A diode array spectrophotometer (Agilent 8453) was used to measure the various absorption spectra and kinetic profiles for the irradiation and calibration experiments. This spectrophotometer was equipped with a 1-cm cuvette sample holder and a Peltier system model Agilent 8453 for temperature control. As such, the sample was kept at 22C, stirred continuously during the reaction monitored experiment, and completely shielded from ambient light. The spectrophotometer was monitored by an Agilent 8453 Chemstation kinetics-software.
Agilent 8453
Manufactured by Agilent Technologies
Sourced in United States, Germany, China
The Agilent 8453 is a diode array spectrophotometer designed for a wide range of applications in analytical chemistry. It is capable of acquiring UV-Vis absorption spectra from 190 to 1100 nanometers with a fast analysis time.
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Lab products found in correlation
Nicolet is50, by Thermo Fisher Scientific (3 mentions)
Xrd dmax 2200 system, by Rigaku (2 mentions)
S 4700, by Hitachi (2 mentions)
Vertex 70, by Bruker (2 mentions)
V 670, by Jasco (2 mentions)
Spectrum 1 model, by PerkinElmer (2 mentions)
F 7000, by Hitachi (2 mentions)
X series 2, by Thermo Fisher Scientific (2 mentions)
Zetasizer nano z, by Malvern Panalytical (2 mentions)
Free base octabutoxyphthalocyanine h2pc obu 8, by Merck Group (2 mentions)
Toluene hplc grade, by Thermo Fisher Scientific (2 mentions)
Qe6500, by OceanOptics (1 mentions)
Nir quest, by OceanOptics (1 mentions)
Zetasizer 3000hs, by Malvern Panalytical (1 mentions)
Ph meter glp 22, by Crison (1 mentions)
Pc hp 845x uv visible system, by Agilent Technologies (1 mentions)
Tensor 27 system fourier transform infrared spectrometer, by Bruker (1 mentions)
S 3000n scanning, by Hitachi (1 mentions)
Rf 5301pc, by Shimadzu (1 mentions)
Asap 2020, by Micromeritics (1 mentions)
112 protocols using agilent 8453
1
Diode Array Spectrophotometry for Kinetics
2
Peroxidase Activity Quantification Using ABTS
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HRP activity was estimated following the technique reported by Sanders et al. [48 (link)], employing ABTS as a reducing substrate—similar to our previous papers [8 (link),35 (link)]. The rate of change in solution absorbance at 405 nm was monitored with an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies Deutschland GmbH, Waldbronn, Germany). The HRP concentration in the quartz spectrophotometric cuvette was 10−9 M, while the concentrations of H2O2 and ABTS were 0.3 mM and 2.5 mM, respectively. Spectrum acquisition was started immediately after the addition of H2O2.
3
Supersaturating Dissolution Studies of Ketoconazole
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Supersaturating non-sink dissolution studies were conducted using the MiniDissolution apparatus, a miniaturized USP II apparatus with a volume of 20 ml (28 (link)). The paddle speed was constant for all experiments at 100 rpm and the temperature was set to 37 °C. One dissolution experiment was conducted at a static pH of 6.8 using phosphate buffer for 3 h. Additionally, a pH-shift dissolution experiment was carried out starting at pH 1 using 0.1 M hydrochloric acid for 1 h, followed by an adjustment to pH 6.8 with 1.9 ml of a buffer concentrate consisting of 0.375 M phosphate buffer and 0.85 M sodium hydroxide. ASDs of each particle size fraction equivalent to 8 mg ketoconazole were used for the dissolution experiments (theoretical concentration of 400 μg/ml). The L100–55 ASD at a drug load of 10% was also filled into enteric capsules (Vcaps Enteric®) prior the pH-shift experiment.
The ketoconazole concentration was measured online using a diode array UV/VIS spectrophotometer (Agilent 8453, Agilent Technologies GmbH, Waldbronn, Germany) with correction for light scattering.
The ketoconazole concentration was measured online using a diode array UV/VIS spectrophotometer (Agilent 8453, Agilent Technologies GmbH, Waldbronn, Germany) with correction for light scattering.
4
Quantification of Residual PVA on NPs
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The residual mass of PVA attached to the NP surface after washing was determined by a colorimetric method based on the reaction with iodine. A sample of freeze-dried NP (2.0 mg) was incubated in 0.5 M NaOH for 15 minutes at 60 °C, neutralised by addition of 1.0 M HCl and the final volume adjusted to 5.0 ml with water. To each NP sample, 3.0 ml of iodine reagent (0.65 M solution of boric acid, 0.5 ml of a solution of I2/KI (0.05 M/0.15 M)) and 1.5 ml of water were added. The absorbance of each sample was measured using visible spectroscopy (Agilent 8453, Agilent Technologies, Palo Alto, CA, USA) at 690 nm after 15 minutes incubation and compared to a standard plot of known PVA concentration (23) . Residual PVA was expressed as a percentage mass fraction of the total NP mass.
5
Photoluminescence Characterization of Quantum Dots
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Photoluminescence spectra were acquired using a fiber fluorescence spectrophotometer (Ocean Optics NIR Quest and QE6500, Ocean Optics, Largo, FL, USA) equipped with a HGIL T250 250 W double-grating monochromator. Absorption spectra were acquired on a UV-Vis spectrophotometer (Agilent 8453, Agilent, Santa Clara, CA, USA). Quantum yield (QY) was calculated using the following equation:
where Ф is the quantum yield, I is the measured integrated emission intensity, η is the refractive index of the solvent, and A is the optical density. The subscript “st” refers to the standard with known quantum yield and “x” refers to the QD sample. Fluorescence spectra were measured at excitation wavelengths below 785 nm. IR-26 dye dissolved in 1,2-dichloroethane was used as a reference.
where Ф is the quantum yield, I is the measured integrated emission intensity, η is the refractive index of the solvent, and A is the optical density. The subscript “st” refers to the standard with known quantum yield and “x” refers to the QD sample. Fluorescence spectra were measured at excitation wavelengths below 785 nm. IR-26 dye dissolved in 1,2-dichloroethane was used as a reference.
6
Dissolution Kinetics of Acetylthiocholine Filaments
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Dissolution studies of B50 and T50 filaments were carried out in a USP Apparatus II (Sotax AT7 smart, Allschwil, Switzerland) with a rotation speed of 50 rpm following the method described by Linares et al. [24 (link)]. 900 mL of distilled water at 37 ± 0.5 °C were employed as dissolution medium. About 250 mg of filament were analyzed by triplicate. 5 mL of filtered samples were withdrawn at specific interval times (15, 30, 45, 60, 120, 180, 240, 300, 360, 420, 480, 1440 min) and no fresh media was added. The percentage of AT released was measured on a UV–Vis spectrophotometer Agilent 8453 (Agilent Technologies, Santa Clara, CA, USA) at 272 nm. Taking into account the quantity of AT in the filaments (125 mg) and the existing volume during the study (considering the volume removed at each time point), less than 10% of the value of AT solubility (11.32 mg/mL) [27 (link)] was present in the medium during the whole study. So, we can confirm that the sink conditions have been maintained.
7
Characterization of Soluplus-Pluronic Micelles
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Micelles were formed by dispersing Soluplus and Pluronic P103 in different concentrations (0.1%, 0.01%, 1%, 2%, 3%, 4%, 5% w/v) in 0.9% NaCl. Also, Soluplus and Pluronic P103 at 10% (w/v) were prepared in pH 6.4 buffer and 0.9% NaCl aq solution. They were kept under magnetic stirring for 12 h. Micelles containing natamycin (0.4 mg drug/mL dispersion) were prepared to compare with unloaded micelles.
Size, zeta potential, and polydispersion index (PDI) were measured using Zetasizer® 3000HS (Malvern Instruments, Malvern, UK). The pH was recorded using pH meter GLP22 (Crison Instruments, L’Hospitalet de Llobregat, Spain). Micelle stability against dilution was recorded for dispersions of Pluronic P103 containing natamycin, which were poured into quartz cells that contained either 0.9% NaCl or pH 6.4 buffer for a sudden 30-fold or 60-fold dilution. The absorbance was recorded at 304 nm every 30 s for 30 min (UV-Vis spectrophotometer Agilent 8453, Waldbronn, Germany). All experiments were carried out in triplicate.
Size, zeta potential, and polydispersion index (PDI) were measured using Zetasizer® 3000HS (Malvern Instruments, Malvern, UK). The pH was recorded using pH meter GLP22 (Crison Instruments, L’Hospitalet de Llobregat, Spain). Micelle stability against dilution was recorded for dispersions of Pluronic P103 containing natamycin, which were poured into quartz cells that contained either 0.9% NaCl or pH 6.4 buffer for a sudden 30-fold or 60-fold dilution. The absorbance was recorded at 304 nm every 30 s for 30 min (UV-Vis spectrophotometer Agilent 8453, Waldbronn, Germany). All experiments were carried out in triplicate.
8
Solubilization of Alpha-Linolenic Acid using AOT and Soluplus
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AOT and Soluplus solutions (2 mL) were placed in Eppendorf tubes and ALA was added in excess (approximately 8 mg for AOT solutions and 50 mg for Soluplus solutions). Solubility in water was also tested. All systems were kept under stirring for 72 h at 650 rpm and 25 C. Then, they were centrifuged at 14,000 rpm and 25 C for 20 min, to separate non-solubilized ALA. Absorbance of the supernatants was measured at 334 nm (UV/VIS spectrophotometer Agilent 8453, Germany). Soluplus systems were previously diluted (0.75/10 mL in ethanol:water 50:50 vol/vol). ALA concentration was calculated from the absorbance using a calibration curve prepared with solutions of ALA in water-ethanol 50:50 vol/vol mixtures.
Data from the solubility study were used to calculate the following parameters 29, 30 :
(a) Molar solubilization capacity, or moles of drug that can be solubilized per mol of copolymer forming micelles.
(b) Micelle-water partition coefficient, which is the ratio of the drug concentration in the micelle to the drug concentration in water.
(c) Molar micelle-water partition coefficient, which eliminates the dependence of P on the copolymer concentration, assigning a copolymer concentration of 1 M.
(d) Standard-free Gibbs energy of solubilization, estimated from the molar micelle-water partition coefficient (PM).
Data from the solubility study were used to calculate the following parameters 29, 30 :
(a) Molar solubilization capacity, or moles of drug that can be solubilized per mol of copolymer forming micelles.
(b) Micelle-water partition coefficient, which is the ratio of the drug concentration in the micelle to the drug concentration in water.
(c) Molar micelle-water partition coefficient, which eliminates the dependence of P on the copolymer concentration, assigning a copolymer concentration of 1 M.
(d) Standard-free Gibbs energy of solubilization, estimated from the molar micelle-water partition coefficient (PM).
9
UV-Vis Spectrophotometric Sample Analysis
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Water-bath, reflux condenser, UV/Vis spectrophotometer Agilent 8453 (Agilent, Karlsruhe, Germany) with PC-HP 845x UV-Visible System (Agilent, Karlsruhe, Germany) and 1 cm quartz cells were used for all absorbance measurements. Filtration of prepared sample solutions was performed by using 0.20 µm Minisart-plus membrane filter (Sartorius AG, Goettingen, Germany).
10
Drug Release Kinetics of DOX-PLGA Nanoparticles
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Triplicate samples of DOX-PLGA, DOX-PLGA (CHI/ALG)1, DOX-PLGA (CHI/ALG)2 and DOX-PLGA (CHI/ALG)3 NPs were respectively suspended in 1 mL of isotonic phosphate-buffered saline (PBS; 0.05 M, pH 7.4) in a microcentrifuge tube. The samples were then incubated on a shaker at a fixed speed of about 100 rpm (SHY-2A, Jin Tan, People’s Republic of China) at 37°C. At defined time intervals, the NPs were centrifuged, the supernatant was removed, and fresh phosphate buffer was replaced. The concentration of each supernatant sample was measured by an ultraviolet spectrophotometer (Agilent 8453; Agilent Technologies, Santa Clara, CA, USA) at 480 nm. The cumulative percentage of the drug released in the first hour was expressed as the initial burst release. The time at which half of the final theoretically encapsulated drug released was expressed as the half release time (t1/2).
Triplicate samples of DOX-PLGA (CHI/ALG)3 NPs were suspended in 1 mL PBS at different pH values (pH 7.4, pH 6.8 and pH 5.0) in a microcentrifuge tube, and other measurements were performed as described above.
Triplicate samples of DOX-PLGA (CHI/ALG)3 NPs were suspended in 1 mL PBS at different pH values (pH 7.4, pH 6.8 and pH 5.0) in a microcentrifuge tube, and other measurements were performed as described above.
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