Ind. J. Pharm. Edu. Res., 2025; 59(3):995-1006.

https://www.ijper.org Original Article

Investigation on the Effect of Alginate Concentration and

Flow Rate on Production of Nanoparticle Loaded with
Naringenin Using Electrospray Method
Fashli Syafiq Abd Razak1,*, Liew Kai Bin1, Shamima Abdul Rahman1, Nurul Izzah Amirah Abdul Rashid1,
Nurul Akmar Abd Malek1, Bontha Venkata Subrahmanya Lokesh2, Wan Hamirul Bahrin Wan Kamal3,
Abd Almonem Dooalaanea4, Mohamed Awang4, Mohamad Faizal Safa’at5, Panairajan Vijayaraj Kumar6
1
Faculty of Pharmacy, University of Cyberjaya, Cyberjaya, MALAYSIA.
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universiti Malaya, Kuala Lumpur, MALAYSIA.
3
Medical Technology Division, Agensi Nuklear Malaysia, MALAYSIA.
4
Faculty of Pharmacy, University College of MAIWP International, Kuala Lumpur, MALAYSIA.
5
Espray Sdn Bhd, Kuala Lumpur, MALAYSIA.
6
Faculty of Pharmaceutical Sciences, UCSI University, Kuala Lumpur, MALAYSIA.

ABSTRACT Correspondence:
Background: Naringenin, a polyphenolic phytochemical belonging to the flavanone class, Mr. Fashli Syafiq Abd Razak
exhibits notable anti-cancer, antioxidant, and anti-inflammatory properties. However, its Faculty of Pharmacy, University of
therapeutic application is constrained by poor aqueous solubility and limited delivery to target Cyberjaya, MALAYSIA.
sites. Purpose: This study aimed to address these challenges by encapsulating naringenin Email: fashlirazak@gmail.com
in alginate nanoparticles using an electrospray method. The effects of varying alginate
concentrations and flow rates on nanoparticle production were investigated. Materials and Received: 05-09-2024;
Methods: Naringenin was mixed with sodium alginate and Tween 80, and the solution was
Revised: 15-11-2024;
electrosprayed into a calcium chloride cross-linking agent. Particle size, Polydispersity Index (PDI),
Accepted: 06-04-2025.
zeta potential, and Encapsulation Efficiency were measured. Results: the research work indicated
that an alginate concentration of 0.5% w/v and a flow rate of 0.2 mL/hr produced nanoparticles
with optimal characteristics, including a particle size of 146 nm, a PDI of 0.331, a zeta potential
of -1.10 mV, and an Encapsulation Efficiency of 92.80%. Conclusion: These findings highlight the
significant influence of alginate concentration and flow rate on nanoparticle formation, providing
insights into developing effective naringenin-loaded nanoparticles for therapeutic applications.

Keywords: Alginate, Electrospray, Encapsulation, Flow Rate, Nanoparticle, Naringenin.

INTRODUCTION
Flavonoids are natural occurring class of compounds as nephroprotective, anticancer, neuroprotective, gastrointestinal
polyphenolic secondary plant metabolites that are mainly present protective, and anti-atherogenic agent.3 Despite possessing
in the food of plant origins such as vegetables, fruits, and nuts.1 various pharmacological properties, naringenin found to be
Naringenin is chemically named as 2,3-dihydro-5,7-dihydroxy-2- highly lipophilic in nature with poor aqueous solubility and low
(4-hydroxyphenyl)- 4H-1-benzopyran-4-one with its molecular bioavailability, thus limiting its biological action and formulation
weight of 272.26 and the melting point of 251ºC is one example development challenges.4
of such a flavonoid compound that belongs to the flavanone
Nanotechnology or nanoparticle-based drug delivery systems has
subclass2 as shown in the chemical structure in Figure 1.
gained so much attention of recent, and it has been extensively
Naringenin has been reported to exert a variety of studied for the treatment application of diseases especially
pharmacological properties such as antioxidant, hepatoprotective, cancer treatment.5 This is due to the fact that nanoparticle-based
cardioprotective, anti-diabetic, anti-inflammatory, drug delivery systems have many potential advantages in terms
of the possibility to modify drug properties such as improving
DOI: 10.5530/ijper.20256682
the solubility of hydrophobic drugs hence bioavailability
owing to their unique characteristics physically and chemically
Copyright Information : which makes them superior compared to conventional drugs.6
Copyright Author (s) 2025 Distributed under Generally, the nanoparticle can be defined as a tiny particle in
Creative Commons CC-BY 4.0
which the size of the particle is within the range from 1 to 100
Publishing Partner : Manuscript Technomedia. [www.mstechnomedia.com] nanometers. Based on their physical characteristics such as

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 Razak, et al.: Investigation on the Effect of Production of Nanoparticle Loaded with Naringenin

morphology and size as well as their chemical characteristics, Preparation of sodium alginate loaded with
nanoparticles can be classified into different classes which are naringenin formulation
lipid-based nanoparticles, polymeric nanoparticles which can be
General nanoparticle formulation preparation was adopted
further classified into nanocapsules and nanospheres, as well as
from Alallam et al., (2020) with modification.16 Nanoparticle
inorganic nanoparticles.7-9
formulation was prepared using materials composed of
Polymer-based nanoparticles are known as a colloidal system sodium alginate solution as a polymer, where naringenin was
built of biocompatible and biodegradable natural or synthetic encapsulated with Tween 80 used as a surfactant that reduced the
polymers in which the active ingredient is encapsulated and surface tension of the alginate solution. Sodium alginate solution
adsorbed onto a polymer matrix.10 Alginate is one of the (1% w/v) was prepared according to the procedure adopted
biodegradable polymers, which has been extensively studied from Cui et al., (2022) with slight modification.17 5 g sodium
for the development of drug delivery systems.11 Alginate is a alginate powder was weighed and dissolved in 500 mL of distilled
naturally occurring anionic polysaccharide made up of various water. The stock solution was then left on stir overnight at room
arrangements of residues of a chain of biodegradable and temperature to achieve a homogenous state and to allow full
biocompatible (1-4)-linked β-D-mannuronic acid and α-L- hydration.18
guluronic acid and it can be found abundantly distributed in the
Firstly, 0.1 g of naringenin powder was weighed to dissolve
cell wall of a brown algae or Phaeophyceae species.11 Alginate
in 10 mL pure ethanol to produce naringenin solution with
has unique biopharmaceutical properties including exhibiting
concentration of 10 mg/mL. The solution was kept in tight-closed
pH sensitivity, biodegradability, biocompatibility as well as
container to prevent evaporation of naringenin solution. 10
mucoadhesive nature and it does not appear to exhibit any
mL of sodium alginate(1% w/v) was measured and taken into
toxicity and immunogenicity, to make this material interesting as
a beaker and while stirring, 10 mL of naringenin solution was
a coating material and as a part of modified-release or controlled
added dropwise into the beaker containing sodium alginate.
release drug formulation.12 Furthermore, it has the capability
Then 0.1 mL of Tween 80 was added into the solution to produce
to create gels through substitution of the sodium ions from
guluronic acids for divalent cations like calcium ion, forming an alginate-naringenin solution up to the concentration of 0.5%
“egg-box” shape.13 v/v. These steps were repeated using the same amount and
concentration of naringenin and Tween 80 but with different
The electrospray method is one of the most effective methods sodium alginate concentrations(0.5% w/v and 0.25% w/v) to
for preparing nanoparticles as it has gain greater advantages produce alginate-naringenin solution to make up to final alginate
including low cost, involve in one step, high reproducibility, concentrations of 0.25% w/v and 0.125% w/v respectively.
and most importantly exhibit high Encapsulation Efficiency.14
Furthermore, it has the capacity to create smaller particles, Nanoparticle production using electrospray method
better size distribution, and less agglomeration. For this, the The preparation of calcium chloride solution was adopted
electrospray technique plays an important role in pharmaceutical from Mehregan Nikoo et al., (2016).18 Firstly, 2 g of calcium
applications.15 The electrospray method works by applying a chloride powder was weighed and then dissolved in 100 mL of
high voltage to a polymeric solution and forcing the polymer to distilled water to produce calcium chloride (2% w/v) solution
emerge from the syringe as nanoparticles.14 Despite this method that acts as a cross-linking agent. Electrospray system was set
is quick and simple, alginate polymer concentration could be one up according to the method adopted from Rutkowski et al.,
of the factors that could affect the production of nanoparticle
(2018) with modification.19 The set up consists of high voltage
using electrospray method. Therefore, this study was done to
power supply unit, syringe pump, a syringe, plastic tube, a metal
investigate the effect of different alginate polymer concentrations
needle serving as nozzle, aluminum foil, positive and negative
and flow rate on the production of nanoparticles loaded with
electrode, and collector as depicted in Figure 2 Briefly, 10 mL of
naringenin using the electrospray method.
alginate-naringenin solution with alginate final concentration
MATERIALS AND METHODS of 0.5% w/v was loaded into 10 mL syringe. 10 mL of 2% w/v
calcium chloride was put into petri dish that act as a collector.
Materials The electrospray was run for 1 hr. The parameters involved in
Sodium Alginate (CAS no. 9005-38-3) was purchased from the preparation of alginate nanoparticle loaded with naringenin
RandM Chemicals (Malaysia), Naringenin from grapeseed using electrospray method were summarized in Table 1 and Table
extract (CAS no. 67604-48-2, purity>98% HPLC) was purchased 2.
from Shaanxi Yuantai Biological Technology Co., Ltd (China),
Ethanol (absolute ethanol, CAS no. 64-17-5), Tween 80 (CAS no. Electrospray mode identification
9005-65-6) and Calcium Chloride (CAS no. 10043-52-4) were The effect of different sodium alginate concentrations on
purchased from Sigma Aldrich (USA). electrospray mode was investigated for alginate-naringenin

996 Indian Journal of Pharmaceutical Education and Research, Vol 59, Issue 3, Jul-Sep, 2025
 Razak, et al.: Investigation on the Effect of Production of Nanoparticle Loaded with Naringenin

solution. The alginate-naringenin solution with sodium alginate A standard curve was obtained by dissolving 50 mg of naringenin
with final concentrations of 0.5% w/v, 0.25% w/v and 0.125% in 50 mL ethanol solution followed by a serial of dilution (0.5, 0.25,
w/v were studied. A camera (Industrial Microscope Camera 0.125, 0.0625, 0.03125 and 0.015625 mg/mL). The absorbance of
with HDMI VGA two output) was set up and a laser was used to six standard samples were measured at max 297 nm. The percent
monitor the formation of the Taylor cone and spray formation.20 Encapsulation Efficiency (EE) was calculated using the formula
Observation of the spray form under continuous light source is below (Equation 1).24
one of the simplest ways of mode identification.21

Particle size analysis and polydispersity index EE=Encapsulation Efficiency,

Particle size and the Polydispersity Index (PDI) of nanoparticle Cf=Free naringenin concentration,
were analyzed by dynamic light scattering using Malvern Zetasizer
Ct=Total naringenin concentration.
Nano series Nano-S (Model ZEN 1600) and Nano-Z (Model
ZEN3600) (Malvern Instruments Ltd., Malvern, Worcestershire, RESULTS
UK). The median particle size (Di50) was evaluated to obtain the
average particle size of nanoparticle, meanwhile Polydispersity Effect of alginate concentrations on the
Index was evaluated to determine the size distribution of the electrospraying mode
nanoparticle in terms of whether the sample is monodispersed or In the absence of voltage, three samples of alginate-naringenin
not.22 Nanoparticle size and Polydispersity Index were measured solutions were pumped at a flow rate of 0.5 mL/hr and exited
at a detection angle of 173º and under the temperature of 25ºC. the needle. The dripping mode could be clearly seen in Figure 3.
The samples were diluted using distilled water at a 1:10 ratio to When the applied voltage was increased slowly to 10 kV to the
ensure optimal concentration. electro sprayed solutions of samples to create an electric field. It
was depicted that the meniscus turned rapidly for sample C to a
Zeta potential stable cone-jet spray mode known as Taylor cone, from which a
jet of liquid emerged and became a stable spray.25 On the other
Zeta potential of nanoparticle was analyzed by dynamic light
hand, the unstable multi-jet spraying mode of sample B and
scattering using Malvern Zetasizer Nano series Nano-S and
sample A respectively, when 10 kV voltage was applied as shown
Nano-Z (Malvern Instruments Ltd., Malvern, Worcestershire, in Figure 4a, 4b and 4c below.
UK) under temperature of 25ºC, where the nanoparticles were
collected from Petri dish containing calcium chloride solution Particle size analysis
(2% w/v) and then transferred to 1-cm path length cuvette for The results were obtained for the measurement of the particle
the measurement. size of nanoparticle for three samples (A, B, C) as shown in Table
3. In this analysis, Di50 and Polydispersity Index (PDI) of all
Encapsulation Efficiency the samples were compared. Sample C has the smallest average
The Encapsulation Efficiency of nanoparticles was measured. The particle size (146 nm). Meanwhile, when lower sodium alginate
method for determining Encapsulation Efficiency was adopted concentration was used, sample A and sample B exhibit large
from Zare Kazemabadi et al., (2019).23 Alginate polymer loaded average particle sizes of 900 nm and 1980 nm respectively.
with naringenin nanoparticle was centrifuged at 14000 rpm Among these samples, sample B has the narrowest distribution as
for 30 min. After nanoparticle was subjected to centrifugation, the sample has the largest average particle size, which might not
a clear supernatant with a non-encapsulated naringenin in the be a good candidate for nanoparticle preparation. On the other
nanocarrier was separated. The absorbance of this phase was hand, PDIs for sample A and sample C exhibit larger particle size
measured using UV-visible spectrophotometer (Shimadzu/ distribution as 0.431 and 0.535 respectively compared to sample
UV-1700, Kyoto, Japan) at 297 nm using a standard curve. B.

Table 1: Parameters involved in the preparation of alginate nanoparticle loaded with naringenin using electrospray method.

Sample Sodium Alginate final Voltage (kV) Flow rate Needle size Tip-to- Calcium chloride
concentration (mL/hr) (Gauge) collector concentration (%w/v)
(%w/v) distance (cm)
A 0.125 10 0.5 27 5 2
B 0.25
C 0.5

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Zeta potential trend of Encapsulation Efficiency with increasing concentration

The results were obtained for the Zeta potential measurement of of sodium alginate.
nanoparticles for different samples (A, B, C), in which sample
Effect of flow rate
C was slightly more anionic (-3.09 mV) compared to sample A
and sample B (-1.10 mV and -1.43 mV) respectively based on the All three formulations were made with the constant concentration
Zeta potential values as shown in Table 4 for different sodium of Alginate-Naringenin of 0.5% and 2% Calcium Chloride.
alginate concentrations. It was interpreted as the concertation of Alginate solution (1% w/v) was prepared to maintain stable
the sodium alginate increased, there was an increasing trend of viscosity according to the procedure mentioned in the previous
zeta potential of nanoparticles. study done and it was left mixing for overnight to get a uniform
formulation.50 Further, Alginate solution was stirred for 3 min
Encapsulation Efficiency to allow fresh distilled water molecules to be in contact with
The results were tabulated for the measurement of Encapsulation Alginate particles resulting in a clump-free solution. While
Efficiency of nanoparticles for different samples (A, B, C), stirring, 10 mL of 10 mg/mL naringenin solution was mixed
in which all three samples were exhibited with % good dropwise into the alginate solution to incorporate solution to
Encapsulation Efficiency in the range of 92.24-92.80 as shown produce Alginate-Naringenin nanoparticles (0.5% v/v) solution.
in Table 5. Among three samples (A, B, C) sample C has shown 0.1 mL of Tween 80 was also added into Alginate-Naringenin
highest percent Encapsulation Efficiency of 92.80 compared to solution as surfactant to reduce the surface tension producing a
encapsulation efficiencies of sample B and sample A (91.07 and stable nanoparticles formulation.
90.24) respectively. It was also noted that there was an increasing
The flow rate effect on Alginate nanoparticles loaded with
Naringenin could be clearly seen and observations were tabulated
in Table 6. The camera images in (a), (b) and (c) showed the
spraying mode of spray solution of Alginate-Naringenin (0.5%
Table 2: Alginate nanoparticles loaded with Naringenin formulations
(T1-T3).

Formulation Process Parameter Flow rate (mL/hr)

T1 0.2
T2 0.3
Figure 1: Chemical structure of Naringenin.
T3 0.5

Figure 2: Electrospray setup.

998 Indian Journal of Pharmaceutical Education and Research, Vol 59, Issue 3, Jul-Sep, 2025
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Table 3: Particle size analysis of alginate nanoparticle loaded with naringenin.

Sample Sodium Alginate final concentration (%w/v) Di50 (nm) PdI

A 0.125% 900 0.535
B 0.25% 1980 0.201
C 0.5% 146 0.431

Table 4: Zeta potential of alginate nanoparticle loaded with naringenin.

Sample Sodium Alginate Zeta Potential

final concentration (mV)
(%)
A 0.125 -1.10
B 0.25 -1.43
C 0.5 -3.09

w/v) solution with different flow rates employed as shown

in Figure 5. Image (a) indicated with a stable Taylor’s cone jet
produced from T1 formulation with flow rate of 0.2 mL/hr,
while image (b) showed an unstable cone jet produced from T2
formulation with flow rate of 0.3 mL/hr. An image (c) from T3
formulation with the flow rate of 0.5 mL/hr was produced with Figure 3: Formation of dripping mode under flow rate of 0.5 mL/hr in the
absence of voltage of all samples.
unstable cone jet and unstable multi jet.
T1-T3 formulation with T1 of 0.2 mL/hr flow rate has 92.80%
Nanoparticle Size Encapsulation Efficiency. On the other hand, formulation T2
Nanoparticle sizes were expressed in Di50 and PDI, known as of 0.3 mL/hr has 92.86% Encapsulation Efficiency meanwhile,
T3 formulation with 0.5 mL/hr has the highest Encapsulation
nanoparticles with median diameter of 50% and polydispersity
Efficiency value of 96.77%.
index. These two parameters are helpful to interpret the
nanoparticles distribution within a given sample. Nanoparticle Calibration curve was done with different known concentrations
size analysis was done on three formulation samples (T1, T2, T3) pure Naringenin using UV-Visible Spectrophotometer at max of
with flow rates of 0.2, 0.3 and 0.5 mL/hr respectively as shown in 297 nm. The linear regression equation of the standard curve was
Table 7. found to be y=2.591x-0.0942 with the correlation coefficient (R2)
of 0.9925. as shown in Figure 6.
It was clearly depicted from the results as the process parameter
flow rate increased, both Di50 nanoparticles size and Polydispersity DISCUSSION
Index increased meanwhile T2 produced Di50 nanoparticles size
of 215 nm with Polydispersity Index for formulations T1-T3. The Alginate nanoparticle loaded with naringenin using
small nanoparticle size (146 nm) was observed with PDI of 0.331 electrospray method
for formulation T1 at a low flow rate of 0.2 mL/hr. It has been reported that naringenin is soluble in the binary
system that consists of ethanol and water.26 10 mg/mL of
Zeta Potential naringenin concentration was chosen to maximize the active
Zeta potential analysis was done on all three formulation samples ingredients being encapsulated in alginate nanoparticle. In this
(T1-T3). T1 formulation with the flow rate of 0.2 mL/hr possessed study, naringenin concentration higher than that resulted in
a zeta potential of -1.10 mV, meanwhile as the flow rate increases cloudy solution.
for formulation (T2-T3) with flow rate of 0.3 mL/hr to 0.5 mL/hr, Alginate-naringenin solution was prepared by employing
the zeta potential (-1.83 to -2.44 mV) value was shown increasing different low sodium alginate solutions with three alginate final
trend as shown in Table 8. concentrations of 0.5%, 0.25% and 0.125%. In electrospray,
the polymer solution is usually employed at low polymer
Encapsulation Efficiency concentrations and low polymer viscosity due to the relatively low
Encapsulation Efficiency, which is an indicator of drug electrostatic stretch over the surface tension during electrospray,
loading efficiency, is one of the most significant parameters in which helps to form nanoparticles. When a very low polymer
developing nanoparticle drug delivery systems. Based on Table viscosity is used, it has resulted in the formation of whipping
9, the Encapsulation Efficiency has an increasing trend from mode, due to instability of charged jet. Meanwhile, when a very

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Table 5: Encapsulation Efficiency of alginate nanoparticle loaded with naringenin.

Sample Sodium Alginate Absorbance Unknown Total Encapsulation

final concentration concentration of concentration of Efficiency
(%) unencapsulated naringenin (%)
naringenin (mg/mL) (mg/mL)
A 0.125 2.4067 0.9760 10 90.24
B 0.25 2.2247 0.8929 10 91.07
C 0.5 1.7710 0.7182 10 92.80

Table 6: Flow rate effect on Alginate nanoparticles loaded with mL/hr to 0.5 mL/hr results in decrease particle size produced and
Naringenin.
when voltage above 8 kV was applied, the decrease in particle size
Formulation Process Observation has also been observed.16
Parameter
Flow rate (mL/ A smaller needle (27G needle) was used as the nozzle to produce
hr) smaller particle size as increasing the needle diameter would lead
T1 0.2 Stable Taylor’s cone jet to increased particle size. Besides that, the distance between the
T2 0.3 Unstable cone jet tip of the nozzle and the collector was kept at 5 cm because the
T3 0.5 Unstable cone jet with distance further than that would lead to loss to the surrounding
unstable multi jet environment, hence reducing the yield of nanoparticle.16

high viscosity of polymers is used, there is a formation of fibers Effect of alginate concentration on electrospray
instead of nanoparticle formation.27 mode
It has been reported that the surface tension of the alginate Solution parameters such as polymer concentration have
solution was significantly reduced when a nonionic surfactant substantial impact on electrospray modes and nanoparticle
was added to the preparation, thus resulting into stable cone-jet production.29 In this study, in the absence of voltage applied, the
spray formation which is an important phenomenon to produce
formation of dripping mode was observed for all the samples.
reduced particle size with narrow size distribution.16 Tween 80
When increasing voltage was applied until 10 kV, the formation
is a nonionic surfactant and considered to add to the mixture to
of stable cone-jet spray mode was observed in sample C as shown
prevent particles agglomeration in addition to surface tension
reduction. In this study, it can be observed that addition of in Figure 4a. Stable cone-jet spray mode is the most favorable
Tween 80 into sample C solution results in the formation of stable electrospray condition for producing near-monodisperse
cone-jet spray mode (Figure 3) thus producing smaller particle polymeric nanoparticles compared to multi-jet spraying mode.30
size with narrow Polydispersity Index as depicted in Table 2. Another study by Noymer and Garel, (2000) reported that
compared to the cone-jet, the multi-jet mode generates finer
After that, the charged droplets were collected in a petri dish
droplets but with a wider size distribution.31
containing a cross-linking solution with 2% w/v calcium chloride
solution to facilitate the formation of alginate nanoparticle loaded However, when the lower alginate concentrations were employed,
with naringenin through ionotropic gelation reactions between the formation of unstable multi-jet spraying mode of both sample
calcium ion and alginate. Calcium alginate gels have a reputation A and B can be seen as depicted in Figure 4b and Figure 4c.
for being an excellent drug carrier due to the ability of alginate's This was due to very low polymer concentrations, where this
salts, such as sodium alginate, to be crosslinked by divalent was clearly supported in the literature, when very low polymer
calcium cations.28 According to a study done by Mehregan Nikoo
concentrations have been used, it resulted with weak viscosity of
et al., (2016), decrease in mean diameter of electrosprayed alginate
the solution thus making electrospray process unstable which in
has been observed with increasing calcium concentration up to
turn increases the particle size.32
2% w/v.18
The process parameters including voltage, flow rate, nozzle On the other hand, it was emphasized clearly that by employing
diameter and distance between the tip of the nozzle and the higher alginate concentrations (more than 1%) makes the solution
collector were kept constant meanwhile the effect of different too viscous to be exited through the nozzle tip due to higher
alginate concentration was studied. Flow rate of 0.5 mL/hr and alginate viscosity, occluding the nozzle tip, therefore preventing
voltage of 10 kV were chosen because it has been reported by the stable cone jet formation.16 Hence, it has been proven in this
Alallam et al., (2020) that upon increasing the flow rate from 0.1 study the importance of alginate concentrations.

1000 Indian Journal of Pharmaceutical Education and Research, Vol 59, Issue 3, Jul-Sep, 2025
 Razak, et al.: Investigation on the Effect of Production of Nanoparticle Loaded with Naringenin

Particle size Sample C was exhibited with the best particle nanoparticle size
(146 nm) compared to sample B and sample A in this study.
A study has reported that 100 nm nanoparticles showed a greater
This difference recorded in particle size were clearly because of
uptake in comparison to 1 µm diameter particles and 10 µm
different alginate concentrations. It was supported in a study
particles. This is due to the larger surface area to volume ratio of
conducted that the size of nanoparticle was reduced significantly
nanoparticle as the particle size becomes smaller and thus, this
when the concentration of polymer was reduced from 0.7% to
will cause faster drug release.33 Ales and Jeffrey M, (2008) has
0.4%.35 However, there was no further decreased observed in
reported that particles with the size of 200 nm or larger have the particle size and even small increase in nanoparticle size was
tendency of getting cleared faster from the circulation because observed when the polymer concentration was reduced from
of lymphatic system activation.34 Therefore, the optimal size 0.4% to 0.1%. Furthermore, it was also reported that electrospray
for a nanoparticle appears to be around 100 nm since this size process becomes unstable when the polymer viscosity is reduced
allows the particle to pass through the blood brain barrier, deliver as a result of decrease in polymer concentration, hence this could
sufficient amounts of drug due to the high surface area to volume explain the increase in observed nanoparticle size for the sample
ratio, and prevent quick clearance by the lymphatic systems.33 B and sample A in this study.
Table 7: Nanoparticle Size Analysis (Data expressed in Di50 and PDI).

Formulation Process Parameter Nanoparticle Size Analysis

Flow rate (mL/r) Di50 (nm) PDI
T1 0.2 146 0.331
T2 0.3 215 0.664
T3 0.5 463 0.97

Figure 4: Formation of stable cone-jet spray mode of sample C (a) stable Taylor cone and (b) spray. (b) Unstable multi-jet spraying
mode of sample B. (c) Unstable multi-jet spraying mode of sample A.

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According to a study, when the concentration of alginate was in which it exhibits negative charge on its surface owing to the
increased from 0.5% to 1%, an increase in particle size from 512 carboxylic acid groups.42,43 As the sodium alginate concentration
nm to 4303 nm was observed due to an increase in the viscosity used increase, the result shows an increasing trend of zeta
of polymer.36 This resulted in wider cone thickness and longer potential of nanoparticle from -1.10 to -3.09. One of the reasons
duration of jet for the droplet to breakup as the viscosity acts in would be due to polysaccharide concentration in which it has
the opposite direction of gravity, hence producing large droplet been reported that polysaccharide concentration was one of the
size.37 most influential factors on zeta potential values where increase
in the polysaccharide concentrations leads to more negative zeta
Polydispersity Index (PDI) potential values.44 Alginate is one of the natural biopolymers that
On the other hand, Polydispersity Index (PDI) is an essential belongs to polysaccharide-based class.
factor in determining nanoparticle size distribution dispersity However, according to Clogston and Patri, (2011), nanoparticles
ranging from 0 to 1, where PDI close to 0 indicates homogenous having a zeta potential between -10 mV and +10 mV are regarded
dispersion or monodisperse system and PDI close to 1 indicates to be approximately neutral.45 The smaller magnitude of the zeta
high polydispersity.38 PDI is also one of the important attribute as potential in this study could be due to an excess of positively
it determines the stability of nano delivery system.39 According charged calcium ions on the particle’s surface, which then could
to Alallam et al., (2020), majority consider PDI values of less impose a positive charge on nanoparticle.46
than or equal to 0.3 to be optimal for a nanoparticle. However,
PDI less than or equal to 0.5 is also reported to be acceptable Encapsulation efficiency
for nanoparticle.16 Sample C and sample B exhibited good Encapsulation Efficiency of the nanoparticle can be defined
Polydispersity Index. Despite good Polydispersity Index, sample as amount of drug encapsulated in nanoparticle divided by
B produced the largest particle size, therefore this concentration initial amount of drug contained in polymer-solvent mixture.47
is not ideal to produce the ideal size of nanoparticle because Encapsulation Efficiency is one of the important parameters
particles with the size of 200 nm or larger have the tendency of of nanoparticle as it determines the percentage of naringenin
getting cleared faster from the circulation. being successfully entrapped in the alginate nanoparticle. All the
samples showed good Encapsulation Efficiency where increasing
Zeta potential the sodium alginate concentration shows an increasing trend of
Zeta potential which refers to the electrical charge on the particle Encapsulation Efficiency of nanoparticle.
surface is one of the parameters assessed for nanoparticle
It has been proven that electrospray has the main advantage
produced as it determines the physical stability of colloidal over conventional encapsulating methods in terms of better
systems.40 In other words, the degree of electrostatic repulsion Encapsulation Efficiency, narrow particle distribution and involves
between similarly charged particles in dispersion is determined by single-step processing which make it convenient.48 According to
the value of zeta potential.41 As a result, samples with a high zeta the Huang et al., (2018), when the polymer concentration was
potential which can be either negative or positive are electrically increased, the Encapsulation Efficiency significantly enhanced.
stable, thus reducing the likelihood of particle aggregation
and floccule formation. Khalifa and Abdul Rasool, (2017) also
reported that normally, particles that have zeta potentials that is Table 8: Zeta Potential Analysis.

more positive than +30 mV or more negative than -30 mV are Formulation Process Parameter Zeta Potential (mV)
considered strongly cationic and strongly anionic, respectively, Flow rate (mL/hr)
and thus, are considered stable.41 T1 0.2 -1.10
In this study, all samples of alginate nanoparticle loaded with T2 0.3 -1.83
naringenin was found to be negatively charged. This could be T3 0.5 -2.44
explained by the usage of sodium alginate as it is anionic polymer
Table 9: Encapsulation Efficiency of Naringenin.

Formulation Process Absorbance Unknown Total concentration Encapsulation

Parameter (nm) concentration of of Naringenin (mg/ Efficiency(%)
Flow rate unencapsulated mL)
(mL/hr) Naringenin (mg/mL)
T1 0.2 1.771 0.7182 10 92.80
T2 0.3 1.7605 0.7141 10 92.86
T3 0.5 0.7442 0.3228 10 96.77

1002 Indian Journal of Pharmaceutical Education and Research, Vol 59, Issue 3, Jul-Sep, 2025
 Razak, et al.: Investigation on the Effect of Production of Nanoparticle Loaded with Naringenin

This is due the risk of particle breaking was significantly lower in that brings strong coulombic forces that increases the intensity
a more viscous polymer solution, which explains the observation of repulsion between adjacent molecules in producing small
that the drug encapsulations were higher at higher polymer nanoparticles droplets size that is accompanied by the optimum
concentrations.49 flow rate.50 Min Wang et al., 2020 investigated that Taylor’s cone
is formed with other factors too such as liquid surface tension,
Flow rate effect on Alginate nanoparticles loaded electrostatic forces and gravity during electrospray mode.
with Naringenin
Based on Figure 6, camera images showed spray mode of different Characterization of Alginate nanoparticles loaded
flow rate of 0.2, 0.3 and 0.5 mL/hr. Different flow rates produced with Naringenin
different stability of cone jet. From (a) Stable Taylor’s cone jet of Nanoparticle Size
T1 formulation was produced with the lowest flow rate of 0.2 mL/
Particle size distribution is the most important parameter
hr. From (b) had unstable cone jet from T2 formulation with flow
of characterization of nanoparticles. The primary use of
rate of 0.3 mL/hr meanwhile (c) showed unstable cone jet with
nanoparticles is in drug release and drug targeting. It has been
multijet from T3 Formulation with flow rate of 0.5 mL/hr.
discovered that particle size influences drug release. Smaller
Xu, Y. et al., 2006 reported the suggested parameters with voltage particles have a greater surface area. Thus, this results in most
supply of 12.5 kV and flow rate of 0.1- 0.2 mL/hr as the optimum of the drug loaded onto them is exposed to the particle surface,
values to produce smaller droplets size into the nanoparticles resulting in rapid drug release. Nanoparticles' small size and large
collector. This can be described by the uneven dispersion of the surface area increase the dissolution rate and solubility of poorly
emulsion at the tip of the needle, which resulted in uncontrolled soluble drugs.50
atomization and the formation of large particles as the flow rate
increases. Media Diameter 50% (Di50)
It can be concluded with flow rate of 0.2 mL/hr and voltage The median diameter 50% tabulated in Table 9 ranging from
supply of 10 kV produces smaller droplets size of nanoparticles 146-463 nm indicates increase of nanoparticle size with increase
as nanoparticles size may be reduced by increase of voltage of flow rate. Based on the result written in Table 9, Di50 of T1

Figure 5: Camera images showing spray mode of Alginate-Naringenin 0.5% w/v solution with different flow rates: (a) Stable
Taylor’s cone jet; (b) Unstable cone jet and (c) Unstable cone jet with unstable multi jet. The constant process parameters were
needle diameter of 27G, applied voltage of 10kV, Alginate-Naringenin concentration of 0.5% and distance between needle tip to
collector of 5 cm.

Indian Journal of Pharmaceutical Education and Research, Vol 59, Issue 3, Jul-Sep, 2025 1003
 Razak, et al.: Investigation on the Effect of Production of Nanoparticle Loaded with Naringenin

Figure 6: Calibration curve of Naringenin in Ethanol.

formulation of 146 nm is the only one that is within the acceptable colloidal stability.56 Numerous investigations have demonstrated
range of 100-200 nm. It has been demonstrated that nanoparticles stable nanoparticles made using non-ionic surfactants that have
with sizes ranging from 100-200 nm have the ideal characteristics low surface charges.
for cellular uptake. An optimal size range can result in improved
For zeta potential, pH is the most crucial variable. For instance,
cellular uptake.51
adding acid to a nanofluid will cause the pH to drop, which will
Polydispersity Index (PDI) increase the positive charges on the particle surface. The zeta
potential will rise. An isoelectric point is the location where there
The PDI values were shown an increasing trend with the
is zero electrophoretic mobility.57
increase of flow rate from 0.2 to 0.5 mL/hr as per Table 9. The
Polydispersity Index (PDI) is crucial in determining the dispersity Thus, in this part of study, the zeta potential of all 3 formulations
of nanoparticles distribution within a given sample.52 The range were within the range of -30 mV to +30 mV regardless of the
of the PDI numerical value is considered 0.0 (for a sample with low surface charges closely to neutral range as nanoparticles
uniform particle size) to 1.0. (For a highly polydisperse sample with non-ionic surfactants such as Tween 80 produced stable
with multiple particle size populations). For polymer-based nanoparticles as it might be one of the factors that leads to the
nanoparticle material, the PDI values 0.2 are most frequently low surface charges.
considered acceptable in practice.18 However, according to a
recent study, the optimum value of PDI is ≤0.3.16 In addition, PDI Encapsulation Efficiency
values are also acceptable ≤0.5.53 Crosslinking is essential in the context of alginate polymer
encapsulation. The degree of cross-linking at the surface of
Zeta Potential the extruded emulsion droplet, as well as emulsion stability,
The surface charge-dependent zeta potential is important for the influenced Encapsulation Efficiency.58 Encapsulation Efficiency
stability of nanoparticles in suspension and is also a major factor all across 3 formulations in Table 9 shows ≥90% in which T1 has
in the initial adsorption of nanoparticles onto the cell membrane. 92.80% efficiency, followed by T2 with 92.86% and the highest
The rate of endocytic uptake after adsorption is determined efficiency showed by T3 with 96.77%.
by particle size. Nanoparticle toxicity is thus affected by zeta
Based on Alallam, B. et al., 2020, high EE was noted in
potential and size.54
electrospraying literature, where it was discovered to be greater
Zeta potentials were shown for all 3 formulations as tabulated than 80%. In addition, electrospray method provides an
in Table 9 ranging from -1.10 mV to -1.83 mV. Zeta potential advantage of producing high EE which can be further explained
determination is a crucial method for characterizing nanocrystals by the strong crosslinking of nanoparticles outer shell. The
that may be used to calculate the surface charge and determine crosslinking is important to evaluate the Encapsulation Efficiency
the physical stability of nanosuspensions.55 In general, it is of nanoparticles with alginate polymers.58 The Encapsulation
believed that a zeta potential value outside of the range of -30 Efficiency of formulation depends on the type of API and
mV to +30 mV has enough repulsive force to improve physical resultant API-polymer interactions.58

1004 Indian Journal of Pharmaceutical Education and Research, Vol 59, Issue 3, Jul-Sep, 2025
 Razak, et al.: Investigation on the Effect of Production of Nanoparticle Loaded with Naringenin

Naringenin is highly soluble in methanol but has a relatively ABBREVIATIONS

limited water solubility.26 In Figure 6, Naringenin showed an
absorbance maximum on 297 nm which was used in the UV PDI: Polydispersity index; EE: Encapsulation Efficiency.
spectrophotometric determinations with a concentration range
SUMMARY
R2=0.9925. Based on a study done by Jha, D. K. et al., 2020, it
showed a linearity in the concentration range of R2>0.99 using Naringenin, a polyphenolic phytochemical with notable
UV spectrophotometer which was chosen to be economical, and pharmacological properties, faces limitations in therapeutic
less time required in comparison with chromatographic analysis application due to its hydrophobic nature, resulting in poor
that is costly and time-consuming. aqueous solubility and delivery to target sites. This study aimed
T1 has the lowest Encapsulation Efficiency with 92.80% in to address these challenges by encapsulating naringenin in
comparison to the T3 that has the highest Encapsulation alginate nanoparticles using an electrospray method. Different
Efficiency of 96.77% even though T1 formulation produced concentrations of alginate and flow rates were investigated
a stable Taylor’s cone shape that represents the optimum to optimize nanoparticle production. Results showed that a
nanoparticle formation. The possible attributing factor that may 0.5% w/v alginate concentration and a flow rate of 0.2 mL/hr
result in lower Encapsulation Efficiency in T1 was due to the slow yielded nanoparticles with optimal characteristics, including a
flow rate contributing to the longer settlement time to encapsulate particle size of 146 nm, narrow Polydispersity Index, high zeta
nanoparticles that may result in increased aggregation. Other potential, and Encapsulation Efficiency of 92.80%. These findings
than that, the friction force produced with higher flow rate in demonstrate the significant influence of alginate concentration
T3 with 0.5 mL/hr that produces nanoparticles that has greatest and flow rate on nanoparticle production, providing insights for
Encapsulation Efficiency. the development of high-quality naringenin nanoparticles for
potential therapeutic applications.
CONCLUSION
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Cite this article: Razak FSA, Bin LK, Rahman SA, Rashid NIAA, Malek NAA, Lokesh BVS, et al. Investigation on the Effect of Alginate Concentration and Flow
Rate on Production of Nanoparticle Loaded with Naringenin Using Electrospray Method. Indian J of Pharmaceutical Education and Research. 2025;59(3):995-
1006.

1006 Indian Journal of Pharmaceutical Education and Research, Vol 59, Issue 3, Jul-Sep, 2025