Chitosan capped MSNs as a carrier of quercetin: Preparation, release kinetic study and cytotoxicity assay on osteosarcoma cancer cell line

doi:10.21203/rs.3.rs-2933417/v1

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Chitosan capped MSNs as a carrier of quercetin: Preparation, release kinetic study and cytotoxicity assay on osteosarcoma cancer cell line

https://doi.org/10.21203/rs.3.rs-2933417/v1

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Mesoporous silica nanoparticles (MSNs) have received particular attention in drug delivery applications during recent years. In this study, we successfully synthesized MCM-41 MSNs by sol-gel method. Chitosan polymer as a pH-sensitive capping was coated on MSNs by (3-Glycidyloxypropyl)trimethoxysilane (GPTMS) crosslinker and quercetin (Que) as a compound with anticancer properties was loaded into prepared nanoparticles (Que@Cs-MSNs) for the first time. The structural characteristics and physicochemical properties were investigated by small-angle X-ray diffraction (SAXS), Nitrogen adsorption-desorption, transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM) and Fourier transform infrared (FTIR) methods. Accordingly, the pore size of the hexagonal silica structure was found to be about 3.5 nm, and the dimensions of the Que@Cs-MSNs were calculated to be about 185 nm. The release behavior and release kinetics were studied at pH of 5 and 7.4 and the results showed that the presence of the chitosan coating due to its pH-sensitive nature, in addition to affecting the release rate, caused a change in the release kinetics due to swelling in acidic pH. Also, the results of the cytotoxicity test using MTT assay demonstrated that Que@Cs-MSNs show a higher anticancer effect compared to free Que against G-292 osteosarcoma cancer cells after 72 hours of incubation.

MCM-41

drug delivery

nanoparticles

mesoporous

Cancer, as one of the threats to human life, is a fatal disease with challenging treatment. Generally, chemotherapy is the common method of cancer therapy and efforts are always being made to develop new pharmaceutical formulations with anticancer properties [1, 2]. However, chemotherapeutic agents are nonspecifically distributed throughout the body, affect normal tissues alongside the cancer cells and cause undesirable side effects [3]. Moreover, most of the new anticancer formulations have poor solubility in aqueous environment that results low bioavailability and efficiency in physiological conditions of body [4, 5]. To overcome these limitations, many studies have been focused to provide novel potential delivery systems for chemotherapeutic agents that are able to release drug formulations, whether water soluble or not, in a targeted and controlled way in cancerous tissues with the least damage to normal cells [6, 7]. In this regard MSNs have become interesting drug carriers due to their unique characteristics such as large surface area and drug loading capacity, tunable morphology and pore sizes, easy surface functionalization, good biocompatibility and low toxicity. MSNs, having pores in sizes of 2 to 50 nm, are capable of encapsulating a variety of pharmaceutical compounds [8–10]. For achieving more controlled release and also promoting targeted drug delivery, stimuli-responsive MSNs systems have been developed in recent years. In such systems, MSNs are modified by stimuli-responsive gatekeepers which can be triggered via environmental stimuluses such as temperature, light, ultrasound, pH, enzymes, redox, etc. [11–14]. For example, in the case of pH-responsive systems, compounds such as hyaluronic acid, chitosan, sodium alginate, polyacrylic acid, polydopamine etc. have been studied [15–18]. These gatekeepers cap the MSNs’ pores containing anticancer drug at neutral pH and carry the drug cargo in the neutral normal physiological environment without leakage. In response to the lower pH of tumor environment and endosome/lysosome of cells (4.5–6.5) compared to normal tissues, uncapping the holes leads to release of the drug cargo specifically into the target tissue thereby reducing the side effects to normal cells [19]. Among the gatekeepers, special attention is paid to chitosan. As a linear polysaccharide, chitosan is composed of D-glucosamine and N-acetyl‐D‐glucosamine units. This natural biopolymer is obtained from chitin deacetylation and has excellent properties for using in biomedical applications such as good biodegradability and biocompatibility [20–22].

On the other hand, the interests in using natural compounds with anti-tumor properties are growing in cancer therapy researches. Quercetin (Que) is a natural flavonoid found in most plants and fruits of the human diet. This polyphenol is known as its strong antioxidant activity that scavenges free radicals [23, 24]. Also, in-vitro studies on various cancer cell lines have proven anti-cancerous effects of Que. Accordingly, Que inhibits the proliferation, growth and migration of cancer cells by inducing apoptosis and arresting the cell cycle (either G2/M or G1) [25–27]. Despite the benefits of this flavonoid, the poor bioavailability, short biological half-life, rapid metabolism and fast clearance from body which are mainly due to the hydrophobic nature, limit its therapeutic efficacy in clinical applications [28, 29].

Hence, in the present work, chitosan capped MCM-41 MSNs (Cs-MSNs) was prepared as a smart delivery system for Que. The drug delivery systems based on chitosan-functionalized MSNs have shown a satisfactory potential for loading anticancer compounds such as curcumin, doxorubicin, 5-fluorouracil, methotrexate, gallic acid, etc. [30–34]. Therefore, we have used this delivery system for Que and, in addition to characterizing, for the first time, we have studied the kinetics of Que release and evaluated its anticancer efficiency on the osteosarcoma cell line model.

Materials

Tetraethyl orthosilicate (TEOS), cetyltrimethylammonium bromide (CTAB), (3-Glycidyloxypropyl) trimethoxysilane (GPTMS), chitosan with a deacetylation degree of 75–85%, quercetin, phosphate-buffered saline (PBS), RPMI-1640 culture medium, trypan blue dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and dialysis tubing were purchased from Sigma-Aldrich (St Louis, MO, USA). Ethanol, methanol, sodium hydroxide, acetic acid, citric acid and hydrochloric acid were purchased from Merck Co, Germany. Fetal bovine serum (FBS), penicillin, streptomycin and trypsin-EDTA (0.25%) were purchased from Gibco™, USA. Human osteosarcoma cancer cell line (G-292) was prepared from Pasteur Institute (Tehran, Iran). All the other solvents and reagents used in tests were analytical grade.

MSNs synthesis

Briefly, CTAB (500 mg) was dissolved in 96 mL distilled water under stirring condition (550 rpm). After obtaining a clear solution, 0.5 mL of NaOH solution (2 M) was added and the temperature adjusted to 80 ℃ and stirred for 5 min. Then 2.5 mL of TEOS was added dropwise to the solution during 10 min and the reaction continued under stirring (550 rpm) at room temperature for 2 h to obtain white precipitate. The resulting solid was collected through filtering and washed 5 times by distilled water and ethanol. For removing the surfactant, the obtained product was refluxed in a solution with 1 mL of HCl in 99 mL of methanol for 6 h, then washed by distilled water and ethanol and dried at room temperature for 24 h. Finally, obtained MSNs were heated at 80 ℃ under vacuum to remove remaining solvents.

Chitosan coating on MSNs

100 mg of MSNs powder was dispersed in 10 mL ethanol through sonicating for 10 min and pH adjusted to 4 by adding acetic acid. subsequently, 100 mg of GPTMS was added to the solution and stirred at room temperature for 3 h. Afterwards, 20 mL of chitosan solution (1% W/V) in citric acid (1.5 wt%) was added to as-prepared reaction solution and stirred for 12 h at room temperature. The obtained chitosan coated MSNs (Cs-MSNs) was centrifuged (10000 rpm, 30 min) and washed by water and ethanol 5 times and dried by freeze-drier.

Drug loading on MSNs and Cs-MSNs

For loading step, 100 mg Que dissolved in 10 mL ethanol and 100 mg of MSNs or Cs-MSNs powder was added gradually under stirring condition. Next, pH was adjusted at 4 by adding HCl (1 M) and the reaction suspension was stirred in darkness at 4℃ for 24 hours. After that, obtained Que loaded nanoparticles (Que@Cs-MSNs) was centrifuged for 30 min (10000 rpm) and washed 3 times by ethanol and water and freeze-dried overnight.

Characterization methods

The surface area and pore characteristics were studied using Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) methods, respectively, by a Belsorp-mini II analyzer. Small angel X-ray scattering (SAXS) was carried out for structural property investigation using Cu Kα radiation (λ = 1.54 A°) at 40 kV by PANalytical X’Pert MPD X-ray diffractometer. FESEM model Mira 3-XMU with accelerating voltage of 15kV was used to identify the morphology and particle size distribution. Also, for more microstructural investigation and observing the nanopores, TEM instrument, Philips CM 200 model with 200 kV voltage was used. FTIR spectra were determined using Brucker Vector33 FTIR spectrometer and KBr discs.

In-vitro drug release

For drug release studies, 100 mg of Que@Cs-MSNs powder was dissolved in 1 mL phosphate buffered saline (0.1 M) and enclosed in dialysis bag with 12 kDa molecular cutoff. The dialysis bag was incubated in 20 mL solutions of 0.1 M PBS under shaking at 37℃. At determined intervals 1 mL of solution was taken out and analyzed by UV-Vis spectrophotometer model Shimadzu UV-2450 model Shimadzu UV-2450 at 370 nm. After each sampling equal amount of fresh medium was added to the solution. The measurements were done at two solution media with pH values of 5 and 7.4 until 36 h. All experiments were performed 3 times. Cumulative release of Que was calculated using the following equation and obtained data were plotted versus time.

Que cumulative release = (the amount of released Que at any time) / (total amount of loaded Que)

Cell culture

For in-vitro evaluations, in brief, human osteosarcoma cancer cell line, G-292, were cultured in 96-well plates containing RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin antibiotics. The cultured cells were placed in an incubator with 5% CO₂ at 37℃. Every three days the culture medium was changed until the cells reached the appropriate density for analysis.

MTT assay

MTT colorimetric assay was used to assess the in-vitro cytotoxicity of Que@Cs-MSNs on G-292 cancer cells. Briefly, G-292 cells at density of 6×10³ cells per well were seeded in 96-well plates. After incubation at 37℃ for 24 h, the cells were treated with free Que and Que@Cs-MSNs in concentrations of 0, 50, 100, 150, 300 and 450 µM. After more incubation for 24 h, the medium was removed and fresh medium containing 0.25 mg/mL MTT solution (tetrazolium salts in RPMI-1640 medium) was added to the wells and incubated again at 37℃ for 4 h. Then the media were removed and the formed formazan crystals in each well were dissolved in 150 µl DMSO for 20 min. Afterwards the absorbance of the supernatant solution was measured by Bio Tek ELx808 microplate reader at 570 nm. The cytotoxicity test was also performed for the treatments incubated for 72 h. The results were reported based on cell viability percentage which obtained using the absorbance of the treated cells, per control cells.

Statistical analysis

One-way ANOVA analysis was used for statistical analysis of data and p < 0.05 was considered statistical significance. Data were presented as means ± standard deviations of three independent experiments.

Synthesis and characterization

MCM-41 MSNs were synthesized through sol-gel method using TEOS and CTAB as silica resource and template surfactant, respectively. The structure of MSNs was evaluated by X-ray diffraction in low angles. According to the diffraction pattern of Fig. 1, three distinct characteristic peaks at 2.2°, 3.9°, 4.6° and a mild peak at 6.1° are observed for MSNs which are attributed to the reflections 100, 110, 200 and 210 planes of the hexagonal ordering [35]. TEM imaging was also used to depict the mesoporous structure of MSNs. Figure 2a and 2b show the formed mesopores of MSNs in two different magnifications. It can be clearly observed the well-ordered porous channels with honeycomb-like hexagonal structures, that verifies the formation of MSNs and X-ray diffraction analysis results.

In order to identify the pores of MSNs and determine the surface area, nitrogen gas absorption and desorption isotherm was obtained based on Brunauer–Emmett–Teller (BET) theory. Figure 3a shows that the nitrogen adsorption-desorption isotherm follows the type IV according to the classification by IUPAC. Also, there is a hysteresis loop in high ratios of P/P0. This result indicates the formation of mesoporous structure and the presence of very narrow and capillary pores of MSNs. According to BET method, the surface area of MSNs was calculated to be about 1023 m²g^− 1. In addition, the average diameter of the pores was obtained 3.50 nm, from BJH study (Fig. 3b).

To covering MSNs with chitosan, GPTMS was used as a crosslinker. In the acidic condition, trimethoxysilane groups of GPTMS hydrolyzed to form silanol groups which condensed in the presence of silica network of MSNs to form a cross-linked structure through Si-O-Si covalent bonds [36, 37]. On the other hand, oxirane group of GPTMS reacted with amino groups of chitosan by the acid-catalyzed addition reaction and chitosan coated MSNs were prepared [38]. To load Que in nanopores, the pH-responsive property of chitosan was utilized. In this way, reducing the pH to the acidic ranges, Cs-MSNs swelled due to protonation of amino groups in chitosan, and Que entered in the pores of MSNs. Readjusting the pH to neutral ranges caused shrinkage in Cs-MSNs structure, capping of the pores due to deprotonating of amino groups of chitosan and entrapment of Que in the pores of MSNs (Scheme. 1).

In addition, the morphology and size distribution of the Que@Cs-MSNs were investigated by FESEM analysis (Fig. 4). The results indicated that particles have nearly spherical shapes and a narrow size distribution with average diameter of 185.09 ± 4.21 nm. Previous studies have shown that nanoparticles with dimensions in the range of 100 to 200 nm can accumulate in the tumor site due to passive targeting caused by enhanced permeability and retention effect (EPR) [39]. Therefore, the dimensions obtained for nanoparticles here can be useful for passive targeting of cancer cells.

To confirm the loading of Que into Cs-MSNs, FTIR analysis was performed on pure Que, Cs-MSNs and Que@Cs-MSNs samples. According to FTIR spectrum of Que in Fig. 5, the characteristic band around 3406 cm^− 1 was attributed to –OH groups, 1666 cm^− 1 to C = O stretching, 1611 cm^− 1 and 1521 cm^− 1 to aromatic C = C stretching vibrations. The C–O stretching of oxygen in the ring was found at 1263 cm^− 1 beside other C–O bands at 1168 cm^− 1 and 1013 cm^− 1. Also the bands at 1450 cm^− 1, 1382 cm^− 1, 1319 cm^− 1, 864 cm^− 1 and 823 cm^− 1 belong to C-H bending vibrations [40–42]. Comparing the spectrum of Que@Cs-MSNs with the pure Que implies that the bands at 1658 cm^− 1, 1512 cm^− 1, 1357 cm^− 1, 1317 cm^− 1 and 819 cm^− 1 belong to Que which confirms the successful loading of Que in Cs-MSNs carriers. The existence of some shifts in the spectrum of Que in nanoparticles can be due to its trapping in the Cs-MSNs pores and overlapping of some peaks by chitosan bands.

In-vitro release behavior

Fig. 6 shows the cumulative release of Que from the Que@Cs-MSNs at pH 5 and 7.4. In addition, to study the effect of pH-sensitive chitosan coating on the release behavior, the release of Que from MSNs without chitosan coating was also investigated and shown in the Fig. 6. According to the results, the release of Que from nanoparticles is burst in the first 5 h, followed by a controlled manner at both pH, but the amount of released drug depends on presence of chitosan coating and pH value. The amount of Que released from Que@Cs-MSNs at pH 7.4 reached about 23.85 % after 36 h, while this amount for uncoated MSNs was about 49.60 %. Indeed, at pH range above 6.5, chitosan caps the pores of MSNs as a gatekeeper due to deprotonation of amine groups and shrinkage, so prevents the release of more Que from MSNs. In acidic pH, although the amount of Que released from uncoated MSNs is equal to 57.38 % after 36 h, this amount reached to 46.02 % for Que@Cs-MSNs. Owing to the pH-sensitive property of chitosan, at lower pH, amine groups are protonated, cause the structure of chitosan to swell and the gate of the pores are opened for the release of Que. This result has also been reported in similar studies [43, 33]. This property is beneficial for drug delivery to the tumor sites. Since the rate of glycolysis is higher in the tumor environment, it has more acidity and the pH value in tumor tissue reaches less than 6.8, while the pH of normal tissues and blood is about 7.4. Therefore, the drug cargo inside the MSNs is protected without leakage into the blood and damage to normal cells, and when it reaches the acidic tumor site, the drug is released by uncapping the holes of MSNs and dissolution of chitosan.

Release kinetic mechanisms

Mathematical kinetic models can be applied to better understand the mechanism and predict the release behavior of drugs in in-vivo conditions using the data obtained from in-vitro studies. Herein, the kinetic mechanism of Que release was evaluated by the Zero-order, First-order, Higuchi and Kormeyer-Peppas mathematical models.

Zero order model

The Zero-order kinetic model is an ideal model for drug release. For drug delivery systems that follow this model, the release rate is independent of drug concentration. In other words, drug release is constant throughout the delivery time in the blood flow [44]. The Zero-order kinetic can be represented by the following equation.

$${Q}_{t}={Q}_{0}+{k}_{0}t$$

Where ${Q}_{0}$ is the initial concentration of drug in release medium which is usually zero, ${Q}_{t}$ is released drug concentration at time $t$ and ${k}_{0}$ is the constant of Zero-order equation.

First-order model

This model is presented based on Fick's law for diffusion and the release rate is dependent on drug concentration. Accordingly, due concentration gradient inside the carrier and outside of it, the drug diffuses to the release medium and as a result, its concentration inside the carrier decreases logarithmically with time [45]. The simplified equation of this model is as follows

$$\text{log}{C}_{t}=\text{log}{C}_{0}-({k}_{1}t/2.303)$$

Where, ${C}_{0}$ is the initial amount of drug in the carrier, ${C}_{t}$ is the amount of drug remaining in the carrier at time t and ${k}_{1}$ is the First-order constant.

Higuchi model

Higuchi developed a widely used model for the release of hydrophilic and hydrophobic compounds loaded in solid and semi-solid matrices with various geometries based on Fick's first law [46]. In Higuchi's simplified model, the amount of drug released () is related to the root of time and is Higuchi release constant.

$${Q}_{t}={k}_{H}\times \sqrt{t}$$

Korsmeyer-Peppas model

Korsmeyer and Peppas presented an empirical kinetic model for release mechanisms, both for Fickian and non-Fickian diffusion phenomena [47]. In this model according to Eq. 4, is the fraction of drug released at time, is Korsmeyer-Peppas constant which depends on structural and geometric characteristics of the system and is release exponent. The values obtained for predict the release mechanism, when the release mechanism corresponds to the Fickian diffusion and non-Fickian or anomalous transport.

$$\text{log}{Q}_{t}/{Q}_{\infty }=\text{log}{k}_{KP}+n\text{log}t$$

The fitting of the experimental data of Que release from Que@MSNs and Que@Cs-MSNs on these kinetic models are shown in the graph of Fig. 7, and the values obtained for the constants along with the coefficient of determination (R²) are given in Table. 1. The R² value being closer to 1 means a better fit of the data with the model, and these values are higher for the Higuchi model in the examined samples. Therefore, diffusion phenomena are involved in the release of Que. On the other hand, the comparison of $n$ values obtained from the Korsmeyer-Peppas model show that for Que@MSNs, the $n$ values are less than 0.5 at both pH of 5 and 7.4, that means the release kinetic follow the Fickian diffusion. However, $n$ values are 0.8850 and 0.6863 for Que@Cs-MSNs at pH of 5 and 7.4, respectively. These results indicate that in the presence of chitosan coating, the release kinetics of Que from MSNs changes and follows the non-Fickian or anomalous transport model. In this case, Que molecules are released both due to the diffusion from the MSNs matrix and the swelling or dissolution of the chitosan coating.

Table 1

R² values and kinetic constants obtained from data fitting with mathematical models
Model	coefficients	Que@MSNs pH = 5	Que@MSNs pH = 7.4	QUE@Cs-MSNs pH = 5	QUE@Cs-MSNs pH = 7.4
Zero order	k₀ R²	1.291 0.9070	1.102 0.8688	1.290 0.8509	0.5805 0.8902
First order	k₁ R²	0.0213 0.9499	0.0166 0.9140	0.0181 0.8904	0.0068 0.9103
Korsmeyer-Peppas	k_KP n R²	1.080 0.4646 0.9613	1.002 0.4860 0.9274	0.4661 0.8850 0.8811	0.4107 0.6863 0.8490
Higuchi	k_H R²	9.413 0.9745	8.148 0.9594	9.580 0.9484	4.255 0.9659

Cytotoxicity study

To study the cytotoxicity, G-292 osteosarcoma cells were treated with different concentrations of Que@Cs-MSNs (0-450 µM) and free Que for 24 and 72 h and evaluated by MTT assay. As shown in Fig. 8, after 24 h of treatment, in the concentration range of 50–150 µM, cell viability is above 88% for both Que@Cs-MSNs and free Que, and cytotoxicity is not significant. However, at higher concentrations, significant cytotoxicity is observed for both formulations compared to the control group. Therefore, cytotoxicity is concentration dependent but free Que and Que@Cs-MSNs does not exhibit a significant cytotoxicity difference up to 450 µM. In 72 h treatments, however, at the concentration of 100 µM and above, Que@Cs-MSNs show significant higher cytotoxicity. In addition, the dependence of cytotoxicity on the concentration after 72 h is more significant so that Que@Cs-MSNs and Que show IC₅₀ values of 280.30 µM and 494.7 µM, respectively. These results demonstrate that firstly, the cytotoxicity of both formulations increase with enhancing incubation time; Secondly, Que loading within Cs-MSNs improves its anticancer property against G-292 osteosarcoma cells. The increased cytotoxicity of Que@Cs-MSNs after 72 h of treatment can be due to the fact that nanoparticles, by improving the limitations of Que caused by low solubility and instability, using specific cell uptake pathways, discharge the drug cargo in a controlled manner into the cells. Meanwhile, due to its hydrophobic nature, Que molecules may undergo agglomeration after 72 h and their cellular uptake will decrease compared to Que@Cs-MSNs.

In addition to the cytotoxicity assessment, optical microscope images of untreated G-292 cells and the cells treated with Que and Que@Cs-MSNs at IC₅₀ concentration of Que@Cs-MSNs were prepared. According to the images shown in Fig. 9 untreated cells have healthy morphology, intact membrane, high density and adherent together on the surface of the plate. However, in cells treated with formulations, cell morphology is completely changed and membrane shrinkage due to the condensation of the cytoplasm is visible. Moreover, the density of the cells is decreased, the adjacent cells are not adhesive to each other and to the surface of the plate. These changes can be caused by the apoptosis induction in the treated cells. Also, the above changes are more significant in cells treated with Que@Cs-MSNs than free Que. Therefore, Que@Cs-MSNs inhibits G-292 cancer cells more effectively probably through apoptosis induction pathways, which is consistent with the results obtained from MTT assay.

In this research, MCM-41 mesoporous silica nanoparticles were successfully synthesized and coated with chitosan polymer. For this purpose, GPTMS was used as a crosslinker and the coating process was performed by creating a covalent bond between the Si-O-Si groups, as well as the reaction between the oxirane and amine groups of chitosan. The prepared formulation was loaded with the hydrophobic Que drug. Evaluation of the release behavior of Que from nanoparticles showed that in the presence of chitosan coating, despite the limitation of drug release in neutral pH, the release increases with the swelling of chitosan in acidic environment. Moreover, the coating of MSNs with chitosan affects the drug release kinetics. Although in uncoated MSNs, the release kinetics follows Fickian diffusion, in coated nanoparticles, a combination of anomalous diffusion and swelling processes control the release kinetics. In addition, the final formulation showed more cytotoxicity against G-292 osteosarcoma cancer cells compared to free Que in 72-hour treatments. Overall, according to present study, chitosan-coated mesoporous silica nanoparticles can be considered as a pH-sensitive carrier candidate for controlled release of Que and improving its anticancer properties.

Acknowledgements

This research was supported by the University of Hormozgan under grant No. 401-D-21821.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

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Chitosan capped MSNs as a carrier of quercetin: Preparation, release kinetic study and cytotoxicity assay on osteosarcoma cancer cell line

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Abstract

Figures

Introduction

Materials and Methods

Materials

MSNs synthesis

Chitosan coating on MSNs

Drug loading on MSNs and Cs-MSNs

Characterization methods

Cell culture

MTT assay

Statistical analysis

Results and discussion

Synthesis and characterization

Release kinetic mechanisms

Cytotoxicity study

Conclusion

Declarations

References

Additional Declarations

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