Abstract
This work reports the preparation, characterization, and a drug release study of mesoporous silica nanoparticles (MNPSiO2) functionalized with folic acid (FA) and loaded with Cis-Pt as a targeted release system to kill glioblastoma cancer cells. The MNPSiO2 were synthesized by the Stöber method using hexadecyltrimethylammonium bromide as the templating agent, which was finally removed by calcination at 550°C. The folic acid was chemically anchored to the silica nanoparticles surface by a carbodiimide reaction. Several physicochemical techniques were used for the MNPSiO2 characterization, and a triplicate in vitro Cis-Pt release test was carried out. The release Cis-Pt experimental values were fitted to different theoretical models to find the Cis-Pt release mechanism. The cytotoxicity evaluation of the MNPSiO2 was performed using LN 18 cells (human GBM cells). Homogeneous and well-defined nanoparticles with well-distributed and homogeneous porosity were obtained. The spectroscopic results show the proper functionalization of the mesoporous nanoparticles; besides, MNPSiO2 showed high surface area and large pore size. High correlation coefficients were obtained. Though the best fitted was the Korsmeyer-Peppas kinetic model, the Higuchi model adjusted better to the results obtained for our system. The MNPSiO2-FA were highly biocompatible, and they increased the cytotoxic effect of Cis-Pt loaded in them.
1 Introduction
Over the last few decades, mesoporous silica nanoparticles (MNPSiO2) have been a focus of interest due to their morphological features such as high surface area (over 700 m2/g), large pore volume (higher than 1cc/g), adjustable pore size (1.6–10 nm), superior surface properties, functionalization surfaces (internal and external), modifiable morphology (particle shape and size), and biocompatibility [1,2,3]. Thereby, MNPSiO2 have significant applications in many research fields such as catalysis, adsorption, separation, sensing, and drug delivery [4, 5]. Mesoporous materials are usually prepared using self-assembled surfactant molecules as templates around which the silica precursors condense [4]. Then, the removal of the template carried out through heat treatment leads to a porous material. Size, morphology, pore size, and pore structure of MNPSiO2 can be designed through the manipulation of the reaction parameters such as temperature, pH, surfactant concentration, and silica precursors, among others [6, 7].
There are mainly four methods to prepare MNPSiO2, namely template-directed method, sol-gel method, microwave-assisted technique, and chemical etching technique [1, 8,9,10,11]. The sol-gel procedure is widely used to develop inorganic oxide networks from the hydrolysis and condensation of inorganic precursors (metal salts or metal alkoxides) [1]. The Stöber method, which is based on sol-gel chemistry, is used to prepare uniform colloidal silica spheres [4, 6, 12, 13]. The Stöber procedure involves the hydrolysis and condensation of tetraethyl orthosilicate (TEOS) in alcohols used as solvents (ethanol, methanol, etc.) in the presence of water and a base-catalyst (ammonia, ammonium hydroxide, etc.) at room temperature. It is suggested that the uniform growth of the particles is based on the base-catalyzed reaction, which causes a rapid formation of nuclei due to the union of the constituent solutes in a kinetically controlled way. The Stöber method is the most common procedure for the synthesis of silica nanoparticles because it allows control of particle size and pore size distribution. MNPSiO2 can be synthesized with different structural features, including particle sizes, shapes, surface areas, nanoscale pore sizes, pore volumes, and surface-modifying groups on their internal or external surfaces. Because of their exceptional textural and their biocompatibility properties, MNPSiO2 have potential biomedical applications in drug delivery for cancer treatment, infectious treatment, and different bone diseases [1, 3, 5, 14,15,16,17,18].
The surface of MNPSiO2 can be modified with cell-specific moieties such as organic molecules, peptides, and antibodies to achieve cell type or tissue specificity [3, 14,15,16, 19]. Specifically, the functionalization of the surface of MNPSiO2 with ligands allows to interact selectively with specific cellular receptors overexpressed in tumor cells [20]. Some of the targeting ligands that can be linked to the MNPSiO2 surface are transferrin, epidermal growth factor, folic acid, concanavalin A, and vascular endothelial growth factor, among others [20,21,22].
The connection of nanotechnology and medicine, specifically in drug delivery for drug targeting, is an essential subject in the biomedical knowledge [23, 24]. This is particularly true for cancer therapies, where the loss of the antitumor drug before it reaches targets results in adverse side effects and limits the drug's effectiveness due to the unspecific drug action on healthy tissue. The functionalization of MNPSiO2 with folate groups gives the nanoparticles the ability to release drugs selectively through their link to receptors on the cell surface [19].
Folate receptors are glycosylphosphatidylinositol proteins, which are expressed in many human cancer cells, where the level of folate receptors expression is related to the stage of cancer [25]. Folate receptors have a high affinity to the FA and its derivatives and mediate the delivery of the 5-methyltetrahydrofolate as physiological folate to the cell. However, non-cancerous cells possess a deficient level of folate receptors, while cancer cells bind to the FA group. Folate receptacles are overexpressed in different types of human cancer cells, such as breast, colorectal, ovarian, brain, epithelial, and lung cancer. As its expression is limited in healthy cells, it could be used as a biomarker for cancer [25, 26].
On the other hand, Glioblastoma (GB) is the most common and most lethal primary brain tumor in adult patients with an average patient survival of 12–15 months. The incidence is in the range of 2–3 cases per 100,000 per year [27] – current treatments include maximal safe re-section, followed by radiotherapy with concomitant and adjuvant temozolomide. Surgical resection is not curative for glioblastoma, and even after gross total resection of the apparent tumor followed by radiotherapy with concurrent and adjuvant chemotherapy, tumor progression occurs [28, 29]. Cisplatin (Cis-Pt) is used as a first-line chemotherapy drug used against various cancers, including glioblastomas, metastatic melanomas, peritoneal, and pleural mesotheliomas [30]. The antitumor properties of Cis-Pt are attributed to the kinetics of its chloride ligand displacement reactions. Cis-Pt interacts with guanine and adenine N7 atoms located in the DNA major groove, leading to DNA bending and interfering with its replication and transcription, as well as other nuclear functions, thus avoiding cancer cell proliferation and tumor growth. However, the intravenous Cis-Pt administration can lead to nephrotoxicity, bone marrow toxicity, intractable vomiting, peripheral neuropathy, deafness, seizures, and blindness. Although Cis-Pt is toxic, it is used successfully in the clinical field.
In a previous work [31] we reported the synthesis of silica-based nanoparticles that served as vehicles for the release of Cis-Pt. In continuation of that previous work, in this present study we are reporting the functionalization of MNPSiO2 with folic acid in order to release Cis-Pt molecules in a targeted way. The standard treatment for GBM consists of surgical removal of as much of the tumor as possible. However, the removal of the tumor is incomplete. Our proposal consists of placing the MNPSiO2-FA/Cis-Pt systems in the cavity where the cancerous tumor was removed, so that the Cis-Pt reaches specifically the remaining cancer cells and eliminates them. Thus, the side effects are reduced, and crossing the blood-brain barrier is avoided, which hinders drugs reaching the brain.
2 Experimental
2.1 Sample preparation
Chemical substances
Sodium hydroxide (NaOH) (SIGMA-ALDRICH), Hexadecyltrimethylammonium bromide (CTAB) (SIGMA), Tetraethyl orthosilicate (TEOS) (ALDRICH, 98%), Dimethyl sulfoxide (DMSO) (SIGMA), Folic acid (AF) (SIGMA, 97%), diclohexylcarbodiimide (DCC) (SIGMA-ALDRICH, 99%), (3-aminopropyl)triethoxysilane (APTES) (SIGMA-ALDRICH), Toluene (SIGMA-ALDRICH), N-hydroxysuccinimide (N-HSA) (ALDRICH, 99%), Cis-Pt (ALDRICH, 99.9%).
MNPSiO2
To prepare this sample we followed the procedure reported by [31]. Brief: 7 mmol of NaOH and 2.74 mmol of CTAB were mixed in 26.6 mol of deionized water, then temperature was raised to 70°C. Subsequently, 0.022 mol of Tetraethyl orthosilicate (TEOS) was slowly added. Once the addition of TEOS was completed, the final mixture was left with moderate agitation for 2h at 70°C. The supernatant was removed from the precipitate, and it was washed with a water/methanol (50:50 v/v) mixture. The sample was dried at 60°C for 24 hours. Finally, the sample was heated at 550°C for 4h in a conventional muffle.
MNPSiO2-FA
A molar ratio of FA:MNPSiO2 of 0.26:1.0 was used, and for the functionalization we followed the procedure reported in [32]. 0.126 mol of DMSO, 0.66mmol of FA, 0.76 mmol of N-HSA, 0.72 mmol of DCC, and 0.84 mmol of APTES were mixed. The entire mixture was kept under stirring for 6h. After this time, 12 ml of toluene and 150mg of MNPSiO2 were mixed. After 6h, the nanoparticles in toluene were added to the mixture containing folic acid. The final mixture was left under stirring at room temperature for 72 h. Finally, the mixture was filtered and the solid was washed with toluene, DMSO, water, and acetone. The resulting powder was dried at 60°C by 12 h.
MNPSiO2-FA/Cis-Pt
A solution of 10 mg of Cis-Pt in 20 ml of water (10% Cis-Pt by weight of MNPSiO2-FA) was added to 100 mg of MNPSiO2-FA. The solution was stirred at room temperature for 24 h, and the water was removed and left to dry at 20°C for three days. The weight percentage of Cis-Pt in MNPSiO2-FA was 1:10.
2.2 Characterization
Scanning Electron Microscopy (SEM)
The SEM images of the samples were obtained using a Quanta 3D FEG Microscope.
Atomic Force Microscopy (AFM)
The AFM-images were acquired in a transmission electron microscope JEM2100 (VEECO) with filament LaB6. This apparatus is an opto-mechanical microscope that allows obtaining topographic images.
High-Resolution Transmission Electron Microscopy (HR-TEM)
A high-resolution transmission electron microscopy analysis was performed on a JEM 2100 microscope with voltages from 80 to 200 kV filament LaB6. For application, the samples were previously suspended in an Isopropanol solution and taken to a sonicator to increase the dispersion. Then samples were placed on a copper plate and introduced into the equipment for reading.
Fourier Transform Infrared spectroscopy (FTIR)
In this technique the KBr method was used, 5mg of the solid samples were mixed and ground with 95 mg of potassium bromide (KBr). The powder was then pressed into a translucent tablet, which was placed in an Affinity-1 Shimadzu spectrophotometer for measurement tasks. The range of analysis was 4000 to 400 cm−1 wavenumbers.
X-Ray Diffraction (XRD)
The samples were characterized by X-ray diffraction (XRD) in Siemens D-500 equipment. The signal strength was measured by scanning in steps in the range of 4 and 80 degrees with a step of 0.03° and a measurement time of 2 s per point.
N2 adsorption-desorption
The specific surface area, average pore diameter, and pore volume were determined from the N2 adsorption–desorption isotherms. The samples were previously thermally treated at 250°C with a vacuum during 12 h. The N2 gas adsorption-desorption was carried out at 77.3 K using the BelsorpII equipment. The specific surface areas were determined using the Brunauer–Emmet-Teller (BET) method, while the average pore size and pore volume were determined from the desorption isotherms using the Barret-Joyner-Halenda method.
2.3 Cis-Pt release test
Simulated cerebrospinal fluid (SCF)
SCF was used as a release medium, and it was prepared according to the information reported by [33].
Calibration curve
To build the calibration curve an initial solution of 10 mg Cis-Pt in 100 ml SCF was made. From this solution, 0.5, 1, 2, 4, and 6 ml aliquots were taken and calibrated to 25 ml each with SCF. Subsequently, a 3 ml aliquot was taken from each solution, and its absorbencies were measured in UV-Vis. From the results, a graph of Cis-Pt concentration versus maximum absorbance at 203 nm was made. Linear regression was performed on the resulting curve, and the molar extinction coefficient was determined.
Drug release test
The profile of the in-situ release kinetics of Cis-Pt was performed according to the procedure reported by [31], using artificial cerebrospinal fluid as the medium and evaluated by UV-Vis by measuring absorbance at 203 nm, which corresponded to Cis-Pt signal. The MNPSiO2-FA/Cis-Pt powder was slightly pressed to form three small cylinders (5–8 mg) which were individually added to 25 mL of SCF (pH = 7.3) maintaining the release medium with moderate stirring at 25°C for 8 h. At predetermined times, an aliquot of 3 mL was removed from the release medium for its measurement at 203 nm. After being measured, the aliquot was returned to the release medium. The Cis-Pt release procedure was made by triplicate.
2.4 Cytotoxicity evaluation
Cell Culture
LN 18 cells (human GBM cell line) [34] were obtained from American Type Culture Collection (ATCC# CRL-2610), and these were maintained in DMEM medium supplemented with 5% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a humidified 5% CO2 atmosphere. The GBM cells were harvested by trypsinization and plated at 1×104 cell/well in 96-well or 1×105 in 35 mm culture plates. The procedure of cell culture was made according to the one described in [35].
Light microscopy
The morphological changes in LN18 cells by the presence of MNPSiO2, MNPSiO2-FA, MNPSiO2-FA/Cis-Pt, and Cis-Pt (100 μg/mL) or by the vehicle (DMSO 1%) in the culture medium were recorded after 48 h, using a light microscope (CKX41, Olympus) at a magnification of 10×.
Tetrazolium reduction assay
After 24h cell growth, the medium was removed and replaced by 100μL fresh medium in the presence of MNPSiO2, MNPSiO2-FA, MNPSiO2-FA/Cis-Pt, and Cis-Pt at different concentrations (10–1000 μg/mL) by 48 h. Later, the medium was removed and washed three times with PBS; and incubated with 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (5mg/mL) at 37°C for 3 h to allow viable cells to convert tetrazolium salts into formazan crystals. The supernatant was removed, and the crystals were dissolved in 100 μL of isopropyl alcohol (acidified 0.04 HCl). Finally, the absorbance was read at 570 nm using a multi-well plate reader (Synergy HT, Biotek). The half inhibitory concentration IC50 values were determined from in vitro dose-response curve. The data were analyzed using GraphPad Prism 8.0 and expressed as mean ± standard deviation. The cytotoxicity was determined using cell viability = (OD of the experiment samples/OD of the control) × 100. MTT assay was based in published protocol [36] but with some modifications.
3 Results and discussion
3.1 Scanning Electron Microscopy and Atomic Force Microscopy
Figure 1 reports the images obtained by atomic force microscopy (a) and scanning electron microscopy (b) of the MNPSiO2 sample. Both figures show the well-defined and homogeneous spherical obtained nanoparticles with a size of approximately 100 nm.
(a) AFM-Image, and (b) SEM-Image of the MNPSiO2 sample took at ×5,000 of magnification with a voltage of 5.0 kV. The bar is equivalent to 1 μm.
3.2 High-Resolution Transmission Electron Microscopy
Figure 2 corresponds to the HR-TEM images of MNPSiO2, MNPSiO2-FA, and MNPSiO2-FA/Cis-Pt samples. The TEM images show features of silica mesoporous nanoparticles, consisting of highly ordered one-dimensional channels in hexagonally packed meso-structures. Nanoparticles smaller than 100 nm in size in the MNPSiO2 sample were found (Figure 2a–c). The porosity confirms that the mesoporous structure of MCM-41 could also be obtained (Figure 2b). The pores had sizes of 3–5 nm (Figure 2c–d). The morphological structure of MNPSiO2 did not change when it was functionalized with folic acid, and when Cis-Pt was loaded (Figures 2e and Figure 2f, respectively).
HR-TEM photographs of (a–d) MNPSiO2, (e) MNPSiO2-FA, and (f) MNPSiO2-FA/Cis-Pt samples.
3.3 FTIR spectroscopy
Figure 3 shows the FT-IR spectra of the MNPSiO2, MNPSiO2-FA, and MNPSiO2-FA/Cis-Pt samples. All samples present the characteristic vibrational modes bands of SiO2, in agreement with the previous literature [31]. The distinctive bands of the MNPSiO2 are observed in the 1020–1110 cm−1 interval (Figure 3c). The band at 1089 cm−1 belongs to the asymmetric stretching vibration of Si-O-Si bonds. The small bands found at 966 cm−1 are related to bending vibrations from Si-O-H bonds. The band at 802 cm−1 is associated with Si-O bending vibrations. Besides, another band was observed at 3323 cm−1, which corresponds to hydroxyl groups from water molecules adsorbed on the MNPSiO2 surface.
FTIR spectra of (a) MNPSiO2-FA/Cis-Pt, (b) MNPSiO2-FA, and (c) MNPSiO2 samples.
The MNPSiO2-FA spectrum shows important bands that allow the identification of the folic acid linked to the surface of MNPSiO2 (Figure 3b). The derived band from this spectrum agrees with our recently reported literature [32]. In brief, the band located at 3319 cm−1 corresponds to the N-H stretching vibration from the pteridine ring. The peak at 2937 cm−1 is attributed to vibrational C-H of CH2 groups from the pteridine ring and glutamic acid group present in the FA. The bands at 1690 and 1608 cm−1 belong to C=O and C=C vibrations in the FA, while the peak at 1526 cm−1 confirmed the formation of an N-MNPSiO2-FA bond during the functionalization of MNPSiO2 with the folate [37].
The MNPSiO2-FA/Cis-Pt spectrum shows the bands related to MNPSiO2 and Folic acid attached to the silica nanoparticles. However, a stretching vibration of the N-H group, from Cis-Pt at 3305 cm−1, is seen (Figure 3c), while the signals at 1700–1320 cm−1 associated with H-N-H asymmetric and symmetric bending vibrations from Cis-Pt overlap with the signs of folic acid and silica.
3.4 X-Ray diffraction
Figure 4 displays the X-Ray diffraction partners of the MNPSiO2, MNPSiO2-FA/Cis-Pt, and Cis-Pt, samples. The MNPSiO2 diffractogram exhibits the characteristic broad X-ray diffraction curve of the amorphous morphology of SiO2. In addition to the broad silica peak at 10–30 degrees observed in the MNPSiO2-FA/Cis-Pt diffractogram, other peaks were seen at 2θ = 7, 14, 16, and 173 degrees, corresponding to the folic acid, which agrees with the data previously reported by [38]. In contrast, the Cis-Pt diffraction pattern contains several thin peaks due to the platinum atom in Cis-Pt. The XRD peaks hat corresponded to Cis-Pt did not appear when Cis-Pt was loaded on MNPSiO2-FA nanoparticles. This result agrees with our previously reported work [31], suggesting that Cis-Pt molecules are dispersed totally into MNPSiO2-FA or that the detection levels of the equipment are not enough to detect the low amount of platinum.
XRD partners of (a) Cis-Pt, (b) MNPSiO2-FA/Cis-Pt, and (C) MNPSiO2 samples. *FA (folic acid).
3.5 N2 adsorption-desorption isotherm
The Nitrogen adsorption-desorption isotherm of the MNPSiO2 sample shows a characteristic type IV isotherm (Figure 5), indicating a uniform mesopore architecture. No hysteresis loop is observed in the sample. The surface area and pore volume values were calculated to be as high as about 1011 m2/g and 1.10 cc/g, respectively. Moreover, the sample has an average pore size of about 4.35 nm, a value consistent with the observed by HR-TEM and pore size distribution (insert in Figure 5), which indicate the existence of uniform mesopores in the MNPSiO2 sample.
Nitrogen adsorption-desorption isotherms of the MNPSiO2 sample. The insert corresponds to its pore size distribution.
3.6 Cis-Pt releases tests
Figure 6a reports the calibration curve obtained by plotting five different known increasing concentrations of Cis-Pt versus the absorbance at 203 nm obtained from the UV spectra shown in the insert. Simulated cerebrospinal fluid (SCF) was the aqueous medium to get solutions with different Cis-Pt concentrations. The Lambert-Beer law was used to determine the molar extinction coefficient of Cis-Pt, as shown in Figure 6a.
(a) Calibration curve constructed from the absorbance at λ=203 nm of the spectra shown in the insert vs. concentration of Cis-Pt, (b) Triplicate in vitro release Cis-Pt profiles, insert shows the characteristic spectra of Cis-Pt obtained during the tests, and (c) Averaged Cis-Pt release profile.
The in vitro Cis-Pt release test was made by triplicate using SCF as the release medium, and Cis-Pt concentrations were obtained from UV-Vis spectroscopy by measuring absorbance at 203 nm (Cis-Pt signal) at each predetermined time.
The cumulative release of Cis-Pt from the MNPSiO2 sample was calculated according to the following equation 1 [39].
where Mt is the Cis-Pt mass released from the MNPSiO2-FA samples at time t, and Mα is the estimated Cis-Pt mass loaded in the MNPSiO2-FA samples.
The experimental Cis-Pt release profiles were obtained plotting the cumulative release (%) values vs. time, as seen in Figure 6b. The insert in Figure 6b shows the spectra taken as a function of time during the release test, where the features Cis-Pt signal can be observed. Figure 6c shows the average Cis-Pt release profile calculated from the experimental results of Figure 6b. A sustained Cis-Pt release as a function of time can be observed. 60% of the Cis-Pt was released approximately after 7h. The primary release mechanism from the mesoporous silica nanoparticles is mainly through a diffusion process, which depends on the porosity properties [31]. In this case, there is no initial release burst due to the large BET surface area, well ordered pores, uniform pore diameter, and high total pore volume of the pure silica nanoparticles, as confirmed by the N2 adsorption-desorption isotherm analysis. A large surface area was suitable to a high Cis-Pt dispersion, while a large pore volume allowed the Cis-Pt to be well housed mostly inside the pores and almost nothing on the nanoparticles’ outer surface. Therefore, the high pores’ homogeneity in the nanoparticles (see Figure 2) allows a Cis-Pt diffusion in a sustained way. Besides, the pore size distribution (insert in Figure 5) shows a narrow pore size distribution, which corroborates what was observed by TEM images. The silica nanoparticles have well-ordered pores that allow a homogeneous suitable diffusion of Cis-Pt from these pores and not from the nanoparticles’ external surface, as it usually happens with other mesoporous materials, that exhibit an initial drug burst.
3.6.1 Theoretical models applied to the experimental Cis-Pt release profiles
To explain the theoretical Cis-Pt release mechanism from the MNPSiO2, the experimental release values were fitted to various kinetic mathematical models: Zero-order, First-order, Higuchi, Korsmeyer -Peppas, and Hixson-Crowell [40,41,42].
(1) Zero-order model
Zero-order kinetics defines a process of constant drug release from a drug delivery system, and the drug level remains constant throughout the delivery process. Drug release from the dosage form can be represented by the (2) and (3) equations.
where Ct is the amount of drug released at time t, C0 is the initial concentration of drug at time t = 0, K0 is the zero-order rate constant.
(2) First-order model
The first-order model is suitable for porous drug carriers with insolubility in water. The release of drug which follows first-order kinetics is represented by the (4) equation.
where, K1 is the first-order rate constant, expressed in time−1 or per hour following linear kinetics. After rearranging and integrating the equation, the following (5) equation remains.
(3) Higuchi model
The Higuchi model concentrates on the drug release from a matrix. The release of a drug from a drug delivery system involves both dissolution and diffusion. Several mathematical equation models describe drug dissolution and release from a drug delivery system. Higuchi equation is a prominent kinetic equation, which evidences drug dissolution studies that are recognized as an important element in drug delivery systems development. Today the Higuchi equation is considered one of the most widely used and the most well-known controlled-release equation. The classical basic Higuchi equation is represented by (6).
where Q is the cumulative amount of drug released in time t per unit area, C0 is the initial drug concentration, CS is the drug solubility in the matrix, and D is the diffusion coefficient of the drug molecule in the matrix.
The simplified Higuchi equation can be represented in the following form (7).
where KH is the Higuchi dissolution constant.
(4) Korsmeyer-Peppas model
To describe the drug release from a polymeric system, Korsmeyer and Peppas use a simple relationship represented by the (8) equation:
where Mt/M∞ is a fraction of drug released at time t.
where Mt is the amount of drug released in time t, M∞ is the amount of drug released after time ∞, n is the diffusional exponent or drug release exponent, and Kkp is the Korsmeyer release rate constant.
In Korsmeyer-Peppas model ‘n’ is the diffusion exponent that explains the drug release mechanism. According to this model, drug release takes place by Fickian diffusion if it is n < 0.43, by Anomalous (non-Fickian) diffusion if it is 0.43 < n < 0.89, by case-II transport if it is n = 0.89 and by Supercase-II transport, if it is n > 0.89.
(5) Hixson-Crowell model
The Hixson-Crowell cube root law describes the release from systems where there is a change in surface area and diameter of particles or tablets. Therefore, particles of regular area are proportional to the cube root of its volume. Crowell established a relationship between drug release and time, which can be represented by the (10) equation.
where W0 is the initial amount of drug in the pharmaceutical dosage form (Amount of drug remaining at time 0); Wt is the remaining amount of drug in the pharmaceutical dosage form at time t; KHC is the Hixson-Crowell constant describing surface volume relation.
Figure 7 shows the fitted experimental values to the different theoretical models, and table 1 summarizes the correlation coefficient (R2) obtained for each model as well as the n value. The correlation coefficient R2 is the criteria commonly used to select the model that most reliably describes the general release drug mechanism. In a reliable prediction model, the R2 value should be as close to 1 as possible. According to the results in Table 1, the First-order, Higuchi, and Korsmeyer-Peppas models showed an R2 greater than 0.95, while the R2 values for the zero-order and Hixson-Crowell model were 0.933 and 0.780, respectively. Therefore, the Cis-Pt release mechanism may follow the rules of any of the three models. The first-order model explains the drug release from the matrix where the drug release rate is concentration-dependent [43]. The Higuchi model describes the release of the drug from the insoluble matrix as a square root of time-dependent process based on Fickian diffusion. The Korsmeyer model derives a simple mathematical relationship that describes the drug release from a polymeric system [43]. As shown in Table 1, the value of the release exponent (n) was 1.0, indicating the presence of a Super Case II, where drug transport type is controlled mainly by the swelling of the drug-loaded systems, and this pattern regularly exhibited a longer period of increased swelling than the period of relaxing [44]. However, this model cannot be used for silica, since this one is not a polymeric matrix that can suffer swelling processes. In our particular case, the Higuchi model effectively describes the cis-Pt release mechanism from MNPSiO2.
Mathematical Models fitted to experimental kinetic release of Cis-Pt. SQRT refers to square root, and CBR to cube root.
Correlation Coefficients (R2) for linear relationship from different mathematical models of MNPSiO2-FA/Cis-Pt sample.
| Mathematical model | R2 |
|---|---|
| Zero-order | 0.93357 |
| First-order | 0.9773 |
| Higuchi | 0.98927 |
| Korsmeyer-Peppas | 1.0, n=1.0 |
| Hixson-Crowell | 0.78096 |
Some postulates of the Higuchi model are [45]:
The initial drug concentration in the matrix must be higher than its solubility in the medium.
The mathematical analysis is based on one-dimensional diffusion.
The drug is considered in a molecularly dispersed state with particles much smaller in diameter than the matrix thickness.
The dissolution of the matrix carrier is negligible.
The drug diffusibility is constant.
These postulates are consistent with the release profile obtained from our Cis-Pt/MNPSiO2 system. Nanoparticles containing Cis-Pt are placed in contact with the liquid medium; the fluid enters through the particle pores; it reaches the Cis-Pt and slowly dissolves it, thus allowing the drug to disperse in the fluid phase [45].
3.7 Cytotoxic effects of MNPs over cell viability
The effects of MNPSiO2, MNPSiO2-FA, MNPSiO2-FA/Cis-Pt, and Cis-Pt on the GBM cancer cells are shown in Figure 8; LN18 cells morphology can be observed (Figure 8a). The presence of MNPSiO2-FA/Cis-Pt and Cis-Pt (100 μg/mL, 48 h) visibly affected cell morphology. With MNPSiO2-FA/Cis-Pt the cell morphology changed slightly, and some cells began to die. Besides, the cell number decreased, and Cis-Pt induced cells to lose their structure and present round shapes. Cytotoxic effects of mesoporous nanoparticles on cell viability was evaluated (Figure 8b). MNPSiO2 showed cytotoxicity only at higher concentrations (100μg/mL), but when the nanoparticles were coupled to folic acid (MNPSiO2-FA), the effect was similar to control cells in all concentrations probed. The MNPSiO2-FA/Cis-Pt and Cis-Pt affected the cells in a dose-dependent manner, and the magnitude of effects varied depending on the doses. This was better observed in a log graph (Figure 8c). The IC50 values were lower for Cis-Pt exposure than to MNPSiO2-FA/Cis-Pt, 78.6 μg/mL, and 149 μg/mL, respectively.
Cytotoxic effects of MNPs over cell viability: (a) LM photographs of LN18 cells. (b) Dose-response of mesoporous nanoparticles, (c) Dose-dependent curves of Cis-Pt (squares), and MNPSiO2-FA/Cis-Pt (circles).
4 Conclusions
MNPSiO2 with well-ordered pores and homogeneous morphology were successfully synthesized and functionalized with folic acid. The SEM, AFM, and TEM results show uniform spherical nanoparticles with sizes less than 100 nm. The infrared spectroscopic results revealed the successful functionalization of the MNPSiO2 with folic acid. Large surface areas (1011 m2/g), mesopores (4.35 nm), and high pore volume (1.10 cc/g) to MNPSiO2 were found. These properties were influential in obtaining a sustained Cis-Pt release profile over time. The drug was released in a homogeneous way from the silica pores, without presenting an initial burst release. When the experimental release results were fitted to the mathematical model, high correlation coefficients (r2) were obtained. The best fitted model was the Korsmeyer-Peppas kinetic model (R2 = 1.0), indicating that a super case-II transport controls the Cis-Pt release mechanism. However, the model that best fitted our system was Higuchi's. The theoretical release mechanism of Cis-Pt consists of the initial leaching of the drug into the fluid medium that enters the matrix phase through the pores. Then, the Cis-Pt slowly dissolves into the fluid phase and is released by diffusion through the solvent-filled pores. The MNPSiO2 functionalized with folic acid were highly bio-compatible. MNPSiO2-FA loaded with cis-Pt increased the cytotoxic effect of the drug, thus MNPSiO2-FA/Cis-Pt may have an essential role in GBM cancer treatment, but further in vivo studies using animal models will be necessary for elucidating the mechanism of their action in cancer cells.
Acknowledgement
The authors thank the National Institute of Neurology and Neurosurgery for the facilities provided for this article.
Authors Contribution: Emma Ortiz-Islas developed the project, was in charge of supervising the activities and writing the article.
Anahí Sosa-Arróniz executed the experimental part on the synthesis of the nanoparticles as well as the obtaining of the infrared spectra and the adsorption-desorption of nitrogen.
Ma Elena Manríquez-Ramírez obtained and interpreted the high-resolution transmission electronic micrographs, atomic force images, and electron scanning figures.
C. Ekaterina Rodríguez-Pérez developed and supervised the in vitro biological tests using glioblastoma cells.
Francisco Tzompantzi supervised and analyzed the infrared and X-ray diffraction spectra.
Juan Manuel Padilla supervised the activities of student Anahi Sosa and helped write the article.
Conflict of Interests: Authors state no conflict of interest
Data availability statement: In this article, there is no data available
References
[1] Tang, F., L. Li, and D. Chen. Mesoporous silica nanoparticles: synthesis, biocompatibility and drug delivery. Advanced materials, Vol. 24, No. 12, 2012, pp. 1504–1534.10.1002/adma.201104763Search in Google Scholar PubMed
[2] Asefa, T., and Z. Tao. Biocompatibility of mesoporous silica nanoparticles. Chemical Research in Toxicology, Vol. 25, No. 11, 2012, pp. 2265–2284.10.1021/tx300166uSearch in Google Scholar PubMed
[3] Thi, T., V.D. Cao, T.N.Q. Nguyen, D.T. Hoang, V.C. Ngo, and D.H. Nguyen. Functionalized mesoporous silica nanoparticles and biomedical applications. Materials Science & Engineering C, Vol. 99, 2019, pp. 631–656.10.1016/j.msec.2019.01.129Search in Google Scholar PubMed
[4] Wu, S.H., C.Y. Mou, and H.P. Lin. Synthesis of mesoporous silica nanoparticles. Chemical Society Reviews, Vol. 42, No. 9, 2013, pp. 3862–3875.10.1039/c3cs35405aSearch in Google Scholar PubMed
[5] Manzano, M., and M. Vallet-Regí. Mesoporous silica nanoparticles in nanomedicine applications. Journal of Materials Science: Materials in Medicine, Vol. 29, No. 5, 2018, pp. 1–14.10.1007/s10856-018-6069-xSearch in Google Scholar PubMed
[6] Shimura, N., and M. Ogawa. Preparation of surfactant templated nanoporous silica spherical particles by the Stöber method. Effect of solvent composition on the particle size. Journal of Materials Science, Vol. 42, No. 14, 2007, pp. 5299–5306.10.1007/s10853-007-1771-ySearch in Google Scholar
[7] Sayani, N., K. Debtosh, and N. Milan Kanti. Synthesis of meso-porous Stöber silica nanoparticles: the effect of secondary and tertiary alkanolamines. Journal of Sol-Gel Science and Technology, Vol. 72, 2014, pp. 49–55.10.1007/s10971-014-3420-7Search in Google Scholar
[8] Dement’eva, O.V., I.N. Senchikhin, M.E. Kartseva, V.A. Ogarev, A.V. Zaitseva, N.N. Matushkina, et al. A new method for loading mesoporous silica nanoparticles with drugs: sol-gel synthesis using drug micelles as a template. Colloid Journal, Vol. 78, 2016, pp. 586–595.10.1134/S1061933X16050045Search in Google Scholar
[9] Tripathy, S.K., A. Mishra, S.K. Jha, R. Wahab, and A.A. Al-Khedhairy. Microwave assisted hydrothermal synthesis of mesoporous SnO2 nanoparticles for ethanol sensing and degradation. Journal of Materials Science: Materials in Electronics, Vol. 24, 2013, pp. 2082–2090.10.1007/s10854-013-1062-0Search in Google Scholar
[10] Li, M., C. Zhang, X.L. Yang, and H.B. Xu. Microfluidization-assisted synthesis of hollow mesoporous silica nanoparticles. Journal of Sol-Gel Science and Technology, Vol. 67, No. 3, 2013, pp. 501–506.10.1007/s10971-013-3107-5Search in Google Scholar
[11] Xin, D., G. Lei, F. Yuqin, L. Dan, and L. Changli. Non-surfactant template-directed synthesis of SiO2-polyimide hybrid self-standing films with ordered mesoporous structure. Polymers for Advanced Technologies, Vol. 22, No. 12, 2011, pp. 2424–2429.10.1002/pat.1779Search in Google Scholar
[12] Wang, X., Y. Zhang, W. Luo, A.A. Elzatahry, X. Cheng, A. Alghamdi, et al. Synthesis of ordered mesoporous silica with tunable morphologies and pore sizes via a nonpolar solvent-assisted Stöber method. Chemistry of Materials, Vol. 28, No. 7, 2016, pp. 2356–2362.10.1021/acs.chemmater.6b00499Search in Google Scholar
[13] Li W., and D. Zhao. Extension of the Stöber method to construct mesoporous SiO2 and TiO2 shells for uniform multifunctional core-shell structures. Advanced Materials, Vol. 25, No. 1, 2013, pp. 142–149.10.1002/adma.201203547Search in Google Scholar PubMed
[14] Vivero-Escoto, J.L., I.I. Slowing, B.G. Trewyn, and V.S.-Y. Lin. Mesoporous silica nanoparticles for intracellular controlled drug delivery. Small, Vol. 6, No. 18, 2010, pp. 1952–1967.10.1002/smll.200901789Search in Google Scholar PubMed
[15] Jafari, S., H. Derakhshankhah, L. Alaei, A. Fattahi, B.S. Varnamkhasti, and A.A. Saboury. Mesoporous silica nanoparticles for therapeutic/diagnostic applications. Biomedicine & Pharmacotherapy, Vol. 109, 2019, pp. 1100–1111.10.1016/j.biopha.2018.10.167Search in Google Scholar PubMed
[16] Wang, Y., Q. Zhao, N. Han, L. Bai, J. Li, J. Liu et al. Mesoporous silica nanoparticles in drug delivery and biomedical applications. Nanomedicine: Nanotechnology, Biology, and Medicine, Vol. 11, No. 2, 2015, pp. 313–327.10.1016/j.nano.2014.09.014Search in Google Scholar PubMed
[17] Izquierdo-Barba, I., M. Colilla, and M. Vallet-Regil. Nanostructured mesoporous silicas for bone tissue regeneration. Journal of Nanomaterials, Vol. 2008, No. 2, 2008, pp.1–14.10.1155/2008/106970Search in Google Scholar
[18] Wang, Y., and H. Gu. Core-shell-type magnetic mesoporous silica nanocomposites for bioimaging and therapeutic agent delivery. Advanced Materials, Vol. 27, No. 3, 2015, pp. 576–585.10.1002/adma.201401124Search in Google Scholar PubMed
[19] Trewyn, B.G., I.I. Slowing, S. Giri, H-T. Chen, and V.S.-Y. Lin. Synthesis and functionalization of a mesoporous silica nanoparticle based on the sol-gel process and applications in controlled release. Accounts of Chemical Research, Vol. 40, No. 9, 2007, pp. 846–853.10.1021/ar600032uSearch in Google Scholar PubMed
[20] Feng, S., H. Zhang, S. Xu, C. Zhi, H. Nakanishi, and X-D. Gao. Folate-conjugated, mesoporous silica functionalized boron nitride nanospheres for targeted delivery of doxorubicin. Materials Science & Engineering C, Vol. 96, 2019, pp. 552–560.10.1016/j.msec.2018.11.063Search in Google Scholar PubMed
[21] Giret, S., M. Wong Chi Man, and C. Carcel. Mesoporous-silica-functionalized nanoparticles for drug delivery. Chemistry A European Journal, Vol. 21, No. 40, 2015, pp.13850–13865.10.1002/chem.201500578Search in Google Scholar PubMed
[22] Xiaodong, S., C. Lijue, L. Chengpeng, H. Canzhong, H. Li, and K. Lingxue. Functionalization of hollow mesoporous silica nanoparticles for improved 5-FU loading. Journal of Nanomaterials, Vol. 2015, 2015, pp. 1–9.Search in Google Scholar
[23] Sanna, V., N. Pala, and M. Sechi. Targeted therapy using nanotechnology: focus on cancer. International Journal of Nanomedicine, Vol. 9, No. 1, 2014, pp. 467–483.10.2147/IJN.S36654Search in Google Scholar PubMed PubMed Central
[24] Bertrand, N., J. Wu, X. Xu, N. Kamaly, and O.C. Farokhzad. Cancer nanotechnology: The impact of passive and active targeting in the era of modern cancer biology. Advanced Drug Delivery Reviews, Vol. 66, 2014, pp. 2–25.10.1016/j.addr.2013.11.009Search in Google Scholar PubMed PubMed Central
[25] Samadian, H., S. Hosseini-Nami, S.K. Kamrava, H. Ghaznavi, and A. Shakeri-Zadeh. Folate-conjugated gold nanoparticles as a new nanoplatform for targeted cancer therapy. Journal of Cancer Research and Clinical Oncology, Vol. 142, No. 11, 2016, pp. 2217–2229.10.1007/s00432-016-2179-3Search in Google Scholar PubMed
[26] Melo-Diogo, D., R. Lima-Sousa, C.G. Alves, E.C. Costa, R.O. Louro, and I.J. Correia. Functionalization of graphene family nanomaterials for application in cancer therapy. Colloids and Surfaces B: Biointerfaces, Vol. 171, 2018, pp. 260–275.10.1016/j.colsurfb.2018.07.030Search in Google Scholar PubMed
[27] Kazerooni, A.F., S. Bakas, H.S. Rad, and C. Davatzikos. Imaging signatures of glioblastoma molecular characteristics: A radiogenomics review. International Society for Magnetic Resonance in Medicine, Vol. 52, No. 1, 2019, pp. 1–16.10.1002/jmri.26907Search in Google Scholar PubMed PubMed Central
[28] Dasari, S., and P.B. Tchounwou. Cisplatin in cancer therapy: molecular mechanisms of action. European Journal of Pharmacology, Vol. 740, 2014, pp. 364–378.10.1016/j.ejphar.2014.07.025Search in Google Scholar PubMed PubMed Central
[29] Go., R.S., and A.A. Adjei. Review of the comparative pharmacology and clinical activity of cisplatin and carboplatin. Journal of Cancer Research and Clinical Oncology, Vol. 17, No. 1, 1999, pp. 409–422.10.1200/JCO.1999.17.1.409Search in Google Scholar PubMed
[30] Ghosh, S. Cisplatin: the first metal based anticancer drug. Bioorganic Chemistry, Vol. 88, 2019, pp. 1–20.10.1016/j.bioorg.2019.102925Search in Google Scholar PubMed
[31] Ortiz-Islas, E., M.E. Manríquez-Ramírez, A. Sosa-Muñoz, P. Almaguer, C. Arias, P. Guevara, et al. Preparation and characterization of silica-based nanoparticles for cisplatin release on cancer brain cells. IET Nanobiotechnology, Vol. 14, No. 3, 2020, pp. 191–197.10.1049/iet-nbt.2019.0239Search in Google Scholar PubMed PubMed Central
[32] Uribe-Robles, M., E. Ortiz-Islas, E. Rodriguez-Perez, T. Lim, and A.A. Martinez-Morales. TiO2 hollow nanospheres functionalized with folic acid and ZnPc for targeted photodynamic therapy in glioblastoma cancer MRS Communications, Vol. 9, 2019, pp. 1242–1248.10.1557/mrc.2019.142Search in Google Scholar
[33] Düzlü, A., B. Günaydın, M. Şüküroğlu, and İ. Değim. Release pattern of liposomal bupivacaine in artificial cerebrospinal fluid. Turkish Journal of Anaesthesiology and Reanimation, Vol. 44, 2016, pp. 1–6.10.5152/TJAR.2016.02438Search in Google Scholar
[34] Diserens, A., N. de Tribolet, A. Martin-Achard, A.C. Gaide, J.F. Schnegg, and S. Carrel. Characterization of an established human malignant glioma cell line: LN-18. Acta Neuropathologica, Vol. 53, No. 1, 1981, pp. 21–28.10.1007/BF00697180Search in Google Scholar
[35] Freshney, R.I. Subculture and cell lines. In culture of animal cells: A manual of basic technique and specialized applications, 7th edn, Freshney, R. I., Ed. Wiley-Blackwell Inc., 2016, pp. 235–258.Search in Google Scholar
[36] Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods, Vol. 65, No. 1-2, 1983, pp. 55–63.10.1016/0022-1759(83)90303-4Search in Google Scholar
[37] Fernandes-Cipreste, M., I. Gonzalez, T. Maria da Mata-Martins, A. Miranda-Goes, W. A. de Almeida-Macedo, and E. Martins Barros de Sousa. Attaching folic acid on hydroxyapatite nanorod surfaces: an investigation of the HA-FA interaction. RSC Advances, Vol. 6, No. 80, 2016, pp. 76390–76400.10.1039/C6RA14068HSearch in Google Scholar
[38] Qi, L., Y. Guo, J. Luan, D. Zhang, Z. Zhao, and Y. Luan. Folate-modified bexarotene-loaded bovine serum albumin nanoparticles as a promising tumortargeting delivery system. Journal of Materials Chemistry B, Vol. 2, 2014, pp. 8361–8371.10.1039/C4TB01102CSearch in Google Scholar PubMed
[39] Eskandari, P., Z. Abousalman-Rezvani, S. Hajebi, H. Roghani-Mamaqani, and M. Salami-Kalajahi. Controlled release of anti-cancer drug from the shell and hollow cavities of poly(N-isopropylacrylamide) hydrogel particles synthesized via reversible addition-fragmentation chain transfer polymerization. European Polymer Journal, Vol. 35, 2020, pp. 1–11.10.1016/j.eurpolymj.2020.109877Search in Google Scholar
[40] González Hurtado, M., J. Rieumont Briones, L.M. Castro González, E. Ortiz-Islas, and I. Zumeta-Dube. Kinetic studies of the release profiles of antiepileptic drug released from a nanostructured TiO2 matrix. Journal of Advances in Chemistry, Vol. 12, No. 4, 2016, pp. 4365–4373.10.24297/jac.v12i4.2176Search in Google Scholar
[41] Ata, S., A. Rasool, A. Islam, I. Bibi, M. Rizwan, M.K. Azeem, et al. Loading of cefixime to pH sensitive chitosan based hydrogel and investigation of controlled release kinetics. International Journal of Biological Macromolecules, Vol. 155, 2020, pp. 1236–1244.10.1016/j.ijbiomac.2019.11.091Search in Google Scholar PubMed
[42] Hajebi, S., A. Abdollahi, H. Roghani-Mamaqani, and M. Salami-Kalajahi. Temperature-responsive poly(N-Isopropylacrylamide) nanogels: The role of hollow cavities and different shell cross-linking densities on doxorubicin loading and release. Langmuir, Vol. 36, 2020, pp. 2683–2694.10.1021/acs.langmuir.9b03892Search in Google Scholar PubMed
[43] Zhou, T., X. Zhao, L. Liu, and P. Liu. Preparation of biodegradable PEGylated pH/reduction dual-stimuli responsive nanohydrogels for controlled release of an anti-cancer drug. Nanoscale, Vol. 7, No. 28, 2015, pp. 12051–12060.10.1039/C5NR00758ESearch in Google Scholar
[44] Bohrey, S., V. Chourasiya, and A. Pandey. Polymeric nanoparticles containing diazepam: preparation, optimization, characterization, in-vitro drug release and release kinetic study. Nano Convergence, Vol. 3, No. 3, 2016, pp. 1–7.10.1186/s40580-016-0061-2Search in Google Scholar PubMed PubMed Central
[45] Souza, K.C., J.D. Ardisson, and E.M.B. Sousa. Study of mesoporous silica/magnetite systemsin drug controlled release. Journal of Materials Science: Materials in Medicine, Vol. 20, 2009, pp. 507–512.10.1007/s10856-008-3592-1Search in Google Scholar PubMed
© 2021 Emma Ortiz-Islas et al., published by De Gruyter
This work is licensed under the Creative Commons Attribution 4.0 International License.
