Cyclohexane

GF19 was loaded in solid silica nanoparticles (SiNPs) as we previously described for its parent peptide, LL37^{33 (link)}. Briefly, 6.7 mL of Triton X-100 (Sigma-Aldrich, Oakville, ON, Canada) was dissolved in 20 mL of cyclohexane (VWR International, Mississauga, ON, Canada) in a round-bottomed flask immersed in oil bath at 50 °C. Then, 8 mg of GF19 was dissolved in 2 mL distilled water and mixed with cyclohexane-Triton X-100 solution to generate reverse w/o microemulsion. Next, 2.5 mL of TEOS (VWR International, Mississauga, ON, Canada) was dropped slowly followed by 0.33 mL of ammonium hydroxide solution. The reactants were stirred for 48 h at 50 °C. The as prepared SiNPs were washed with ethanol (ChapTec, Montreal, QC, Canada) thrice followed by distilled water and then lyophilized for storage. They were reconstituted with PBS for further use.

Acetic anhydride, acetic acid, sulphuric acid (96%), iron(III) chloride, potassium iodide and mercury(II) chloride were purchased from Kemika (Zagreb, Croatia). Methanol, dichloromethane, ethyl acetate, cyclohexane and potassium peroxodisulphate were obtained from VWR Chemicals (Radnor, PA, USA) while chloroform was obtained from Honeywell (Charlotte, NC, USA). ABTS and linoleic acid were purchased from Alfa Aesar (Kandel, Germany). Trolox^® was obtained from Acros Organics (Geel, Belgium). DPPH, β-carotene and lipoxidase type I-B from soybean were purchased from Sigma-Aldrich (Darmstadt, Germany). Finally, Dulbecco’s modified Eagle medium (DMEM), foetal bovine serum (FBS), L-glutamine, penicillin and streptomycin were obtained from Capricorn Scientific (Ebsdorfergrund, Germany).

PFO, synthesized by Suzuki coupling, was supplied by Cambridge Display Technology and used as received. The polymer had a number‐average molecular weight
Mn = 18 × 10³ g mol⁻¹ and a polydispersity index = 2.7, as determined by polystyrene‐equivalent gel permeation chromatography. The absolute molecular weight was calculated by scaling
Mn down by a factor of 2.7 due to the higher relative chain stiffness of PFO,17 giving an estimate of 17 fluorene repeat units per average chain. Toluene (HPLC grade, >99.7%, VWR), decahydronaphthalene (“decalin”; reagent grade, mixture of cis and trans isomers, Sigma‐Aldrich), cyclohexane (HPLC grade, >99%, VWR), and 2‐(iso‐)propanol (“IPA”; >99.5%, Sigma‐Aldrich) were used as received.

Chemicals such as ammonium hydroxide, cyclohexane, and tetraethyl orthosilicate (TEOS), were purchased from VWR International (Mississauga, Ontario, Canada). Triton X-100 detergent was acquired from Sigma-Aldrich. Ultra-pure water (Millipore Elix Essential Water Purification System) was used for all experiments.
GF19 was encapsulated within silica nanoparticles (SiNPs) as we described for its parent peptide, LL37^{21 (link)}. In a round-bottomed flask, immersed in an oil bath at a temperature of 50 °C, a solution was prepared by dissolving 4 mL of Triton X-100 in 12 mL of cyclohexane. Next, a solution containing 4.8 mg of GF19 was dissolved in 2 mL of distilled water and subsequently combined with a cyclohexane-Triton X-100 solution in order to produce a reverse water-in-oil (w/o) microemulsion. Subsequently, a gradual addition of 1.5 mL of TEOS was performed, followed by the addition of 0.2 mL of ammonium hydroxide solution. The reactants underwent agitation for a duration of 48 h at a temperature of 50 ℃. SiNPs were initially treated with ethanol three times, followed by rinsing with distilled water and then lyophilization for the purpose of preservation.

The dimensional distribution of lactose samples and APIs was determined by laser light diffraction using a Spraytec analyzer (Malvern, UK). Two different procedures were adopted.
In the first case, a supersaturated solution of lactose in ethanol was prepared by adding 5 g of lactose to 1 L of ethanol (96% v/v). The solution was stirred overnight and then filtered under vacuum through a nylon membrane filter (0.45 µm, Ø 47 mm, Whatman^®, GE Healthcare Life Sciences, Little Chanfont, UK). The filtered solution was used as dispersing liquid for the analysis. Furthermore, 100 mg of each sample were dispersed in 10 mL of the lactose-saturated ethanol solution and put in an ultrasound bath (40kHz) for 5 min before the analysis. Each measurement was performed in triplicate with an 8% obscuration threshold. To analyze the APIs, cyclohexane (VWR, Ismaning, Germany) was chosen as dispersing liquid. Then, 10 mg of drug were dispersed in a 0.1% w/v solution of Span^® 85 Croda Inc., Newark, NJ, USA) in cyclohexane and sonicated for 5 min before being analyzed. Each measurement was repeated three times with an 8% obscuration threshold.