Insight on the Dependence of the Drug Delivery Applications of Mesoporous Silica Nanoparticles on Their Physical Properties

Mesoporous silica nanoparticles (MSNs) are fascinating due to their interesting properties and applications. The optimization of MSNs for drug delivery applications was achieved by preparing different formulations of MSNs using different concentrations of ammonium hydroxide (NH4OH) (0.7, 1.4, 2.8, 4.2, and 5.6 mg/ml for MSN1, MSN2, MSN3, MSN4, and MSN5, respectively). In the synthesis of MSNs, NH4OH was used as a catalyst while tetraethyl orthosilicate were used as a source of silica. Transmission electron microscopy (TEM) image revealed a linear increase in the size of the formed MSNs with increase in catalyst concentration. TEM images showed that all investigated nanoparticles were dispersed and spherical (changed to oval on addition of higher concentration of NH4OH). The hydrodynamic sizes of prepared MSNs were (64.18 ± 6.8, 90.46 ± 7.1, 118.98 ± 7.01, 152.7 ± 1.7, and 173.9 ± 9.36 nm for MSN1, MSN2, MSN3, MSN4, and MSN5, respectively) assessed using the dynamic light scattering (DLS) technique. The negative values of zeta potential indicated high surface stability of the formed MSNs. N2-isotherm revealed that the pore volume of MSNs decreased with increase in the size of MSNs. In vitro drug release showed that all MSNs exhibited high encapsulation efficiency of doxorubicin. The encapsulation efficiency were 92.2%, 82.8%, 72.2%, 72.1% and 71.9%for MSN1, MSN2, MSN3, MSN4, and MSN5, respectively. MSN1 and MSN2, with sizes of 64.18 ± 6.8 and 90.46 ± 7.1 nm, pore volume of 0.89 and 0.356 cc/g, encapsulation efficiency of 92.2% and 82.8%, and adequate drug release profiles, were probably the best choices for a drug carrier in drug delivery applications.


Introduction
Nanoparticles attracted incredible interest due to the gap between bulk materials and atomic or sub-atomic structures. Particles at the nanoscale have different chemical, physical, optical and mechanical properties. So, they have vastapplications in drug delivery as nano-drug carriers.Among these are,eliminating drug overdose, increasing solubility, decreasing immunogenicity, reducing side effects, providingspecific reach of the drug to the target cell [1],and beating the pharmacokinetics impediments [2]. This provides advantages that help treat diseases that were previously difficult to treat [3].
Mesoporous silica nanoparticles (MSNs) are important for drug delivery applications [4]. MSNs are spherical nanoparticles consisting of several pores isolated from each other by a solid skeleton [5]. MSNs have distinguished properties, such as a large surface area, uniform and customizable pore size, and large pore volume [5][6][7]. These attractive features make MSNs applicable in many areas, including hyperthermia treatment, diagnostics,catalysis,adsorbents,antireflection coating,sensing, and drug delivery [8][9][10][11][12][13][14][15][16]. Based on these features, MSNs are the most important nanoparticles used as drug carriers in medicate delivery. The vital characteristic This manuscript was written with contributions from all authors.
* Mohamed M. Fathy mfathy@sci.cu.edu.eg for enhancing drug delivery to a target cell is the size of MSNs. The cellular uptake of MSNs is affected by particle size, which affects the interaction of MSNs with the cell membrane [17][18][19][20]. The uptake by target cells increases as nanoparticles decrease [21]. The electrical, magnetic, optical, thermodynamic, and mechanical properties of nanomaterials are size-dependent; therefore, an accurate determination of the size of nanoparticles is essential [22,23]. The most common method to synthesize MSNs is templating [24,25]. It involves synthesizing MSNs using surfactants (pore producing agents) as templates, allowing the synthesis of MSNs with different morphologies, mesostructures, and dimensions by controlling the reaction conditions [26,27]. Researchers have used various strategies to tailor the properties of MSNs with different textural properties. Some studies have used different templating agents to synthesize MSNs, producing MSNs with less than 4 nm [28][29][30][31][32]. Moreover, the effect of the presence and absence of hexanol, as a cosolvent in the reactant solution, on the morphology and mesostructure of MSNswas studied. With hexanol, the sizes of the particles become larger [33]. Some studies investigated the impact of not stirring [34,35] or stirring many times [36,37] on the MSN preparation. Other works studied the effect of xylene, toluene, and trimethylbenzene (TMB) (as different swelling operators), time and temperature of synthesis, different silica sources [tetraethyl orthosilicate (TEOS) versus tetramethylorthosilicate], and silica/surfactant ratio on the synthesis of MSNs [38,39]. Nooney et al. [40] synthesized MSNs using different ratios of TEOS-surfactant under dilute conditions, and their results showed that the synthesized MSNs ranged from 65 to 740 nm. Vazquez et al. [41] examined the impact of distinct molar proportions of NH 3 /TEOS, water/TEOS, and surfactant cetyltrimethyl-ammonium bromide (CTAB) on the morphology, pore size, and surface area of synthesized MSNs.
Different characterization techniques were used to estimate the different properties of the nanoparticles. These properties guide researchers to the possible use of nanoparticles in specific applications. Some of the previous studies combined different techniques to characterize nanoparticles, such as using small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) in the study of oxidesupported platinum nanoparticle solution [42]. Using SAXS, TEM, and DLS in the characterization of shell crosslinked nanoparticles [43],surface-functionalized gold nanoparticles [44], and the formation of silica nanoparticles in different suspensions [45,46] are all reported. Moreover, Sponchiaet al. used different characterization techniques, SAXS, BET, X-ray powder diffraction (XRD), and TEM, to characterize the pore size of MSNs [7]. Pabischet al. combined SAXS, DLS, Brunauer-Emmett-Teller (BET), XRD, and TEM to characterize oxide nanoparticles [47].
This study focuses on preparing and characterizing several MSN formulations using different concentrations of ammonium hydroxide (NH4OH) as a catalyst and studying their applicability in the in vitro delivery of anticancer drug doxorubicin (DOX). The characterization is performed using different biophysical techniques: TEM, DLS, SAXS, WAXS, gas isotherm (N 2 -isotherm), and atomic force microscopy (AFM).
Although most of the previous research were focused on using various strategies for tailoring the properties of MSNs with different textural properties, there is still a lack of reports discussing, in depth, the effect of internal properties of nanoparticles on drug encapsulation and release. This work aimed at fulfilling this gap through employment of various characterization techniques providing deep insight on the structural properties of the investigated MSNs and their relation to drug encapsulation and release. The control of these parameters would significantly promote the usage of MSNs in drug delivery applications.

Preparation of MSNs
Mesoporous silica nanoparticles were synthesized according to Chen et al. [48]. CTAB (0.5 g) was dissolved in 70-ml deionized water, and then 0.7-mg/ml NH 4 OH and 30-ml 2-ethoxyethanol (cosolvent) were added to the solution. After 30 min of stirring at room temperature, 2.5ml of TEOS was added. The white precipitate was collected and washed using ethyl alcohol and deionized water. To remove CTAB residuals, the sample was calcined at 600 °C for 6 h.
To study the impact of ammonia concentration on the physical characteristics of the prepared MSNs, different samples of MSNs were prepared using the same steps at different concentrations of NH 4 OH: 0.7, 1.4, 2.8, 4.2, and 5.6 mg/ml. The samples were named MSN1, MSN2, MSN3, MSN4, and MSN5, respectively.

Characterization
The morphology and average size of MSNs were analyzed using transmission electron microscopy (TEM) (JEM 1230 electron microscope Jeol, Tokyo, Japan). The mean hydrodynamic diameter and the number of surface charges (zeta potential) were determined using the ZetasizerNanoseries (Nano ZS, Malvern Instruments, UK). SAXS was used to analyze the prepared MSNnanoformulations using XPERT PRO -PANalytical -Netherland. The target is a CuK α producing X-rays at a wavelength of λ = 1.54 Å. All powder samples were analyzed at angle 2θ, ranging from 0.1°to 5°(scattering vector (q) ranges from 0.007 to 0.355 1/Å) where q = (4π sin θ / λ) with a step size 2θ = 0.0° at a temperature of 25 °C. WAXS of all MSNs was measured using the same X-ray diffractometer that was used to measure SAXS and LAXS. All powder samples were analyzed at angle 2θ, ranging from 10° to 80° with a step size of 2θ = 0.02° at a temperature of 25 °C. Nitrogenadsorption/desorption isotherms (N 2 -isotherm) of the prepared MSN samples were obtained using Quantachrome Nova Win-Data Acquisition and Reduction for NOVA instruments ©1994-2016, Quantachrome Instruments (version 11.04). The MSNs were degassed at − 200 °C before measurements. The specific surface area of the MSNs was determined using Brunauer-Emmett-Teller (BET) method. Barrett-Joyner-Halenda (BJH) method was used for pore analysis. AFM was used to examine the samples' surface properties (roughness) in a noncontact mode using Wet-SPM9600 (scanning probe microscope) (Shimadzu, Japan). The area of the resultant scanning images was 5 μm × 5 μm and obtained scanning rate was 0.8 Hz/s.

Loading of DOX on MSNs
The samples (40 g each) (MSN1, MSN2, MSN3, MSN4, and MSN5) were dissolved in 3-ml deionized water, and then 1-ml DOX (2 mg/ml) was added. The suspensions were left in a bath shaker for 24 h at 37 °C and 100 rpm. After 24 h, the suspensions were centrifuged at 5000 rpm for 0.5 h. The encapsulation efficiency (EE) was determined using the formula below:

Release of DOX from MSNs
In vitro drug release of DOX from MSNs was assessed. For all MSN samples, the pellet produced by centrifugation in the DOX loading step was resuspended in 5-ml PBS pH 7.4. The suspension was transferred into a dialysis bag suspended in 20-ml PBS in closed tubes. Finally, the tubes were shaken for 48 h at 37 °C. The cumulative release of DOX from MSN samples was determined as the concentration of the released DOX divided by the concentration of DOX in the nanoparticles.

MSN Characterization Using TEM
TEM images (Fig. 1a, b, c, & d) demonstrated that the MSN1, MSN2, MSN3, and MSN4 samples, respectively, were composed of spherical nanoparticles [49], whereas the MSN5 sample (Fig. 1e) [49]. The images showed welldispersed nanoparticles for MSN1, MSN2, and MSN3 samples (Fig. 1a, b, & c). However, MSN4 and MSN5 samples ( Fig. 1d and e) showed an increase in the aggregation of MSNs due to the increased pH. The latter resulted in a strong electrostatic interaction between silica and cationic surfactant (CTAB), as well as fast silica condensation rate and fast assembling and growth of silica with surfactant [50].

MSN Characterization Using DLS
Dynamic light scattering assessment showed an increase in the average hydrodynamic size of MSNs with increasing NH 4 OH, from 0.7 to 5.6 mg/ml (with increased pH) (Fig. 2) Fig. 1d and e). The distribution of the silanol group on the surface of MSNs made their surfaces negatively charged [53,54]. The variation in the values of the ZP is due to the distribution and concentration variations of silanol groups on the surface of the MSNs [55].

X-ray Scattering Characterization
SAXS measurements were performed for all samples. The scattering profiles were recorded at a scattering angle (2θ) ranging from 0.1° to 5.0°. Measured SAXS profiles (scattering intensity versus scattering vector, q) for all prepared samples are shown in Fig. 3. The scattering profiles for MSN1, MSN2, and MSN3 were similar; however, there was an observed shift in the shoulder at large values of scattering vector (from 0.15 to 0.22 Å −1 ) for MSN4 and MSN5 samples, respectively. This shift in the shoulder position could be attributed to the corresponding change in shape (turning slightly oval) and aggregation of nanoparticles as disclosed in the TEM (Fig. 1d and e). The presence of oscillations at high q values (from 0.2 to 0.355 Å −1 ) in all samples was due to the spherical shape of nanoparticles. Moreover, it indicated a degree of monodispersity [56].
WAXS was performed for all samples. The WAXS spectra of samples were plotted (Fig. 4) and showed one characteristic peak at approximately 2θ = 22.5°. WAXS profiles of the investigated samples showed no detectable differences. WAXS could not detect the changes in the internal molecular structure of prepared MSNs.

Gas Isotherm (N 2 -Isotherm) of MSNs
Nitrogen adsorption/desorption on the surface of the nanoparticles were performed to obtain the pore volume and specific surface area of MSNs from the analysis of the isotherm of each sample. Figure 5 shows the isotherm plots for samples MSN1, MSN2, MSN3, MSN4, and MSN5. From the figure, the type of isotherm is IV isotherm for the investigated samples, regardless of increasing in size. The IV isotherm appeared due to the mesoporous nature of the nanoparticles [57,58].
The specif ic sur face areas obtained using Brunauer-Emmett-Teller (BET) method for isotherm showed fluctuation in values. This could be due to the aggregation discussed earlier in the TEM image (Fig. 1) [7]. By the Barrett-Joyner-Halenda (BJH) method, the pore volume is derived from the adsorption branch of isotherms of MSN samples. The volume of pores was found to be 0.897, 0.356, 0.243, 0.180, and 0.142 (cc/g) for MSN1, MSN2, MSN3, MSN4, and MSN5, respectively. The pore volume of nanoparticles decreased with size.

Roughness of Prepared MSNs Using AFM
AFM was conducted to investigate the surface topography of all MSN samples. The 3D images of the surfaces of nanoparticles are shown in Fig. 6a, b, c, d, and e for samples MSN1, MSN2, MSN3, MSN4, and MSN5, respectively. The surface roughness were 2.29 ± 0.993, 1.69 ± 0.448, 1.66 ± 0.471, 1.524 ± 0.43, and 1.79 ± 0.618 μm, respectively. For samples MSN1, MSN2, MSN3, and MSN4, as the size of MSNs increased, the roughness of nanoparticle surfaces decreased, except in sample MSN5, where the roughness increased. The increase in roughness for sample MSN5 can be attributed to the aggregation found in MSN5, as illustrated in the TEM image (Fig. 1e).

Loading of DOX on MSNs
The encapsulation efficiency (EE) of DOX into the investigated MSNs was determined. As the size of nanoparticles increased, the loading efficiency decreased. EEs were 92.2%, 82.8%, 72.2%, 72.1%, and 71.9% for MSN1, MSN2, MSN3, MSN4, and MSN5, respectively. This is due to a decrease in the pore volume of nanoparticles as the size increased (like the data previously produced using the N 2 -isotherm). For samples MSN3, MSN4, and MSN5, the EEs are the same as their nanoparticles had comparable pore volumes.

Release of DOX from MSNs
The synthesized nanoparticles were loaded using DOX to optimize the best preparation conditions for drug delivery applications. The cumulative drug release for all samples is shown in Fig. 7. Sample MSN5 (with size 173.9 ± 9.36 nm, pore volume 0.142 cc/g, roughness 1.79 μm, and smallest EE, 71.9%) showed the highest drug release. This implied that it would not be preferred for drugdelivery applications because drugs cannot be stored in its pores for a long time. Therefore, the drug might be released into the biological system before reaching the target cell.
However, the MSN1 sample (with the largest pore volume 0.897 cc/g, roughness 2.29, EE 92.2%, and smallest size 64 ± 6.8 nm) showed the lowest drug release profile at the assigned time intervals. This indicated that the MSN1 sample released the drug in minimal amounts despite its high EE. The low rate of drug release seemed to be due to the association of the drug and tiny silica pores of MSN1. These features would be favorable, except that the release rate might be insufficient for efficient drug delivery. However, samples MSN2, MSN3, and MSN4 represented a compromise between the high encapsulation and release as in MSN5 and the low encapsulation and release as in MSN1. A choice of these three MSNs for drug delivery purposes would be reliable.
The important characteristic for enhancing drug delivery to target cells is the size of MSNs. The particle size affects the cellular uptake of MSNs as it affects the interaction between MSNs and the cell membrane [17][18][19][20]. When the sizes of nanoparticles are greater than 100 nm each and aggregated, toxicity may increase [29,59] and cause fast uptake of the reticuloendothelial system (RES) [32,60,61]. Therefore, samples MSN3, MSN4, and MSM5 would not be preferred for drug delivery applications because their particle size was more significant than 100 nm (118.98 ± 7.01, 152.7 ± 1.7, and 173.9 ± 9.36 nm, respectively). Moreover, aggregation was reported for MSN4 and MSN5, as shown in TEM images ( Fig. 1d and e).
Small-sized nanoparticles have advantages as nanocarriers for drug delivery applications due to their ability to overcome biological system barriers, showing significant uptake by target cells and biocompatibility [5,[62][63][64]. MSNs less than 100 nm would be the best choice of the investigated formulations as carriers of drugs in drug delivery systems. MSN1 sample with small size (64.18 ± 6.8 nm), large pore volume (0.89 cc/g), and high EE (92.2%) is the best choice for biological applications. MSN2 sample with a small size (90.46 ± 7.1 nm), large pore volume (0.356 cc/g), and high EE (82.8%) is another choice for biological applications. According to the application, MSN1 or MSN2 samples can be used. If an application needs a high drug release rate, MSN2 is the best choice. However, MSN1 is used when the application requires a low drug release rate. The large pore volume has the advantage of easy biodegradation as the thickness of the silica wall becomes thinner with increased pore volume [7,65].

Conclusions
The study of MSNs for drug nanocarriers in drug delivery applications was achieved by preparing different formulations of MSNs. MSN samples were synthesized by varying the concentration of ammonium hydroxide as a catalyst while keeping all other synthetic conditions constant. These MSNs were characterized using different techniques. The preparation of MSNs with different properties (size, porevolume, roughness, and zeta potential) was feasible. It would probably affect their capability as nanocarriers and would allow the use of MSNs in different biological and medical applications. All MSN samples exhibited a high EE of DOX. The drug release profile for all helped determine the best formulation for drug delivery applications. Considering all investigated parameters, we found that the best formulations for drug delivery applications are the MSN1 and MSN2 samples. Data Availability All data generated or analyzed during this study are included in this published article (the raw data will be available in case required them from the authors).

Declarations
Competing Interests 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.

Consent to Participate
Informed consent was obtained from all individual participants included in the study.

Consent for Publication
All authors agreed to publish this study at the silicon journal.

Research Involving Human Participants and/or Animals This study
were not included neither animals nor human.
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