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Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) ISSN: 2663-2187
https://doi.org/10.48047/AFJBS.6.15.2024.11691-11715
Induction of cytotoxicity and cell cycle arrest in human prostate cancer cells
by chitosan nanoparticles encapsulating Andrographis serpyllifolia (Rottler ex
Vahl) Wight extract.
Hemalatha Ravikumar1, Kavimani Thangasamy1, Sradha Sajeev1, Anju Rani George1,
Menaga Suriyakanthan1, Madhu Priya Govindhan Anbazhagan1, Natesan Geetha1*
DEPARTMENT AND INSTITUTION: Research Scholar, Department of Botany, Bharathiar University,
Coimbatore- 641046, Tamil Nadu, India.
Email id: kavieswari007@gmail.com, sajeevsradha@gmail.com, anjuranigeorge94@gmail.com,
menaga2620@gmail.com, madhumaha2919@gmail.com.
CORRESPONDING AUTHOR: Natesan Geetha1*
Address: Professor, Department of Botany, Bharathiar University, Coimbatore- 641046, Tamil Nadu,
India.
*Email id: ngeethaptc@gmail.com
Phone no: 7010663900
Volume 6, Issue 15, Sep 2024
Received: 15 July 2024
Accepted: 25 Aug 2024
Published: 25 Sep 2024
doi: 10.48047/AFJBS.6.15.2024.11691-11715
ABSTRACT
Prostate cancer is the second most common cause of death for men
worldwide. The encapsulation of herbal extract in polymeric
nanoparticles is a prominent therapeutic remedy and targeted drug
delivery for cancer. The aim of the present study was to investigate
the cytotoxicity and cell cycle arrest of human prostate cancer cells by
using chitosan nanoparticles encapsulated Andrographis serpyllifolia
extract. Aqueous extract of A. serpyllifolia (ALE) at various
concentrations were encapsulated with 2% (w/v) chitosan
nanoparticles (ALE CSNPs). Initially, entrapment efficiency of ALE
by CSNPs and drug release percentage from CSNPs were determined
and then ALE CSNPs were subjected to various spectroscopic
characterization. The effect of ALE CSNPs on PC3 cells was
examined by MTT assay and flow cytometry DNA analysis. Among,
various concentrations of ALE CSNPs, 0.25 mg/mL concentration
exhibited higher entrapment percentage (87.73%). In vitro drug
release study showed a controlled and sustained crude drug release
from ALE CSNPs (91.30%) within 5.5 hours. The addition of plant
extract onto chitosan nanoparticles increases the size of the
nanoparticles of ALE CSNPs i.e. 22.3 nm compared to control i.e.
29.3 nm which is confirmed by XRD. SEM image revealed the
synthesized ALE CSNPs had huge surface area. Compared to CSNPs,
ALE CSNPs showed decreased cell viability and increased percentage
of obstructed cells at G0/G1 cell cycle phase effectively. In
conclusion, ALE CSNPs act as a promising drug delivery system
using chitosan nanocarrier for targeted release of A. serpyllifolia leaf
aqueous extract to PC3 cell lines.
Keywords: Prostate cancer, chitosan nanoparticles, Andrographis
serpyllifolia, cytotoxicity, cell cycle arrest.
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11692 to 10
INTRODUCTION
Prostate cancer is the second most commonly second eventuating cancer in men and the fourth
most common cancer altogether. Nanoscale drug-delivery systems for cancer therapeutics are
rapidly evolving and may provide a novel strategy [1]. In cancer chemotherapy, nanoparticle-
based drug carriers have garnered a lot of interest due to their numerous advantages, which
include improved chemo drug solubility in water, longer blood circulation times, higher cellular
uptake, and improved accumulation in tumors. In recent years, nanotechnology that combines
with polymers has a tremendous interest in pharmaceuticals industry along with therapeutic
innovations. Polymeric nanoparticles offer a new advancement in drug discovery and can be
prepared either from synthetic or natural polymers. Chitosan nanoparticles (ChNPs) are the drug
carriers that have the capacity to release the encapsulated drug gradually due to their high
stability, biodegradability and quick intracellular transit. Because of its chemical makeup,
chitosan is a material that shows promise for use in polyphenol nano-delivery systems and as a
safe agent for drug loading and release [2]. Biologically active compounds with a synergistic
effect are produced when polyphenols, which have at least one aromatic ring and –OH groups
with at least one ring–are combined with chitosan nanoparticles [3].
Andrographis serpyllifolia (Rottler ex Vahl) Wight, a member of the family Acanthaceae, is a
valuable medicinal plant with significant therapeutic activity and is found incorporated in several
ethnobotanical formulations. A. serpyllifolia is densely hispid prostrate herb endemic to
peninsular India. A. serpyllifolia has evolved into a ground hugging prostrate, perennial,
geophyte that successfully survives multiple geo-ecological challenges and grazing threats year
after year. The bioactive compounds of A. serpyllifolia are related to have important biological
applications such as antiulcer, anti-diabetic, [4] antipyretic, anti-inflammatory, antimicrobial,
anticancer and antidote [5]. According to [6] phenolics and andrographolide present in aqueous
extract of A. serpyllifolia may be the factor responsible for anticancer properties. Effective
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11693 to 10
antiproliferative and DNA protective activities are due to free and bound phenolics of A.
serpyllifolia [7]. Based on previous literature, there is no study on cytotoxicity and cell cycle
arrest of human prostate (PC3) cell line induced by A. serpyllifolia aqueous leaf extract loaded
with chitosan nanoparticles. Therefore, the present investigation was undertaken.
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11694 to 10
MATERIALS AND METHODS
Chemicals
Medium molecular weight chitosan (190-310 kDa MW with 75-85% degree of deacetylation)
and dialysis membrane bags were purchased from Sigma-Aldrich. Glacial acetic acid, Sodium
tripolyphosphate and phosphate buffer (pH 7.4) were procured from Sisco Research Laboratories
(Mumbai, India).
Collection, Identification and Extraction process of the plant
A. serpyllifolia was collected from Dharmapuri district, Tamil Nadu India. The identification and
authentification of the plant was done in Botanical survey of India Coimbatore district, Tamil
Nadu (BSI/SRC/5/23/2023/Tech). Leaf samples were collected from mature plant and cut into
small pieces and washed under running water to remove adhering debris. Then the samples were
dried under shade and ground into fine powder and stored at 4oC. 10 grams of leaf powder were
taken and mixed in 100 ml of distilled water. The mixture was boiled at 80°C for 30 minutes in
water bath and cooled at room temperature. Then, the extract was filtered through Whatman No.
1 filter paper. The collected filtrate was dried and stored in refrigerator till further use.
Synthesis of ALE encapsulated chitosan nanoparticles (ALE CSNPs)
ALE CSNPs were prepared according to the ionic gelation method [8]. Briefly, chitosan (2%
w/v) was dissolved in dilute acetic acid (1% v/v) overnight at room temperature to form a 2 mg/2
mL concentration solution and pH was adjusted to 5 using 1 M NaOH. Sodium tripolyphosphate
(TPP) was dissolved in water to reach a final concentration of 1 mg/mL. ALE solution was
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11695 to 10
prepared by dissolving the filtrate at a concentration of 10 mg/10 mL. Different concentrations of
ALE (0.25, 0.75, 0.5 and 1.0 mg) was mixed with 1 mL of TPP separately. Next, this mixture
was added to 1 ml of chitosan solution to reach a final mass ratio of 3:1 (chitosan:TPP) and then
left to stir for 45 min. The mixtures were centrifuged at 10000 rpm for 15 min and washed twice
with de-ionized water. After centrifugation, the supernatants were discarded and the pellets
containing ALE CSNPs were stored in the refrigerator at 4°C for further use.
Determination of Entrapment Efficiency (EE %)
The amount of ALE encapsulated within the CSNPs was determined by indirect method, through
calculating the amount of unencapsulated drug. After adding the TPP, the mixture was
centrifuged at 10000 rpm for 15 min and the clear supernatant containing the free unencapsulated
drug was collected, diluted with distilled water and measured spectrophotometrically at 273 nm
[9]. The drug (ALE) entrapment efficiency in CSNPs was determined using the following
equation.
EE% = (Total amount of ALE added- free ALE in supernatant) X 100
Total ALE added
Determination of in vitro drug release percentage
ALE release percentage from CSNPs was determined using the technique of dialysis tube
analysis (12,000-14,000 molecular weight). In brief, the dialysis membrane was washed with
lukewarm double distilled water (70ºC) for 1hr and rinsed thoroughly (thrice) to eliminate
glycerin. ALE CSNPs with higher entrapment efficiency (87.73%.) was placed in a dialysis bag
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11696 to 10
which was sealed and immersed in 50 mL of phosphate buffer (pH 7.4) at room temperature with
stirring at 1000 rpm for 6 h. 3 mL of the solution was withdrawn every half an hour and replaced
with an equivalent volume of fresh solution. This process was repeated upto 3.5 hours. The
withdrawn samples were analyzed using UV/visible spectroscopy at 265 nm and the amount of
ALE release pattern from CSNPs was determined [10]. The drug release percentage was
determined by using the following formula.
Drug release [%] = C (t)/C (0) × 100
where C(t) is the absorbance of ALE CSNPs at 265 nm at time t.
In vitro drug release kinetics
Various release kinetic models such as zero order, first order, Higuchi model and Korsmeyer–
Peppas have been used to fit the cumulative in vitro drug release data and to describe the drug
release kinetics [11]. The best release pattern is explained using the coefficient of determination
(R2) value. Model with the highest R2 is considered as the best one [12].
Characterization of drug loaded chitosan nanoparticles
CSNPs and ALE CSNPs were subjected to UV-vis spectrophotometry and Fourier transform
infrared spectroscopy (FTIR) to study optical properties and to identify functional groups,
respectively. Crystallinity patterns of CSNPs and ALE CSNPs were determined by X-ray
diffraction (Shimadzu LabX- XRD 1600). Particle size, the charge on the surface of the
nanoparticles and poly dispersity index (PDI) were measured through Dynamic Light Scattering
(DLS) with zetasizer (Malvern analytical, Chennai India). Topography and elemental
composition of CSNPs and ALE CSNPs were investigated through Scanning electron
microscopy and Energy Dispersive analysis of X-Ray (Quanta 400 ESEM).
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11697 to 10
In vitro anti-prostate activity
Human prostate cell lines (PC3) were obtained from American Type Culture Collection (ATCC)
10801. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) media
supplemented with 10% (v/v) fetal bovin serum and 1% (v/v), 100 U/Ml penicillin and 100
µg/mL streptomycin. Cells were cultured in an incubator at 37°C and 5% CO2 humidified
atmosphere. Medium was changed every 2 -3 days. Reagents and media for cell culture were
purchased from Sigma-aldrich (Merck) and Sisco Research Laboratories Pvt. Ltd., Mumbai, India.
The cellular toxicity on cultured cells was measured using MTT [3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyl tetrazolium bromide] assay. Cells were grown overnight in a 96-well plate at a
density of 1 × 104 cells per well. Then, cells were treated with different concentrations of test
samples such as ALE and ALE CSNPs (0, 10, 20, 40, 80, 160 and 320 μg/mL) and anticancer
standard Doxorubicin (3.125, 6.25, 12.5, 25, 50, 100µM) and incubated at 37°C for 24 h. Later,
cells were washed twice with phosphate buffer saline (PBS). MTT solution was added to each
well (0.5 mg/mL) and the plate was incubated for 4h at 37° C in 5% CO2 atmosphere. Finally,
the medium was replaced by DMSO to solubilize the formazan. The absorbance was measured
using a microplate reader at 590 nm. IC50 value for cytotoxicity tests were derived from a
nonlinear regression analysis based sigmoid dose response curve and calculated using prism
Graph Pad Prism 6 (Graph pad, SanDiego, CA, USA) [13].
The percentage growth inhibition was calculated using the following formula:
% inhibition = (control abs - sample abs) / (control abs) × 100
Cell morphological analysis
PC3 cancer cells were grown and incubated with CSNPs, Doxorubicin, ALE and ALE CSNPs at
their IC50 concentration and cells were taken and observed under an inverted phase contrast
microscope to study the cell morphology at 40× magnification (Vivek et al., 2012).
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11698 to 10
Flow Cytometric Analysis of Cell Cycle Distribution
Cell cycle distribution and ploidy status of cells after treatment with ALE and ALE CSNPs were
determined by flow cytometry DNA analysis. At the end of treatments, cells were detached from
the plates by the addition of 0.25% trypsin, washed in PBS, fixed in 70% ethanol at 4oC and
treated with 10 mg/ml RNAse for 30 minutes at 37oC. The DNA content was evaluated in a
FACS can flow cytometer (Becton Dickinson, Franklin Lakes, NJ) after staining the cells with
50 mg/ml propidium iodide for 15 minutes in the dark at room temperature and analyzed using
Cell Quest software. For cell cycle analysis, only single cells were considered [14].
Statistical analysis
All statistical analysis was performed using Prism 5 (GraphPad Software, Inc.). Data are
presented as the mean ± SEM. One-way ANOVA with Dunnett's post hoc test was used to
compare across multiple treatments. Experiments were repeated three times with at least
triplicate wells per condition. Results with P < 0.05 was considered to indicate a statistically
significant difference. The SPSS software (ver. 22.0, SPSS Inc., United States) was used to
analyze the experimental data.
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11699 to 10
0
20
40
60
80
100
120
0.25 0.5 0.75 1
Entrapment efficiency (%)
Concentration of ALE (mg\mL)
Entrapment efficiency
RESULTS
Entrapment efficiency
The percentage of entrapment efficiency is illustrated in Fig 1. Among various concentrations of
ALE CSNPs (0.25, 0.5, 0.75 and 1.0 mg/mL), 0.25 mg showed higher percentage of entrapment
efficiency (87.73%) within CSNPs.
Fig. 1: Entrapment efficiency (%)
In vitro drug release study
The cumulative release of the ALE from CSNPs was studied in dialysis bag containing PBS
solution at pH 7.4. The released amount of ALE was calculated by comparing the absorbance of
the drug at 265 nm by UV–vis spectroscopy with the earlier measured calibration curves with a
dilution series. Finally, the cumulative percent of drug released from CSNPs was plotted against
time. In vitro drug release studies showed a controlled and sustained release of ALE from CSNPs
(91.3%) within five and half hours (Fig.2).
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11700 to 10
0
10
20
30
40
50
60
70
80
90
100
0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5
Drug release percentage (%)
Time (hrs)
Percentage (%) of drug release
Fig. 2: In vitro drug release percentage of ALE from ALE CSNPs
In vitro drug release kinetics
In vitro drug release profile of ALE CSNPs indicates that 50% of the drug was released within 2
hours. After five and half hours, 91.3 % of the drug was released with slow and sustained
manner. In order to analyze the in vitro release data and to evaluate the drug release kinetics,
four mathematical models such as zero order, first order, Higuchi model and Korsmeyer–Peppas
have been used (Fig. 3). The values of correlation coefficient of kinetics models are presented in
Table 1. The R2 value obtained from the Higuchi model is found to be greater (0.99) than those
of other models. It indicates the release mechanism of ALE from CSNPs has been taking place
following dissolution process.
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11701 to 10
y = 40.201x - 1.5486
R² = 0.9961
0
20
40
60
80
100
0.5 1 1.5 2 2.5
Percentage of drug release
Sq.root of time
Higuchi Model
y = -0.6806x + 1.9971
R² = 0.6145
0
0.5
1
1.5
2
2.5
-0.5 0 0.5 1
Cumulative Percentage (%)
Log time
Korsmeyer-Peppas Model
y = -0.1562x + 2.2004
R² = 0.8507
0
0.5
1
1.5
2
2.5
0.5 2.5 4.5 6.5
Cumilative percentage (%)
Time (hrs)
First Order Kinetics
y = 12.571x + 27.278
R² = 0.9671
0
20
40
60
80
100
120
0.5 2.5 4.5 6.5
Percentage (%) of drug release
Time (hrs)
Zero Order Kinetics
Fig 3: In vitro drug release kinetics plots
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11702 to 10
Table 1: Correlation coefficients and kinetic constants of different kinetics models for
ALE CSNPs.
Kinetic Models
Encapsulated ALE CSNPs
R2
Slope
Intercept
Zero order kinetics
0.9671
12.571
27.278
First order kinetics
0.8507
-0.1562
2.2004
Higuchi Model
0.9961
40.201
1.5486
Korsmeyer-Peppas Model
0.6145
-0.6806
1.9971
Ultraviolet Visible (UV – Vis)
To identify the absorbance peaks of both CSNPs and ALE CSNPs, a UV/Vis spectrophotometer
scans were taken over the wavelength range of 200 to 800 nm. The strong surface plasmon
resonance (SPR) centered at 250 nm was attributed to both CSNPs and ALE CSNPs (Fig.4A).
Fourier Transform Infrared (FTIR) Spectroscopy
FTIR analysis was carried out to identify functional groups present in the ALE CSNPs complex.
FTIR spectrum of ALE CSNPs is compared with the FTIR spectrum of CSNPs (Fig. 4B). The
spectra obtained for CSNPs and ALE CSNPs showed thirteen and eighteen peaks with various
functional groups, respectively. Seven functional groups were found common for both. The
FTIR spectrum of CSNPs exhibits characteristic bands assigned to the δ-lactone at the
wavelength of 1742.05 cm-1, aldehyde at the wavelength of 1385.85 cm-1, alkyl aryl ether at the
wavelength of 1222.65 cm-1 and 1,2,4-trisubstituted aromatic compound at the wavelength of
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11703 to 10
886.131 cm-1. There were six characteristic bands unique to ALE CSNPs such as conjugated acid
halide at the wavelength of 1747.19 cm-1, imine/oxime at the wavelength of 1644.98 cm-1,
carboxylic acid at the wavelength of 1420.32 cm-1, phenol at the wavelength of 1375.96cm-1,
sulfonamide at the wavelength of 1314.25 cm-1 and anhydride at the wavelength of 1045.94 cm-1.
X-ray diffraction (XRD)
The XRD pattern of CSNPs showed the highest peaks in 2θ range with various crystal planes i.e
20.96° (223), 21.96° (390), 22.60° (455), 23.04° (399) and 25.10° (683). ALE CSNPs exhibited
the presence of several crystal peaks i.e 21.98° (389), 22.26° (403), 24.04° (733), 29.98° (560)
and 30.08° (643) situated at angles (2θ). The amorphous nature of the XRD pattern was observed
for both samples. The average size of the CSNPs and ALE CSNPs were 22.3 nm and 29.3 nm,
respectively, which was determined using the Debye-Scherrer equation (Fig. 4C).
Zeta potential, Dynamic light scattering and Polydispersity index
The zetapotential of CSNPs and ALE CSNPs were + 10.67 mV and + 28.40 mV, respectively
(Fig. 4D). The mean diameter and poly dispersity index (PDI) of the tested nanoparticles in the
water medium were observed by the DLS method and the results are described in fig.4E. The
average diameter of CSNPs and ALE CSNPs are found to be 240 ± 0.5 nm and 260 ± 1.5 nm,
respectively. PDI for CSNPs and ALE CSNPs were 0.22 and 0.35, respectively.
Scanning Electron Microscopy (SEM)
The surface morphology of the CSNPs and ALE CSNPs was determined by SEM analysis. There
was a large amount of spherical shaped and well separated nanoparticles found for both the
samples (Fig.4F). The size of crystalline structure was calculated using Debye–Scherrer
equation. The mean size of CSNPs was found smaller (56.88 nm) than the ALE loaded CSNPs
(64.53 nm).
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11704 to 10
Energy-dispersive X-ray (EDAX)
The EDAX spectra analysis of CSNPs and ALE CSNPs showed the presence of carbon (C),
oxygen (O) and nitrogen (N) as main elements (Fig. 4G). The presence of signals for sliver (Ag)
and copper (Cu) indicates the impurities of the samples.
Fig. 4: A) UV Visible spectra of CSNPs and ALE CSNPs; B) FTIR spectra CSNPs and
ALE CSNPs; C) X-ray diffraction Pattern of CSNPs and ALE CSNPs; D) Zeta potential of
CSNPs and ALE CSNPs; E) DLS of CSNPs and ALE CSNPs; F) SEM micrograph of
CSNPs and ALE CSNPs and G) EDAX analysis of CSNPs and ALE CSNPs.
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11705 to 10
Cell inhibition and Cell morphological analysis
In this study, the effect of different concentrations of ALE CSNPs on cancerous cells was
assessed by both morphological analysis and MTT assay. After 24 h treatment, of various
concentrations of ALE CSNPs, the higher cell inhibitory percentage i.e 92.9% against PC3
cell line was found at 100 µg/mL concentration compared to standard Doxorubicin (Fig. 5-
I). At this same concentration, notable impact on cancer cell morphology such as the cell
membrane disruption and nuclear condensation were observed in cells treated with ALE
CSNPs compared to CSNPs, standard Doxorubicin and ALE treated cancer cells (Fig. 5-II
a-b).
Fig.5: I) Effect of ALE CSNPs and standard Doxorubicin on PC3 cells inhibition percentage.
PC3 cells were treated with 20, 40, 60, 80 and 100 µg/mL of ALE CSNPs for 24 hr and their
viability were examined by MTT assay. Data are reported as the mean ± SEM (n=3) ; II) a-b
Morphological changes of PC3 cell line treated with CSNPs, Doxorubicin, ALE and ALE
CSNPs for 24 hrs.
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11706 to 10
Cell cycle analysis by Flow cytometry
Flow Cytometric analysis revealed that PC3 cells treated with ALE CSNPs exhibited significantly
increased percentage of obstructed cells at G0/G1 phase after 24 h, whereas the percentages were
significantly decreased at S and G2/M phases. These results indicated that following exposure to ALE
CSNPs, cells were impeded in their cycle progression (Fig. 6 A-D).
Fig 6: Cell cycle analysis of (A) CSNPs (B) Doxorubicin (C) Plant extract (D) ALE CSNPs.
Quantification of the effects of treatment on the cell cycle distribution as determined via flow cytometry.
* P<0.05 vs. control and Chitosan nanoparticles.
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11707 to 10
Discussion
Cancer is one of the major causes of death in various countries and is one of the main problems
in the current century [15]. Conventional radiation and chemotherapies are associated with
severe side effects in addition to being unduly expensive. Herbal medicines have acquired very
high demand since the early civilization because of their effectuality against several diseases
comprising cancer. Research conducted by different people suggests the extensive potential of
various plant extracts used in traditional as well as folk medicine and phytochemicals thereof
with a peculiar mode of action as an effective remedy for detrimental diseases like prostate
cancer [16,17] In the current study, we investigated the cytotoxicity and cell cycle arrest of
human prostate cancer cells by using chitosan nanoparticles encapsulated A. serpyllifolia
aqueous extract.
In general, A. serpyllifolia claims to treat antibacterial, anti-dote, anticancer, anti-ulcer, anti-
inflammatory [18] and anti-diabetic activities [19]. Phenolics and andrographolide of A.
serpyllifolia are the main bioactive compounds acting on its anticancer property [20]. Chitosan is
a linear co-polymer composed of β-(1–4) linked D-glucosamine and N-acetyl-D-glucosamine
units and is currently drawing attention as a promising raw material in pharmaceutical, medicinal
and agricultural applications [21]. In the present investigation, aqueous extract of A. serpyllifolia
(ALE) at various concentrations were encapsulated with 2% (w/v) chitosan nanoparticles (ALE
CSNPs). The encapsulation or entrapment percentage was ranged from 43.39% to 87.73%. The
highest EE% was obtained for 0.25 mg/mL concentration of ALE CSNPs. Increasing the
concentration of plant extract caused a decrease of EE% from 0.5 to 1.0 mg/mL. The reason
behind is the excess level of plant extract cannot be absorbed by chitosan nanoparticles due to
accomplishment of saturation level. Previous studies have also reported similar results [22]. In
general, drugs with higher entrapment efficiency are able to enter nanocarrier systems more
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11708 to 10
easily due to their hydrophobic nature. It is also due to ionic interaction between the CSNPs and
TPP [23].
Our investigation on in vitro drug release studies reveals that a characteristic biphasic
release tendency i.e release of ALE from CSNPS was 57.8 ± % within 2 hours (initial burst) and
was 91.3± % within five and half hours (sustained drug release). These results are in parallel
with those reported in a recent study [24]. In order to analyze the in vitro release data and to
evaluate the drug release kinetics, four mathematical models such as zero order, first order,
Higuchi model and Korsmeyer–Peppas have been used. The mechanism of drug release from
CSNPs involves various mechanisms such as desorption, erosion, degradation, reabsorption and
diffusion [25,26]. The drug release kinetics from drug carrier is vital in preclinical development
and will serve as the basis for evaluation of drug formulations and regulatory approvals.
Prediction of in vivo drug release through in vitro techniques for nano formulations is becoming
widely developed [27]. The diffusion-controlled release kinetics was explored using the zero-
order, first-order, and Higuchi models. Mathematical models have many advantages, involving
predicting drug release mechanisms, helping in formulation development and fabricating
controlled drug release systems [28]. In the present study, the R2 value obtained from the
Higuchi model is found to be greater (0.99) than those of other models. This result suggested that
ALE release at pH 7.4 from ALE CSNPs complex follows the Higuchi model kinetics. It also
indicates that ALE is released by diffusion process [29].
To identify the absorbance peaks of both CSNPs and ALE CSNPs, a UV/Vis spectrophotometer
scans were taken over the wavelength range of 200 to 800 nm. The strong surface plasmon
resonance (SPR) centered at 250 nm was ascribed to both CSNPs and ALE CSNPs (Fig. 4A ). It
was earlier reported that the UV–visible spectrum of chitosan nanoparticles was ranged between
200 and 322 nm due to the presence of the C=O functional group [30].
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11709 to 10
FTIR analysis shows the characteristic bands for chitosan nanoparticles (δ-lactone, Aldehyde,
Alkyl aryl ether and 1,2,4-trisubstituted aromatic compound) and for ALE CSNPs (Conjugated
acid halide, Imine/oxime, Carboxylic acid, Phenol, Sulfonamide and Anhydride). The presence
of carboxylic acid and phenols in the plant extract are involved in the interaction of phosphoric
ammonium ions of chitosan nanoparticles [31]. XRD analysis show some characteristic bands
for CSNPs (455, 399, 683) and ALE CSNPs (733, 560, 643) in their diffractograms at different
angles. No significant difference was found between diffractograms of both. However,
broadened peaks were found in ALE CSNPs compared to CSNPs which confirmed the
amorphous nature of CSNPs and successful encapsulation of ALE within CSNPs. A similar
observation was made by Piran [32]. who showed the increased antioxidant activity of green tea
extract encapsulated in chitosan-citrate nanogel. In the present study, free chitosan nanoparticles
showed a particle size of 240±0.5 nm and the size of ALE encapsulated chitosan nanoparticles is
of 260± 1.5 nm during DLS test. The size of the particles increases due to interaction of plant
extract with chitosan nanoparticles. Zeta potential value indicates the surface charge of the
nanoparticles. In the present study, ALE CSNPs has higher positive zeta potential value
(+ 28.40 mV) compared to CSNPs (+ 10.67 mV). The higher zeta potential value indicates the
stability of the chitosan nanoparticles. Polydispersity index (PDI) value, ranging from 0 to 1
determines the homogeneity of the nanoparticles. In the present investigation, PDI value
observed for CSNPs and ALE CSNPs are 0.22 and 0.35 respectively. (Manne et al., 2020;
Mondéjar-López et al., 2022). In this study, the SEM analysis shows the topography of CSNPs
and ALE loaded CSNPs. The unloaded separated free chitosan nanoparticles have a smaller size
whereas plant extract loaded unglued chitosan nanoparticles have a larger size which may due to
encapsulation of plant extract. These results are found similar with the findings of previous
researchers [33,34]. Energy-dispersive X-ray spectroscopy (EDAX) analysis was used to
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11710 to 10
investigate the chemical composition and principal constituents of biosynthesized CSNPs [35In
this present study, EDAX results inferred the number of different constituents (carbon, oxygen
and nitrogen) present in the nanocomposite.
In the present study, it is clear that PC3 cancer cells treated with various concentrations of ALE
CSNPs lose their capacity to proliferate in a dose dependent manner after 24 hrs during MTT
assay. The higher cell inhibitory percentage was found at 100 µg/ml concentration with 24
µg/mL IC50 value. These findings are in agreement with earlier studies describing genistein and
anti-inflammatory drug celecoxib loaded nanoliposome formulation [36], curcumin loaded lipid
nanoparticles [37] and sodium butyrate loaded PEG, folic acid and chitosan mediated nano
complex [38] are found to be suppressing prostate cancer cell growth via induction of apoptosis
and autophagy during cellular phases.
Cell cycle is a series of events that controls the self-replication of cells. One or more cell-cycle
checkpoint defects are involved in most of the cancer types including prostate cancer [39] The
results of our study showed that the anti-tumor effect of ALE CSNPs significantly hampered the
PC3 cell lines in S- phase and led to less accumulation of obstructed cell lines in G2-M phase
[40].
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11711 to 10
CONCLUSION
In conclusion, our results indicate that ALE CSNPs has cytotoxic activity in PC3 cancerous cells. This
present study has successfully developed the ALE CSNPs as a promising drug delivery system using
chitosan nanocarrier for targeted release of A. serpyllifolia leaf aqueous extract to PC3 cell lines. The
characterization studies of plant extract encapsulated chitosan nanoparticles confirmed the successful
loading of plant drug onto CSNPs. Some biomedical tests like MTT assay and analytical DNA flow
cytometry have been applied for analyzing the prostate cancer cell growth destruction. Outputs have
noticeably confirmed the potential performance of ALE CSNPs in the inhibition of PC3 cancer cells. The
outcome of the investigation provided a ground base to initiate in vivo experiments to assess the
efficiency of ALE CSNPs on animal models and also to perform some advanced studies to understand the
effective molecular mechanisms of ALE CSNPs behind prostate cancer suppression.
Acknowledgement
The authors duly acknowledge funding agencies i.e DST-FIST and DST-PURSE, India for
providing all the instrumentation facilities for carrying out the research work in the Dept. of
Botany, Bharathiar University, Coimbatore, Tamil Nadu, India.
Conflicts of interest
The authors declare that there is no conflict of interest.
Funding Source
Nil
Hemalatha Ravikumar /Afr.J.Bio.Sc. 6(15) (2024) Page 11712 to 10
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