(2022) 16:110
Asariha et al. BMC Chemistry
https://doi.org/10.1186/s13065-022-00895-x

BMC Chemistry
Open Access

RESEARCH

Extended‑release of doxorubicinthrough green surface modification of gold
nanoparticles: in vitro and in ovo assessment
Maryam Asariha1, Seyed Hossein Kiaie2,3*, Sepideh Izadi4, Faezeh H. Pirhayati5, Mehdi Fouladi5 andMaryam Gholamhosseinpour5

Abstract
In the present study, a green surface modification of gold nanoparticles (GNPs) using chondroitin sulfate (CHS) andchitosan (CS) to deliver an extended-release of doxorubicin (DOX) was proposed. Following synthesis of each stepof unconjugated counterpart, including CHS-GNPs, DOX–CHS–GNP, and conjugated construct DOX–CHS–GNP-CS,
physicochemical properties of the nanoparticles (NPs) were characterized by FT-IR, DLS, and TEM analyses, and therelease of DOX was determined by using UV–Vis spectrometry. Then, NPs were effectively taken up by MDA-MB-468,
βTC-3, and human fibroblast (HFb) cell lines with high release percent and without significant cytotoxicity. The DOX–
CHS–GNPs and DOX–CHS–GNP-CS NPs showed a mean size of 175.8 ± 1.94 and 208.9 ± 2.08 nm; furthermore, a zeta
potential of − 34 ± 5.6 and − 25.7 ± 5.9 mV, respectively. The highest release of DOX was 73.37% after 45 h, while in
the absence of CS, the release of DOX was 76.05% for 24 h. Compared to CHS-GNPs, the presence of CS decreased
the rate of sustained release of DOX and improved the drug release efficiency. The results demonstrated an excellent
release and negligible cytotoxicity at high concentrations of CHS-GNP-CS. Consequently, in ovo assessment corroborated the efficacy of the green fabricated NPs proposed effective targeted delivery of DOX for anti-tumor therapy
in vitro.
Keywords: Chondroitin sulfate, Gold nanoparticles, Chitosan, Doxorubicin, Cytotoxicity, Green synthesis, DrugdeliveryGraphical Abstract*Correspondence: hosseinkiaie@gmail.com
2
Nano Drug Delivery Research Center, Health Technology Institute,
Kermanshah University of Medical Sciences, Kermanshah, Iran
Full list of author information is available at the end of the article

Introduction
Doxorubicin (DOX) is an efficient anthracycline chemotherapeutic for treating solid tumors, particularly desmoplastic cancers such as breast and pancreatic cancers.
Two main pathways for the DOX mechanism of action© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​
mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Asariha et al. BMC Chemistry(2022) 16:110

are considered: intercalating with DNA leading to topoisomerase II inhibition and the generation of reactive oxygen species leading to plasma membrane damage [1–4].
Despite superb performance in cancer treatment, undesirable cytotoxicity reported on different tissue, including the heart and gut, has limited the systemic delivery of
DOX. For instance, irreversible cardiomyopathy has beenreported due to the cumulative drug dose and consequent cell damage resulting from the enhanced free radical in myocytes [5, 6].
Nanoparticles (NPs) have shown an exciting capacity for drug delivery systems (DDS) with adjustable andsustained profile release of drugs and devising platforms
for targeted delivery with minimal side effects [7]. ManyDDS types have been introduced through encapsulation or conjugation of different NPs for targeted delivery of DOX with low cytotoxicity and enhanced profile
extended-release on tumor sites [8]. In addition to FDAapproved liposomal forms of DOX delivery [9], a wide
variety of NPs have been formulated for systematized
release of the drug, including liposome [10], inorganicNPs [11–13], polymeric NPs [14, 15], conjugates [14–17],
micelles [18, 19], silica NPs [20] and exosomes [21, 22].
Gold nanoparticles (GNPs) have shown promising resultsfor DDS purposes. Appropriate biocompatibility, tunable surface chemistry, capability to conjugate with different bioactive molecules, and adjustable particle size
have turned this nanomaterial into a reliable candidatefor designing novel tailored-made carriers for therapeuticand diagnostic applications [23–26]. Surface modification is an essential step in GNPs preparation to improvestability within an aqueous solution, minimize NPs
aggregation, and restrain existing positive surface charges[27–29]. Importantly, detailed surface modification of
GNPs is critical for tissue-specific delivery of solubilized
formulations through the circulatory system while showing minimum toxicity [30–32].
Chondroitin sulfate (CHS) is a naturally occurring
polysaccharide with repeating disaccharide units of
b-1,4-linked d-glucuronic acid and b-1,3-linked N-acetyl
galactosamine [33]. This glycosaminoglycan has beensuccessfully employed to prevent the aggregation of
GNPs. The polyanionic property of CHS reduces the
positive charges of the gold atom, encloses GNPs’ surface, and subsequently forms an efficient capping agent
for enhancing GNPs’ stability, biocompatibility, and solubility [34, 35]. Moreover, chitosan (CS) is accepted as apromising drug delivery vector due to the primary aminogroups’ presence on this biodegradable polysaccharide[36]. The bioadhesive properties of CS can prolong theretention time of NPs in the bloodstream, thus improvethe cellular internalization of NPs and the release rateof pharmaceutics [37, 38]. Although the conjugation ofPage 2 of 10

GNPs with various ligand moieties for efficient deliveryof Dox was successful, the efficient loading and sustained
release of targeted GNPs for Dox delivery were unsolved.
In a targeted case study, Aptamers (Apts)-CS-GNPs
using nucleolin aptamers (AS1411) were developed forco-delivery Dox, and FOXM1 aptamer has efficiently
enhanced mortality in 4T1 (breast) and A549 (lung) cancer cells [39]. A combination of Dox-loaded thiol-modified chitosan GNPs and photothermal therapy against
cancer cells demonstrated good biocompatibility and low
cytotoxicity of the construct [40]. Finally, an efficient pHdependent targeted DOX delivery was utilized to cancer cells through immobilization of Dox on GNPs wascapped with carboxymethyl chitosan (CMC). The systemdemonstrated pH-triggered drug release to overcome
drug resistance in HeLa (Human cervical cancer) [41].
Moreover, GNPs functionalized by sodium 3-mercaptopropane sulfonate (GNP-3MPS) were synthesized to evaluate the release kinetics of dexamethasone(DXM) for inhibition of cell proliferation and apoptosis on a human lymphoma cell line, and upregulation of
DXM-inducible programmed cell death-1 (PD-1) molecule on activated mouse T lymphocytes [42]. In anotherresearch study, a controlled-release anticancer therapy
was assessed by GNPs with two different copper (I) complexes, including [Cu(PTA)4] + ­[BF4] (loading 90% andslow release) and [HB(pz)Cu(PCN)] (loading 10%) was
demonstrated [43]. Comparing other mentioned targeted
ones, the release profile of Dox with GNPs NPs not only
must supply reproducible and flexible sustain release ofDox but also corroborates GNPs as a potential alternative
for the functionalized carrier with at least burst release.
In this study, a novel and effective strategy for tumortargeted DOX delivery is introduced based on the functionalization of GNPs with CHS and loading DOX
molecules on this stable and nontoxic compound. The
drug sustain release was achieved by adding CS as thedelivery vector. Our approach in DOX delivery can minimize the occurrence of cardiac toxicity. The efficacy ofthe suggested green fabricated GNPs was evaluatedby examining the response of three different cell lines,
including MDA-MB-468 (human breast cancer cells),
βTC-3 (mouse pancreatic β-cells), and human fibroblast(HFb) cell lines.

Materials and methods
Materials

Chondroitin 4-sulfate sodium salt from a bovine trachea, low molecular weight chitosan (75–85% DDA and
50–190 kDa), and chloroauric acid 99.99% ­(HAuCl4) werepurchased from Sigma-Aldrich (St. Louis, MO, USA).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) was purchased from Roche (Mannheim,

Asariha et al. BMC Chemistry(2022) 16:110

Germany). Doxorubicin HCl (DOX) was obtained from
Actoverco pharmaceutical company (Tehran, Iran). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine
serum (FBS), antibiotic solutions, and phosphate-buffered saline (PBS) were obtained from Gibco (NY, USA).
All other materials were purchased from Merck (Darmstadt, Germany) and were analytical reagent grade. Chitosan was further purified by the precipitation procedure[44].
Cell lines and culture

Briefly, MDA-MB-468 cells were cultured in DMEM
supplemented with 10% FBS, 100 IU ­mL−1 penicillin,−1
100 IU ­mL streptomycin at 37 °C in a humidified incubator with 5% ­CO2 levels. Then, the growth patterns andmorphology of cells were regularly examined using an
inverted microscope [45]. After 48 h incubation, the cells
were harvested by trypsinization to perform cell passaging and backup cultures. Furthermore, βTC3 cells in thepresence of 2 mM l-glutamine were cultured similarly ina proper culture medium [45, 46]. Human fibroblast as
the normal cell was isolated by the procedure describedin our previous work [45, 47]. All the cell culture ingredients were supplied from Auto-cell Co. (Warsaw, Poland).
Synthesis and preparation of NPs
Green synthesis of GNPs capped with CHS (CHS‑GNPs)

20 ml of H
­ AuCl4 (0.39 mg ­mL−1) with 10 mL of CHS(5 mg ­mL−1) were reacted (in a 2:1 ratio v/v %) while
the pH of the solution was maintained at 7.5 by NaOH
solution (0.1 N) and HCl solution (0.1 N). The solution
was stirred at 60 °C for 70 min until a deep-red color
appeared. After three times centrifuging (8000 rpm,
30 min), the reaction was performed in a stirring aqueous environment at room temperature without the needfor UV light, autoclave, microwave, and laser irradiation(CHS-GNPs) [34, 48].
Conjugation of DOX and CHS‑GNPs

The conjugation of DOX onto CHS-GNPs was performed by mixing 2 mg of CHS-GNPs with 20 µL DOX(1% w/v) in distilled water with a ratio of 1:10 w/w. The
product was shaken at 37 °C and 100 rpm for 30 min. It is
assumed that positively charged DOX can be complexedwith negatively charged CHS-coated GNPs through electrostatic interaction. Eventually, the solution was centrifuged at 14,000 rpm for 20 min. The yielded pellet wasdispersed in 300 μL of distilled water to determine thepercentage of CHS-GNPs loaded with DOX.
Modification of DOX‑CHS‑GNPs by CS

For this purpose, 3 µL of CS (2 w/v %) was added dropwise to 300 µL of DOX-CHS-GNPs solution (in a ratio

Page 3 of 10

of 1:100 v/v %). Then, the mixture was shaken for 3 min,incubated at room temperature for 30 min, and centrifuged at 14,000 rpm for 20 min. The DOX loading andsupernatant concentrations were determined. Different
DOX concentrations (3.125, 6.25, 12.5, 25, 60, 80, and100 μgmL−1) in distilled water were prepared to evaluate
the amount of drug-loaded on the surface of synthesized
NPs. The DOX concentration in the supernatant andthe precipitate was determined using UV–Vis spectroscopy at 480 nm. A step-by-step schematic procedure forpreparing DOX-CHS-GNPs-CS by the green synthesis
method was presented in Additional file 1: Fig. S1.
Physicochemical properties of NPs

UV–Vis spectroscopy Cary 100 (Varian) is a reliable technique to evaluate the formation, and crystal growth ofNPs. [48]. To this end, the solution of DOX-CHS-GNPsCS with Dox concentration 20 μg ­mL−1 was diluted by
distilled water in a 2:1 ratio v/v % at pH 7 and preparedusing UV–vis spectrophotometry.
To verify the DOX-CHS-GNPs-CS synthesis, FT-IR
spectra were recorded by Prestige-21 Shimadzu Spectrometer (Kyoto, Japan). The suitable amount of samples,
including CHS, CHS-GNPs, DOX, DOX-CHS-GNPs, CS,
and DOX-CHS-GNPs-CS, was compressed into KBr, andsamples were then tested in the range of 400–4000 ­cm−1with a resolution of 4 ­cm−1. Furthermore, particle size,
zetapotential and polydispersity index of CHS-GNPs,
DOX-CHS-GNPs, and DOX-CHS-GNPs-CS were evaluated using DLS following distilled water in a 1:9 ratio v/v% at pH 7.
Transmission electron microscopy (TEM) was used to
identify the morphology and particle size of CHS-GNPs
and DOX-CHS-GNPs by using LEO 906 E microscope(Carl Zeiss, Germany) with an accelerating voltage of
100 kV. Before analysis, GNPs were deposited on carbon-copper grids. In addition, the surface morphology
of DOX-CHS-GNPs-CS was investigated through TEM.
Finally, GNPs represent consistent stability between − 30
and + 30 mV of their surface potential values because
ambient ionic liquids can produce electrostatic forces toincrease GNPs stability which was quite evident fromhigher zeta potential value versus aqueous extract value[49, 50].
X‑ray diffraction (XRD) analysis

To confirm the crystalline nature of the synthesized
CHS-GNPs, XRD analysis was carried out (Xpert pro,Panalytical, Holland), which was operated at a voltage of
40 kV and a current of 30 mA with cu ­k−1 radiation.

Asariha et al. BMC Chemistry(2022) 16:110

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Fig. 1 FT-IR spectrum of CHS (A), CHS-GNPs (B), DOX (C), DOX-CHS-GNPs (D), CS (E), and DOX-CHS-GNPs-CS (F)In vitro cytotoxicity assay

The MTT assay was utilized to evaluate the cytotoxicity of free DOX and NPs-DOX on MDA-MB-468, βTC3, and HFb cell lines, as described previously [46, 51].
To evaluate cell viability, 1 × ­104 cells were cultured in a
24-well plate with the proper culture medium for 24 h.
Then, 20 µL of treatment solutions of DOX, DOX-CHSGNPs, and DOX-CHS-GNPs-CS, were added at different
concentrations into each well for another 48 h. Finally,
the cell viability was evaluated by analyzing optical densities (ODs) obtained from the SpectraMax 190 absorbance microplate reader (Bio Tek Instruments, USA) at
570 nm.
In vitro release study of DOX

In order to evaluate the release of DOX at the surface of
DOX-CHS-GNPs and DOX-CHS-GNPs-CS, cellulose
acetate dialysis membranes (Spectra, MWCO 12 kDa)
in phosphorus buffer solution (PBS) at pH 7.4 wereused [46, 52]. DOX-CHS-GNPs and DOX-CHS-GNPsCS were located inside the membrane separately andwere dialyzed against 7 mL PBS for continuous stirring
at 100 rpm. Every hour, to read the absorbance of DOX,a total solution of 7 mL of PBS dialysis solution was
replaced completely with 7 mL of fresh PBS. The amountof released DOX was analyzed by determining theabsorbance at 481 nm, and the release of DOX was calculated according to the standard equation of the released
drug. The released DOX amounts from DOX-CHS-GNPs
and DOX-CHS-GNPs-CS were measured in the different

time intervals (0.25, 0.5, 1, 2, 3, 4, 5, 6, 18, 24, 27, 30, 42,
and 45 h). Finally, the results were repeated three times
for each sample, and the calibration curve of DOX in the
incubation medium was calculated.
CAM assay

Chick chorioallantoic membrane (CAM) assay was used
to evaluate the effect of different therapeutic groups on
reducing angiogenesis and tumor growth. In summary,fertilized hen’s eggs were incubated at 37 °C and 60%
humidity. After 10 days, an incision (square dimension:
0.5 ­cm2) was made on the eggshell under sterile conditions to access the CAM layer. Subsequently, 0.5–1 × ­106
cells (10 µl of the cell suspension) treated with DOXCHS-GNPs-CS, DOX-CHS-GNPs, free DOX, and CSCHS-GNPs were injected onto the CAM surface. The
incised area was then covered, and the eggs were reincubated for 7 days. Finally, the shell was opened, and CAM
was extracted to assess tumor weight, size, and angiogenesis, and images were taken.

Results and discussion
Structural and physicochemical analyses
FT‑IR assessment

As shown in Fig. 1A, the FT-IR spectrum of CHS depicts
the broad peaks in the region around 2600–3650 ­cm−1
and 2924 ­cm−1, which indicates the stretching vibrationof hydroxyl groups (OH) and ­CH2 and ­CH3 asymmetricstretching vibration aliphatic. Furthermore, the peaks at
1190 and 1064 ­cm−1 show two C–O stretching vibrations

Asariha et al. BMC Chemistry(2022) 16:110

of O–H, and the other peaks at 3425 and 1566 ­cm−1 indicate N–H stretching vibration and bending vibration of
the secondary amine of CHS, respectively [53, 54]. Subsequently, the peaks at 1242 ­cm−1 and 1651 ­cm−1 show
N–C stretching vibration and C=O, the carboxylic acidcarbonyl stretching vibration. As shown in Fig. 1B, the
FT-IR spectrum of CHS-GNPs confirmed the wavelength
decreasing of the C=O group from 1651 to 1694 ­cm−1
due to the presence of gold molecules, the carboxylic acidgroup of CHS has reduced to the ketone [48].
The FT-IR spectrum of DOX (Fig. 1C) depicts a broad
peak at 2700–3650 ­cm−1 for -OH stretching vibrationand two peaks at 3414 and 1624 ­cm−1 for N–H stretching vibration and bending vibration of the primaryamine, respectively. Furthermore, the peaks at 1215 and1072 ­cm−1 indicate to C–O stretching vibration of DOX,
and the peaks at 1577 and 1454 ­cm−1 show C=C stretching vibrations of the aromatic ring.
The FT-IR spectrum of DOX-CHS-GNPs (Fig. 1D)
depicts the peaks at 3437, 1627, and 1583 ­cm−1 that areassigned to N–H stretching vibration, and bending vibration of the primary and secondary amine of DOX-CHS,
respectively. In addition, the peak at 1689 ­cm−1 shows
C=O stretching vibrations of DOX and CHS units from
DOX-CHS-GNPs. Finally, the shifts in the peak positions from 3425 and 3414 to 3437 ­cm−1 and also 1651
and 1708 to 1689 ­cm−1 confirmed the presence of DOX
and CHS in the spectrum of DOX-CHS-GNPs. As
shown in Fig. 1D, the intensity of the peaks in the spectrum of DOX-CHS-GNPs was lower than DOX andCHS peaks. The FT-IR spectrum of CS (Fig. 1E) depicts
the peaks at 2924 and 2893 ­cm−1 related to C–H asymmetric stretching vibrations. The peak at 1523 ­cm−1 indicates the bending vibration of the primary amine. The
peak at 1072 ­cm−1 shows the C–O stretching vibrationof CS [48, 55]. The FT-IR spectrum of DOX-CHS-GNPsCS (Fig. 1F) depicts the sharp peaks at 3429, 1627, and1570 ­cm−1 representing the N–H stretching vibration of
DOX and bending vibration of the primary amine of CS
and secondary amine of CHS, respectively. These results
confirmed the presence of DOX, CS, and CHS in the
spectrum of synthesized NPs.
XRD analysis

The XRD analysis recognizes the purity and crystal structure of the central construct of synthesized NPs. Figure 2
shows the XRD pattern of the synthesized CHS-GNPs,
which revealed four well-defined characteristic peaksin the 2θ range (10–80°). The diffraction peaks of CHSGNPs were detected at 37.8°, 44.6°, 64.6°, and 77.5°, whichcorresponded to (111), (200), (220), and (311) crystalline
planes, respectively. This presented a reflection of theface center cubic structure of CHS-GNPs, showing that

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Fig. 2 XRD spectrum of synthesized GNPs

the synthesized GNPs are composed of crystalline. The
XRD pattern showed no additional peak, which refers to
the high purity of CHS-GNPs.
NPs characterization

Following the successful synthesis and preparation of
DOX–CHS–GNP–CS, DLS was used to evaluate of NPs
characterization. To that end, the DLS illustrates theaverage hydrodynamic diameter (Dh) of CHS-GNPs and
DOX-CHS-GNPs 93.75 ± 2.52 nm and 175.8 ± 1.94 nm,
respectively (Fig. 3A). Furthermore, Fig. 3B shows the
TEM image of the synthetic NPs, which demonstrates anentirely dense and homogeneous morphology.
As shown in Table 1, the average size of DOX-CHSGNPs is larger than CHS-GNPs due to the swelling of
DOX around CHS-GNPs. PDI of CHS-GNPs and DOXCHS-GNPs is 0.241 ± 0.06 and 0.244 ± 0.08, which demonstrate highly polydisperse [56]. The surface charge orzeta potential (stability) of DOX-CHS-GNPs and DOXCHS-GNPs-CS were − 34 ± 5.7 and − 25.70 ± 5.90 mv,
respectively, which confirms their proper stability. The
presence of negative charges on CHS minimized theaccumulation of CHS-GNPs, leading to stabilized NPs
dispersion. As seen in Fig. 3A, B, the diameter of CHSGNPs was larger after DOX loading, indicating successful DOX loading. It is noteworthy that the size of NPs,
measured by the TEM, is different from what the DLS
reported due to the differences in the way they work [57].
DLS shows the particles’ hydrodynamic diameter (Dh),including the inorganic core and any coating developedaround the core, such as the hydration layer. On the other
hand, TEM estimates the projected area pore diameter (Pd), which is the equivalent hard-core diameter ofthe particle [58]. Hence, the particle size from the DLS
technique is larger than TEM due to the presence o the
CHS compound around the core of GNPs. Moreover, theAsariha et al. BMC Chemistry(2022) 16:110

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Fig. 3 A Particle size distribution of CHS-GNPs (Blue line), DOX-CHS-GNPs (Green line), and DOX-CHS-GNPs-CS (Red line), B TEM image of
DOX-CHS-GNPs, and C drug release of free DOX from DOX-CHS-GNPs and DOX-CHS-GNPs-CS in PBS (pH 7.4)

Table 1 Physicochemical characteristics of CHS-GNPs, DOX-CHS-GNPs, and DOX-CHS-GNPs-CS
FormulaSize (nm) (DLS)

CHS-GNPs

93.75 ± 2.52

DOX-CHS-GNPs
DOX-CHS-GNPs-CS

175.8 ± 1.94

208.9 ± 2.08

Size (nm)(TEM)

Zeta potential (mV)

PDI

Yield
(%)

DOX loading (%)

83 ± 5.0− 15.50 ± 5.09

0.241 ± 0.06

86.11 ± 2.0–

191 ± 5.0

− 25.70 ± 5.90

0.12 ± 0.03–

84.07 ± 1.15

147 ± 5.0

− 34.00 ± 5.67

surface morphology of CS-CHS-GNPs with loaded DOX
was investigated.
Drug loading and release studies

The amount of drug loading was tested through the massratio changes of 10, 20, and 30%; therefore, the optimized
mass ratio of 10% was selected. At this ratio, the masspercentage of the drug was loaded about 89%. Moreover, the ability to selectively release drugs demonstrates
the optimal synthesis route for preparation with the welldefined characterization of the NPs [46, 47]. The releasestudies were performed under ambient conditions forthe first 6 h of treatment with NPs. As shown in Fig. 3C,
the release of DOX amounts from DOX-CHS-GNPs and
DOX-CHS-GNPs-CS in PBS was illustrated. In the first
6 h of treatment with NPs, 54% of DOX from DOX-CHSGNPs was released, while at the end of 24 h, the releasereached 76.05%. However, the release of the drug from
DOX-CHS-GNPs-CS was slower than DOX-CHS-GNPs
due to the presence of CS, at the end of the first 6 h oftreatment with NPs, 43.8% of DOX was released from
DOX-CHS-GNPs-CS. The highest DOX release from
DOX-CHS-GNPs-CS was indicated at 73.37% after 48 h,
while in the absence of CS, the DOX release was 76.05%
for 24 h. Due to the presence of CS at the surface of
DOX-CHS-GNPs, the process of releasing DOX was prolonged over time, which improved efficient drug release.

0.244 ± 0.08

89.73 ± 5.0

89.10 ± 3.02

Cell viability assay

MDA-MB-468 and βTC-3, and HFb cells were exposed
to free DOX, DOX-CHS-GNPs, and DOX-CHS-GNPsCS at different concentrations of 12.5, 25, 50, 100, and150 μg ­mL−1 over 48 h (Fig. 4). Furthermore, a fluorescence microscopy comparison of HFb cells, MDAMB-468, and βTC-3 cells with DOX-CHS-GNPs-CS
treatment was shown in Additional file 1: Fig. S2.
Fibroblasts exhibited the highest viability after exposure to DOX-CHS-GNPs-CS compared to two cancer
cell lines, which means less toxicity of DOX-NPs on thenormal cells. MDA-MB-468 and βTC-3 cells showed
the lowest viability in exposure to DOX-CHS-GNPsCS compared to DOX-CHS-GNPs. On the other hand,
DOX-CHS-GNPs-CS had the least effect on normal cells
than cancer cells, proving targeted therapy with fewerside effects. The toxicity of DOX-CHS-GNPs-CS innormal fibroblasts significantly increased at concentrations above 12.5 μg ­mL−1, and thus, the viability rate was
reduced. DOX-CHS-GNPs-CS showed a lower effect on
the βTC-3 compared to MDA-MB-468 cells. The findings indicate that βTC-3 cells are less responsive to DOX
chemotherapy. It can be concluded that the low internalization of NPs by this cell line resulted in a milder
response of the cells in comparison with treated cellswith free DOX. The comparison of ­IC50 values is summarized in Table 2.

Asariha et al. BMC Chemistry(2022) 16:110

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Fig. 4 In vitro cytotoxic of DOX, DOX-CHS-GNPs, and DOX-CHS-GNPs-CS against human fibroblast, MDA-MB-468, and βTC-3 cells at various
concentrations after 48 h. ­IC50 value was measured by plotting the log10 percentage of proliferation values versus DOX concentrations

Table 2 IC50 values following treatment of free DOX, DOX-CHSGNPs, and DOX-CHS-GNPs-CS in the cell lines
FormulaIC50 (µg ­mL )−1

MDA-MD-468

βTC

Fibroblast

Free DOX< 12.5< 12.5= 12.5> 12.5

DOX-CHS-GNPs
DOX-CHS-GNPs-CS< 12.5> 12.5> 12.5= 12.5= 12.5

IC50; The half-maximal inhibitory concentration

The difference in the effect of DOX-CHS-GNPs-CS
on normal cells and tumor cells could be related to CHS
interaction with CD44 receptors in the cancer cell membrane. Although CD44 is widely expressed on various celllines, the level of distribution, variety in isoforms, andnature of cell lines explain the differences in MTT results.
Our results comply with previous studies showing MDAMB-468 cells are more susceptible to CHS-GNPs mainly
because of CD44-high expression [59–61].
The effect of DOX‑CHS‑GNPs‑CS on angiogenesisof studied cells

The CAM assay was used to evaluate the potential of
DOX-encapsulated CS-CHS-GNPs in suppressing
angiogenesis and reducing tumor size. As illustrated inFig. 5, the angiogenesis rate in cells treated with DOXCHS-GNPs-CS and DOX-CHS-GNPs (compared withfree DOX and control group) was significantly reduced.
Besides, treatment with DOX-loaded CS-CHS-GNPs
dramatically reduced the size and weight of tumors
formed on the CAM layer (Fig. 6). According to the evidence, the impact of pharmaceutic groups on the growth
and angiogenesis of MDA-MB-468 cells is more significant than βTC3 and normal cells, respectively, which may
be due to the higher cellular uptake of NPs by these cells.

Statistical analysis

All experiments were assessed in triplicate wells
and repeated twice, and data have been shown asmean ± standard error for at least three independent
samples. Data were analyzed by using the student’s t-test
with SPSS software (version 17.0). The statistical comparison between groups and within groups was performed
using one-way ANOVA (*p-values < 0.05).

Conclusion
Development of efficient delivery requires sustained drug
release at a programmed rate which remains a prolonged
period for the precise release of the drug. Manipulationof various biocompatible nanomaterial facilities the controlled and extended-release, likewise selection of CS asa biodegradable, regularly shaped polyhedron and semicrystallisation polymer demonstrated controlled release of
the DDS. The presence of CHS, as an excellent stabilizing
agent, increases the stability of CHS-GNPs. The increasein size and zeta potential with keeping PDI exhibited
controllable release and optimized preparation of NPs
for Dox delivery. Additionally, the cytotoxicity activityof DOX-CHS-GNPs-CS on MDA-MB-468, βTC-3, andHFb cells demonstrates that the construct promise a safe,
sustained release profile and efficient DDS for soluble inwater chemotherapy drugs. Considering these significant
features, the green synthesized CS-CHS-GNPs hold tremendous potential for DOX delivery to the cancer cellswith a lower damage effect on normal cells. Subsequently,
in ovo studies corroborated that treating malignant cellswith DOX-CHS-GNPs-CS could significantly inhibit
tumor growth and angiogenesis to mimic an in vivo
model. Taken together, the findings suggest GNPs as aneffective drug delivery system for the treatment of breastand pancreatic cancers that should be further evaluatedin the future.

Asariha et al. BMC Chemistry(2022) 16:110

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Fig. 5 CAM assay was utilized to investigate the effect of combination therapy on the angiogenesis rate as well as the size and weight of thetumors (A–C)

Fig. 6 The tumor size (left) and tumor weight (right) of CHS-GNPs, DOX-CHS-GNPs, and DOX-CHS-GNPs-CS for release of DOX

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s13065-​022-​00895-x.

Additional file 1: Figure S1. The step-by-step schematic procedure forsynthesized process of DOX-CS-CHS-GNPs. Figure S2. Fluorescence microscopy comparison of human fibroblast, MDA-MB-468, and βTC-3 with DOXCS-CHS-GNPs treatment at various concentrations after 48 h.

Asariha et al. BMC Chemistry(2022) 16:110

Acknowledgements
Not applicable.
Author contributions
SHK and MA accomplished the conception or design of the work. MA and SHK
contributed to the preparation, characterization, and functional analysis of thework. FHP, MF, and MG carried out MTT and in vitro assays. Drafting the article
was prepared by SHK and MA. SI performed in ovo assay. MA and SHK did thedesign and generation of the figures. Critical revision of the article and final
approval of the version to be published SHK. All authors read and approved
the final manuscript.
Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
Availability of data and materials
The datasets generated and/or analyzed during the current study are notpublicly available due feasibility of the study to begin a clinical trial; however,they are available from the corresponding author upon reasonable request.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
All authors give consent for the publication of the manuscript in the Journal of
BMC Chemistry.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Medical Biology Research Center, Health Technology Institute, Kermanshah
University of Medical Sciences, Kermanshah, Iran. 2 Nano Drug Delivery
Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran. 3 Department of Pharmaceutics, Facultyof Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. 4 Department
of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences,
Tabriz, Iran. 5 Pharmaceutical Sciences Research Center, School of Pharmacy,
Kermanshah University of Medical Sciences, Kermanshah, Iran.
Received: 29 April 2022 Accepted: 3 November 2022

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