Materials Today: Proceedings 42 (2021) 3041–3045

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Materials Today: Proceedings

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Biocompatibility of gold nanoparticles: In-vitro and In-vivo study

Rua J. Kadhim, Esraa H. Karsh, Zainab J. Taqi, Majid S. Jabir ⇑
Applied Science Department-University of Technology, Baghdad, Iraq

a r t i c l e i n f o a b s t r a c t

Article history: Gold nanoparticles have unique characters such as chemical, physical, and optical properties. It have used
Available online 10 February 2021 in biomedical, pharmaceutical, industrial, and other sciences. Thus, for their safe and effective applica-
tions, much responsiveness has been given to the side effect and toxicity of gold nanoparticles in biolog-
Keywords: ical and medical systems. In this study, we investigated the toxicity of Gold nanoparticles (GNPs) using
GNPs In-viro and In-vivo study. MTT assay was used to investigate cytotoxic activity of GNPs against Rate
Biocompatible embryonic fibroblast (REF) cell line. While, In-vivo model the GNPs were intraperitoneal injected in mice
MTT assay
at concertation 100 mg/Kg. Then, mice body weight and histopathological changes were studied after
REF
Histopathology
4 weeks of injection. GNPs were characterized and confirmed using UV-spectrum assay. Cytotoxicity of
GNPs against REF cells showed no toxic effect and no morphological changes in REF cells after treated
with GNPs at concentration 1, 5, and 10 mg/ml. While, histopathological results showed no changes were
addressed. The findings demonstrated that the GNPs were biocompatible materials In-vitro and In-vivo.
Ó 2021 Elsevier Ltd. All rights reserved.
Selection and peer-review under responsibility of the scientific committee of the 3rd International Con-
ference on Materials Engineering & Science

1. Introduction tems. A studies [6,7] has designed NIR-responsive drug delivery

Nanoparticles (NPs) are substances the diameter of which does
not exceed 100 nm. This size provides them with physical and
chemical properties different from materials usually found in envi-
ronment [1,2]. Several types of NPs can be distinguished. Natural
ones are found in soils, natural waters, or volcanic dust. Both geo-
logical and biological processes generate them. Even when toxic,
numerous organisms can adapt and evolve in environments rich
in natural NPs [3]. NPs are produced by industries and used in
science, agriculture, electronics, medicine, pharmacy, cosmetology,
and so forth [4]. Colloidal gold nanoparticles have been proposed
for diverse biomedical applications due to their unique surface,
electronic, and optical properties. Because of the strong and size-
tunable surface plasmon resonance, fluorescence, and easy-
surface functionalization, gold nanoparticles have been widely
used in many applications such as antimicrobial agent, immune
modulator, biosensors, cancer cell imaging, photothermal therapy,
and drug delivery. In the medical applications GNPs were used to
treat rheumatoid arthritis [5]. GNPs are very attractive in biomed-
icals applications studies. Previous studies are demonstrated the
applications of gold nanomaterials in biological and medical sys-
⇑ Corresponding author.
E-mail address: 100131@uotechnology.edu.iq (M.S. Jabir).
systems based on gold nanoparticles to achieve the purpose of
combined photothermal-chemotherapy. Many other applications
include
great potential for NIR-responsive controlled release [8]. Other
studies used GNPs in delivery of chemical drugs and genes [9],
and other refer to importance of GNPs in theranostic application
[10,11]. [12] used GNPs as a promising agents in bio-imaging.
While, other studies reported the role of GNPs in bio-sensing and
as immunotherapy carriers [13,14]. The quick development of nan-
otechnology creates a subsequent rise in generation and utilizing
of engineered nanoparticles and therefore increased the danger
of exposure and toxicity [15,16].This study was designed to esti-
mate the toxicity of GNPs In-vitro and In-vivo models.
2. Material and method
2.1. Chemicals and reagents
Acridine orange (AO), Propidium iodide (PI), Trypsin-EDTA, fetal
bovine serum, DMSO, MTT, and crystal violate stain were obtained
from Sigma (St. Louis, MO, USA).
RPMI-1640 medium was purchased from Gibco (USA). Gold
nanoparticles purchased from Sigma Aldrich (Germany). PBS

https://doi.org/10.1016/j.matpr.2020.12.826
2214-7853/Ó 2021 Elsevier Ltd. All rights reserved.
Selection and peer-review under responsibility of the scientific committee of the 3rd International Conference on Materials Engineering & Science
R.J. Kadhim, E.H. Karsh, Z.J. Taqi et al. Materials Today: Proceedings 42 (2021) 3041–3045

Fig. 1. UV- spectrum of GNPs.

2.2. Characterization of nanoparticles

Gold nanoparticles were characterized using UV-spectrum

technique, and SEM [17–19].

2.3. Maintenance of cell cultures

REF cells were maintained in RPMI-1640 supplemented with

10% Fetal bovine serum, 100 unit’s/mL penicillin, and 100 mg/mL
streptomycin [20]. Cells were passaged using Trypsin-EDTA
reseeded at 80% confluence twice a week, and incubated at 37 °C
[21,22].

2.4. Cytotoxicity assays (MTT assay)

Cytotoxicity assay was done according to previous methods

[23,24]. REF cells were seeded at 1  104 cells mL 1 in wells of
Fig. 2. SEM image of GNPs. microtiter plates supplied with RPMI and allowed to adhere over-
night. The GNPs solutions were added in triplicate and incubated
for 48 hrs. Thereafter, the MTT solution was added to the cells. Fol-
lowing the time of incubation, the medium was aspirated and the
DMSO solution was applied to the wells. The absorbance for each
well was determined under a wavelength of 492 nm on a micro-
plate reader. Regression analysis was used to extract the concen-
tration for 50% inhibition of cell growth (IC50) [25,26].

2.5. Acridine orange (AO) – propidium iodide (PI) assay

The ability of GNPs to induce apoptosis was performed using

AO/PI. Briefly, after 24 hrs of seeding cells on 12 well glass slides,
they were exposed to the IC50 of the tested GNPs for 24 [27]. After
washing twice with PBS, dual fluorescent dyes were added into
each well. Finally, the cells were observed using fluorescent micro-
scope [28].

2.6. Animals model

To investigate the toxicity of GNPs, male mice weighting

Fig. 3. Anti-proliferative activity of GNPs against REF cell line. The results were between (25–30 g) was used in this study. First group was
represented as mean ± SD. intraperitoneal injected with 250 ml of PBS as a control group. Sec-
ond group was injected intraperitoneal with GNPs at concentration
100 mg/ kg. The experimental time was four weeks. The mice were
obtained from OXOID (England). Penicillin and streptomycin from anesthetized by exposure to the Chloroform [29,30]. The samples
(Sigma). of liver, lungs, and spleen tissues were collected from each group.
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R.J. Kadhim, E.H. Karsh, Z.J. Taqi et al.
Fig. 4. AO/PI double stain in REF cells after treated with GNPs. Magnification power 40x.
Fig. 5. Effect of GNPs in body weight of mice after intraperitoneal injected as indicated. The results were represented as mean ± SD. n.s. mean non-significant.
These organs were hold on in formalin (10%). The histopathological
section was done according to standard protocol. [31,32].
2.7. Statistical analysis
The statistical analysis was done using GraphPad prism version-
6 [33]. The results are represented as mean ± SD [34].
3. Result and discussion
3.1. Characterization of gold nanoparticles
The GNPs were analyzed by UV–visible (UV–VIS) spectroscopic
analysis between 200 and 1200 nm wavelengths for their absorp-
tion values and were found exhibiting the kmax absorption values
at 528 nm as seen in Fig. 1, the peak of GNPs was observed almost
528 nm. The morphology of GNPs was studied using SEM, as in
Fig. 2. GNPs was found to be spherical shape.
3.2. Anti-proliferative activity of GNPs
In the current study, we focused on assessing whether GNPs
have cytotoxicity on REF cells. REF cells were seeded as 1104 cells
/ well in 96 well plats and after 24 h, when the cells become con-
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Materials Today: Proceedings 42 (2021) 3041–3045
fluent monolayer, they were exposed GNPs at concentrations (1, 5,
10) ug/ml and incubated in 37 °C for 48hr. then stained by MTT
stain and calculated the percentage of cytotoxicity. The results of
current study showed that anti-proliferative activity of GNPs
against REF cell line very low and the highest inhibition of the cell
culture was found in the concentration (10 mg/ml) and the toxicity
of these nanoprticles gradually decreases to reach The lowest inhi-
bition in the concentration (1 mg/ml) as shown in Fig. 2, The results
was demonstrated that the no big significant destruction and no
damage in nanoparticles treated cells as well as no reduce in the
number of cancer treated cells with GNPs for 48 h as in Fig. 3.
Apoptotic cells have elevated plasma membrane permeability
to fluorescence stains. After treating the REF cell line with the
IC50 concentrations of GNPs for 24hrs, the dual cell staining and
visualization under fluorescence microscope was performed to
detect the changes in the nuclear morphology. As shown in
Fig. 4. The results showed there is no morphological alterations
in the treated REFcells.
3.3. Histopathology changes induced by GNPs
Based on the in-vitro results, the in-vivo experiments were fur-
ther investigated to evaluate the toxicity of GNPs using mice model
(Figs. 5 and 6). It can be seen that injection of GNPs at dose 100 mg/
R.J. Kadhim, E.H. Karsh, Z.J. Taqi et al.
Fig. 6. Histopathology sections in mice after intraperitoneal injected with GNPs as indicated.
Kg for 28 days did not cause mortality, and no statistically signifi-
cant differences (P  0.05) in body weight of mice were observed.
The results focus on side effect of GNPs on body weight, liver, lung
and spleen of injected mice. The mice were intraperitoneal injected
with GNPs for (4) weeks. The results of this study demonstrated
that the used GNPs had no side effect and there is no changes
accrued in animals body weight as well as mice organs. The results
showed on significant changes in interlobular vein and liver hepa-
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Materials Today: Proceedings 42 (2021) 3041–3045
tocytes. In addition, the red pulp surface and follicles structure of
spleen have been showed as normal structure after GNPs were
intraperitoneal injected. The same results are showed for lung after
4 weeks injection of GNPs. These results demonstrated that the
GNPs had on toxic effects at these specific concentrations. There-
fore, injected of the animals with the GNPs at concertation
100 mg/kg did not induces any histopathological changes in the
liver, spleen and lung.
R.J. Kadhim, E.H. Karsh, Z.J. Taqi et al. Materials Today: Proceedings 42 (2021) 3041–3045

4. Conclusions [13] L.G. Xu, Y. Liu, Z.Y. Chen, Nano Lett. 12 (2012) 2003–2012.
[14] A.M. Mohammed, Chin. Chem. Lett. 27 (2016) 801–806.
[15] R.R. Schumann, Biochem. Soc. Trans. 39 (2011) 989–993.
In conclusion, the toxic effect of GNPs has been investigated [16] A. Liu, H. Fang, W. Wei, Histochem. Cell Biol. 142 (2014) 667.
using In-vitro and In-vivo model. The results of cytotoxicity of GNPs [17] E.H. Karsh, R.J. Kadhim, M.S. Jabir, In AIP Conference Proceedings. 2213 (2020)
020145. AIP Publishing LLC.
against REF cells using MTT assay, and the results of histopathology
[18] Z.J. Taqi, M.A. Hamad, M.S. Jabir, Resh. J. Biotechnol. 14 (2019) 156–159.
of injected mice indicate that the GNPs don’t exhibit toxic effect in [19] K.S. Khashan, M.S. Jabir, F.A. Abdulameer, Surface Rev. Lett. (SRL) 26 (2019) 1–
both models. Taken together, the results demonstrated that the 8.
[20] M.S. Jabir, U.M. Nayef, K.H. Jawad, Z.J. Taqi, N.R. Ahmed, IOP Conf. Ser. Mater.
GNPs were biocompatible materials In-vitro and In-vivo when use
Sci. Eng. 454 (2018) 012077.
them at low concentrations. [21] Z.U. Ali, M.S. Jabir, A.M. Al-Shammari, Res. J. Biotechnol. 14 (2019) 79–82.
[22] M.S. Jabir, U.M. Nayef, W.K. Abdulkadhim, G.M. Sulaiman, Mater. Res. Express
Declaration of Competing Interest 6 (2019) 115412.
[23] M.M. Radhi, H.N. Abdullah, M.S. Jabir, A.J. Emad, Nano Biomed. Eng. 91 (2017)
103–106.
The authors declare that they have no known competing finan- [24] W.K. Kadhim, U.M. Nayef, M.S. Jabir, Surf. Rev. Lett. (2019) 1950079.
cial interests or personal relationships that could have appeared [25] H.N.K. Al-Salman, E.T. Ali, M. Jabir, G.M. Sulaiman, S.A. Al-Jadaan, Eur. Food
Res. Technol. (2020).
to influence the work reported in this paper. [26] H.A. Kadhem, S.A. Ibraheem, M.S. Jabir, A.A. Kadhim, Z.J. Taqi, Nano Biomed.
Eng. 1 (2019) 35–43.
References [27] G.M. Sulaiman, H.M. Waheeb, M.S. Jabir, S.H. Khazaal, Y.H. Dewir, Y. Naidoo,
Sci. Rep. 10 (2020) 1–16.
[28] K.S. Khashan, G.M. Sulaiman, S.A. Hussain, T.R. Marzoog, M.S. Jabir, J. Inorg.
[1] M.C. Danieland, D. Astruc, Chem. Rev. 104 (2004) 293–346.
Organomet. Polym Mater. (2020) 1–17.
[2] R.D. Handy, R. Owen, E. Valsami-Jones, Ecotoxicology 17 (2008) 315–325.
[29] M.S. Jabir, U.M. Nayef, W.K.A. Kadhim, Nano Biomed. Eng. 11 (2019) 18–27.
[3] V. Matranga, I. Corsi, Marine Environ. Res. 76 (2012) 32–40.
[30] S. Albukhaty, H. Naderi-Manesh, T. Taki, M.S. Jabir, Artif. Cells Nanomed.
[4] C. Alexander, E.T. Rietschel, J. Endotoxin Res. 7 (2001) 167–202.
Biotechnol. 46 (2018) S125–S132.
[5] C.F. Shaw III, Chem. Rev. 99 (1999) 2589–2600.
[31] W.K.A. Kadhim, U.M. Nayef, M.S. Jabir, Surf. Rev. Lett. 26 (2019) 1950079.
[6] J.F. Liao, W.T. Li, J.R. Peng, Theranostics 5 (2015) 345–356.
[32] S.H. Kareem, A.M. Naji, Z.J. Taqi, M.S. Jabir, J. Inorg. Organomet. Polym Mater.
[7] J.R. Peng, T.T. Qi, J.F. Liao, et al., Theranostics 4 (2014) 678–692.
(2020) 1–15.
[8] M.S. Yavuz, Y.Y. Cheng, J.Y. Chen, Nat. Mater. 8 (2009) 935–939.
[33] I.H. Ali, M.S. Jabir, H. Al-Shmgani, G.M. Sulaiman, A.H. Sadoon, J. Phys. Conf.
[9] J.D. Wu, B. Liu, H.M. Wu, J. Biomed. Nanotechnol. 12 (2016) 800–810.
Ser. 1003 (2018) (2018) 012009.
[10] B. Cheng, H.C. He, T. Huang, J. Biomed. Nanotechnol. 12 (2016) 435–449.
[34] A.H. Younus, S. Ahmer, M.S. Jabir, Res. J. Biotechnol. 14 (2019) 131–133.
[11] L. Sun, D.Y. Joh, A. Al-Zaki, J. Biomed. Nanotechnol. 12 (2016) 347–356.
[12] D. Kim, S. Park, J.H. Lee, Y.Y. Jeong, S. Jon, J. Am. Chem. Soc. 129 (2007) 7661–
7665.

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