Ibuprofen sodium salt

Ibuprofen sodium salt and
poly(vinyl alcohol) 9–10 kDa were purchased from Sigma-Aldrich
(Dorset, U.K.). Itraconazole (purity, 98%) and Tween 80 were purchased
from Tokyo Chemical Industry (Oxford, U.K.). Lutrol F108 and Pluronic
F88 Pastille were provided by BASF (Ludwigshafen, Germany). Poly(vinyl
pyrrolidone) (PVP) 58 kDa and PVP 90 kDa were provided by Ashland
(Kidderminster, U.K.). Ultrapure water was obtained from a water purification
system (Elga PURELAB DV 25, Veolia Water Systems, Dublin, Ireland).
All other chemicals and materials were of analytical grade and purchased
from Sigma-Aldrich (Dorset, U.K.) or Fisher Scientific (Loughborough,
U.K.). Full-thickness neonatal porcine skin was obtained from stillborn
piglets in less than 24 h post-mortem and stored
at −20 °C until use.

Arthritis was induced with two i.p. injections of 100 μl of K/BxN serum on days 0 and 2. Disease was monitored daily until day 8 by assessing the paw volume using a plethysmometer (Ugo Basile, Comerio, Italy), disease incidence (mice showing any signs of arthritis) and clinical score (score per paw: 0 = no signs of inflammation; 1 = subtle inflammation, localized; 2 = easily identified inflammation but localized; 3 = evident inflammation, not localized; max score = 12 per mouse) [23 (link)]. Pharmacological treatments were administered i.p. twice daily from day 2 until the end of the experiment. Fenoprofen calcium salt hydrate and ibuprofen sodium salt were obtained from Sigma-Aldrich (Poole, UK).

Poly(vinyl
alcohol) (PVA)
Mw 85,000–124,000, 99% hydrolyzed; poly(vinyl methyl ether-alt-maleic anhydride) (PVME-MA) Mw 216,000; poly(vinyl methyl
ether) (PVME) 50 wt % in water; multiwalled carbon nanotubes (MWCNT)
(≥98%, Aldrich, OD × ID × L: 10
± 1 nm × 4.5 ± 0.5 nm × 3–6 μm);
sodium dodecyl sulfate (SDS); 5-fluorouracil (5-FU) 99%; and ibuprofen
sodium salt ≥98% were purchased from Sigma-Aldrich. The PVME-MA
reagent was dissolved in water and thermally treated at 70 °C
for 6 h to obtain poly(vinyl methyl ether-alt-maleic
acid) (COP).^{20 (link)} Functionalized multiwalled
carbon nanotubes (MWCNT-f) were obtained by the microwave-assisted
treatment of MWCNT with a strong acid mixture, as reported previously
by our research group.^{27 (link)} In that work,
TEM images revealed that the nanotubes preserved their integrity without
severe changes in their surface morphology and length. All aqueous
solutions were prepared with deionized water (DI), which was obtained
by a Milli-Q Organex system (Millipore).

All reagents used in the study were commercially available and used without prior purification. L-Phenylalanine (99%) was provided by Alfa Aesar (Ward Hill, MA, USA). Chlorotrimethylsilane (TMSCl) (99%), ibuprofen sodium salt (98%), and acetonitrile for HPLC (99%) were provided by Sigma-Aldrich (Darmstadt, Germany). Pol-Aura (Morąg, Poland) provided ethanol (96%). The ammonium hydroxide (25%) was provided by P.P.H. Stanlab (Lublin, Polska) sp. j. Diethyl ether (99.5%) and anhydrous sodium sulfate (Na₂SO₄) (99%) were provided by Chempur (Piekary Śląskie, Polska). Ibuprofen (98%) was supplied by AmBeed (Arlington Heights, IL, USA), chloroform (≥99.9%) and triethanolamine (≥99.9%) were supplied by Eurochem BGD sp. z.o.o (Tarnów, Poland), and Efavit provided hemp oil. Span 80 and Tween 60 were purchased by Croda. Carbomer (Carbopol^® 940 NF Polymer) was purchased from Lubrizol (Wickliffe, OH, USA). The commercial products (in gel and cream form) were purchased (Dolorgiet Pharmaceuticals, Sankt Augustin, Germany).

To thoroughly investigate the relationship occurring between drug loading and changes in micelle hydrodynamic diameter, dynamic light scattering (DLS) measurements were performed on CHP407 samples loaded or not-loaded with drugs characterised by a different wettability. Specifically, CHP407 was first dissolved at 0.5% w/V in physiological saline solution (0.9% NaCl); then Ibuprofen (IBU, Sigma Aldrich, Italy) or Ibuprofen Sodium Salt (IBUSS, Sigma Aldrich, Italy), as hydrophobic or hydrophilic drug, respectively, was added to PEU solution at 2, 5, 10, or 20 mg/mL concentration. Analyses were performed at 25, 30, and 37°C, according to the protocol recently published by Laurano et al. (2020) (link), using a Zetasizer Nano S90 (Malvern Instruments, Worcestershire, United Kingdom) instrument. Before starting, samples were equilibrated at the test temperature for 15 min and then analysed according to Pradal et al. (2013) (link). The reported hydrodynamic diameters resulted from the average of three different analysed samples. Data are reported as mean ± standard deviation.
Hereafter, drug loaded-CHP407 aqueous solutions (0.5% w/V) will be referred to as CHP407_IBU_Xmg/mL and CHP407_IBUSS_Xmg/mL, where X stands for the amount of encapsulated drug.

Twenty-four hours before the experiment, 5.10⁴ EC or 2.10⁴ FB were seeded in each well of a 24-well plate. On the day of the experiment, cells were washed twice with PBS and exposed to Pg-LPS stimulation at a concentration of 1 μg/mL. Ibuprofen sodium salt (Sigma, St-Quentin, France) was used as the experimental drug at a concentration of 50 µg/mL.