Evaluation of sonication on stability-indicating properties of optimized pilocarpine hydrochloride-loaded niosomes in ocular drug delivery

Abstract

Niosomes are increasingly explored for enhancing drug penetration and retention in ocular tissues for both posterior and anterior eye delivery. They have been employed in encapsulating both hydrophilic and hydrophobic drugs, but their use is still plagued with challenges of stability and poor entrapment efficiency particularly with hydrophilic drugs. As a result, focus is on understanding the parameters that affect their stability and their optimization for improved results. Pilocarpine hydrochloride (HCl), a hydrophilic drug is used in the management of intraocular pressure in glaucoma. We aimed at optimizing pilocarpine HCl niosomes and evaluating the effect of sonication on its stability-indicating properties such as particle size, polydispersity index (PDI), zeta potential and entrapment efficiency. Pilocarpine niosomes were prepared by ether injection method. Composition concentrations were varied and the effects of these variations on niosomal properties were evaluated. The effects of sonication on niosomes were determined by sonicating optimized drug-loaded formulations for 30 min and 60 min. Tween 60 was confirmed to be more suitable over Span 60 for encapsulating hydrophilic drugs, resulting in the highest entrapment efficiency (EE) and better polydispersity and particle size indices. Optimum sonication duration as a process variable was determined to be 30 min which increased EE from 24.5% to 42% and zeta potential from (−)14.39 ± 8.55 mV to (−)18.92 ± 7.53 mV. In addition to selecting the appropriate surfactants and varying product composition concentrations, optimizing sonication parameters can be used to fine-tune niosomal properties to those most desirable for extended eye retainment and maintenance of long term stability.

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Progress in Biomaterials
https://doi.org/10.1007/s40204-021-00164-5
ORIGINAL RESEARCH
Evaluation ofsonication onstability‑indicating properties
ofoptimized pilocarpine hydrochloride‑loaded niosomes inocular
drug delivery
K.Owodeha‑Ashaka1 · M.Ilomuanya2· A.Iyire1
Received: 8 June 2021 / Accepted: 9 September 2021
© The Author(s) 2021
Abstract
Niosomes are increasingly explored for enhancing drug penetration and retention in ocular tissues for both posterior and
anterior eye delivery. They have been employed in encapsulating both hydrophilic and hydrophobic drugs, but their use is
still plagued with challenges of stability and poor entrapment efficiency particularly with hydrophilic drugs. As a result, focus
is on understanding the parameters that affect their stability and theiroptimization for improved results. Pilocarpine hydro-
chloride (HCl), a hydrophilic drug is used in the management of intraocular pressure in glaucoma. We aimed at optimizing
pilocarpine HCl niosomes and evaluating the effect of sonication on its stability-indicating properties such as particle size,
polydispersity index (PDI), zeta potential and entrapment efficiency. Pilocarpine niosomes were prepared by ether injection
method. Composition concentrations were varied and the effects of these variations on niosomal properties were evaluated.
The effects of sonication on niosomes were determined by sonicating optimized drug-loaded formulations for 30min and
60min. Tween 60 was confirmed to be more suitable over Span 60 for encapsulating hydrophilic drugs, resulting in the high-
est entrapment efficiency (EE) and better polydispersity and particle size indices. Optimum sonication duration as a process
variable was determined to be 30min which increased EE from 24.5% to 42% and zeta potential from (−)14.39 ± 8.55mV
to (−)18.92 ± 7.53mV. In addition to selecting the appropriate surfactants and varying product composition concentrations,
optimizing sonication parameters can be used to fine-tune niosomal properties to those most desirable for extended eye
retainment and maintenance of long term stability.
Keywords Niosomes· Sonication· Stability· Non-ionic surfactants
Introduction
Optimum drug delivery to the eye is especially difficult as
the eye possesses intrinsic anatomical and physiological
properties that pose barriers. There is therefore the problem
of poor bioavailability and an associated need for frequent
administrations to achieve and maintain optimum ocular
concentrations. Pilocarpine hydrochloride is used for manag-
ing intraocular pressure (IOP) in the treatment of glaucoma.
It is a miotic drug that acts as a muscarinic agonist, causing
ciliary muscle contraction that opens up the trabecular mesh-
work which allows aqueous humour drainage and a resultant
reduction in IOP (Jain and Verma 2020). It is hydrophilic in
nature and its use as a conventional eye drop is faced with
the challenges highlighted prior, leadingto short retention
times, low bioavailability and reduced efficacy (Keipert etal.
1996). It is currently available in other dosage forms as oral
tablets, ocular inserts, which are associated with limitations
such as poor bioavailability and invasiveness due to inserts.
In solving the above problems, many different formula-
tion strategies are being explored to improve drug solubility,
precorneal absorption and retention time in the eye. Nanomi-
celles, microemulsions, insitu gels and liposomes are some
nanotechnology-based systems that have been investigated
for pilocarpine delivery (Anumolu etal. 2009; Cholkar
etal. 2013; Naveh etal. 1994). Of note are niosomes which
serve as nanocarriers and are fundamentally composed of
amphiphilic non-ionic surfactants with a polar head and a
* K. Owodeha-Ashaka
oashkruga@gmail.com
1 Aston Pharmacy School, College ofLife andHealth
Sciences, Aston University, BirminghamB47ET, UK
2 Faculty ofPharmacy, University ofLagos, Yaba, LagosState,
Nigeria

Progress in Biomaterials
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non-polar tail, lipids like cholesterol, a hydration medium
and other additives (Bhardwaj etal. 2020). They are formed
as a result of partitioning upon tensile interactions of the
aqueous solution and the lipophilic tails of the amphiphi-
lic non-ionic surfactants, causing the tails to associate and
leaving the polar hydrophilic heads pointing outwards in
contact with the aqueous phase (Seleci etal. 2016). It is
able to achieve localized controlled and sustained release
in addition to protection of the drug from degradation by
metabolic enzymes resident in the eye (Sahoo etal. 2014).
Because it is a lipid vesicular system, absorption is increased
and reducedsystemic drainage results in longer drug contact
time, and therefore bioavailability is improved compared to
conventional drug solutions (Sahoo etal. 2014).
Different classes of surfactants have been used in the
preparation of niosomes. Their properties—size, structure,
hydrophilic/lipophilic balance (HLB) value and physical
state, and phase transition temperature (Tc)—as well as the
concentrations in which they are used, influence vesicle size,
polydispersity index (PDI) encapsulation efficiency, charge
and stability (Bnyan etal. 2018). Cholesterol interacts with
the hydrophobic alkyl end of the surfactant producing an
increase in vesicle transition temperature and alteration of
bilayer fluidity which stabilizes the membrane (Chen etal.
2019). Cholesterol affects the vesicle’s permeability and
drug release, membrane rigidity, encapsulation efficiency,
toxicity and stability (Bhardwaj etal. 2020).
Size reduction is beneficial for preventing ocular irritation
and inflammation, enhancing pharmacokinetics/drug bio-
distribution by increased surface area to volume ratio, pro-
moting intracellular delivery and increasing retention time
(Prabhu etal. 2010; Nowroozi etal. 2018). Several methods
for modifying size reduction to meet desired parameters
exist. They include sonication (bath and probe), microfluidi-
zation, high-pressure homogenization and extrusion through
filters (Uchegbu and Vyas 1998). Sonication is the applica-
tion of sound energy to a liquid containing particles and
has been known for its effects on lipid membranes to pro-
duce nano-sized vesicles (Essa 2010). Frequencies greater
than 20Hz are usually used so it is referred to “ultrasonica-
tion”. It is a commonly used method for effectual creation
of smaller unilamellar vesicles from larger multilamellar
vesicles in a lamellar dispersion (Zasadzinski etal.2011).
A major challenge that continually hinders progress in
theclinical application of niosomes is the issue of their
stability. As such, extensive research has gone into inves-
tigating formulation and process parameters that influence
stability, evaluating niosomal characteristics such as par-
ticle size, polydispersity index, zeta potential, and entrap-
ment efficiency, which are often indicative of the relatively
unchanged nature of a formulation (Seleci etal. 2016; Chen
etal. 2019). Formulations containing charge inducers that
ensure adequate electrostatic repulsion between vesicles to
prevent aggregation, have shown promise (Bhardwaj etal.
2020). Since the composition and manufacturing process
affect product properties, a successful optimization of these
parameters give great promise for finally arriving at nio-
somal formulations that maintain optimum characteristics
throughout the cycle of preparation, movement, storage and
final use. We will attempt to shed more light on how sonica-
tion in the formulation process could play an important role
in optimizing pilocarpine hydrochloride niosomal properties
that affect their bioavailability and stability. In this work, we
formulate, optimize and evaluate the characteristics of pilo-
carpine hydrochloride-loaded niosomes prepared by ether
injection method and determine the effect of sonication time
on the above properties.
Materials
Methanol 99.8% (Fisher Scientific, UK), pilocarpine hydro-
chloride (HCl) (Sigma Aldrich, Brazil), deionized water,
sodium chloride (Sigma Aldrich, Switzerland), potassium
chloride (Sigma Aldrich, Spain), sodium hydrogen dibasic
phosphate (Sigma Aldrich), potassium dihydrogen phos-
phate (Fisher Scientific, UK), Tween 60 from (CRODA,
UK), Span 60 (CRODA, UK), cholesterol (Sigma Aldrich,
USA), diethyl ether 99.7% (Sigma Aldrich, Germany), etha-
nol 99.8% (Fisher Scientific, UK). All reagents used were
of analytical grade.
Methods
Reverse‑phase HPLC method validation
An Agilent Technologies 1220 Infinity II LC system was
used in pilocarpine HCl quantification based on a method
developed by Fan etal. (1996) and outlined by El Deeb etal.
(2006). The mobile phase was a mixture of solution A, con-
taining 13.5mL phosphoric acid, 3mL trimethylamine and
983.5mL deionized water, and solution B as methanol in
a ratio of 98:2. A standard Gemini® 5μm C18 110A LC
column 150 × 4.6mm was used under ambient experimental
conditions (18–21°C). Flow rate was set at 1.5mL/min and
injection volume was 20μL. The UV absorbance wavelength
of pilocarpine hydrochloride was determined to be 215nm
and as such, UV detection was done at this wavelength. Vali-
dation was carried out according to ICH guidelines Q2R1
(2005) over a linearity range of 7.8125–500µg/mL with
coefficient of variation r2 = 0.9999. The method was pre-
cise with repeatability giving 1.1% RSD; mean % recovery
ranging from 78.38 to 103.93%; andlimits of detection and
quantification at 0.158μg/mL and 0.528μg/mL respectively.
All but one of the mean recovery RSD% values were under

Progress in Biomaterials
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the acceptable15% upper limit for pharmaceutical analysis
(Iyire etal. 2018). Recovery at the lowest concentration in
the range gave the lowest recovery, informing the use of
theindirect method for quantifying the amount of pilocar-
pine hydrochloride entrapped in the niosomes.
Compatibility studies
Compatibility studies between pilocarpine hydrochloride
and cholesterol were previously done in the lab using FTIR
and DSC and the data are already published by Alyami etal.
(2020). DSC thermograms and FTIR spectra obtained estab-
lished the absence of drug-excipient interactions or potential
for incompatibilities in the formulation.
Preparation ofniosomes
Niosomes were prepared by ether injection method as
described by Ravalika and Krishna (2017) with slight modi-
fications. Composition ratios are shown in Table1. Ethanol
was used instead of methanol and the drug was dissolved
in the aqueous phase (phosphate buffer) as it was insolu-
ble in the organic solvents at the quantities used. Briefly,
the surfactant(s) together with cholesterol were accurately
weighed into a beaker. A mixture of 2mL ethanol and 6mL
diethyl ether was added to the beaker and gently agitated to
facilitate dissolution. The beaker was covered with parafilm
to reduce solvent evaporation to the barest minimum. Upon
complete dissolution, the solution was withdrawn using a
syringe fitted with a 23G needle. To a sample vial contain-
ing 40mg pilocarpine HCl dissolved in 10mL phosphate
buffer previously warmed to and maintained at 60–62°C in
a water bath, the solution was slowly injected at 0.8mL/min
while magnetically stirring at 100rpm. Care was taken to
ensure the solution was injected into the phosphate buffer
and not above it. The resulting suspension was continually
stirred for 45min to allow for solvent evaporation and a well
dispersed suspension.
Characterisation ofniosomes
Visual inspection andmorphology
The formulated suspensions were inspected for their physi-
cal appearance and colour. Redispersed niosomal suspension
was viewed on a glass slide under a Carl Zeiss Analytical
Microscope (Germany) using Axiovision software through
lenses × 10, × 40 and × 100. The viewing was adjusted to
acquire as clear an image as possible which was captured
by an attached camera.
Particle size, polydispersity index (PDI) andzeta potential
These parameters were evaluated according to a procedure
adopted from Sankhyan and Pawar (2013). Particle size and
PDI were determined using dynamic light scattering (DLS)
technique in a Brookhaven Zetasizer, model Nanobrook
90plus zeta (USA) with BIC particle solutions software.
Electrophoretic light scattering (ELS) technique in the same
zetasizer was used to determine zeta potential at a tempera-
ture of 25°C.
Entrapment efficiency
This was carried out as described by Verma etal. (2019)
with slight modifications. 1mL of the niosomal suspension
was measured into a 1.5mL capacity Eppendorf tube and
placed in a cooling centrifuge, Prism R model from Lab-
net International Inc. (USA), ensuring proper centrifuge
balance. The centrifugation parameters were set at 4°C,
11,000 × g force and run time of 1h. After centrifugation,
the supernatant was carefully drawn up into appropriately
labelled containers and the separated pellets were washed.
Washing was done by adding1mL of pH 7.4 phosphate
Table 1 Niosomal for-
mulation compositions
showing concentration of
drug,surfactant(s)andcholes-
terolused
Formulation Tween60(mg) Span60(mg) Cholesterol(mg) Pilocar-
pineHCl
(mg)
K1 100 – 100 40
K2 – 100 100 40
K3 50 50 100 40
K4 50 – 100 40
K5 – 50 100 40
K6 25 25 100 40
K7 100 – 50 40
K8 – 100 50 40
K9 50 50 50 40

Progress in Biomaterials
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buffer to the solute in the tube and mixing. The mixture
was centrifuged for another1 h and all the supernatant was
carefully collected and stored in foil-wrapped sample vials
to protect pilocarpine HCl from light degradation.
The quantity of total supernatant recovered was deter-
mined by measurement after which the supernatant was
filtered and analyzed using an AgilentTechnologies1220
Infinity II LCHPLC system to determine the amount of
free unencapsulated drug. Indirect determination of EE was
carried using Eq.(1):
Statistical analysis
GraphPad Prism software, version 8.4.3 from GraphPad
(USA) was used in the statistical analysis of results obtained.
Data was compared using one-way analysis of variance
(ANOVA) and two-way ANOVA followed by Tukey or
Sidak post-test as indicated. Differences with p < 0.05 were
considered significant. All particle sizes, PDI and zeta
potentials were taken in repetitions (n = 6) and were pre-
sented as mean ± standard deviation.
Results
Visual inspection andmorphology
The formulated niosomal suspensions were milky/cloudy
with a well dispersed fluid consistency. The formulations
containing Span 60 were observed to be cloudier than those
with Tween 60. On standing, a greater separation of the Span
60 niosomal suspension was observed, with the settling of
vesicles at the bottom of the vial and a clear aqueous phase
above. Tween 60 niosomes did not show the same degree of
separation as the supernatant above remained significantly
cloudy (Fig.1).
Nisosomes formed were spherical (Fig.2D), correspond-
ing with other studies in which Span 60 and Tween 60 were
used (Junyaprasert etal. 2012). Vesicles were unilamellar
as is characteristic of niosomes formed by ether injection
method. Span 60 niosomes were seen as ‘densely’ packed
aggregates with a few disperse vesicles and having a greater
proportion of visibly larger lamellar vesicles compared
with Tween 60. For some Span 60 containing formula-
tions as seen in Fig.2A, incompletely formed vesicles were
observed.
Elongated vesicles and tubules were observed in some
Span 60 formulations, Fig.2B, as has also been reported by
(1)
Total drug
−
Free drug
Total drug
×
100
Barakat etal. (2014), Marwa etal. (2013) and Rangasamy
etal. (2008). The formulations containing Tween 60 were
composed of smallerspherical disperse unilamellar vesicles,
with some showing small crystal-like structures (Fig.2C).
Particle size andpolydispersity index
Tween 60 produced particle sizes generally smaller
than those of Span 60. K2 had the largest size at
1,229.87 ± 277.24nm and was significantly larger than
other formulations, p < 0.0001 (Table2). The correspond-
ing K1 formulation containing Tween 60 had smaller sizes
of 516.69 ± 30.22 nm. K4 (Tween 60:cholesterol, 0.5:1)
was the smallest in size at 318.90 ± 26.97nm, which was
significantly different compared with other formulations
p ≤ 0.0220.
Increased surfactant concentrations gave mostly larger
sized vesicles for both Span 60 and Tween 60 niosomes,
with Span 60’s influence being more significant, p < 0.0001
(Table2). Similarly, for corresponding concentrations of
both surfactants, lower cholesterol concentrations in the
composition ratio produced smaller sized niosomes. Upon
drug loading, formulations containing Span 60 registered
increase in particle size (p ˂0.0001, two-way ANOVA,
Sidak’s post-test), presented in Table3.
The PDI for all the blank formulations ranged from 0.16
to as high as 2.52 (Table2), although this high value was
seen only in formulation K8. Other formulations had accept-
able PDIs from 0.16 to 0.51 indicating a homogenous disper-
sion (Rehman etal. 2018). K8 with 2.52 translated to a dis-
persion lacking homogeneity as seen by the distribution of
particle sizes, some of which are up to twice the size of the
lowest size recorded. Tween 60 formulations had the low-
est values and it can be said that smaller particle sizes gave
Fig. 1 Visual observation of niosomal formulations K5 (0.5:1, Span
60:cholesterol) and K4 (0.5:1, Tween 60:cholesterol) on standing

Progress in Biomaterials
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better PDI (Kaur etal. 2016). For both the Tween 60 and
Span 60 singly composed formulations, higher surfactant
concentrations gave lower PDIs, an observation in line with
that made by Sharma etal. (2016). By an analysis using
one-way ANOVA followed by Tukey post-test, K8 showed
significant differences (p ≤ 0.0002) from the other formu-
lations. Following drug loading, Span-only niosomes were
seen to have generally higher PDIs, only running as low as
0.76 which is typically not considered desirable (Table3).
Zeta potential
In this study, Tween 60 containing formulations had lower
values (p < 0.0001, one-way ANOVA followed by Tukey
post-test) than their counterpart Span 60 vesicles. As
Fig. 2 Micrographs of drug
loaded formulations A
(K8) × 100 showing malformed
niosomes, B (K2) drug-
loaded × 100 showing tubules,
C (K4) blank × 40 showing
crystal structure, and D (K4)
blank × 10 showing well formed
niosomes
Table 2 Summary of particle
size analysis and PDI for blank
niosomes
All formulations gave homogenous dispersions except K8 (Span 60:cholesterol, 1:0.5) with a PDI greater
than 0.7 showing statistically significant differences (p < 0.0001) by one-way ANOVA. Data is presented as
mean of six determinations ± standard deviation
* 0.05; 0.01 to 0.05 - Significant
** ≤ 0.01; 0.001 to 0.01 – Very significant
*** ≤ 0.001; 0.0001 to 0.001 – Extremely significant
**** ≤ 0.0001 – Extremely significant
Blank formulation Particle size (nm)
(mean ± SD)
Polydispersityindex
(mean ± SD)
K1; Tween 1: Chol 1 516.69 ± 30.22 0.21 ± 0.14
K2; Span 1: Chol 1 1229.87 ± 277.24 0.16 ± 0.12
K3; Tween 0.5/Span 0.5: Chol 1 556.56 ± 76.73 0.23 ± 0.21
K4; Tween 0.5: Chol 1 318.90 ± 26.97 0.36 ± 0.08
K5; Span 0.5: Chol 1 819.87 ± 232.99 0.51 ± 0.69
K6; Tween 0.25/Span 0.25: Chol 1 730.73 ± 91.69 0.47 ± 0.18
K7; Tween 1: Chol 0.5 484.49 ± 15.00 0.28 ± 0.18
K8; Span 1: Chol 0.5 535.15 ± 125.84 2.52 ± 1.88****
K9; Tween 0.5/Span 0.5: Chol 0.5 844.22 ± 112.79 0.24 ± 0.15

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shown in Table3, K2 (Span 60:cholesterol, 1:1) recorded
the highest zeta potential of − 54.66 ± 4.26mV, while K8
(Span 60:cholesterol, 1:0.5) had a slightly lower value of
− 41.36 ± 5.76mV and K5 (Span 60:cholesterol, 0.5:1) gave
− 26.68 ± 10.93mV, just about half that of K2's. This same
trend was observed in the Tween 60 containing formulations
with K1 giving zeta values of − 24.16 ± 3.38mV, K7 giv-
ing − 13.06 ± 2.66mV, and K4 giving − 10.61 ± 3.59mV.
Zeta potential was seen to increase (more negative) with
an increase in surfactant ratio in Span 60 formulations from
− 22.68 ± 8.2mV in K5 to − 54.66 ± 4.26mV in K2. Tween
60 as well as the co-surfactant formulations followed the
same trend. For the surfactant combinations, K3 with equal
surfactant:cholesterol ratio gave the highest zeta value of
− 33.13 ± 10.71mV, while K9 with half the cholesterol con-
centration giving a slightly lower − 26.19 ± 8.37mV and
K6 with half the surfactant concentration giving the lowest
value at − 22.68 ± 8.2mV. These results are a seemingly
good demonstration of the effects of different surfactant
types as has been described by other researchers (Bnyan
etal. 2018). Summarily, K2 showed statistically signifi-
cant differences in zeta potential value (p ≤ 0.001, one-way
ANOVA followed by Tukey test) compared to other formu-
lations except K8 where there was no significant difference
(p > 0.05), which is understandable as it had the same Span
60 concentration as K2.
Entrapment efficiency
K1 containing equal Tween 60 and cholesterol gave the high-
est EE% of 34% compared with K2 having Span 60 which
gave 21.9%, Table3. EE was seen to increase for Tween 60
formulations from 20.6% in K4 to 34% in K1 as surfactant
concentration increased, translating to a larger aqueous
space so more drug uptake. The co-surfactant formulations
followed the same trend as K3 with a higher total surfactant
concentration produced a slightly higher EE, 26.5% than
K6, 24.2%.
Comparing K1 and K7, K2 and K8 and K3 and K9 to
demonstrate the effect of cholesterol concentration, the
higher cholesterol containing formulations all gave higher
EE. With half the cholesterol concentration, formulations K7
with Tween 60, K9 combining Span 60 and Tween 60 and
K8 with Span 60 gave EE of 24.2%, 23% and 18.3% respec-
tively. The effect of increase in cholesterol concentration
was clearly observed when comparing K1 and K7, where
K1 having the higher concentration gave an EE of 34% and
K7 gave 24.2%.
In these experiments, as with results obtained by Palozza
etal. in (2006) using β-carotene niosomes, EE did not cor-
relate with size particularly with Tween 60 formulations as
K1 with the smallest size had a higher EE than K2 with a
much larger size. However, the size and EE for K2 and K5
containing Span 60did correlate, with the larger K5 show-
ing the higher EE.
Effects ofsonication onniosomal properties
K7 containing Tween 60 was chosen to evaluate the effect
of sonication on the niosomal dispersion because it gave
good cumulative properties in terms of particle size, poly-
dispersity index and zeta potential. For good comparability,
K8 containing the same concentrations of cholesterol and
surfactant, in this case Span 60 was also chosen. This was
to determine any possible influence the type of surfactant
would have on the effect of sonication.
Sonication was carried out using a Fisherbrand bath
sonicator and the sample vial was placed in an ice bath to
maintain a cool temperature as the process usually results in
generation of heat. This process was described by Mavaddati
etal. (2015) as they investigated its effects on the physical
Table 3 Summary of particle size analysis, PDI, zeta potential (data
is presented as mean ± SD for 6 determinations) and entrapment
efficiency for pilocarpine HCl-loaded niosomes. K2 (Span 60:cho-
lesterol, 1:1) showed statistically significant differences (****;
p < 0.0001, one-way ANOVA) in zeta potential compared with other
formulations except K8 (Span 60:cholesterol, 1:0.5)
PDI for K2 was also significantly larger than other formulations except K5 (Span 60:cholesterol, 0.5:1) (p < 0.0007)
Drug loaded Formulation Particle size (mean ± SD) Polydispersity
index(mean ± SD)
Zeta Potential (mV)
(mean ± SD)
% EE using
free drug
K1; Tween 1: Chol 1 294.37 ± 14.73 0.09 ± 0.07 − 24.16 ± 3.38 34.0
K2; Span 1: Chol 1 1124.61 ± 389.28 2.43 ± 1.73*** − 54.66 ± 4.26**** 21.9
K3; Tween 0.5/Span 0.5: Chol 1 520.76 ± 37.01 0.25 ± 0.27 − 33.13 ± 10.71 26.5
K4; Tween 0.5: Chol 1 335.42 ± 18.23 0.17 ± 0.06 − 10.61 ± 3.59 20.6
K5; Span 0.5: Chol 1 1644.92 ± 254.23 1.69 ± 1.20 − 26.68 ± 10.93 25.1
K6; Tween 0.25/Span 0.25: Chol 1 713.77 ± 94.11 0.28 ± 0.15 − 22.68 ± 8.20 24.2
K7; Tween 1: Chol 0.5 433.41 ± 41.58 0.07 ± 0.03 − 13.06 ± 2.66 24.2
K8; Span 1: Chol 0.5 1007.81 ± 178.95 0.76 ± 1.00 − 41.36 ± 5.76 18.3
K9; Tween 0.5/Span 0.5: Chol 0.5 1054.69 ± 157.68 0.25 ± 0.09 − 26.19 ± 8.37 23.0

Progress in Biomaterials
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character of niosomes of dexamethasone. It is based on the
principle of generation and oscillation of formed bubbles
i.e., cavitation in liquids by ultrasound mechanical waves.
Application of a frequency of resonant size leads to the non-
linear oscillation and eventual collapse of bubbles with sizes
near those of the frequency applied. The collapse results in
generation of extremely high temperatures, shock waves and
high pressures. Larger vesicles are then randomly but uni-
formly broken down by the ultrasonic high energy to small
discoid fragments which fold up to form thermodynamically
stable vesicles.
Visual inspection andmorphology
For both formulations, the separation of solvent and solute
phases was more evident and marked by a sediment below
and a clear phosphate buffer solution above. Large unilamel-
lar vesicles were produced for both formulations as earlier
noted. Especially for K8 containing Span 60, longer soni-
cation time and the associated particle size reduction led
to the vesicles becoming more discrete. K7 with Tween 60
also behaved in this way but to a lesser degree that could be
observed. De etal. (2018) made similar observations after
probe sonicating niosomes of temozolomide.
Particle size andpolydispersity index (PDI)
Particle sizes generally reduced after sonication with a
more significant decrease after 60min for both Span 60
and Tween 60 formulations (Table4). With no sonica-
tion, K7a containing Tween 60 had an average size of
338.74 ± 14.37nm only reducing slightly after 30min of
sonication (K7b) to 334.94 ± 19.80nm. After 60min (K7c),
there was a more noticeable decrease in particle size to
270.35 ± 17.21nm. For K8a, with no sonication, average
size was 1,900.54 ± 610nm. After 30min of sonication
(K8b), there was a decrease to 1,308.55 ± 310.90nm and
sonication for 60min (K8c) resulted in a 75.6% decrease
in size to 462.89 ± 42.47nm. The difference in size of K8a
and K8b from the other formulations and between them-
selves was statistically significant (p < 0.0001, by two-way
ANOVA followed by Tukey test). There was no statisti-
cally significant difference between K8c and K7a, b and
c (p > 0.05) as the sizes were close in range as shown in
Table4.
As shown in Table4 above, there was not much change
in the PDI of the K7 through the sonication process. For
K8 containing Span 60, the PDI was seen to consistently
reduce in value from 0.41 with no sonication (K8a) to
0.27 60min after sonication (K8c). Although changes in
PDI values can be seen, they were not considered statisti-
cally significant (p > 0.05, one-way ANOVA followed by
Tukey test).
Zeta potential
As previously determined in non-sonicated drug-loaded
vesicles, the Span 60 containing formulation had a higher
(more negative) zeta potential than its Tween 60 counter-
part. For K8, zeta potential consistently increased from
-33.34mV at 0min of sonication to − 43.94mV at 30min
to − 51.49 at 60min, Table4. Tween 60 containing K7
showed an increase in zeta potential from − 14.39mVprior
to sonicating to − 18.92mV after 30 min. It remained
significantly unchanged after 60min at a potential of
− 17.78mV.
Entrapment efficiency
For K7, EE was seen to increase from 26.5% at no sonication
to 42.7% after sonicating for 30min, Table4. On further
sonication up to 60min as seen with K7c, EE reduced to
35.8%. For K8 a decrease from 23% before sonication to
20% after 30min of sonication, followed by a statistically
insignificant increase to 21.6% after 60min was observed.
Table 4 Effects of sonication
on niosomal character from
0-60min
Data presented as mean ± SD where indicated are for 6 determinations. K8a and b showed statistically sig-
nificant differences (****; p < 0.0001, two-way ANOVA)
Formulation Particle size (nm) (mean ± SD) Polydisper-
sity index
(mean ± SD)
Zeta poten-
tial (mV)
(mean ± SD)
%EE using
free drug
K7a No sonication 338.74 ± 14.37 0.18 ± 0.05 − 14.39 ± 8.55 26.5
K7b 30min 334.94 ± 19.80 0.18 ± 0.02 − 18.92 ± 7.53 42.7
K7c 60min 270.35 ± 17.21 0.20 ± 0.11 − 17.78 ± 7.62 35.8
K8a No sonication 1900.54 ± 610.39**** 0.41 ± 0.52 − 33.34 ± 12.91 23.0
K8b 30min 1308.55 ± 310.90**** 0.30 ± 0.07 − 43.94 ± 12.68 20.6
K8c 60min 462.89 ± 42.47 0.27 ± 0.08 − 51.49 ± 6.10 21.6

Progress in Biomaterials
1 3
Discussion
Effect ofSurfactant andcholesterol concentration
onniosomal properties
The use of pilocarpine HCl is a long-standing therapeutic
strategy in the management of open-angle glaucoma and
acute angle-closure glaucoma. As a hydrophilic drug, the
corneal epithelium being lipophilic is the major barrier to
its permeation after topical administration, retarding the
passage of up to 90% of administered drug (Loftsson etal.
2008). It is still most widely available as conventional oph-
thalmic solutions for topical administration in drops, as well
as suspensions and gel-based formulations. The fundamental
challenges with these dosage forms are the associated poor
ocular retention, limited precorneal absorption and loss due
to nasolacrimal drainage, necessitating the need for frequent
administrations which negatively affect patient compliance
and ultimately, treatment outcomes (Gaudana etal. 2009).
Niosomes have shown great potential for addressing these
challenges in ocular delivery due to the nature of their struc-
ture and composition which offers the advantage of being
biodegradable, biocompatible and non-immunogenic (Sahoo
etal. 2014).
The cloudy appearance of formulated niosomes was
typical and consistent with reports by Shah etal. (2020).
Clear differences in the degree of separation on standing
can be attributed to the intrinsic physical character of the
surfactants. Span 60 being more hydrophobic (HLB value
of 4.7) than Tween 60 (HLB value of 14.9) would exhibit
less interaction with the aqueous phase. It is well known that
the method of preparation influences the type of resulting
niosomes. The unilamellar vesicles obtained were consist-
ent with the results obtained by Marwa etal. (2013) who
formulated diclofenac sodium niosomes using ether injection
method. As suggested by Uchegbu and Vyas (1998), incom-
pletely formed bilayers could be the effect of residual etha-
nol from the formulation process which causes an additional
phase transition, thus affecting membrane rigidity. It could
also be attributed to the formulation having a higher ratio of
cholesterol which is known to have great impact on bilayer
integrity. Crystals observed in Tween 60 formulations could
be cholesterol or Tween 60; determining this conclusively
will require other characterization techniques that show the
interaction among various niosomal constituents like Fourier
transform infrared spectroscopy (FTIR), X-ray diffraction
analysis or differential scanning calorimetry (DSC) (Van-
kayala etal. 2018; Taymouri and Varshosaz 2016; Ruckmani
and Sankar 2010).
Particle sizes contrasted with those previously obtained
by Yoshioka etal. (1994) and a number of other research-
ers (Ghafelehbashi etal. 2019; Nowroozi etal. 2018). They
had established that the surfactant with a higher HLB value
and larger head group resulted in vesicles of larger sizes,
with those of Tween 60 (14.7) being greater than Span 60
(4.7). Results obtainedhere where Span 60 formed larger
sized niosomes than Tween 60have however also been noted
by Junyaprasert etal. (2012) with niosomes of ellagic acid,
Bayindir and Yuksel (2010) with paclitaxel, and Ruckmani
and Sankar (2010) with zidovudine. In their study with
Tween 80 and Span 80, Nadzir etal. (2017) showed that
lower HLB values of surfactants composition could indeed
result in larger sized niosomes. Basiri etal. (2017) noted that
increased Span 60 in the surfactant ratio of Span 60:Tween
60 combination increased particle size; on the contrary the
formulation with the highest total quantity of surfactant as
well as highest Tween 60 ratio in the experiment gave the
smallest vesicles. This was said to be influenced by the larger
hydrophilic head group and high HLB value of Tween 60,
while the larger sizes were as a result of Span 60 being more
hydrophobic and having a higher critical packing param-
eter (CPP) than Tween 60. Generally, higher concentrations
of both components gave larger sized vesicles. Gugleva
etal. (2019) made the same observations with niosomes
of doxycycline, as well as Taymouri and Varshosaz (2016)
with carvedilol nano-niosomes. According to Gugleva etal.
(2019), increased surfactant concentration would occupy a
larger membrane area together with the aryl chain leading
to chain distortion, increased membrane fluidity and vesicle
size.
Changes in cholesterol concentrations seemed to have
more impact on particle size than changes in surfactant
concentrations, as well as more effect on Span 60 formula-
tions than Tween 60 formulations similar to observations
made by Akbari etal. (2015) and Nowroozi etal. (2018).
This was attributed to the hydrophilicity of Tween 60 and
the increase in cholesterol being insufficient in affecting the
hydrophobicity of the bilayer. Comparing formulations with
combined and single surfactant(s), the effect of surfactant
type can be clearly seen. For the three groups of cholesterol
concentrations, all the Tween 60 containing formulations
had the smallest particle sizes, the co-surfactant formula-
tions were larger and the Span 60 only formulations had the
largest sizes. This is similar to the observations made by
Naderinezhad etal. (2017).
Drug loading would ideally cause an increase in particle
size as depicted by most of the Span 60 formulations which
is in line with results that have been reported by various
authors (Kaur etal. 2016; Taymouri and Varshosaz 2016).
It is due to space taken up by the added drug molecules after
drug loading, although others have also recorded no change
in particle size due to drug loading (Tavano etal. 2013).
In contrast, a slight decrease for Tween 60 formulations
was observed with K1 although exhibiting the highest EE
showing a reduction in particle size. A possible reason for

Progress in Biomaterials
1 3
this is that despite pilocarpine hydrochloride being a hydro-
philic drug and preferentially entrapped in the aqueous core,
some of the drug could be deposited in the hydrophobic
bilayer as a result of hydrophobic/hydrophobic interactions
between the drug, cholesterol and surfactants as explained
by Ghafelehbashi etal. (2019) in the niosomal encapsula-
tion of cephalexin. Studies conducted by García-Manrique
etal. (2020) corroborated these findings when they showed
that hydrophilic drugs sometimes interact with the lipid
bilayer membrane and the drug is incorporated there lead-
ing to reduced surfactant curvature and a consequent reduc-
tion in vesicle size. Similar results were reported by Tavano
etal. (2013) with doxorubicin niosomes where particle size
reduced after drug loading which was attributed to elec-
trostatic attractions between the drug and bilayer causing
increased vesicle cohesion, a closely packed membrane con-
figuration and increased membrane curvature. Akbari etal.
(2013) with ciprofloxacin-loaded nano-niosomes, and Lu
etal. (2019) also noted the same reduction in size.
Zeta potential measures particle surface charge. The
technique employed here was electrophoretic light scat-
tering (ELS) which measures electrophoretic mobility, a
function of zeta potential. Electrophoretic mobility is the
velocity of particles moving through an electric field and
is obtained by determining the frequency change of laser
light scattered as they move (Wilson etal. 2001). Although
pilocarpine HCl is a cationic drug, zeta potentials deter-
mined for all formulations regardless of cholesterol con-
centration, surfactant type and concentration were indica-
tive of negatively charged vesicles. This could be due to
cholesterol which inputs a negative surface charge on the
vesicle as demonstrated by Farmoudeh etal. (2020) with
methylene blue-loaded niosomes. They also showed that
higher cholesterol concentrations resulted in increased zeta
potential which supports the results obtained here. Manos-
roi etal. (2010) also confirmed the negative charge induc-
ing effect of cholesterol in their study with niosomes of the
cationic drug gallidermin and attributed it to uneven polar-
ity distribution of cholesterol’s hydroxyl group. Increased
surfactant ratio saw an increase in electrical conductivity.
This is in accordance with determinations made by Dukhin
and Goetz (2006) and Smith and Eastoe (2013) in their
studies related to conductivity of surfactants in non-polar
liquids.
The higher negativity of the Span 60 formulations may
be attributed to ionic dissociation with resultant ionic impu-
rities as suggested by Dukhin and Goetz (2006) and Guo
etal. (2010). Results by Sadeghi etal. (2020) implicated the
phosphate buffer as a contributor to the negative charge on
the particles. They stated that in phosphate buffer, niosomal
formulations of cationic lysozymes were surrounded by lay-
ers of ‘counter-ions’ with opposite charges to those of the
niosomes. The implication of these zeta potential values is
that on long term storage, the Span 60 formulations would
be expected to show less tendency for aggregation, hence
greater stability. This is because having higher values, there
is more electrostatic repulsion and stabilization resulting in
a lesser tendency for particle aggregation in the colloidal
system (Seleci etal. 2016; Uchegbu and Vyas 1998). These
results are supported by those obtained by Gugleva etal.
(2019) where zeta potentials were negative across board,
with Span 60 formulations being more negative than equiva-
lent Tween 60 formulations.
Generally, zeta potentials of greater than − 30 mV
or + 30mV are said to be acceptable indicators of good sta-
bility (Cho etal. 2013; Khan etal. 2017). Zeta potential and
associated stability could be improved particularly for the
Tween 60 formulations by adding a negative charge inducer
such as dicetyl phosphate (DCP) that has been widely
applied in niosomal formulations and has been established
to be effective in improving stability (Okore etal. 2011;
Sezgin-Bayindir and Yuksel 2012; Nayak etal. 2020).
Low EE values were possibly due to the hydrophilic
nature of pilocarpine hydrochloride as it is well-established
that better entrapment efficiency is generally achieved with
more hydrophobic drugs than hydrophilic drugs (Hashemi
Dehaghi etal. 2017; Bhardwaj etal. 2020). With the hydro-
philic drug being soluble in the aqueous phase, during vesi-
cle formation the amount of aqueous phase encapsulated
in the core is much less than that outside the lipid bilayer,
resulting in lower percentage entrapment compared with
hydrophobic drugs that have preference for the bilayer (Joshi
etal. 2020). It is also known that for hydrophilic drugs, the
Tween series of surfactants give the best entrapment effi-
ciency as seen from workdone by Kumar and Rajeshwarrao
(2011). Tween 60 possesses a larger hydrophilic head with
long alkyl chain length and hydrophilic drugs are typically
entrapped in the polar aqueous core, so it enables more solu-
bilization and entrapment of the drug (Naderinezhad etal.
2017; Manosroi etal. 2003). This was seen in work done by
Ghafelehbashi etal. (2019) with cephalexin and Gugleva
etal. (2019) with doxycycline where Tween 60 alone gave
a higher EE compared with Span 60 alone. Span 60 on the
other hand although having the same chain length (C18) as
Tween 60 has a smaller hydrophilic head group so does not
take up as much of the hydrophilic drug (Bhardwaj etal.
2020; Wang and Gao 2018). Manosroi etal. (2003) studied
the characteristics of vesicles formed with various non-ionic
surfactants and cholesterol mixtures, showing that the Tween
with a C18 alkyl chain and a higher HLB value gave a better
EE than the equivalent C18 Span due to a higher hydration
of the polar head of the Tween. Another reason suggested
by Gugleva etal. (2019) is that as the surfactant:cholesterol
molar ratio increased, the cholesterol saturation limit of Span
60 was reached resulting in bilayer disruption and drug loss.
For Tween 60 due to its high HLB, this saturation limit was

Progress in Biomaterials
1 3
not reached. A number of researchers have noted that the use
of the most suitable surfactant and cholesterol in a 1:1 ratio
was desirable in achieving the needed enhanced bilayer com-
pactness and increased entrapment efficiency (Balakrishnan
etal. 2009; Barakat etal. 2014; Bayindir and Yuksel 2010).
This was, however, not the case for the Span 60 containing
counterpart comparing K2 having more surfactant than K5
but EE of 21.9% and 25.1%, respectively.
Co-surfactant formulations gave better EE than those with
only Span 60 and were second only to those with Tween 60
alone, demonstrating well the effect of surfactant combi-
nation. Naderinezhad etal.(2017) had similar results with
Tween 60, Tween 60/Span 60 combination and Span 60 giv-
ing the highest to lowest EE of doxorubicin and curcumin in
that order. In our experiments, there was, however, an excep-
tion to this trend in the group containing half the surfactant
concentration, with K5 containing Span 60 alone having a
higher EE than the combination but still maintaining a lower
EE than Tween 60 alone. From results obtained by Barakat
etal. (2014) combining a hydrophilic and hydrophobic non-
ionic surfactant in niosomal formulations of hydrophilic
vancomycin hydrochloride, increased EE with co-surfactant
was as a result of integration into the bilayer structure by
mainly hydrophobic molecular interactions of the surfactants
alkyl tails as well as hydrogen bonding between the closely
packed polar head groups. This resulted in increased hydro-
philicity of the bilayer, hence the increased entrapment of
hydrophilic vancomycin.
Cholesterol gave more stability to the bilayer, increas-
ing rigidity and reducing permeability (as Tweens typically
need cholesterol to form stable vesicles), hence more drug
is retained in the vesicle (Manosroi etal. 2003). Hashemi
Dehaghi etal. (2017) made similar observations with
hydrophilic dorzolamide-loaded niosomes. Basiri etal.
(2017) demonstrated the role of increased concentrations
of cholesterol in improving EE in niosomes. It was noted
to do this by increasing the chain order of bilayers in liquid
state, thereby abolishing the phase transition of the system.
The conclusion can be drawn thus, that in using the same
surfactant and cholesterol concentration ratio in K1, it was
sufficient to increase cohesion of non-polar portions in the
bilayer thereby inhibiting drug leakage (Di Marzio etal.
2011). This was particularly observed in the difference in EE
be Tween K1 and K7 as highlighted above. Similar results
were obtained by Guinedi etal. (2005) with acetazolamide-
loaded niosomes prepared by reverse-phase evaporation and
thin film hydration.
Effect ofsonication onniosomal properties
It was established that indeed sonication reduced vesicle
size and that longer durations produced smaller vesicle
sizes. This is in line with reports on the effect of sonication
observed by Sezgin-Bayindir and Yuksel (2012) who
showed that optimum size reduction in their experiment
was obtained after probe sonication for 60min. For Tween
60 niosomes which had a generally lower size range, there
was overall a smaller degree of size reduction than the Span
60 formulation. This can be attributed to the fact that the
Tween 60 formulation had more thermodynamic stability
and achieved equilibrium quickly with minimal size reduc-
tion compared to the Span 60 formulation (Diskaeva 2018).
As size reduced, the PDI was also seen to reduce, indicating
that the dispersion became more homogenous with sonica-
tion. Nowroozi etal. (2018) obtained similar results after
bath sonication of niosomal formulations prepared with
Span 60, as well as other studies investigating the effects of
sonication by Akbari etal. (2013), Pereira-Lachataignerais
etal.(2006), and Yeo etal. (2019).
Overall, there was more impact on the zeta potential of
the Span 60 formulation than the Tween 60 formulation.
This is possibly due to the occurrence of only minimal
changes in size with the Tween 60 formulation, suggesting
that there might be some relationship between changes in
particle size and zeta potential (Shi etal. 2018). Formula-
tion K8's behaviour was explained by Nakatuka etal. (2015)
stating that smaller particles are more easily affected by sur-
rounding particles and random fluid flow movement in a
Brownian diffusion effect, hence there is easy collision with
other particles. Smaller particles therefore have a relatively
greater surface charge than larger particles. These results
correspond to those reported when Akbari etal. (2013) that
determined the effect of increased sonication durations on
zeta potential, noting an increase in potential with increase
in time as particle size reduced.
Ultrasonic effects on the lipid membrane result in open-
ing and shutting of niosomes in a process of reformula-
tion explained by Widayanti etal. (2017), so more drug
is entrapped within the aqueous core with each opening.
This can account for the increase in EE with K7. Decrease
after 60min is similar to what Mavaddati etal. (2015)
observed and was probably due to vesicle destruction lead-
ing to drug leakage (Khan etal. 2017; Zhang etal. 2020).
This trend was also observed by Anbarasan etal. (2013)
with capecitabine-loaded niosomes. Considering that drug
loading increased particle size for K8, the reduced EE after
30min of sonicating could be as a result of smaller vesicles
forming similar to observations by Shete etal. (2012). Some
studies have shown progressive reduction in EE with longer
sonication times due to reduction in particle size (Nayak
etal. 2020). However, it was noted that further sonication
to 60min slightly increased EE although there was a contin-
ued decrease in size. A possible explanation for this is that
along with the dispersion gaining thermodynamic stability,
the bath sonication process also facilitates hydration by the
aqueous buffer containing pilocarpine HCl so encourages

Progress in Biomaterials
1 3
drug entrapment (Mavaddati etal. 2015). A similar effect
on EE attributed to increased hydration time was noted by
(Yeo etal. 2019).
The two formulations were observed to exhibit opposite
trends; the K7 increasing after 30min and then decreas-
ing after 60min, and K8 the reverse. A possible reason for
this can be tied to the characteristics of the surfactants, with
Span 60 known to form a more cohesive/stable lipid mem-
brane with cholesterol due to its hydrophobicity compared
to Tween 60, hence would be less likely to be destroyed
under the same conditions as Tween 60 formed membrane
(Bagheri etal. 2014).
Conclusion
Tween 60 formulations gave more homogenous dispersions,
more desirable particle sizes for ocular delivery and bet-
ter EE than Span 60 formulations. Sonication was seen to
reduce particle size, improve PDI and increase zeta poten-
tial by approximately 28% and EE by 61%, with optimum
sonication time for this study pegged at 30min. These
results are specific to the conditions used in this experi-
ment and cannot be exhaustively and broadly generalized
as there were no replicates. They, however, suggest that in
addition to carefully selecting and varying surfactant types
and concentrations, it is possible to use sonication time as
a process parameter for formulating optimized niosomes of
hydrophilic drugs with properties that indicate homogene-
ity, alow propensity for aggregation and high entrapment
efficiency. Further work would be required to exhaustively
determine the influence surfactant type has on the effect of
sonication on entrapment efficiency. This could be useful in
overcoming stability challenges and maximally exploiting
niosomes advantages ofgreater permeability, longer ocular
retention,and drugprotection from metabolic degradation
over conventional formulations and systems for pilocarpine
hydrochloride delivery to the eye.
Authors contributions Design–AI, KO; formal analysis, investigation,
writing: original draft preparation—KO; conceptualisation and super-
vision—AI; writing: review and editing: MI, AI, KO. All authors read
and approved the final manuscript.
Funding Partly supported by the Allan and Nesta Ferguson Charitable
Trust Scholarship at Aston University.
Availability of data and materials Available upon request.
Declarations
Conflict of interest The authors declare they have no competing in-
terests.
Ethical approval and consent to participate Not applicable.
Consent for publication Not applicable.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other 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 this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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