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

Buket Bakan
Buket Bakan
, 
Fatih Oltulu
Fatih Oltulu
, 
Yeliz Yıldırım
Yeliz Yıldırım
, 
Altuğ Yavaşoğlu
Altuğ Yavaşoğlu
, 
Sinan Akgöl
Sinan Akgöl
 and 
Nefise Ülkü Karabay Yavaşoğlu
Nefise Ülkü Karabay Yavaşoğlu
   | Sep 30, 2023
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Article Category: Original article
Published Online: Sep 30, 2023
Page range: 207 - 217
Received: Aug 01, 2023
Accepted: Sep 01, 2023
DOI: https://doi.org/10.2478/aiht-2023-74-3768
© 2023 Buket Bakan et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

Body weight changes in control mice and those exposed to 25, 50, or 100 mg/kg bw of L-glutamic-g-p(HEMA) for five days a week for four weeks. A – female mice; B – male mice
Body weight changes in control mice and those exposed to 25, 50, or 100 mg/kg bw of L-glutamic-g-p(HEMA) for five days a week for four weeks. A – female mice; B – male mice

Figure 2

Brain histology with haematoxylin & eosin staining in female and male mice receiving L-glutamic-g-p(HEMA) polymeric nanoparticles as follows: 25 mg/kg (A); 50 mg/kg (B); 100 mg/kg (C); negative control (D). The arrows represent glia (g), neuron (n), and pia mater (p) (20× magnification)
Brain histology with haematoxylin & eosin staining in female and male mice receiving L-glutamic-g-p(HEMA) polymeric nanoparticles as follows: 25 mg/kg (A); 50 mg/kg (B); 100 mg/kg (C); negative control (D). The arrows represent glia (g), neuron (n), and pia mater (p) (20× magnification)

Figure 3

Liver histology with haematoxylin & eosin staining in female and male mice receiving L-glutamic-g-p(HEMA) polymeric nanoparticles as follows: 25 mg/kg (A); 50 mg/kg (B); 100 mg/kg (C); negative control (D). The arrows represent hepatocytes (h), vena centralis (v), ductus biliferi interlobularis (s), and vena porta interlobularis (vp) (20× magnification)
Liver histology with haematoxylin & eosin staining in female and male mice receiving L-glutamic-g-p(HEMA) polymeric nanoparticles as follows: 25 mg/kg (A); 50 mg/kg (B); 100 mg/kg (C); negative control (D). The arrows represent hepatocytes (h), vena centralis (v), ductus biliferi interlobularis (s), and vena porta interlobularis (vp) (20× magnification)

Figure 4

Kidney histology with haematoxylin & eosin staining in female and male mice receiving L-glutamic-g-p(HEMA) polymeric nanoparticles as follows: 25 mg/kg (A); 50 mg/kg (B); 100 mg/kg (C); negative control (D). The arrows represent glomeruli (g), Bowman’s capsule (b), distal tubule (d), proximal tubule (p), collective tubule (c), and Henle’s handle (h) (20× magnification)
Kidney histology with haematoxylin & eosin staining in female and male mice receiving L-glutamic-g-p(HEMA) polymeric nanoparticles as follows: 25 mg/kg (A); 50 mg/kg (B); 100 mg/kg (C); negative control (D). The arrows represent glomeruli (g), Bowman’s capsule (b), distal tubule (d), proximal tubule (p), collective tubule (c), and Henle’s handle (h) (20× magnification)

Figure 5

A. Time-course fluorescence imaging in living mice after intravenous injection of L-glutamic-g-p(HEMA) polymeric nanoparticle (n=3); B. Ex vivo imaging of major organs of mice at 6, 12, and 24 h; C. Optical imaging of bone marrow cells by IVIS® spectrum at 6, 12, and 24 h (excitation at 745 nm, emission at 850 nm)
A. Time-course fluorescence imaging in living mice after intravenous injection of L-glutamic-g-p(HEMA) polymeric nanoparticle (n=3); B. Ex vivo imaging of major organs of mice at 6, 12, and 24 h; C. Optical imaging of bone marrow cells by IVIS® spectrum at 6, 12, and 24 h (excitation at 745 nm, emission at 850 nm)

Figure 6

Images of bone marrow cells at 6, 12, and 24 h under the light (left side) and fluorescence (right side) microscope (40× magnification). Fluorescent microscopy revealed a fluorescent signal at hour 6 only.
Images of bone marrow cells at 6, 12, and 24 h under the light (left side) and fluorescence (right side) microscope (40× magnification). Fluorescent microscopy revealed a fluorescent signal at hour 6 only.

Organ and relative organ weights in female (n=5) and male mice (n=5)

  Absolute organ weights (Mean±SD) Relative organ weights (%)
Lung Liver Kidney* Brain Lung Liver Kidney* Brain
Female mice
Control 0.183±0.02 1.095±0.04 0.318±0.04 0.362±0.02 0.0083 0.0073 0.0074 0.0095
25 mg/kg 0.173±0.03 1.322±0.16 0.312±0.01 0.37±0.09 0.0497 0.0563 0.0518 0.0551
50 mg/kg 0.186±0.03 1.286±0.13 0.295±0.03 0.378±0.05 0.0144 0.0133 0.0118 0.0123
100 mg/kg 0.248±0.08 1.436±0.05 0.322±0.03 0.408±0.04 0.0164 0.0157 0.0152 0.0156
Male mice
Control 0.222±0.03 1.536±0.11 0.47±0.05 0.37±0.06 0.0084 0.0077 0.0077 0.0069
25 mg/kg 0.228±0.05 1.818±0.21 0.468±0.05 0.374±0.03 0.0581 0.0615 0.0550 0.0548
50 mg/kg 0.225±0.04 1.596±0.17 0.462±0.04 0.358±0.06 0.0178 0.0158 0.0159 0.0157
100 mg/kg 0.205±0.03 1.615±0.27 0.465±0.06 0.397±0.03 0.0140 0.0126 0.0123 0.0134

Biochemical parameters in BALB/C mice (n=5) after acute single-dose exposure to 2000 mg/kg of L-glutamic acid-g-p(HEMA) nanoparticles

Biochemical parameters Baseline (Mean±SD) Day 14 post-exposure (Mean±SD)
ALB (g/dL) 3.6±0.4 3.3±0.1
ALP (U/L) 64±0.4 60±5.5
ALT (U/L) 36±0.4 35±2.5
TBIL (mg/dL) 5.0±0.4 5.0±0.5
BUN (mg/dL) 9.0±0.4 8.5±1.6
Ca (mg/dL) 2.37±0.4 2.75±0.3
PHOS (mg/dL) 2.88±0.4 3.35±0.7
CRE (mg/dL) 2.90±0.4 2.20±0.1
GLU (mg/dL) 136±0.4 136±8
K (mmol/L) 5.3±0.4 8.0±0.1
TP (g/dL) 4.5±0.4 5.5±0.4
GLOB (g/dL) 1.2±0.4 2.2±0.3

Haematological test results in BALB/C mice (n=5) after acute single-dose exposure to 2000 mg/kg of L-glutamic acid-g-p(HEMA) nanoparticles

Haematological parameters Baseline (Mean±SD) Day 14 post-exposure (Mean±SD)
WBC (109 cells/L) 8.68±2.1 3.21±1.1*
LYM (109 cells/L) 5.43±0.8 2.41±0.9
MON (109 cells/L) 0.23±0.1 0.10±0.1
NEU (109 cells/L) 1.36±1.02 0.70±0.9
RBC (1012 cells/L) 8.02±1.3 10.41±1.2
HGB (g/L) 14.52±1.3 14.5±1.1
HCT (%) 38.63±3.9 45.65±4.1
MCV (fl) 48.0±2.9 44.0±2.8
MCH (pg) 12.26±0.7 13.90±0.7
MCHC (pg) 28.04±1.1 31.70±0.8
PLT (109 cells/L) 294.60±25.1 975±35.8*

Changes in biochemical parameters (means ± SD) in female (n=5) and male (n=5) mice after exposure to L-glutamic acid-g-p(HEMA) polymeric nanoparticles over 28 days

Biochemical parameters Groups
Control Group 1 (25 mg/kg) Group 2 (50 mg/kg) Group 3 (100 mg/kg)
Baseline Post-exposure Baseline Post-exposure Baseline Post-exposure Baseline Post-exposure
Female mice
ALB (g/dL) 3.9±0.6 3.4±0.30 3.7±0.4 3.3±0.3 4.1±0.1 3.5±0.30 4.1±0.2 3.6±0.23
ALP (U/L) 81.2±4.7 65±15.8 82.2±5.2 54±6.02 50±9.1 53±8.50 50.6±4.1 51±19.4
ALT (U/L) 61.0±7.5 37±5 57.0±12.9 31±15.8 44±16.1 45.5±16.9 40.6±14.6 35±7.54
TBIL (mg/dL) 0.3±0.1 5±0.57 0.3±0.1 5±0.57 3.0±0.1 5±0.57 3.0±0.1 5±0.57
BUN (mg/dl) 12.0±1.8 9.0±1.19 12.2±1.6 8.7±0.1 6.0±3.2 7±1.17 7.1±2.7 6.4±0.40
Ca (mg/dL) 10.3±0.8 2.37±0.6 4.0±0.6 2.36±0.4 2.51±0.8 2.48±0.11 3.9±0.4 2.41±0.8
PHOS (mg/dL) 8.3±1.4 2.88±0.2 8.1±1.6 2.51±0.2 8.2±1.3 2.36±0.21 3.8±1.4 2.04±0.4
CRE (mg/dL) 0.2±0.1 2.9±0.95 0.2±0.1 1.8±1.2 0.3±0.0 2.15±0.20 2.5±0.0 3.6±0.41
GLU (mg/dL) 214±53 136±31.0 216±19 112±60 214±16 135±26.7 215±54 118±6.6
K (mmol/L) 6.1±1.6 5.3±0.23 6.1±1.8 4.8±0.5 6.0±1.4 5.4±0.95 4.0±2.0 3.9±1.12
TP (g/dL) 5.4±0.4 4.5±0.37 5.3±0.5 5.0±0.1 5.7±0.4 4.9±0.20 5.5±0.5 4.9±0.32
GLOB (g/dL) 2.2±0.7 1.2±0.4 2.3±0.6 1.7±0.2 2.4±0.5 1.4±0.25 2.8±0.4 1.2±0.32
Male mice
ALB (g/dL) 3.7±0.6 3.0±0.30 3.3±0.1 2.9±0.37 3.0±0.1 3.0±0.30 3.0±0.4 36±0.23
ALP (U/L) 62.2±3.7 56±15.8 87.2±2.7 53±6.02 86.4±6.6 48±8.50 84.8±5.7 57±19.4
ALT (U/L) 31.0±6.5 29.5±5 57.4±2.0 58.5±15 56.6±2.5 39±16.9 55.6±4.3 42.5±7.5
TBIL (mg/dL) 3.4±0.8 4.5±0.57 0.2±0.1 4.5±0.57 0.2±0.1 6±0.57 0.2±0.05 5±0.57
BUN (mg/dl) 7.50±1.8 6.95±1.1 10.2±1.6 8.7±0.15 10.8±1.3 7.7±1.17 10.6±0.8 6±0.40
Ca (mg/dL) 3.58±1.7 2.47±0.6 10.6±0.3 2.43±0.4 10.2±0.5 2.42±0.1 9.8±0.4 2.47±0.8
PHOS (mg/dL) 4.3±1.4 2.5±0.26 8.1±0.7 2.86±0.2 8.2±1.6 2.22±0.2 8.1±0.8 2.33±0.4
CRE (mg/dL) 21.8±0.98 27.5±0.9 0.1±0.1 32±1.20 0.2±0.1 18±0.20 0.2±0.1 35±0.41
GLU (mg/dL) 214±53 157±31 223±8 101±6.0 227±17 117±26.7 226±9 118±6.6
K (mmol/L) 6.1±1.6 4.9±0.23 5.2±0.1 4.7±0.50 5.2±0.1 4.7±0.95 5.2±0.3 5±1.12
TP (g/dL) 5.4±0.4 4.7±0.37 6.2±0.2 4.8±0.17 6.2±0.4 4.8±0.20 6.3±0.2 5.2±0.32
GLOB (g/dL) 2.2±0.7 1.8±0.4 2.8±0.5 1.9±0.20 2.8±0.3 1.8±0.25 2.8±0.4 1.5±0.32

Micronucleus test results in mice (5 per group) after exposure to L-glutamic acid-g-p(HEMA) polymeric nanoparticles over 28 days

Groups Micronuclei (% per 1,000 erythrocytes per animal)
Negative control (saline) 3
Group 1 (25 mg/kg) 2
Group 2 (50 mg/kg) 3
Group 3 (100 mg/kg) 5
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