Hollow mesoporous silica nanoparticles were successfully fabricated and functionalized with appropriate silanes. After modifications, amine, carboxyl, cyano, and methyl groups were grafted onto the nanoparticles and all functionalized hollow mesoporous silica nanoparticles maintained a spherical and hollow structure with a mean diameter of ~120 nm and a shell thickness of ~10 nm. The loading capacity of the hollow mesoporous silica nanoaprticles to the anticancer drug, 5-fluorouracil, can be controlled via precise functionalization. The presence of amine groups on the surface of nanoparticles resulted in the highest loading capacity of 28.89%, due to the amine functionalized nanoparticles having a similar hydrophilicity but reverse charge to the drug. In addition, the change in pH leads to the variation of the intensity of electrostatic force between nanoparticles and the drug, which finally affects the loading capacity of amine functionalized hollow mesoporous silica nanoparticles to some extent. Higher drug loading was observed at pH of 7.4 and 8.5 as 5-fluorouracil becomes more deprotonated in alkaline conditions. The improved drug loading capacity by amine functionalized hollow mesoporous silica nanoparticles has demonstrated that they can become potential intracellular 5-fluorouracil delivery vehicles for cancers.
Nanoparticle based targeted drug delivery systems hold great promise for cancer therapy because of their targeting functions [
However, even though with a high surface area and a high pore volume, MSNs showed a relative low loading capacity of 5-fluorouracil (5-FU), a standalone therapeutic for the treatment of colon cancer, which may be due to the electrostatic repulsion between the drug and the carriers. In this case, in comparison to those carriers with a higher drug loading capacity, more MSNs were required to be taken up by cancer cells to achieve an effective cure concentration for cancer therapy. From a technical point of view, it is more preferable to deliver a smaller quantity of MSNs to minimise the side effects to patients [
The capacity to encapsulate drugs in nanoparticles can be improved by functionalizing or optimizing the drug encapsulation conditions [
In this paper, HMSNs were modified for enhanced 5-FU loading. The partition coefficient of 5-FU was investigated to provide information about the degree of the hydrophilicity of 5-FU, which is crucial for selecting chemical groups for the subsequent functionalization. Amine, carboxyl, methyl, and cyano groups were introduced onto HMSNs. The structure of plain and functionalized HMSNs was characterized and the 5-FU loading capacity of HMSNs with and without chemical groups was investigated and compared.
Tetraethyl orthosilicate (TEOS, Si(OCH2CH3)4), Triton X-100 (TX100), sulphuric acid, octadecyltrimethoxysilane (OTMS), (3-aminopropyl)triethoxysilane (APTES), 3-cyanopropyltriethoxysilane (CPTES), and fluorinated pyrimidine 5-fluorouracil (5-FU) were all purchased from Sigma Aldrich (Sydney, Australia). Eudragit S100 which is made in Germany was purchased from Shenzhen Youpuhui Pharmaceutical Co. Ltd (Shenzhen, China).
For the formation of the mesoporous silica shell, the Eudragit S-100 nanoparticle solution was ultrasonically dispersed for 3 min, followed by addition of 1.0 g TX100 under stirring. After TX100 was completely dissolved, 2.3 mL TEOS was added and this solution was left to proceed for 24 h before being transferred into a sealed container and hydrothermally treated overnight under 100°C. The resulting nanoparticles (as-synthesized HMSNs) were collected by centrifugation and redispersed in acetone for the removal of Eudragit core and TX100 templates. The particles were then washed 3 times with ethanol and deionized water, respectively, before finally freeze-drying.
For amine functionalization (HMSN-NH2), 50 mg HMSNs were ultrasonically dispersed in 50 mL ethanol; 25
To prepare carboxyl-modified MSNs (HMSN-COOH), fifty milligrams of HMSNs was ultrasonically dispersed in 50 mL ethanol; 25
Measurement of 5-FU oil : water partition coefficients was conducted via shake flash method according to OECD guideline 107 [
The loading of 5-FU was carried out by soaking HMSNs in a concentrated drug solution at room temperature for 24 h while stirring to ensure the diffusion of the drug molecules through the mesopores. Firstly, 5-FU was dissolved in deionized water to a concentration of 3 mg/mL. 150 mg of HMSNs was ultrasonically dispersed in 50 mL of the 5-FU solution. The mixture was stirred at room temperature. At timed intervals of 0, 2, 4, 6, 8, 12, and 24 h, 0.5 mL of the solution was extracted and centrifuged to collect the supernatant for UV-Vis analysis at a wavelength of 266 nm while the 5-FU-loaded HMSNs precipitate was placed back into the drug solution. The 5-FU loading in the HMSNs was calculated by subtracting the amount of 5-FU in the supernatant from the amount in the original drug loading solution. The drug loading capacity was calculated using the following equation:
The morphology of HMSNs was studied using scanning electron microscopy (SEM, Carl Zeiss AG SEM Supra 55VP) and transmission electron microscopy (TEM, LaB6 JEOL JEM-2100). Before imaging under SEM, all the samples were coated with carbon through a BAL-TEC SCD 050 sputter coater (Leica Microsystems, Australia). TEM imaging was conducted under 200 kV. Nitrogen absorption and desorption isotherms of HMSNs were conducted on a Micromeritics Tristar 3000 (Particle & Surface Science, UK) equipment at 77 K. Before measurements, samples were degassed at 523 K for 1 h under nitrogen. The specific surface areas of HMSNs were calculated using the Brunauer-Emmett-Teller (BET) method. The Barrett-Joyner-Halenda (BJH) model was utilized to obtain the pore size distributions from the desorption branch of isotherms. Thermogravimetric analysis (TGA) of HMSNs was conducted using a TG Simultaneous Thermal Analyser (STA 449C, ZETZSCH, Germany). Samples with a weight of ~5 mg were placed into aluminium oxide crucibles and heated under nitrogen condition at a heating rate of 10°C/min from room temperature to 600°C. Fourier transform infrared (FTIR) spectra were measured with a Vertex 70 spectrometer (Bruker, Germany). All the samples were compressed into KBr pellets and recorded at 64 scans from 4000 cm−1 to 400 cm−1 with a resolution of 4 cm−1.
The partition coefficient (
The shake flask method is one of the common and standard experimental procedures adopted for
According to OECD guideline 107 [
Partition coefficient of 5-FU and relevant parameters.
1 : 2 (O : W) | 1 : 1 (O : W) | 2 : 1 (O : W) | |
---|---|---|---|
Water volume (mL) | 26.67 | 20 | 13.33 |
Oil volume (mL) | 13.33 | 20 | 26.67 |
|
7.747 | 9.925 | 13.17 |
|
2.279 | 2.380 | 2.629 |
|
0.2942 | 0.2398 | 0.1996 |
|
−0.5314 | −0.6201 | −0.6997 |
The weight percentages of 5-FU dissolved in each phase with varying octanol to water ratio were calculated. As seen in Figure
Percentages of 5-FU dissolved in each phase with varying octanol to water ratio.
Surface-functionalized HMSNs were synthesized by postgrafting method where chemical groups were introduced to the surface of as-synthesized HMSNs using appropriate silanes. CPTES, APTES, and TEMS were used to prepare HMSN-CN, HMSN-NH2, and HMSN-CH3 while HMSN-COOH was obtained by oxidizing HMSN-CN. In all cases, plain HMSNs without any modification were used as a control. The morphology of modified and plain HMSNs was characterized by TEM. As shown in Figure
TEM images of (a) HMSNs; (b) HMSN-NH2; (c) HMSN-COOH; (d) HMSN-CN; and (e) HMSN-CH3.
The presence of mesoporous structure in all HMSNs was confirmed by BET (Figure
N2 adsorption-desorption isotherms (a) and the corresponding pore size distributions (b) of functionalized and plain HMSNs.
Structural parameters of the HMSNs with and without modifications were summarized in Table
Structure parameters of functionalized and nonfunctionalized HMSNs.
HMSNs | HMSN-CH3 | HMSN-CN | HMSN-NH2 | HMSN-COOH | Plain HMSNs |
---|---|---|---|---|---|
Pore size (nm) | 2.51 | 2.28 | 2.47 | 2.61 | 2.78 |
Pore volume (cm3/g) | 0.43 | 0.50 | 0.43 | 0.47 | 0.53 |
Surface area (m2/g) | 434 | 514 | 413 | 498 | 820 |
Different chemical groups of –NH2, –CN, –COOH, and –CH3 were grafted onto the surface of HMSNs separately and the successful grafting was demonstrated from FTIR spectra (Figure
FTIR spectra of plain and functionalized HMSNs.
Thermogravimetric analysis (TGA) was carried out to further confirm the effective surface modification of functionalized HMSNs. All samples were tested under nitrogen condition at the temperature ranging from room temperature up to 650°C. The weight loss of samples obtained from TGA arose from the decomposition of chemical groups; thereby a difference in the weight loss between samples can indicate the relative amount of chemical groups grafted onto particles. As seen from Figure
TGA curves of functionalized HMSNs and plain HMSNs.
The 5-FU loading capacities of functionalized and nonfunctionalized HMSNs have been monitored using a UV spectrophotometer. An initial drug concentration of 3 mg/mL was applied and the concentration change after 24 hours of loading was tracked. The loading capacity of all HMSNs were calculated and presented in Figure
Loading capacities of functionalized and nonfunctionalized HMSNs.
5-FU was absorbed in both the pores and the hollow cavity and the external surface area. Firstly, 5-FU is attached on the surface of nanoparticles; as the loading process goes on, 5-FU can pass through the pores (2-3 nm) and get into the cavity (~100 nm). As the pores are much bigger than the drug molecules (~0.5 nm) and can provide enough room for the free transportation of the drug, the drug can be encapsulated into the pores and the cavity of HMSNs.
The presence of different functional groups on the external area of nanoparticles will affect the loading capacity of the nanoparticles. As expected, a difference in the amount of 5-FU loading after surface functionalization was observed, resulting from the different hydrophobicity and hydrophilicity of the functional groups [
Loading capacities of functionalized and nonfunctionalized HMSNs.
HMSNs | HMSN-CH3 | HMSN-CN | HMSN-NH2 | HMSN-COOH | Plain HMSNs |
---|---|---|---|---|---|
Loading capacity (mg 5-FU/g HMSNs) | 127.3 | 225.4 | 288.9 | 207.3 | 183.4 |
Furthermore, –CH3 functionalization decreases the loading capacity of HMSNs to some extent (from 18.34% to 12.73%). 5-FU is a water-soluble drug; therefore, it is harder for it to approach HMSNs functionalized with methyl groups which are slightly more hydrophobic. On the other hand, –CN and –COOH functionalized HMSNs showed a moderate improvement in loading with a loading percentage of 22.54% and 20.73%, respectively. Although HMSN-COOH is hydrophilic which is similar to 5-FU, its loading capacity is inferior to HMSN-NH2. This can be explained by the different surface charge of HMSN-NH2 and HMSN-COOH. Given that 5-FU is negatively charged, it is much easier for 5-FU to get inside the positively charged HMSN-NH2 via electrostatic attractions. However, the negative charge of –COOH groups may repel 5-FU molecules and decrease the 5-FU loading capacity of HMSN-COOH.
The –NH2 on HMSNs can significantly increase the amount of 5-FU encapsulated into nanoparticles due to the similar hydrophilicity of the HMSN-NH to drug and the existence of positive charge provided by –NH2 groups. Therefore, the control of the intensity of electrostatic force between nanoparticles and 5-FU may further regulate 5-FU loading capacity of particles.
In this regard, the loading of 5-FU by HMSN-NH2 was carried out in 4 different pHs, 4.0, 5.5, 7.4, and 8.5 (Figure
5-FU loading capacity of HMSNs and HMSN-NH2 at different pH values.
5-FU anions (AN1 and AN3) and dianion.
On the other hand, no significant differences can be seen in the loading capacity of HMSNs at various pHs (Figure
HMSNs were successfully functionalized with appropriate silanes. After functionalization, HMSNs with different chemical groups on the surface maintained the intact hollow structure and no significant changes were found with the pore size and pore volume. However, due to the coverage of functional groups, the surface area of functionalized samples decreased when compared to plain HMSNs, which were consistent with similar studies. The surface modification of functionalized HMSNs was proven by FTIR and TGA to be effective and the relative amount of grafted functional groups was ~12% (w/w). The loading capacity of HMSNs specific to 5-FU can be improved via precise functionalization. The presence of –NH2 groups on the surface of nanoparticles resulted in the highest 5-FU loading capacity of 28.89%. The similar hydrophilicity of the HMSN-NH2 and the drug and the presence of reverse charge of the drug give rise to this highest loading capacity of HMSN-NH2. In addition, when compared to that of nonhollow MSNs reported in a very similar study, the loading amount of 5-FU by HMSN-NH2 was much higher, suggesting that, besides functionalization, 5-FU loading could be improved by designing and fabricating MSNs of different structure such as the hollow MSNs. The change in pH leads to the variation of the intensity of electrostatic force between nanoparticles and 5-FU, which finally affect the loading capacity of HMSN-NH2 to some extent. Higher drug loading of HMSN-NH2 was observed at pH of 7.4 and 8.5 due to the fact that 5-FU becomes more deprotonated in alkaline conditions. By incorporating targeting molecules such as folic acid and epidermal growth factor (EGF), HMSN-NH2 with improved loading of 5-FU will definitely render the current drug delivery system more versatile for applications in cancer therapy.
The authors declare that there is no conflict of interests regarding the publication of this paper.