Nanoparticles (NPs) carrying PFP and ICG were successfully synthesized using DSPG, DSPE-PEG2000, DPPC, and cholesterol, and were called Lip-ICG-PFP-cRGD, generating a US-mediated cavitation effect. The diameter, zeta potential, and polydispersity index (PDI) of different kinds of NPs were measured by Zetasizer Nano ZS unit (Malvern Instruments, Malvern, UK) and their morphology and structure were observed by transmission electron microscopy (TEM). Ultraviolet–visible (UV–vis) spectrophotometer with scanning wavelength ranging from 200 to 900 nm was used to determine the absorption spectra of different nanoparticles. UV–vis spectrophotometer (260-Bio, Thermo Fisher Scientific) was used to evaluate the entrapment efficiency and loading of ICG, and entrapment efficiency and loading content were determined based on Eqs. (1) and (2) :
260 bio
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 260-Bio is a laboratory instrument designed for the measurement of nucleic acid concentration and purity. It utilizes UV spectrophotometry to determine the optical density of a sample at 260 nanometers, which is the wavelength at which nucleic acids (DNA and RNA) absorb light. This information can be used to calculate the concentration of nucleic acids in the sample.
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6 protocols using 260 bio
1
Synthesis and Characterization of Multifunctional Nanoparticles for Ultrasound-Mediated Therapy
2
Cellular ATP Modulation by L-Arginine and Nanoparticles
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Cellular ATP levels were measured with an ATP assay kit. First, we investigated whether free L-Arg could decrease cellular ATP levels. MDA-MB-231 cells were grown in 6-well plates with a density of 2×105 per well and after 24 h incubation they were treated for 12 h with different concentrations of free L-Arg (5, 10, 20, 40 and 80 μg/mL). Finally, cells were lysed followed by centrifugation (12,000 g for 5 min) at 4 °C for luminometer detection. To measure the ATP level of cells treated with NPs, cells were incubated for 12 h with PBS, L-Arg@Ce6P@P NPs (Ce6:10 μg/mL) or Ce6@P NPs (Ce6:10 μg/mL) in a normoxic environment. The hypoxia group was covered with liquid paraffin for another 2 h. Finally, cells were collected and centrifuged, and the supernatant was used for RLU detection by a luminometer with multimode reader (260-Bio, Thermo Fisher Scientific, USA).
3
Quantification of NO Release from NPs
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Griess reagent was used to detect released NO in vitro. 1 mL solution of L-Arg@Ce6@P NPs (20 mg/mL) or Ce6@P NPs (20 mg/mL) dissolved in PBS were dialyzed (cut-off MW: 2,500 Da) against 10 mL PBS (pH = 7.4) containing H2O2 (10 mM) and incubated at 37 °C. The standard curve of NO was obtained from NaNO2 in the kit. The NO released from NPs at different time points was quantified by a multimode reader (260-Bio, Thermo Fisher Scientific, USA) at 540 nm.
4
Quantifying Intracellular ROS with L-Arg@Ce6@P NPs
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ROS levels in vitro were measured with SOSG (λex/λem = 504 nm/525 nm). Briefly, different concentrations of L-Arg@Ce6@P NPs and SOSG (5 μM) were added into cuvettes, followed by irradiation with a 660 nm NIR laser (Stone Laser, China) at a power density of 5 mW/cm2 for different time intervals. Fluorescence intensity changes of SOSG were observed by a multimode reader (260-Bio, Thermo Fisher Scientific, USA).
Intracellular ROS levels in MDA-MB-231 cells were detected with a typical fluorescent probe DCFH-A (λex/λem = 488 nm/530 nm). Cells were grown in cell-culture dishes with a density of 1×105 per dish. After 24 h incubation, the medium was replaced by fresh DMEM medium containing either PBS, L-Arg@Ce6@P NPs (Ce6:10 μg/mL) or Ce6@P NPs (Ce6:10 μg/mL) and incubated for 12 h, with or without covering with liquid paraffin. After incubation, cells in the laser group received irradiation with a 660 nm laser at a power density of 5 mW/cm2 for 3 min. Samples were fixed in 4% formaldehyde for 20 min. Formaldehyde was removed and cells were dyed with DAPI for another 15 min. CLSM was used to observe intracellular ROS levels. Collected cells were used for flow cytometry analysis.
Intracellular ROS levels in MDA-MB-231 cells were detected with a typical fluorescent probe DCFH-A (λex/λem = 488 nm/530 nm). Cells were grown in cell-culture dishes with a density of 1×105 per dish. After 24 h incubation, the medium was replaced by fresh DMEM medium containing either PBS, L-Arg@Ce6@P NPs (Ce6:10 μg/mL) or Ce6@P NPs (Ce6:10 μg/mL) and incubated for 12 h, with or without covering with liquid paraffin. After incubation, cells in the laser group received irradiation with a 660 nm laser at a power density of 5 mW/cm2 for 3 min. Samples were fixed in 4% formaldehyde for 20 min. Formaldehyde was removed and cells were dyed with DAPI for another 15 min. CLSM was used to observe intracellular ROS levels. Collected cells were used for flow cytometry analysis.
5
Quantifying Nanoparticle Entrapment Efficiency
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The entrapment efficiency and the content of ICG loaded in NPs were determined in triplicate by an ultraviolet–visible (UV–Vis) spectrophotometer (260-Bio, Thermo Fisher Scientific). The supernatant of centrifugation and washing process was collected and the concentration of ICG in the supernatant was quantified by measuring the absorbance at 780 nm and comparing the reading to a standard concentration curve of free ICG in the same solvent. The entrapment efficiency and the content were calculated as follows: entrapment efficiency (%) = ([ICG used in formulation – ICG in supernatant]/ICG used in formulation) ×100; content (% w/w) = ([ICG used in formulation – ICG in supernatant]/mass of NPs) ×100.
6
Liposomal Nanocarrier Characterization and Release
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The size distribution of Lipo/pB-DOX/ICG, Lipo/pB-DOX, and Lipo/ICG was measured using a Malvern Zetasizer Nano Series instrument (Nano ZS, Malvern Instruments, UK), and their morphology was observed by transmission electron microscopy (TEM, Titan G2-F20, FEI, USA). The aforementioned formulations were monitored by UV-vis-NIR spectroscopy (260-Bio, Thermo Fisher Scientific). After ultrafiltration centrifugation and organic solvent extraction, the encapsulation efficiencies (EE) and loading contents (LC) of pB-DOX and ICG in the liposomes were quantified and calculated using HPLC and UV-vis-NIR spectroscopy, respectively. UV-vis-NIR absorption at 784 nm was used to determine the ICG concentration.
To study the release behavior of pB-DOX and ICG in the liposomes, nanosuspension for each group (1 mL, containing 235 µg of pB-DOX and/or 150 µg of ICG) was sealed in a dialysis bag (MWCO: 3.5 kDa ) and dialyzed in 30 mL PBS with acidic pH (5.5) or 0.2 % Tween 80. At certain intervals, a 100 µL aliquot of the solution outside the bag was withdrawn for HPLC and UV-vis-NIR analyses.
To study the release behavior of pB-DOX and ICG in the liposomes, nanosuspension for each group (1 mL, containing 235 µg of pB-DOX and/or 150 µg of ICG) was sealed in a dialysis bag (MWCO: 3.5 kDa ) and dialyzed in 30 mL PBS with acidic pH (5.5) or 0.2 % Tween 80. At certain intervals, a 100 µL aliquot of the solution outside the bag was withdrawn for HPLC and UV-vis-NIR analyses.
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