was unable to retain drug under sink dialysis conditions (n = 3, mean ± SD).

Article Snippet: FSI–CF and ISR–Rho were mixed (100 μ M each) on a 35 mm glass-bottom dish (MatTek, MA) and imaged at 30 °C using a Zeiss LSM 510 Meta NLO confocal microscopy (Thornwood, NY) with an Instec HCS60 temperature control stage (Denver, CO; ).

Techniques:

Journal: Bioconjugate chemistry

Article Title: Bifunctional Elastin-like Polypeptide Nanoparticles Bind Rapamycin and Integrins and Suppress Tumor Growth in Vivo

doi: 10.1021/acs.bioconjchem.7b00469

Figure Lengend Snippet: FSI–Rapa and ISR/FSI–Rapa potency in inhibiting MDA-MB-468 proliferation in vitro compared to free Rapa. (a) The inhibitory concentration corresponding to 50% suppression of proliferation, IC50, for FSI–Rapa, ISR/FSI–Rapa, and free Rapa were determined by nonlinear regression as 0.21 ± 0.02, 0.32 ± 0.16, and 1.81 ± 0.96 nM, respectively (n = 3–4, mean ± SD). (b) Tukey’s post-hoc analysis revealed statistically significant differences in IC50 between free Rapa and FSI–Rapa and ISR/FSI–Rapa each (α = 0.05; *, p = 0.0004; **, p = 0.001).

Article Snippet: FSI–CF and ISR–Rho were mixed (100 μ M each) on a 35 mm glass-bottom dish (MatTek, MA) and imaged at 30 °C using a Zeiss LSM 510 Meta NLO confocal microscopy (Thornwood, NY) with an Instec HCS60 temperature control stage (Denver, CO; ).

Techniques: In Vitro, Concentration Assay

Journal: Bioconjugate chemistry

Article Title: Bifunctional Elastin-like Polypeptide Nanoparticles Bind Rapamycin and Integrins and Suppress Tumor Growth in Vivo

doi: 10.1021/acs.bioconjchem.7b00469

Figure Lengend Snippet: Tumor regression study demonstration of greater antitumor efficacy for the bifunctional nanoparticle than FSI–Rapa or free Rapa in breast tumor xenografts. Athymic nude mice with orthotopic 50–100 mm3 MDA-MB-468 tumors were treated three times a week intravenously. (a) SI–Rapa, free Rapa, and FSI–Rapa were compared at 0.25 mg/kg. Simultaneously, FSI–Rapa was evaluated for dose escalation from 0.0075–0.075 mg/ kg, which revealed intermediate tumor suppression at the 0.075 mg/kg dose. The comparisons between all of the untargeted formulations gave significantly different tumor volumes on the last day of treatment only between PBS and FSI–Rapa at 0.25 mg/kg (Tukey’s post-hoc analysis; α = 0.05; ***, p = 0.001). (b) Based on the prior study, a comparative study was performed between the bifunctional and the untargeted formulations at an intermediate Rapa dose of 0.075 mg/kg. The bifunctional nanoparticle gave significant differences in tumor volume on the last day of treatment compared to PBS or free Rapa (Tukey’s post-hoc analysis; α = 0.05; **, p = 0.003; *, p = 0.02, respectively). (c) Analysis of this study using nonparametric survival analysis also demonstrated a significant extension of survival for the bifunctional ISR/FSI–Rapa treatment compared with PBS, free Rapa, and FSI–Rapa (log-rank post-hoc analysis; α = 0.008; p = 0.001, 0.001, and 0.002, respectively). FSI–Rapa treatment also significantly prolonged survival compared to PBS (α = 0.008, p = 0.005).

Article Snippet: FSI–CF and ISR–Rho were mixed (100 μ M each) on a 35 mm glass-bottom dish (MatTek, MA) and imaged at 30 °C using a Zeiss LSM 510 Meta NLO confocal microscopy (Thornwood, NY) with an Instec HCS60 temperature control stage (Denver, CO; ).

Techniques:

Journal: Bioconjugate chemistry

Article Title: Bifunctional Elastin-like Polypeptide Nanoparticles Bind Rapamycin and Integrins and Suppress Tumor Growth in Vivo

doi: 10.1021/acs.bioconjchem.7b00469

Figure Lengend Snippet: Intratumoral accumulation of bifunctional ISR/FSI nanoparticles compared with controls. Athymic nude mice with orthotopic 50–150 mm3 MDA-MB-468 tumors were injected intravenously with 0.15 mg/kg rhodamine tagged FSI or ISR/FSI nanoparticles, and tumor accumulation was studied by quantifying fluorescence in tumor tissue sections excised at 2 and 8 h (n = 3 mice per group). (a) Tumor tissue sections at 2 h showed no rhodamine (red) fluorescence above PBS background. Distinct fluorescence was observed only for the bifunctional nanoparticle at 8 h relative to PBS, as indicated by white arrows. Nuclei were stained with DAPI (blue). A representative image is shown from each group. Scale bar = 20 μm. (b) The quantified rhodamine fluorescence (n = 6–12, mean ± 95% CI) showed no significant difference above PBS background at 2 h. In contrast, the rhodamine fluorescence of ISR/FSI nanoparticles at 8 h was significantly greater compared with PBS, FSI at 2 h, and ISR/FSI at 2 h each (Tukey’s post-hoc analysis; α = 0.05; *, p = 0.013; **, p = 0.004; and ○○, p = 0.001).

Article Snippet: FSI–CF and ISR–Rho were mixed (100 μ M each) on a 35 mm glass-bottom dish (MatTek, MA) and imaged at 30 °C using a Zeiss LSM 510 Meta NLO confocal microscopy (Thornwood, NY) with an Instec HCS60 temperature control stage (Denver, CO; ).

Techniques: Injection, Fluorescence, Staining