L-glutamic acid-g-poly hydroxyethyl methacrylate nanoparticles: acute and sub-acute toxicity and biodistribution potential in mice

We used the L-glutamic acid-g-p(HEMA) nanoparticles synthesised and characterised in our previous study (6). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA).

For the study we used 6–8 weeks old BALB/c albino mice (n=44) obtained from the Bornova Veterinary Control and Research Institute, Izmir, Turkey. During the 7 days of acclimation and throughout the experiment, the mice had free access to standard laboratory pellet feed (Arden Araştırma & Deney, Ankara, Turkey) and drinking water under the following controlled ambient conditions: temperature 22±2 °C, humidity 55±5 %, and 12/12 h day-night light cycles. The protocol was approved by the Ege University Ethics Committee for Animal Experiments (approval No. 2015–044 of 25 November 2015).

To determine the acute oral lethal dose (LD₅₀) of the L-glutamic acid-g-p(HEMA) nanoparticles, we followed the OECD Guideline 425: Acute Oral Toxicity: Up-and-Down Procedure (10). First, we administered 2.000 mg/kg per kg of body weight (bw) of L-glutamic acid-g-p(HEMA) dissolved in saline to one mouse by gavage, and 24 h later to the remaining three mice in a 24-hour sequence (two male and two female mice weighing 15–20 g each). At the end of the experiment (Day 14), blood was collected (100–150 µL) with cardiac puncture from all mice under the ketamine+xylazine (50+10 mg/kg ip) anaesthesia, and their organs were removed and weighed to calculate relative organ weights. Collected blood was analysed immediately after collection using the VetScan VS2 analyser (Abaxis Inc. Union City, CA, USA) for biochemical parameters and VetScan HM5 analyser (Abaxis Inc.) for haematological parameters.

Repeated-dose toxicity testing included 6–8 weeks BALB/c albino mice (n=40) and followed the OECD Guideline 407 (11). The mice were randomised in three exposed groups (10 mice each; 5 male and 5 female) and one unexposed control group. The exposed groups received oral doses of either 25 mg/kg, 50 mg/kg, or 100 mg/kg bw [calculated based on oral LD₅₀ (>2.000 mg/kg bw) and body weight] by gavage five days a week for 28 days (four weeks). The control group was receiving saline (0.1 mL), also by gavage.

The mice were weighed each week before and after the current round of 5-day exposure. On Day 29, the mice were sacrificed under ketamine+xylazine (50 +10 mg/kg ip) anaesthesia by taking 0.7– 1.0 mL of blood from the heart. The organs were removed and weighed.

Blood biochemistry was determined for 12 parameters at baseline and upon sacrifice with a Vetscan VS2 analyser as follows: albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), total bilirubin (TBIL), blood urea nitrogen (BUN), calcium (Ca), phosphate (PHOS), creatinine (CRE), glucose (GLU), potassium (K), total protein (TP), and globulin (GLOB).

Brain, liver, and kidney tissues for microscopy were fixed in 4 % paraformaldehyde for 24 h and processed for embedding in paraffin. Histological sections were cut into 5 µm thick slices and stained with haematoxylin and eosin (H&E). Tissue images were then taken with an Olympus C-5050 digital camera (Tokyo, Japan) mounted on an Olympus BX5 microscope (20× magnification).

The micronucleus (MN) test followed the OECD Guideline 474 (12) and procedure described by Schmid, with slight modifications (13). For it we took bone marrow cells, removed from the posterior foot tibia and femur with 1 mL of foetal calf serum. The cells were then pipetted into tubes and centrifuged at 450 g for 10 min and the supernatant discarded. The pellet was carefully resuspended, and one drop was placed and spread on a clean slide. After overnight drying, the slides were stained in May Grünwald solution for 3 min and then in May Grünwald + demineralised water (1:1 v/v) solution for another 2 min. Followed washing with distilled water, staining in 10 % Giemsa solution for 10 min, and washing again. After air-drying, the slides were fixed in methanol for 10 min and mounted with entellan. At least 1000 polychromatic erythrocytes per animal were scored for the presence of micronuclei.

L-glutamic acid-g-p(HEMA) was labelled with IRD800-CW (Perkin Elmer Inc., Waltham, MA, USA) dye following the protocol described by Wu et al. (14) with minor changes. The nanoparticles (1 mg/mL) in PBS (pH 7.5) and 50 µL IRD800-CW (5 mg/mL) were incubated in the dark at room temperature for 3 h, centrifuged at 10,000 g for 10 min to remove unbound dye, and washed with 5 mL of PBS three times.

Then we injected 0.2 mL of stained nanoparticles into the tail vein of mice for optical imaging under isoflurane anaesthesia using the IVIS^® Spectrum in vivo imaging system (Perkin Elmer Inc.). After imaging, the animals were sacrificed with a ketamine/xylazine overdose and the liver, kidney, heart, lung, spleen, stomach, and small and large intestine were imaged ex vivo (7). We also imaged bone marrow cells and examined them under an Olympus BX5 fluorescence microscope (Tokyo, Japan) (40× magnification).

Statistical analyses were run on the SPSS for Windows 10.0 (IBM Corp., Armonk, New York, USA) and GraphPad Prism 7 (GraphPad Software, LLC, Boston, MA, USA) statistical analysis programs. Total and relative organ weight and biochemical values were calculated, and results compared with the control group using one-way analysis of variance (ANOVA) followed by the LSD test. All values are expressed as means ± standard deviation (SD). Statistical significance was set to p<0.05.

Acute exposure to nanoparticles did not cause any lethal effect or significant changes in total body and (relative) organ weights (data not shown) or biochemical parameters (Table 1). The same is true for the haematological parameters with the exception of a significant decrease in white blood cell and significant increase in platelet counts (Table 2). Rodents show variations in many clinical chemistry values. White blood cell counts in mice vary between 2 and 10 × 10³/mm³ (20). For this reason, we disregarded the difference between the baseline and final (day 14) WBC counts in the single-dose toxicity test, as they were within the reference range.

The 28-day dosing with 25, 50, or 100 mg/kg bw of polymeric nanoparticles for five days a week did not cause any morphological or behavioural adverse effects, and the study was completed with the initial number of mice. Their body weights increased in a similar way in all groups, including control (Figure 1). No significant difference in (relative) organ weights (Table 3) or biochemical parameters (Table 4) was found between control and any of the exposed groups.

Figure 1

Furthermore, we found no significant histological changes in the exposed groups. Brain tissue (Figure 2) showed no abnormalities; the pia mater, the cortex, and medulla underneath appeared normal, as did the neurons and neuroglia. Liver tissue (Figure 3) had normal stromal and parenchymal areas and cells. The same is true for the kidneys (Figure 4), whose glomeruli, proximal and distal tubules, and other parenchymal, stromal, and perivascular areas and cells appeared normal.

Figure 2

Figure 3

Figure 4

These results are well in line with Malonne et al. (21), who reported no differences between control mice and those receiving 2.000 mg/kg bw of PNIPAAm, PNIPAAm-co-NVA, or PNIPAAm-co-AAc nanoparticles a day over 28 days.

In contrast to polymeric nanoparticles, metallic and metal-oxide nanoparticles have been reported to deposit in the brain and disturb the normal metabolism of neurotransmitters (22) or induce liver and spleen injury, lung inflammation, and cardiac toxicity (23).

The bone marrow micronucleus test found no significant differences in MN counts between controls and exposed animals (Table 5). These results are supported by Karlsson et al. (24).

Intravenously injected nanoparticles were absorbed by the organs in a time-dependent manner and most of them eliminated between 12 and 24 h after dosing (Figure 5A). Ex vivo images show relatively high nanoparticle amounts in the liver and intestine at 6 h after dosing. The signal became weaker at 12 h, and completely disappeared 24 h after dosing (Figure 5B). Although IVIS^® spectral imaging showed no fluorescent signal in the bone marrow (Figure 5C), fluorescent microscopy revealed a fluorescent signal at hour 6 (Figure 6).

Figure 5

Figure 6

Intravenous L-glutamic acid-g-p(HEMA) polymeric nanoparticles seem to enter the organs by hour 6 of exposure and are eliminated from between hours 12 and 24. This may point to their metabolism, as suggested by Durner et al. (25) and elimination by macrophages, as suggested by He et al. (17) and Arnida et al. (26).