Ovarian Accumulation of Nanoemulsions: Impact of Mice Age and Particle Size

Abstract

Nanotechnology in the field of drug delivery comes with great benefits due to the unique physicochemical properties of newly developed nanocarriers. However, they may come as well with severe toxicological side effects because of unwanted accumulation in organs outside of their targeted site of actions. Several studies showed an unintended accumulation of various nanocarriers in female sex organs, especially in the ovaries. Some led to inflammation, fibrosis, or decreasing follicle numbers. However, none of these studies investigated ovarian accumulation in context to both reproductive aging and particle size. Besides the influences of particle size, the biodistribution profile may be altered as well by reproductive aging because of reduced capacities of the reticuloendothelial system (RES), changes in sex steroid hormone levels as well as altering ovarian stromal blood flow. This systematic investigation of the biodistribution of intravenously (i.v) injected nanoemulsions revealed significant dependencies on the two parameters particle size and age starting from juvenile prepubescent to senescent mice. Using fluorescent in vivo and ex vivo imaging, prepubescent mice showed nearly no accumulation of nanoemulsion in their uteri and ovaries, but high accumulations in the organs of the RES liver and spleen independently of the particle size. In fertile adult mice, the accumulation increased significantly in the ovaries with an increased particle size of the nanoemulsions by nearly doubling the portion of the average radiant efficiency (PARE) to ~10% of the total measured signal of all excised organs. With reproductive aging and hence loss of fertility in senescent mice, the accumulation decreased again to moderate levels, again independently of the particle size. In conclusion, the ovarian accumulation of these nanocarriers depended on both the age plus the particle size during maturity.

Keywords: accumulation; biodistribution; nanoemulsion; nanotoxicology; optical imaging; ovaries; reproductive aging.

Figure 1

Composition of the chosen nanoemulsion for this in vivo study.

Figure 2

Physicochemical properties of the four different sized nanoemulsions, determined by three individual produced batches.

Figure 3

IC50 as total mass nanoemulsion (DiR loaded MCT, Kolliphor^® HS 15 + aqueous phase) per ml cell culture media on 3T3 and NHDF fibroblasts after 24 h of incubation, determined by three individual incubated batches (eight replicates per run).

Figure 4

PARE in plasma and blood cell fractions after 4 h of incubation, determined by three individual incubated batches.

Figure 5

Noninvasive in vivo fluorescence images of representative mice directly after (5–10 min) and 24 h after i.v. injection in the tail vein of the four nanoemulsions in juvenile prepubescent (age 3–4 weeks), adult (age 12–39 weeks), and senescent mice (age > 48 weeks).

Figure 6

Combined 3D bar chart (mean PARE of the five mice per group) and scatter chart (individual PARE values of each mouse) of the ex vivo excised organs and blood, 24 h after i.v. injection of the four nanoemulsions in juvenile prepubescent (age 3–4 weeks), adult (age 12–39 weeks), and senescent mice (age > 48 weeks).

Figure 7

Logarithmic contour plots of the mean PARE for the organs with the highest accumulations ((a) liver, (b) spleen, (c) uterus + ovaries, (d) heart, (e) stomach, (f) kidneys, and (g) caecum) plotted against the particle size of the i.v. injected nanoemulsions and the exact age of the mice displayed as ●.

Figure 8

Ex vivo fluorescence images of the excised organs with the highest accumulation (liver, spleen, uterus +ovaries, heart, stomach, kidneys, and caecum) of the representative mice.

Figure 9

Experimental design of the animal trials: (a) time points of the single-dose i.v. injections with the nanoemulsions in the age groups juvenile, adult, and senescent, including the onset of puberty [24,25,26], the onset of declining fertility, and nearly ceased fertility of female mice [21,23,28,29,30,31,32,33]; (b) general experimental procedure of the animal trials.