The Use of Electrohydrodynamic Spraying to Disperse Hydrophobic Compounds in Aqueous Media

Figures & data

TABLE 1 Summary of cholesterol formulations investigated

Formulation ID	Content
ChoPlu	Cholesterol particles produced by electrospray were collected in 1% Pluronic F68 dissolved in 1X PBS
ChoCyc	Cholesterol particles produced by electrospray were collected in 25 mg/mL methyl-β -cyclodextrin dissolved in 1X PBS
ChoCycPlu	Cholesterol particles produced by electrospray were collected in 25 mg/mL methyl-β -cyclodextrin and 1% Pluronic F68 dissolved in 1X PBS
ChoCyc Mix	Cholesterol powder (Sigma C3045) was dissolved in 25 mg/mL methyl-β -cyclodextrin dissolved in 1X PBS by bulk mixing
WatCho Mix	Water soluble cholesterol powder (Sigma C4951) was dissolved in 1X PBS by bulk mixing
SyntheChol	SyntheChol™ NS0 supplement (Sigma S5442)

TABLE 2 Appearance of the cholesterol formulations

	Appearance
Formulation ID	Before filtration	After filtration
ChoPlu	milky	Slightly milky
ChoCyc	clear	clear
ChoCycPlu	clear	clear
ChoCyc Mix	clear	clear
WatCho Mix	clear*	clear*
SyntheChol	clear	clear

TABLE 3 Cholesterol nanoparticle sizes of all formulations before and after filtration

	Size distribution
	Before filtration		After filtration		A CR (avg.) at pinhole = 200 (kcps)
Formulation ID	Dp, effective (nm)	Poly-dispersity	Dp, effective (nm)	Poly-dispersity	Before filtration	After filtration
1% Pluronic in 1X PBS	870 ± 5	0.62 ± 0.04	NA ^a	NA ^a	27 ± 1	9 ± 0.1
25 mg/mL methyl-β-cyclodextrin in 1X PBS	1443 ± 242	0.67 ± 0.07	NA ^a	NA ^a	12 ± 2	5 ± 0.2
ChoPlu	284 ± 51	0.31 ± 0.02	149 ± 14	0.10 ± 0.04	5000 ± 37	1250 ± 24
ChoCyc	3159 ± 1007	0.20 ± 0.13	NA ^a	NA ^a	500 ± 9	9 ± 1
ChoCycPlu	254 ± 26	0.18 ± 0.02	188 ± 19	0.15 ± 0.03	1200 ± 13	330 ± 2
ChoCyc Mix	629 ± 19	0.54 ± 0.05	NA ^a	NA ^a	2500 ± 14	8.5 ± 0.2
WatCho Mix	NA ^a	NA ^a	NA ^a	NA ^a	NA ^a	NA ^a
SyntheChol	NA ^a	NA ^a	NA ^a	NA ^a	NA ^a	NA ^a

FIG. 1 A typical size distribution of cholesterol nanoparticles collected in 1% Pluronic F68/ 1X PBS (Dp, effective = 137 nm after sterilization with a 0.22 μ m PVDF filter, polydispersity = 0.066).

TABLE 4 Cholesterol concentration before and after filtration

	Cholesterol concentration (μ g/mL)
Formulation	Before	After	difference
ID	filtration	filtration	(μ g/mL)
ChoPlu	180*	150	30 (16.7%)
ChoCyc	199*	183*	26 (8.0%)
ChoCycPlu	147*	134*	38 (8.8%)
ChoCyc Mix	535	548	−13 (−2.4%)
WatCho Mix	1757	1679	78 (4.44%)
SyntheChol	NA ^+	1500	NA ^+

FIG. 2 (a) Growth of NS0 cells supplemented with SyntheChol, ChoPlu, ChoCycPlu, and ChoCyc (3 replicates). (b) Growth of NS0 cells supplemented with SyntheChol, ChoCyc Mix, and WatCho Mix (3 replicates). The * indicates that the correction factor of 10 was applied.

FIG. 3 Change of cholesterol concentration in medium before (– 24 h to 0– h) and after (0+ h till 144 h) NS0 cells were seeded. “–24”: right after adding supplements into T-flasks without seeding any NS0 cells. “–19”: 5 hours after adding supplements into T-flasks without seeding any NS0 cells. “0–”: 24 hours after adding supplements into T-flasks without seeding any NS0 cells. “0+”: right after NS0 cells were seeded. “24”: 24 hours after NS0 cells were seeded (Day 1). “48”: 48 hours after NS0 cells were seeded (Day 2). “72”: 72 hours after NS0 cells were seeded (Day 3). “96”: 96 hours after NS0 cells were seeded (Day 4). “120”: 120 hours after NS0 cells were seeded (Day 5). “144”: 144 hours after NS0 cells were seeded (Day 6). (a) Supplements were SyntheChol, ChoPlu, ChoCycPlu, and ChoCyc. (b) Supplements were SyntheChol, ChoCyc Mix and WatCho Mix. Each had 3 replicates. *Correction factor of 10 was applied and values have been multiplied by a factor of 5 for clarity.

FIG. 4 Growth of NS0 cells in 10 passages supplemented with 3 μ g/mL SyntheChol and 0.6 μ g/mL ChoCycPlu (3 replicates). Cholesterol nanoparticles provide cholesterol to NS0 cells and maintain long-term NS0 cell growth. *Correction factor of 10 was applied.

FIG. 5 Growth (a) and viability (b) of NS0 cells during the 10th passage in medium supplemented with 3 μ g/mL SyntheChol and 0.6 μ g/mL ChoCycPlu (3 replicates). *Correction factor of 10 was applied.