Formation of ion pairing as an alternative to improve encapsulation and anticancer activity of all-trans retinoic acid loaded in solid lipid nanoparticles

Figures & data

Figure 1 Chemical structure of RA (A), benethamine (B), stearylamine (C), and triethylamine (D).

Abbreviation: RA, all-trans retinoic acid.

Table 1 Influence of the amine type on the particle size, polydis-persion index, zeta potential and EE of RA in SLNs

Parameter	Without amine	SA	TA	BA
Mean diameter (nm)	155 ± 1	104 ± 4	96 ± 1	95 ± 1
Polydispersion index	0.24 ± 0	0.24 ± 0	0.3 ± 0.1	0.24 ± 0.01
Zeta potential (mV)	−30 ± 4	20 ± 2	−33 ± 4	−39 ± 0.2
EE (%)	36 ± 6	97 ± 2	90 ± 2	100 ± 1

Note: Data are shown as mean ± SD (n = 3).

Abbreviations: BA, benethamine; EE, encapsulation efficiency; RA, all-trans retinoic acid; SLN, solid lipid nanoparticle; SA, stearylamine; SD, standard abbreviation; TA, triethylamine.

Figure 2 Stability of encapsulation efficiency (EE) of RA in SLNs prepared with triethylamine (TA) or benethamine (BA).

Note: Data represent mean ± SD (n = 3).

Abbreviations: RA, all-trans retinoic acid; SD, standard deviation; SLN, solid lipid nanoparticle.

Figure 3 Cell viability studies, as evaluated by MTT assay, of blank SLNs, RA-SA-loaded SLNs, and free RA in cancer and normal cells, MCF-7 (A), Jurkat (B), HCT-116 (C), and PBMCs (D), after 48 hours of exposure.

Notes: Data were expressed as mean ± SD of three independent experiments. The blank SLNs were diluted at the same proportion as RA-loaded SLNs.

Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; RA, all-trans retinoic acid; PBMC, peripheral blood mononuclear cells; SA, stearylamine; SD, standard deviation; SLN, solid lipid nanoparticle.

Figure 4 Cell viability studies, as evaluated by MTT assay, of blank SLNs, RA-TA-loaded SLNs, RA-BA-loaded SLNs and free RA in MCF-7 cells after 48-hour exposure.

Notes: Data were expressed as mean ± SD of three independent experiments. The data for blank SLNs, which were diluted at the same proportion as RA-loaded SLNs, represent overlapping values for TA-based SLNs and BA-based SLNs. ^aSignificant difference compared to free RA; ^bsignificant difference between TA-based SLNs and BA-based SLNs.

Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; BA, benethamine; RA, all-trans retinoic acid; SLN, solid lipid nanoparticle; TA, triethylamine.

Table 2 Effects of different treatments of MCF-7 cancer cells on DNA fragmentation and cell-cycle stage distribution

Sample	Subdiploid content (%)	G0/G1 (%)	S (%)	G2/M (%)
Control	0.2 ± 0.1	60.5 ± 1.0	14.8 ± 0.7	24.2 ± 0.4
Free RA	0.4 ± 0.1	70.6 ± 0.5	10.8 ± 0.1	16.2 ± 0.3
SLNs with TA
Blank SLNs	0.4 ± 0.2	64.0 ± 0.6	16.8 ± 0.0	17.5 ± 0.7
RA-loaded SLNs	0.5 ± 0.2	73.0 ± 0.5	12.4 ± 0.6	14.1 ± 0.3
SLNs with BA
Blank SLNs	0.7 ± 0.1	62.8 ± 0.8	17.1 ± 0.4	19.3 ± 1.0
RA-loaded SLNs	6.1 ± 0.7	73.8 ± 0.6	6.8 ± 0.2	13.3 ± 0.9

Notes: MCF-7 cells were treated with RA-loaded SLNs, free RA, and unloaded (blank) SLNs, as indicated in the methods section. Cell-cycle distributions were determined after 48 hours of treatment. The data are representative of three independent experiments. Mean ± SD is shown.

Abbreviations: BA, benethamine; RA, all-trans retinoic acid; SLN, solid lipid nanoparticle; SD, standard deviation; TA, triethylamine.

Figure 5 DNA fluorescence histograms of PI-stained MCF-7 cells after 48 hours of incubation under various experimental conditions: medium alone (A), blank SLNs (B), free RA (C), or BA-RA-loaded SLNs (D).

Notes: RA was used at 25 μM. BA-RA-loaded SLNs induce cell-cycle arrest accompanied by reduction in the S phase in MCF-7 cells. Data from one representative experiment that represents 20,000 events (cells) are shown.

Abbreviations: BA, benethamine; PI, propidium iodide; RA, all-trans retinoic acid; SLN, solid lipid nanoparticle.