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

Buket Bakan; Fatih Oltulu; Yeliz Yıldırım; Altuğ Yavaşoğlu; Sinan Akgöl; Nefise Ülkü Karabay Yavaşoğlu

doi:10.2478/aiht-2023-74-3768

Figures & Tables

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

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)

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)

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)

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)

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

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

Figures & Tables

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

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

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

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

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

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