Opalescence

For host range determination, a double agar overlay plaque assay method [29] was employed. Phage isolation, plating and titering were carried out as described previously [30] . Further purification using a CsCl step gradient was performed [31] with few modifications. The phage suspension was deposited on the top of CsCl step gradient (densities: 1.1 g/ml, 0.9 g/ml, 0.7 g/ml, 0.5 g/ml) and centrifuged in a Spinco SW39 rotor for 3 h at 24,000 rpm, 11°C. The resulting phage band with the highest opalescence was collected with a syringe and dialyzed against three changes of SM buffer (100 mM NaCl, 8 mM MgSO₄, 50mM Tris-HCl, pH 7.5) at 4°C. The adsorption tests and one-step growth experiments were carried out as described by Khusainov et al. [32] and Carlson and Miller [33] , respectively. Meanwhile determination of the efficiency of plating (e.o.p.) was performed as described previously [34] (link). High-titer phage stocks were diluted and plated in duplicate. Plates incubated at 18, 22, 24, 26, 28, 30, 32, 34 and 36°C were read after 24–48 hours of incubation. The temperature at which the largest number of plaques were formed was taken as the standard for the e.o.p. calculation.

ST-CS NP were prepared using the innovative technique called “cold dilution of microemulsion” [25 ]. This technique involves the preparation of an O/W microemulsion (µE) using a partially water-soluble organic solvent as disperse oil phase. Following the dilution of µE with water, the solubilization of the organic solvent in water occurs, with the consequent NP precipitation. In our experimental conditions, butyl lactate (BL) was chosen as partially water-soluble organic solvent.
Different O/W µE were prepared using cholesterol (CHOL) dissolved in water-saturated BL (s-BL) as internal phase, BL-saturated water (s-water) as external phase, and various surfactants and cosurfactants. ST-CS was pre-solubilized in benzyl alcohol (BA) and then added in the dispersed phase. The resulting µE compositions are reported in Table 1. µE were diluted with water to obtain the precipitation of ST-CS NP.
ST-CS NP suspensions were purified by gel chromatography using agarose cross-linked gel (Sepharose^® CL 4B) as stationary phase. Briefly, 1 mL ST-CS NP was introduced at the head of a 10 mL-column and eluted by gravity with hypertonic PBS (8.0 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na₂HPO₄ 2H₂O, 0.24 g/L KH₂PO₄). Fractions of 1 mL each were collected. The opalescent fractions containing purified ST-CS NP were concentrated under nitrogen up to 1 mL final volume.
To verify the effective presence of ST-CS on NP surface, FITC-ST-CS NP were prepared introducing FITC-ST-CS in µE1.

Aortas, carotid arteries and SMA were dissected and incubated for NO production for 30 min in Krebs–Hepes buffer containing: BSA (20.5 g/L), CaCl₂ (3 mM) and L-Arginine (0.8 mM) (Sigma-Aldrich). NaDETC (1.5 mM) and FeSO₄.7H₂O (1.5 mM) (Sigma-Aldrich) were separately dissolved under argon gas bubbling in 10 mL volumes of ice-cold Krebs–Hepes buffer. These were rapidly mixed to obtain a pale yellow-brown opalescent colloid Fe(DETC)₂ solution (0.4 mM), which was used immediately. The colloid Fe(DETC)₂ solution was added to vessels and incubated for 45 minutes at 37°C. Then, arteries were immediately frozen in plastic tubes using liquid N₂. NO measurement was performed on a table-top x-band spectrometer Miniscope (Magnettech, MS200; Berlin, Germany). Recordings were made at 77°K, using a Dewar flask. Instrument settings were 10 mW of microwave power, 1 mT of amplitude modulation, 100 kHz of modulation frequency, 150 s of sweep time and 3 scans. Signals were quantified by measuring the total amplitude, after correction of baseline as done previously [23] (link). Values are expressed in unit/mg weight of dried tissue.
For O₂⁻ spin-trapping, aortas, carotid arteries and SMA were dissected and allowed to equilibrate in deferoxamine-chelated Krebs-Hepes solution containing 1-hydroxy-3methoxycarbonyl-2,2,5,5-tetramethylpyrrolidin (CMH, Noxygen; Denzlingen, Germany) (500 µM), deferoxamin (25 µM, Sigma-Aldrich) and DETC (5 µM, Sigma-Aldrich) under constant temperature (37°C) for 60 minutes. Then, they were frozen in liquid N₂ and analyzed in a Dewar flask by EPR. Values are expressed in unit/mg weight of dried tissue.

Three milligrams of RH, 9.42 mg of BCL, 331 mg of Labrafil M 1944CS, 580 mg of HS15, 193 mg of PEG400, and 122 mg of Bor-PEG₄₀₀-LA were stirred strongly at 37 °C overnight. 5 mL of ultrapure water was added dropwise until the solution became homogeneous and transparent with opalescence [21 (link)]. Sd III-M and Bor/C6-M were prepared as the similar methods mentioned above, with the exception of replacing the drugs with corresponding probes. The particle size and zeta potential of different microemulsion formulations were measured by dynamic light scattering (DLS, Nano ZS, Malvern, UK). The morphology was evaluated by transmission electron microscopy (TEM, Tecnai 12, Philips, Amsterdam, Netherlands) following previously established methods. Briefly, 15 μL of each sample was deposited on a carbon-coated copper mesh and stained with 1% (v%) phosphotungstic acid. After being dried under infrared light, the sample was observed by TEM. The encapsulation efficiency (EE) and drug loading capacity (LC) of RH and BCL were calculated by the following equations, $$Encapsulationefficiency\left(E,E,\%\right)=\frac{W_{\left(t,e,s,t,e,d,,d,r,u,g\right)}}{W_{\left(f,e,e,d,i,n,g,,d,r,u,g\right)}}\times 100\%;$$ $$Loadingcapacity\left(LC\%\right)=\frac{W_{\left(testeddrug\right)}}{W_{\left(totalmicroemulsion\right)}}\times 100\%,$$ where W_{tested drug}, W_{feeding drug}, and W_{total microemulsion} represent the weight of the tested drug, initial feeding drug, and total microemulsion, respectively. Bor/RB-M were loaded into centrifuge tubes and centrifuged at 13,000 rpm for 10 min to observe any stratification of the microemulsion. Bor/RB-M was placed in PBS at pH 7.4 and the temperature was set at 25 °C. The changes in particle size and PDI of the microemulsions were measured with the DLS on day 1, 3, 5, and 9 to evaluate the stability of the microemulsions. RH and BCL were quantified by high-performance liquid chromatography (HPLC) by the chromatographic conditions reported previously [22 (link)].

The optical parameters—translucency (TP), contrast ratio (CR), and opalescence (OP)—were recorded under D65 standard illumination on polished and glazed surfaces before and after aging using a spectrophotometer (Easyshade IV; Vita Zahnfabrik, Bad Säckingen, Germany); it is a clinical device that only works in “tooth mode” [37 (link)]. The instrument was calibrated before each measurement, and the probe tip was held at 90° to the surface of the sample. The recording was accepted when two identical consecutive readings were obtained on each area (cervical, incisal, medial) in three randomly selected zones of the polished or glazed surfaces. The measurements were performed by a single operator.
To determine the CIE L*a*b* coordinates—the black (b) and white (w) background of the grey card WhiBal G7 (White Balance Pocket Card) was used.
L* is the lightness coordinate (L* = 0 perfect black, L* = 100 perfect white); a* = is the chromatic coordinate in the red (positive value)/green (negative value) axis, and b* = is the chromatic coordinate in the yellow (positive value)/blue (negative value) axis [38 ,39 (link),40 ].
TP values result by calculating the color difference on black and white backgrounds according to the formula:
TP values may range from 0 (totally opaque) to 100 (totally transparent).
CR defines the opacity and is calculated over a black and white background according to the formula:
CR values may range from 0 (totally transparent) to 1 (totally opaque).
OP values estimate the difference in red–green and yellow–blue color coordinates between transmitted and reflected light; the values are obtained using the formula:
ΔE*—The total color change value, signifying the color difference between two stages, was achieved using the formula:
The recordings were performed for each group in three areas (cervical, medium, incisal).
To report the color change to a clinical standard, ΔE* was converted to NBS units (NBS—The National Bureau of Standards) according to the formula: NBS = ΔE* × 0.92 [34 ,35 ,36 (link),37 (link)]. In conformity with NBS, the levels of color changes (expressed in NBS units) are: extremely slight change (0.0–0.5), slight change (0.5–1.5), perceivable (1.5–3.0), marked change (3.0–6.0), extremely marked change (6.0–12.0), and change to another color (12.0–more).

VS, PDI, and ZP are crucial parameters for the selection of the optimized transferosomal formulation, and they have an impact on the stability and quality of the formulated transferosomes. These parameters were measured using ZetaSizer Nano ZS (Malvern Instrument Ltd., Worcestershire, UK). Each of the prepared formulations was diluted with distilled water (1:10 v/v) until obtaining a faint opalescent appearance with suitable intensity for light scattering. The measurements were performed in duplicates, and the outcomes were expressed as mean ± SD.