Materials Today Communications 35 (2023) 105632

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Materials Today Communications

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Tuning the size and concentration of functional molecules coated onto

mesoporous silica nanoparticles for efficient therapeutic protein delivery
Parvaneh Esmaeilnejad-Ahranjani a, *, Sayed Ali Maboudi b
a
Department of Anaerobic Bacterial Vaccine Research and Production, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension
Organization (AREEO), P.O. Box: 31975/148, Karaj, Iran
b
Iran Food and Drug Administration, P.O. Box: 1314715311, Tehran, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: In this work, the importance of the size and concentration of binding sites in mesoporous nanostructures on their
Mesoporous silica nanoparticles performance for protein delivery is reported. Various types of carboxyl-functionalized mesoporous silica (MS)
Polyacrylic acid nanoparticles, i.e., SA(x)-MS, PA1(x)-MS and PA2(x)-MS, where x = 5, 10, 15, 20 and 30 mg, were synthesized
Functional molecules size
by using succinic anhydride (SA) and polyacrylic acid with two molecular weights (i.e., 1800 (PA1) and 100,000
Binding sites concentration
Protein delivery
(PA2)), respectively. The particles were thoroughly characterized by TGA, FTIR, STEM and zeta potential
measurement techniques and then, their biomedical performance was investigated by using a model therapeutic
protein, lysozyme. The carboxyl functional molecules concentration (CCOOH) on the particles was increased as a
function of the functional molecules type and quantity in the synthesis reaction environment. The maximum
CCOOH values were recorded for SA(10)-MS, PA1(15)-MS and PA2(15)-MS, being 827, 1662 and 2137 µmol.g− 1,
respectively. However, SA(10)-MS, PA1(15)-MS and PA2(10)-MS particles led to the highest lysozyme loading
yield values of 63.1%, 90.0% and 71.5% as well as the loading capacities of 631, 900 and 715 mg.g− 1,
respectively. The controlled lysozyme release rate and some protein conformational changes favored the in vitro
antibacterial activity of lysozyme molecules carried by the particles, which followed the order of PA1(15)-MS >
PA2(10)-MS > SA(10)-MS.

against Escherichia coli in vitro and in vivo. In another report, the effect
of silica particles size on the lysozyme loading and antibacterial activity
1. Introduction
was investigated [16]. The best antibacterial performance was reported
for the particles with the average size of 79 nm and pore size of 22.4 nm,
Owing to promising developments in pharmaceutical sciences, the
as compared to the large particles (160 nm) having smaller pore size
design and application of nanostructures have been one of the main
(2.4 nm). Yu et al. [17] reported the poly(N-isopropylacrylamide)-­
topics of researches [1,2]. Generally, the aim of using nanomaterials in
coated core-shell Fe3O4/mesoporous silica particles as a potential
the medical fields has been to load and deliver significant quantities of
nanocarrier of lysozyme to fight against Bacillus cereus and Micrococcus
therapeutic agents to the target cells and reduce the side effects [3–5].
luteus, where the lysozyme release was depended to the thermal alter­
For this purpose, various nanocarriers, including organic, inorganic, and
ations. Moreover, the ability of the isobutyramide-modified mesoporous
hybrid types of nanoparticles, have been extensively developed [6–8].
silica for the delivery of various therapeutic biomolecules such as serum
Among various nanomaterials, because of the low cost, easy prepara­
albumin, peroxidase, immunoglobulin and polylysine, was investigated
tion, high surface area, excellent biocompatibility, and high thermal and
[18]. The results demonstrated the higher loading capacity of particles
chemical stabilities, silica has been of utmost interest for the therapeutic
for serum albumin and peroxidase compared to others, coupled with the
biomolecules delivery [9,10]. It has been reported that the capacity of
sustainable release rate and high biological activity of the immobilized
the mesoporous silica for loading any therapeutic agent and its release
biomolecules. Recently, the influence of silica structure on the encap­
remarkably depends on the physico-chemical properties of particles
sulation and release of a model bone morphogenetic proteins (soybean
[11–14]. Li et al. [15] reported that the excellent performance of
trypsin inhibitor) has been reported [19]. Compared to the ordered
carboxylated mesoporous silica particles as a carrier of lysozyme phys­
structured mesoporous silica, the unstructured sample showed higher
ically adsorbed onto them to act as an efficient antibacterial agent

* Corresponding author.
E-mail address: p.esmaeilnejad@rvsri.ac.ir (P. Esmaeilnejad-Ahranjani).

https://doi.org/10.1016/j.mtcomm.2023.105632
Received 7 December 2022; Received in revised form 17 January 2023; Accepted 13 February 2023
Available online 14 February 2023
2352-4928/© 2023 Elsevier Ltd. All rights reserved.
P. Esmaeilnejad-Ahranjani and S.A. Maboudi
Nomenclature
SA Succinic anhydride.
PA1 Low-molecular-weight polyacrylic acid.
PA2 High-molecular-weight polyacrylic acid.
MS Mesoporous silica nanoparticles.
APS-MS Amine-functionalized MS nanoparticles.
SA(x)-MS SA-functionalized MS nanoparticles.
PA1(x)-MS PA1-functionalized MS nanoparticles.
PA2(x)-MS PA2-functionalized MS nanoparticles.
L-SA(x)-MS Lysozyme loaded onto SA(x)-MS.
L-PA1(x)-MS Lysozyme loaded onto PA1(x)-MS.
L-PA2(x)-MS Lysozyme loaded onto PA2(x)-MS.
x Initial amount of the applied functional molecules (mg).
Y Mass loss observed in TGA profiles (%).
mF Mass ratio of the functional molecules attached on
particles (mg.g− 1).
CF Concentration of functional molecules attached on
particles (µmol.g− 1).
CCOOH Concentration of carboxyl groups on functionalized
particles surface (µmol.g-1).
YLP Lysozyme loading yield (mg.mg− 1%).
CLP Lysozyme loading capacity of particles (mg.g− 1).
PRP Lysozyme release percentage (mg.mg− 1%).
protein loading and release values, fairly explained by the difference in
the pore interconnectivity within the particle and its hydrophobic
property. Yang et al. [20] studied the adjustment of pore size of
polymer-silica nanostructures to obtain high adsorption capacity for
bovine serum albumin, where the particles with ultra-large pore size (27
nm) showed the protein absorption capacity of 166 mg/g, being twice
that of the particles with smaller pore size (11.6 nm). Beside of the
particles structural properties, it should be considered that the
physico-chemical characteristics of environment (e.g., temperature,
buffer type and pH) can highly affect the lysozyme adsorption and
release from mesoporous silica particles, which was comprehensively
studied in the literature [10]. In our previous report, the effect of protein
corona formation method, i.e., physical and covalent, onto the bare and
succinic anhydride functionalized mesoporous silica particles on the
lysozyme activity was investigated [21]. It was shown that physically
adsorbed lysozyme onto the functionalized particles can lead to
extremely high activity, which was remarkably increased once a
pH-shock was induced to them.
Despite numerous studies, the effects of the size and concentration of
binding sites on the capacity of mesoporous silica particles for protein
delivery as well as on the conformation and activity of the proteins have
been overlooked. The aim of this work is to develop carboxyl-
functionalized MS particles with optimized structure for the efficient
delivery of proteins. Lysozyme was used as a model therapeutic protein,
because of its unique characteristics of antimicrobial, anti-
inflammatory, antifungal and antiviral activities, making it a potential
candidate for the pharmaceutical applications [22–24]. The
as-synthesized MS particles were coated with succinic anhydride (SA)
and low-molecular-weight polyacrylic acid (PA1) and
high-molecular-weight polyacrylic acid (PA2) via the covalent attach­
ment method and then lysozyme was loaded by the physically adsorp­
tion method. The effects of the size and initial concentration of coated
functional molecules on the concentration of carboxyl groups on parti­
cles and lysozyme loading capacity (CLP) as well as the conformational
and antibacterial properties of the lysozyme molecules were discussed
comprehensively.
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Materials Today Communications 35 (2023) 105632
2. Materials and methods
2.1. Materials
N-(2-Aminoethyl)− 3-aminopropyltrimethoxysilane (APS), N,N-
dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetic acid,
succinic anhydride (SA) and brain-heart infusion (BHI) were purchased
from Merck. Lysozyme, sodium hydroxide, ethanol, N-hydrox­
ysuccinimide (NHS), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide
hydrochloride (EDC) and polyacrylic acid (two molecular weights of
1800 (PA1) and 100000 (PA2)), were obtained from Sigma-Aldrich.
2.2. Synthesis of carboxyl-functionalized mesoporous silica nanoparticles
Mesoporous silica (MS) nanoparticles were synthesized via the
template removing method and then functionalized with amine groups
using APS to prepare APS-MS particles, as according to the procedure
reported previously [21,25,26]. Afterwards, the SA(x)-, PA1(x)- and
PA2(x)-coated MS particles, which are hereafter referred to as SA(x)-MS,
PA1(x)-MS and PA2(x)-MS, were prepared by the application of various
initial amounts (x) of functional molecules, which were 5, 10, 20 and 30
mg. Briefly, for the preparation of SA(x)-MS particles, SA was dissolved
in 35 mL DMF by stirring at room temperature, 100 rpm for 5 min, which
was continuously outgassed by N2. Afterwards, 100 mg APS-MS particles
dispersed in 5 mL of DMF was added to the as-mentioned solution and
stirred at 100 rpm for 14 h. The particles were collected through
washing with DMF, ethanol and deionized water by using a centrifuge
(13,000 rpm, 20 min).
In order to covalently attach the PA1 and PA2 onto the APS-MS
particles, first it needs to activate the carboxyl groups of these func­
tional molecules. Therefore, PA1 and PA2 were separately dissolved in
25 mL deionized water, then added to a mixed solution (50 mL) of EDC
(10.5 mmol.L− 1) and NHS (10.5 mmol.L− 1), following mixing at 60 rpm,
4 ◦ C for 10 min. Then, the corresponding solutions were immediately
added to 25 mL solution of APS-MS particles in deionized water (4 mg.
mL− 1) and shaken for 2 h, at room temperature. Finally, the as-prepared
PA1(x)-MS and PA2(x)-MS particles were comprehensively washed with
the deionized water by centrifugation (13,000 rpm, 20 min).
2.3. Characterization methods
To evaluate the successful attachment of functional molecules onto
the particles, thermogravimetric (TGA) analysis was performed by using
TG/DTA6300 instrument over the temperature range of 25–600 ◦ C with
a heating rate of 10 ◦ C.min− 1 and an air flow rate of 20 mL.min− 1. The
mass ratio of functional molecules attached onto MS particles (mF, mg.
g− 1) was calculated using the mass loss values related to the organic/
polymer layer (Y) observed in the TGA profiles (Eq. (1)). Additionally,
the concentration of SA, PA1 and PA2 functional molecules (CF, µmol.
g− 1) as well as the corresponding carboxyl groups concentration (CCOOH,
µmol.g− 1) onto the SA(x)-MS, PA1(x)-MS and PA2(x)-MS particles were
estimated using Eq. (2) and Eq. (3), respectively.
mF =
Y
× 1000 (1)
1− Y
mF × 1000
CF = (2)
Mw,F
mF × 1000
CCOOH = × nCOOH (3)
Mw,F
where Mw,F is the molecular weight of SA, PA1 and PA2 molecules, and
nCOOH is the number of carboxyl functional groups in one molecule of
SA, PA1 and PA2.
The particles’ surface functional molecules were further analyzed by
a Bruker Tensor 27 Fourier-transform infrared (FTIR)
P. Esmaeilnejad-Ahranjani and S.A. Maboudi
spectrophotometer. The zeta (ζ) potential and hydrodynamic diameter
of particles were estimated by a Malvern Zetasizer Nano instrument
(S90, UK) equipped with the dynamic light scattering (DLS) system.
Scanning transmission electron microscopy (STEM) was carried out by a
Hitachi S-5500 microscope. The Quantachrome CHEMBET-3000 in­
strument was used to measure the BET surface areas. Prior to the anal­
ysis, the samples were outgassed with a gas mixed of N2 and He, at
300 ◦ C for 2 h. The pore volume and diameter were determined by BJH
model.
2.4. Loading and release of protein
In a typical procedure, 4 mg of the prepared carboxyl-functionalized
MS particles dispersed in 2 mL sodium phosphate buffer solution (pH
7.4, 50 mM) was added to 2 mL of the lysozyme solution (2 mg.mL− 1) in
the sodium phosphate buffer solution (pH 7.4, 50 mM). After shaking at
200 rpm for various contact times (30, 60, 90 and 120 min), lysozyme-
loaded particles were centrifuged (13,000 rpm, 20 min) and washed two
times with the sodium phosphate buffer solution (pH 7.4, 50 mM). The
lysozyme-loaded SA(x)-MS, PA1(x)-MS and PA2(x)-MS particles were
named as L-SA(x)-MS, L-PA1(x)-MS and L-PA2(x)-MS, respectively. The
amount of loaded lysozyme (MLP, mg) onto the particles (MNP, g) was
determined by subtracting the amount of lysozyme remained in the
solutions (MRP, mg) from the applied initial lysozyme (MIP, mg), which
was analyzed by using the spectrophotometry method at 280 nm. The
lysozyme loading yield (YLP, mg.mg− 1%) and the loading capacity (CLP,
mg.g− 1) of particles were determined using Eq. (4) and Eq. (5),
respectively.
YLP =
MLP
× 100 (4)
MIP
MLP
CLP = (5)
MNP
To study the in vitro protein release, the lysozyme-loaded particles
were dispersed in sodium phosphate buffer solution (50 mM, pH 7.4)
containing KCl (0.5 M), following by shaking at 180 rpm for various
time periods. After separation of particles through centrifugation, the
amount of released lysozyme (MDP) in the supernatant was estimated at
280 nm using the spectrophotometer. The lysozyme release percentage
(PRP, mg.mg− 1%) was calculated using Eq. (6).
MDP
PRP = × 100 (6)
MLP
The secondary structure of protein was evaluated by the circular
dichroism (CD) spectroscopy technique using the J-715 spec­
tropolarimeter (Jasco Co.). The analysis were performed at the wave­
length range of 190–260 nm on 0.2 mg.mL− 1 solution of either fresh free
lysozyme or separated lysozyme with the background corrected by
blank spectra of buffer solution.
2.5. Antimicrobial activity
The antibacterial activity of the free lysozyme and the ones loaded
onto the particles were valuated via the minimum inhibitory concen­
tration (MIC) using Staphylococcus aureus. To do the experiments,
various concentrations of lysozyme (1000–3000 μg.mL− 1) being either
in free form or loaded onto the particles were added to the solution of
1 × 106 CFU.mL− 1 S. aureus in 20 mL BHI medium, and incubated at
37 ◦ C and 200 rpm for 24 h. The bacterial cell viability was measured by
recording the optical density (OD) of samples at 610 nm using a spec­
trophotometer. For the background correction through the OD mea­
surement, the BHI media including the free lysozyme and/or lysozyme
loaded particles with the content exactly equal to the ones used in MIC
assays were applied. The experiment was performed in triplicate and
mean standard deviation values were reported.
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Materials Today Communications 35 (2023) 105632
3. Results and discussion
3.1. Characterization of nanoparticles
The coating of functional molecules onto the surface of particles was
evaluated using TGA technique (Fig. 1a). For all the samples, the
desorption of physically adsorbed water molecules below 120 ◦ C makes
a mass loss of about 2.96%, which is in agreement with the literature
[25,26]. For the bare MS particles, no further mass loss is observed,
revealing that the possible decomposition of silica may occur at tem­
peratures above 600 ◦ C [27]. For APS-MS particles, the mass loss be­
tween 120 and 250 ◦ C can be due to the thermal decomposition of the
corresponding organic component [28]. The further mass losses for all
SA(x)-MS particles are associated with the thermal decomposition of the
SA bonded to APS, which is in agreement with the literature [28]. For
both PA1(x)-MS and PA2(x)-MS particles, the mass losses observed in
the range of 120–350 ◦ C can be related the thermal decomposition of
PA1 and PA2 molecules covalently attached to the APS-MS particles [29,
30]. The corresponding mF, CF and CCOOH values calculated from TGA
profiles and using Eq. (1), Eq. (2) and Eq. (3), respectively, are reported
in Table 1. From Table 1, it is observed that by the increase in the initial
concentration of SA, the mF, CF and CCOOH values for the corresponding
particles gradually enhance and then level off at x = 10. Therefore, at
this point, the maximum mF, CF and CCOOH values of 82.8 mg.g− 1,
827.8 µmol.g− 1 and 827.8 µmol.g− 1 are achieved for SA(10)-MS parti­
cles, respectively.
However, for PA1(x)-MS and PA2(x)-MS particles, mF continuously
increases by the enhancement of the initial polymers concentration,
showing the maximum values of 130.0 and 166.8 mg.g− 1 for PA1(15)-
MS and PA2(15)-MS, respectively, and then starts to decrease by
further enhancement of x. The corresponding CF and CCOOH values for
PA1(15)-MS are 72.26 and 1.668 µmol.g− 1, where they are 1662.0 and
2137.5 µmol.g− 1 for PA2(15)-MS particles, respectively. Additionally,
the rate of decrease in the attachment of polymers by the enhancement
of x, is much higher for PA2(x)-MS in comparison to PA1(x)-MS.
Perhaps, the formation of polymers aggregates at higher polymer con­
centrations in the solution may limited the attachment of polymers onto
the particles. Additionally, the lower CF value for PA2(x)-MS particles
surface compared to that of PA1(x)-MS can be related to the severe steric
and electrostatic repulsions between polymer molecules that hinder the
PA2 molecules to attach closely on the particles surface. However, the
higher CCOOH value for PA2(x)-MS in comparison to that of PA1(x)-MS
can be due to the higher number of carboxyl functional groups (about 55
folds) in one molecule of PA2 compared to that of PA1.
The FTIR spectra of the particles are illustrated in Fig. 1b. The bands
at 810, 973 and 1099 cm− 1 are related to the stretching vibration of
Si–O–Si, Si–OH and bending vibration of Si–O bonds, respectively [28].
For the SA(10)-MS, PA1(15)-MS and PA2(15)-MS particles, the coated
carboxyl groups make two absorption peaks at 1410 and 1725 cm− 1
associated to the bending vibration of C− H bonds and stretching vi­
bration of C− O bonds, respectively [30]. The absorption peaks at 1560
and 1647 cm− 1 are associated with the stretching vibrations of amide
(C− N) bonds, fairly confirming the formation of covalent bonds be­
tween the carboxyl groups of SA, PA1 and PA2 molecules and amine
groups onto the APS-MS particles [31].
The surface charges (ζ potential value) of the prepared particles
recorded in an aqueous solutions with pH 7.4 are reported in Table 1.
The ζ potential value of bare MS particles was − 28.8 ± 2.3 mV,
implying the presence of the hydroxyl groups (-OH) on silica surface
[28,32]. After coating the particles with the functional molecules, the
surface charges of SA(x)-MS, PA1(x)-MS and PA2(x)-MS particles were
decreased and, as it can be expected, by the increase of the CCOOH values
of particles, the surface charges of particles get more negative (Table 1).
The STEM images revealed the spherical morphologies with the
average diameter of 92 ± 10 nm for all the MS, SA(10)-MS, PA1(15)-MS
and PA2(15)-MS particles (Fig. 2). It is observed that through coating of
P. Esmaeilnejad-Ahranjani and S.A. Maboudi Materials Today Communications 35 (2023) 105632

Fig. 1. TGA profiles (a) and FTIR spectra (b) of the prepared particles.

3.2. Protein loading and release

Table 1
Surface characteristics of carboxyl-functionalized MS nanoparticles.
The YLP and CLP values of the prepared particles are shown in Fig. 3.
Samples Ya (%) mbF (mg. CcF (µmol. CdCOOH (µmol. ζe Referring to our previous report and other literature [21,34,35], because
g− 1) g− 1) g− 1) (mV)
of the proteins denaturation at acidic pH values, the higher activity of
SA(5)-MS 4.99 52.52 524.84 524.84 -32.3 lysozyme molecules at pH 7.4, and their higher positive charge at pH 7.4
SA(10)-MS 7.65 82.83 827.79 827.79 -40.2
(pI 9.5), the lysozyme loading process was carried out at pH 7.4. It is
SA(15)-MS 7.64 82.72 826.62 826.62 -40.0
SA(20)-MS 7.63 82.60 825.45 825.45 -41.1 worth to mention that all the particles have a negative surface charge
SA(30)-MS 7.66 82.95 828.96 828.96 -40.9 (see Table 1). From Fig. 3a,b,c, it is revealed that the loading of lysozyme
PA1(5)-MS 5.00 52.63 29.24 672.51 -34.5 is rapidly occurred by using PA1(x)-MS and PA2(x)-MS particles within
PA1(10)- 9.99 110.98 61.66 1418.17 -46.2 15 min, while the contact time of 30 min is required for SA(x)-MS par­
MS
ticles to obtain the maximum lysozyme loading. It is noted that using for
PA1(15)- 11.51 130.07 72.26 1662.02 -53.7
MS the bare MS particles, the YLP and CLP values of 82.3% and 823 mg.g− 1
PA1(20)- 8.77 96.13 53.41 1228.33 -45.4 were obtained at the contact time of 120 min, which was optimized in
MS our previous study [21], respectively. For SA(x)-MS particles, the YLP
PA1(30)- 7.08 76.20 42.33 973.60 -42.6
and CLP values increase in the order of SA(30)-MS = SA(20)-MS = SA
MS
PA2(5)-MS 4.99 52.52 0.52 672.79 -36.1
(15)-MS = SA(10)-MS > SA(5)-MS, which is according to the order of CF
PA2(10)- 10.00 111.11 1.11 1423.33 -47.9 and CCOOH values as they were remained constant for x > 10 mg (see
MS Table 1). In contrary, for PA1(x)-MS samples, the order of PA1(15)-MS
PA2(15)- 14.3 166.86 1.67 2137.49 -59.9 > PA1(10)-MS > PA1(20)-MS > PA1(30)-MS > PA1(5)-MS, and for PA2
MS
(x)-MS particles, such an order of PA2(10)-MS > PA2(15)-MS > PA2
PA2(20)- 9.78 108.40 1.08 1388.63 -47.6
MS (20)-MS > PA2(30)-MS > PA2(5)-MS are observed for YLP and CLP
PA2(30)- 6.52 69.75 0.70 893.47 -42.3 values. These results for PA1(x)-MS is reasonable as it is according the
MS order of CF and CCOOH values (Table 1). In other words, by the
a
Mass loss from TGA profiles; bMass ratio of functional molecules coated onto enhancement of the coating PA1 molecules and consequently the in­
MS particles; cConcentration of functional molecules on particles; dConcentra­ crease of CCOOH on this type of particles, the better environment is
tion of carboxyl functional groups on particles; eZeta potential at pH 7.4 provided for protein loading. However, aforementioned lysozyme
loading capacities for PA2(x)-MS particles are not exactly according to
PA1 and PA2 molecules on the MS particles, the particles are agglom­ the order of CCOOH values of particles (Table 1). The higher values of YLP
erated to some extent. The hydrodynamic diameters of 103, 111, 175 and CLP for PA2(10)-MS compared to PA2(15)-MS while the former
and 193 nm were estimated for MS, SA(10)-MS, PA1(15)-MS and PA2 showed lower mF and CF values compared to the later, may exhibit that
(15)-MS particles, respectively. According to the literature [33], the the high amounts of large PA2 molecules on PA2(15)-MS hinder the
difference between the particles diameters values obtained using SEM access and loading of lysozyme molecules. Additionally, the protein
and DLS analyses can be due to the swelling of organic/polymer layers in molecules adsorbed on the outer regions of the polymer layers in the
the aqueous solutions and the fact that the DLS analysis measures the particles may impose a steric barrier against the other protein molecules
hydrodynamic size of particles in liquid media, which is usually larger in the solution, resulting in the reduction of the diffusion and adsorption
than the actual size of particles. The specific BET surface areas of MS, SA of protein into the inner regions of the polymer layers. This event may be
(10)-MS, PA1(15)-MS and PA2(15)-MS particles were 882.1, 715.0, more sever for PA2 in comparison to PA1 polymer. Overall, using the
540.6 and 381.5 m2.g− 1, respectively. Also, pore size values of 9.1, 8.0, optimized structures of SA(10)-MS, PA1(15)-MS and PA2(10)-MS, the
6.2 and 4.3 nm were calculated for MS, SA(10)-MS, PA1(15)-MS and YLP values of 63.1%, 90.0% and 71.5%, and the CLP values of 631, 900
PA2(15)-MS particles, respectively. Such decreases in the surface area and 715 mg.g− 1 were obtained, respectively. The reason of better per­
and pore size of functionalized particles, can be due the aggregation of formance of PA1(x)-MS and PA2(x)-MS particles for protein loading in
the coated functional molecules through drying of particles prior to comparison to SA(x)-MS ones can be more likely due to the difference
performing these analyses. among the CF values of particles and their surface charge values

4
P. Esmaeilnejad-Ahranjani and S.A. Maboudi
Fig. 2. STEM images of (a) MS, (b) SA(10)-MS, (c) PA1(15)-MS and (d) PA2(10)-MS particles.
influencing the electrostatic interactions between the positively charged
lysozyme and the negatively charged particles (see Table 1).
This work intends to develop a system to carry and deliver proteins,
thus the release of the loaded lysozyme molecules was analyzed. From
the desorption profiles (Fig. 4), upon 24 h treatment in the buffer so­
lution, about 32.5%, 16.2% and 8.3% of the lysozyme molecules release
from the L-SA(10)-MS, L-PA1(15)-MS and L-PA2(10)-MS particles,
respectively. The slow lysozyme release can be attributed to the cationic
charge of lysozyme (pI 9.5) at pH 7.4, which in turn leads to the
reduction of the electrostatic repulsion between the particles and pro­
tein molecules [36,37]. In addition, as the particles surface gets more
negative, this repulsions decrease and therefore, desorption rate in­
creases in the order of SA(10)-MS > PA1(15)-MS > PA2(10)-MS.
3.3. Secondary structure of the lysozyme delivered by nanoparticles
Before suggesting the optimum structures of the prepared particles, it
is important to study whether proteins structures can be affected by the
type of carboxyl groups coated onto the particles. The secondary
conformation of lysozyme loaded onto particles was studied using CD
spectroscopy technique. The contents of structural elements are re­
ported in Table 2. The results show that, for the lysozyme loaded onto
the particles, the content of α-helical and β-sheet elements decreases and
the unordered elements increase in the order of L-PA2(10)-MS > L-PA1
(15)-MS > L-SA(10)-MS, in comparison to the free lysozyme. The
alteration of proteins’ structure is the typical future the processes of the
adsorption of proteins onto the particles [36–38]. Here, as reported in
the previous sections, by the increase of functional molecules size
attached onto the particles (i.e., PA2 > PA1 > SA), the CCOOH values of
the particles increased leading to the reduction of ζ potential values, see
Table 1. Therefore, it can be concluded that as the surface charge of
particles gets more negative, the electrostatic interactions between the
protein molecules and particles increase, thus the higher conformational
changes of protein in L-PA2(10)-MS compared to L-PA1(15)-MS and
both of them in comparison to L-SA(10)-MS are observed.
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Materials Today Communications 35 (2023) 105632
3.4. Antibacterial activity of the protein-loaded nanoparticles
The in vitro antibacterial efficacy of the samples against S. aureus are
shown in Fig. 5. The bare particles do not show any activity, which is in
agreement with the literature [21]. L-SA(10)-MS, L-PA1(15)-MS and
L-PA2(10)-MS show MIC values of 1000, 350 and 550 µg.mL− 1, which
are 2.1, 6 and 3.8 folds higher than that of the free lysozyme (2100 µg.
mL− 1), respectively. According to the literature [35,36], the enhance­
ment in the antibacterial efficacy of lysozyme through loading onto the
particles can be originated from the improvement of the interaction
between the adsorbed lysozyme and the bacterial cell wall [35,36].
Therefore, the reason of the order of L-PA1(15)-MS > L-PA2(10)-MS
> L-SA(10)-MS for the antibacterial activity can be related to the
mechanism of protein release and the conformational changes of pro­
tein. From Fig. 4, it was shown that PRP values increase in the order of
L-SA(10)-MS > L-PA1(15)-MS > L-PA2(10)-MS. In addition, CD analysis
results showed that the conformation of the lysozyme changes in the
order of L-PA2(10)-MS > L-PA1(15)-MS > L-SA(10)-MS (Table 2). From
these results, it can be concluded that the protein release from L-SA
(10)-MS is fast and even not controllable, thus it shows lower antibac­
terial performance compared to two other prepared samples, though this
loaded lysozyme showed the negligible conformational changes
(Table 2). In contrary, the almost low PRP values for L-PA1(15)-MS and
L-PA2(10)-MS may be favorable to introduce high antibacterial activ­
ities. The lower activity of L-PA2(10)-MS in comparison to that of L-PA1
(15)-MS can be related to the very slower lysozyme release rate and
higher conformational changes of the former. Moreover, perhaps, some
changes in the lysozyme conformation through loading, particularly by
loading onto the PA1(15)-MS particle, is favorable for its activity.
4. Conclusion
To develop carboxyl-functionalized mesoporous silica nanoparticles
with the optimized size and concentration of binding sites, different
structures of SA(x)-MS, PA1(x)-MS and PA2(x)-MS, where x = 5, 10, 15,
20 and 30 mg, were synthesized and applied for the therapeutic protein
delivery. The size and initial concentration of the applied functional
molecule had a significant effect on the concentration of carboxyl groups
P. Esmaeilnejad-Ahranjani and S.A. Maboudi Materials Today Communications 35 (2023) 105632

Fig. 3. The YLP values of (a) SA(x)-MS, (b) PA1(x)-MS and (c) PA2(x)-MS; and CLP values of (d) SA(x)-MS, (e) PA1(x)-MS and (f) PA2(x)-MS at pH 7.4 using the
protein initial concentrations of 2 mg.mL− 1.

on particles and consequently on the YLP and CLP values. Among the
particles prepared by SA, PA1 and PA2 functional molecules, the SA
(10)-MS, PA1(15)-MS and PA2(15)-MS particles exhibited high CCOOH
values, respectively. However, the YLP and CLP values of particles
increased in the order of PA1(15)-MS > PA2(10)-MS > SA(10)-MS.
Additionally, the release rate, PRP values, secondary structure and
antibacterial activity of the loaded protein depended to the type of
particles. The lysozyme release rate increased in the order of L-SA(10)-
MS > L-PA1(15)-MS > L-PA2(10)-MS. The protein conformational
change increased in the order of L-PA2(10)-MS > L-PA1(15)-MS > L-SA

6
P. Esmaeilnejad-Ahranjani and S.A. Maboudi Materials Today Communications 35 (2023) 105632

editing, Supervision. Sayed Ali Maboudi: Writing – review & editing,

Investigation.

Declaration of Competing Interest

The authors declare that they have no known competing financial

interests or personal relationships that could have appeared to influence
the work reported in this paper.

Data Availability

Data will be made available on request.

Acknowledgment

Dr. Ayyoob Arpanaei is thankfully acknowledged for useful discus­

sions and providing synthesis and instrumentation facilities at the Na­
Fig. 4. Profiles of protein release from L-SA(10)-MS, L-PA1(15)-MS and L-PA2 tional Institute of the Genetic Engineering and Biotechnology of Iran.
(10)-MS nanoparticles at pH 7.4.
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