DOX-Fe₃O₄@mSiO₂-PO-FA nanocomposite for synergistic chemo- and photothermal therapy

Abstract

The integration of multiple therapies into one nanoplatform was considered to be the most efficient method to give an enhanced therapeutic effect for anti-cancer treatments. In this research, pH sensitive drug release and near-infrared (NIR)-induced photothermal therapy (PTT) were placed in one nanocomposite using iron(II,III) oxide@meso-silica (Fe₃O₄@mSiO₂) NPs as the host. Then, a sensitive ketal modified silane coupling agent (PO linker) was prepared to encapsulate the anti-cancer drug, doxorubicin hydrochloride (DOX), into the mSiO₂ shell, ensuring the few leakage. Based on the different pH conditions of normal and cancer cells, the DOX-Fe₃O₄@mSiO₂-PO nanocomposite exhibits the specific drug release in cancer cells (acid conditions) before that the few leakage makes sure there are a low number of side effects. Furthermore, the release kinetics can also be easily adjusted by varying the amount of PO linker. Under NIR (808 nm) irradiation, the photothermal effect derived from magnetic Fe₃O₄ NPs can also induce the apoptosis of the cancer cells which is how PTT is achieved. HeLa cells (cervical cancer cell line) were used as the typical cancer cell and the detailed cell experiments further display the enhanced specific cytotoxicity of the method to HeLa cells. The multifunctional nanocomposite has great potential as an application for anti-cancer therapy.

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Introduction

Based on their various superior properties, some nanoparticles [(NPs) polymers, micelles, liposomes, gold and zinc oxide NPs and so on] are adopted as nanocarriers to prolong the blood circulation of antitumor drugs to offer an opportunity for drug accumulation in the tumor and these give exciting results in anti-cancer therapy.^1–5 Among these materials, mesoporous silica nanoparticles (MSN) have attracted great interest because of their advantageous properties, including large surface and pore volume, outstanding biocompatibility, accessible modification, tunable particle size at the nanoscale.^6–10 To further control the release behavior, stimuli responsive MSNs were developed to make sure that the drug molecules were localized to the immediate tumor environment and to reduce the side effects of the drug. Currently, stimuli responsive drug release can be regulated by external stimuli (thermal, light, electric, and magnetism) or by internal stimuli (enzymes and pH).^11–15 Because of the pH difference between the normal (neutral/alkaline conditions) and cancer cells (weak acid conditions), the pH sensitive drug release is considered as a feasible means to insure specific chemotherapy.^14,16–19

In addition, photothermal therapy (PTT) has become an appealing strategy for cancer treatment.^20,21 The use of light as a highly orthogonal external stimulus allows for spatial and temporal exposure in the tumor region to maximize the treatment efficacy without causing extensive damage to the surrounding normal tissue. Among those photoabsorbers, iron(II,III) oxide (Fe₃O₄) NPs have received tremendous attention because of their excellent magnetism, biocompatibility, highly targeting ability and photothermal conversion efficiency.^22–24 Furthermore, Fe₃O₄ NPs can absorb near-infrared (NIR) light to induce an increase of temperature.²⁵ Compared with ultraviolet (UV) and visible light, NIR light is an attractive stimulus because of its non-invasive and deep tissue penetration. This further makes Fe₃O₄ NPs as the desired photoabsorber to be used in PTT for deep tumor therapy. However, pure Fe₃O₄ NPs are prone to aggregation because of anisotropic dipolar attraction and rapid biodegradation when they are exposed directly to biological systems.^23,26,27 Lately, much effort has been devoted to the fabrication of multifunctional Fe₃O₄ nanocomposites to overcome the previously mentioned defect and also to combine some other functions. Lin et al. synthesized Fe₃O₄@polydopamine core–shell nanocomposites using an in situ self-polymerization method for intracellular messenger ribonucleic acid (mRNA) detection and multimodal imaging-guided PTT.²⁸ Xu et al. used calcium phosphate cement containing Fe₃O₄ NPs for minimally invasive and efficient magnetic hyperthermia ablation of tumors.²⁹ However, the tedious assembly process, the instability, and the lack of porous structure have inhibited the further use of functional Fe₃O₄ NPs in multi-therapy for anti-cancer treatment.

In this research, Fe₃O₄ NPs were adopted as a core coated with a mesoporous silica (mSiO₂) shell to prepare Fe₃O₄@mSiO₂ core–shell NPs as the nanocarrier (Scheme 1). Then, the sensitive organic linker, 3,3′-(propane-2,2-diyl bis(oxy))bis(2,2-dimethylpropanoic acid) (PO linker), was designed and synthesized. Using a simple silane coupling reaction, the PO linker can be grafted outside the NPs to act as a “gate” to the anti-cancer drug, doxorubicin hydrochloride (DOX), encapsulated inside the mSiO₂. The acid cleavage ketal group in the PO linker causes its degradation under acid conditions and still blocks the pores under neutral/basic conditions. This is significant for the specific drug release to cancer cells because of their acid internal environment, and before that few leakage ensures the low number of side effects. It is a simple way to make sure the chemotherapy is targeted. Under NIR (808 nm) irradiation, the photothermal effect derived from using Fe₃O₄ NPs cannot only induce the PTT but can also enhance the intercellular drug release. The synergistic effect of chemotherapy and PTT is expected to reveal enhanced cytotoxicity to HeLa cells.

Scheme 1 Schematic illustration of the synthesis and the controlled release process.

Materials and methods

Materials

Cetyltrimethyl ammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), DOX, N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), (3-aminopropyl)triethoxysilane (APTES), fluorescein isothiocyanate (FITC) and folic acid (FA) were obtained from Aladdin (China). Hydroxy-2,2-dimethylpropionic acid was purchased from the Tokyo Chemical Industry (TCI).

Instruments

Samples were characterized using transmission electron microscopy (TEM; Hitachi H-8100). X-ray patterns (XRD; Siemens D5005 X-ray diffractometer) were recorded using Cu Kα radiation (40 kV, 30 mA). Fourier transform infrared (FTIR; PerkinElmer 580B infrared spectrophotometer) spectra were recorded using the potassium bromide pellet technique. The nitrogen (N₂) adsorption/desorption isotherms were measured using a Micromeritics ASAP 2010 M system. The surface areas were obtained by using the Brunauer–Emmett–Teller (BET) method, the pore parameters were obtained using the Barrett–Joyner–Halenda method. A ultraviolet-visible (UV-Vis) spectrum was used to determine the amount of drug loading and release (Shimadzu UV-2550 spectrophotometer). Zeta potential measurements were carried out using a Brookhaven Instruments NanoBrook ZetaPALS zeta potential analyzer.

Synthesis of Fe₃O₄@mSiO₂ nanoparticles

NPs of Fe₃O₄@mSiO₂ were synthesized using a method previously reported by Yang et al.³⁰ Firstly, 500 μL of the Fe₃O₄ nanocrystals in chloroform (5 mg mL⁻¹) were poured into 4 mL of 0.2 M aqueous CTAB solution and the resulting solution was stirred vigorously at a temperature of 32 °C. Secondly, the mixture was heated up to 60 °C for about 1 h, to evaporate the chloroform. Then, 4 mL of distilled water was added to the black solution obtained and the pH value of the mixture was adjusted to 9–10 using 0.1 M sodium hydroxide. After that, 100 μL of 20% TEOS in ethanol was injected into the mixture, three times at 30 min intervals. The reaction mixture was reacted for 24 h under vigorous stirring. The Fe₃O₄@mSiO₂ obtained was centrifuged and washed with ethanol several times to remove the excess precursors. Then, the products collected were extracted for 3 h with a 1 wt% solution of sodium chloride in methanol at room temperature to remove the CTAB template.

Photothermal testing

A portion (1.5 mL) of Fe₃O₄@mSiO₂ (0.25 mg mL⁻¹) aqueous solution was added to a cuvette and irradiated with a laser at 808 nm at 2, 4 and 6 W cm⁻². The temperatures were recorded at 2 min intervals using a fiber coupling laser system (FC-W-808, Changchun New Industries Optoelectronics Technology Co., Ltd. China).

Drug loading

Fe₃O₄@mSiO₂ (150 mg) and DOX (10 mg) were added to the ethanol solution (10 mL) and stirred at 25 °C for 12 h. Then, different amounts (100, 200, or 300 μL) of the PO linker (Fig. S1 and S2; ESI†) were added to the mixed solution for 6 h and then washed once with ethanol solution and dried at room temperature under vacuum. The loading amount of DOX was determined using UV-Vis measurement at 480 nm. The experiment was repeated three times. The encapsulation efficiency (EE) and loading efficiency (LE) was calculated based on eqn (1) and (2):

Synthesis of FA-labeled DOX-Fe₃O₄@mSiO₂-PO

Firstly, 1 mmol of FA dissolved in 2 mL of dimethyl sulfoxide (DMSO). Then, 1 mmol of EDC, 1 mmol of NHS and 2 mmol of APTES were added and stirred at room temperature for 24 h to obtain APTES–FA. Subsequently, 60 mg of DOX-Fe₃O₄@mSiO₂-PO was dispersed in ethanol (5 mL) and the APTES–FA solution (100 μL) was added and the mixture was stirred for 12 h.

Drug release

Briefly, DOX-Fe₃O₄@mSiO₂-PO (30 mg) was dispersed in phosphate buffered saline (PBS; 5 mL) at pH 5.0 or 7.4 and then sealed in a dialysis bag, which was submerged in 50 mL of media solution at 37 °C with gentle shaking. At selected time intervals, a portion of the solution was taken out to determine the amount of drug released using UV-Vis analysis at 480 nm.

Cell culture

HeLa cells were grown in a monolayer in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum and penicillin/streptomycin (100 U mL⁻¹ and 100 μg mL⁻¹, Gibco) in a humidified 5% carbon dioxide atmosphere at 37 °C.

Cell viability

The viability of cells in the presence of NPs was investigated by using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The assay was carried out in quintuplicate in the following manner. For the MTT assay, HeLa cells were seeded into 96-well plates at a density of 1 × 10⁴ per well in 100 μL of media and grown overnight. The cells were then incubated with various concentrations of samples for 24 h. Following this incubation, cells were incubated in the media that contained MTT (0.5 mg mL⁻¹) for 4 h. The medium was replaced with 150 μL of DMSO per well and the absorbance was monitored with a microplate reader (Bio-Tek ELx800) at a wavelength of 490 nm. The cytotoxicity was expressed as the percentage of cell viability relative to the untreated control cells.

Cellular uptake

To check cellular uptake and DOX release, HeLa cells were cultured in cover glass bottom dishes (35 mm × 10 mm) in incubation medium (DMEM) for 24 h and then treated with FITC modified NPs at the same final concentration of 500 μg mL⁻¹. After incubation for 6 h, the media were removed, and the cells were then washed twice with PBS. A portion of 4′,6-diamidino-2-phenylindole (DAPI; 0.5 mL) in PBS (10%) was added and incubated for 15 min to stain the nuclei and fix the cells. After the incubation, the cells were washed gently six times with PBS to remove the excess DAPI. Then, 1 mL of the PBS solution was added and the cells were visualized using a Leica Microsystems DFC450 C digital microscope camera with a 10× objective.

Results and discussion

TEM was used to reveal the microstructure of the samples. As shown in Fig. 1A, it was revealed that the Fe₃O₄ NPs had a monodisperse spherical morphology with a unified size of 20 nm. After coating the silica shell, the core–shell structure of Fe₃O₄@mSiO₂ can be seen clearly in Fig. 1B and all the NPs reveal a mesoporous silica shell of about 15 nm in size. Additionally, the hydrodynamic diameter of Fe₃O₄@mSiO₂ centers were found to be ∼87 nm using dynamic light scattering (DLS) measurements (Fig. S4, ESI†). This is larger than that observed from TEM, and results from the hydrate layer of Fe₃O₄@mSiO₂ in an aqueous environment. In Fig. 1C, the wide-angle XRD pattern of Fe₃O₄@mSiO₂ exhibits a broad peak at 22.6°, which is assigned to the amorphous SiO₂ network. All the rest of the diffraction peaks are in good agreement with those of standard Fe₃O₄ (JCPDS card no. 65-3107). The low-angle XRD pattern of Fe₃O₄@mSiO₂ in Fig. 1D(a) displays the three diffraction peaks corresponding to the (100), (110) and (200) planes of a two-dimensional hexagonal mesoporous structure. Furthermore, after the drug loading and the grafting of the PO linker, the diffraction intensities of DOX-Fe₃O₄@mSiO₂-POs undergo an obvious decrease that is consistent with that recoded in previous reports.^17,31,32 Increasing the amount of PO linker, the diffraction intensity decreases (Fig. 1D).

Fig. 1 (A) TEM image of Fe₃O₄ and high resolution TEM image of Fe₃O₄. (B) Typical TEM image of Fe₃O₄@mSiO₂. (C) Wide-angle XRD pattern of Fe₃O₄@mSiO₂. (D) Low-angle XRD patterns of Fe₃O₄@mSiO₂ (a), DOX-Fe₃O₄@mSiO₂-PO1 (b), DOX-Fe₃O₄@mSiO₂-PO2 (c) and DOX-Fe₃O₄@mSiO₂-PO3 (d).

N₂ adsorption–desorption was carried out to further investigate the porous structure of these samples. The corresponding isotherms and pore size distribution curves are shown in Fig. 2. From Fig. 2A, all samples show the typical type IV isotherm curves with H1 hysteresis loops, suggesting the uniform mesoporous structure that agrees with the previously discussed TEM and XRD data. Additionally, DOX-Fe₃O₄@mSiO₂-POs exhibit decreased adsorption compared with that of Fe₃O₄@mSiO₂ because of the drug loading and PO graft. That also induces the decline of pore size from 2.72 nm of Fe₃O₄@mSiO₂ to 2.33 nm of DOX-Fe₃O₄@mSiO₂-PO3 as displayed in Fig. 2B. Also, the BET surface area and pore volume decreases from 614 m² g⁻¹ and 0.18 cm³ g⁻¹ of Fe₃O₄@mSiO₂ to 224 m² g⁻¹ and 0.14 cm³ g⁻¹ of DOX-Fe₃O₄@mSiO₂-PO1, to 180 cm³ g⁻¹ and 0.13 cm³ g⁻¹ of DOX-Fe₃O₄@mSiO₂-PO2, to 160 cm³ g⁻¹ and to 0.10 cm³ g⁻¹ of DOX-Fe₃O₄@mSiO₂-PO3, respectively. With most amounts of PO linker, DOX-Fe₃O₄@mSiO₂-PO3 encapsulates most of the DOX, which gives the lowest porous parameters (Table 1).

Fig. 2 (A) N₂ adsorption–desorption isotherms and (B) pore diameter distribution cures of Fe₃O₄@mSiO₂ (a), DOX-Fe₃O₄@mSiO₂-PO1 (b), DOX-Fe₃O₄@mSiO₂-PO2 (c) and DOX-Fe₃O₄@mSiO₂-PO3 (d).

Table 1 The porous parameters of the samples

Sample	EE (wt%)	LE (wt%)	BET (m^2 g^−1)	Pore volume (cm^3 g^−1)	Pore diameter (nm)
Fe3O4@mSiO2			614	0.18	2.72
DOX-Fe3O4@mSiO2-PO1	40.1 ± 1.2	3.8 ± 0.3	224	0.14	2.69
DOX-Fe3O4@mSiO2-PO2	41.6 ± 1.0	4.2 ± 0.4	180	0.13	2.44
DOX-Fe3O4@mSiO2-PO3	44.8 ± 1.3	4.5 ± 0.4	160	0.10	2.33

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FTIR spectrometry was used to characterize the functional modification and drug loading of the system. From Fig. 3A(a) and (c), the peaks at 1091, 799 and 466 cm⁻¹ can be ascribed to the stretching and deformation vibrations of Si–O.³³ In Fig. 3A(b), the peak at 1725 and 909 cm⁻¹ corresponds to the C=O stretching vibration and O–H bending vibration of carboxylic acid. The absorption band at 1094 cm⁻¹ is caused by the C–O–C stretching vibrations. It is further confirmed that the PO linker has been modified on Fe₃O₄@mSiO₂. Furthermore, the typical band at 1394 cm⁻¹ because of the C=C stretching vibration of benzene, implies the loading of DOX as shown in Fig. 3A(c). In addition, the zeta potentials of the NPs were determined to further monitor the surface change.³⁴ Fe₃O₄@mSiO₂ shows the negative zeta potential (−11.17 ± 0.65 mV) which arises from the surface of the Si–OH which has a negative charge. After the graft of the PO linker, the zeta potential increases to −1.74 ± 0.65, 1.29 ± 1.67, and 4.27 ± 0.73 mV for DOX-Fe₃O₄@mSiO₂-PO1, DOX-Fe₃O₄@mSiO₂-PO2, and DOX-Fe₃O₄@mSiO₂-PO3, respectively, because of the replacement of the surface Si–OH with the PO linker.

Fig. 3 (A) FTIR spectra of Fe₃O₄@mSiO₂ (a), 3,3′-(propane-2,2-diyl bis(oxy))bis(2,2-dimethylpropanoic acid) (b) and DOX-Fe₃O₄@mSiO₂-PO (c). (B) Zeta potential of Fe₃O₄@mSiO₂ (S₀), DOX-Fe₃O₄@mSiO₂-PO1 (S₁), DOX-Fe₃O₄@mSiO₂-PO2 (S₂), DOX-Fe₃O₄@mSiO₂-PO3 (S₃) and S₁, S₂, S₃ in different media.

Magnetization characterization of these samples was carried out at 300 K (Fig. 4). All the samples possessed superparamagnetism, and the as-synthesized Fe₃O₄ NPs show the highest saturation magnetization of 60.12 emu g⁻¹. After the assembly process, the saturation magnetization drops to 43.66 (Fe₃O₄@mSiO₂), 34.92 (DOX-Fe₃O₄@mSiO₂-PO1), 29.17 (DOX-Fe₃O₄@mSiO₂-PO2), 23.49 emu g⁻¹ (DOX-Fe₃O₄@mSiO₂-PO3) because of the assembly of non-magnetic components, including the DOX and PO linker. Considering the good superparamagnetism obtained, these NPs can be used in targeting the point of drug delivery.

Fig. 4 Magnetization curves of Fe₃O₄ (a), Fe₃O₄@mSiO₂ (b), DOX-Fe₃O₄@mSiO₂-PO1 (c), DOX-Fe₃O₄@mSiO₂-PO2 (d) and DOX-Fe₃O₄@mSiO₂-PO3 (e).

DOX was used as the model drug to evaluate the loading and the pH sensitive controlled release kinetics. The DOX release profiles of DOX-Fe₃O₄@mSiO₂-POs nanocomposites under different pH conditions (pH 5.0 and 7.4) are displayed in Fig. 5. From Fig. 5A, the release is inhibited at pH 7.4. After 48 h, the release is about 10.30–24.14% for DOX-Fe₃O₄@mSiO₂-POs. When the pH value decreases to 5.0, the release is markedly enhanced. As shown in Fig. 5A, after about 6 h the DOX release increases to 30.20%, 26.97%, and 20.20% for DOX-Fe₃O₄@mSiO₂-PO1, DOX-Fe₃O₄@mSiO₂-PO2, and DOX-Fe₃O₄@mSiO₂-PO3, respectively, at pH 5.0. After about 48 h, those values can reach 80.23%, 70.23%, and 58.82%, DOX-Fe₃O₄@mSiO₂-PO1, DOX-Fe₃O₄@mSiO₂-PO2, and DOX-Fe₃O₄@mSiO₂-PO3, respectively. In order to further investigate the pH sensitive drug release, the release process of DOX from DOX-Fe₃O₄@mSiO₂-PO2 at pH 7.4 for the first 24 h and then at pH 5.0 was also studied and the results of this are displayed in Fig. 5B. In the first 24 h, the release just reaches 15.23%, after that the release is improved to 70.23% at 48 h. Furthermore, the release behaviors of DOX-Fe₃O₄@mSiO₂ have also been studied. From Fig. S3 (ESI†), without the PO linker coating DOX was released more freely (84.34% at 48 h), and the distinct difference of release between pH 5.0 and pH 7.4 cannot be found. Thus, the acid enhanced DOX release can be ascribed to the PO linker. As shown in Scheme 1, PO linkers possess a pH sensitive (ketal) group and their cleavage under acid conditions insures that the “gate” is open and drug can be released. However, at alkaline conditions, the stability of the PO linker prevents drug diffusion. The acid enhanced drug release is significant for specific chemotherapy for anti-cancer treatment, because there are acid conditions in most cancer cells. Before that, the few leakage also means that there are low side effects. In addition, the pH sensitive performances of these nanocomposites were further monitored using zeta potential measurement. From Fig. 3B, the zeta potential of DOX-Fe₃O₄@mSiO₂-PO1, DOX-Fe₃O₄@mSiO₂-PO2, and DOX-Fe₃O₄@mSiO₂-PO3 decreases to −6.94, −5.50, and −4.04 mV, respectively, after treatment under acidic media (pH 5.0) because of the broken bond of PO linker which leaves a negative –OH outside (Scheme 1). Nevertheless, compared with the as-synthesized samples, the negligible change of the zeta potentials can be seen at pH 7.4, implying the stability of PO linker in alkaline media.

Fig. 5 Release profiles of DOX from (A) DOX-Fe₃O₄@mSiO₂-PO1 (a and d), DOX-Fe₃O₄@mSiO₂-PO2 (b and e), DOX-Fe₃O₄@mSiO₂-PO3 (c and f) at pH 5.0 (a–c) and 7.4 (d–f). (B) Cumulative release of DOX from DOX-Fe₃O₄@mSiO₂-PO2 at pH 7.4 for the first 24 h and then at pH 5.0. (C) Temperature change of Fe₃O₄@mSiO₂ irradiated by NIR laser with different powers (2, 4, or 6 W cm⁻²). (D) Cumulative release rates of DOX from DOX-Fe₃O₄@mSiO₂-PO2 at different pH media with NIR irradiation (808 nm, 2.0 W cm⁻², on/off) and dark conditions.

As mentioned previously, it is PO linker which makes sure that the pH sensitive drug is released. The amount of PO linker can also influence the release performance. From Fig. 5A, it can be seen that by increasing the PO amount, the DOX release slows down. Too much PO linker induces a small leakage of DOX-Fe₃O₄@mSiO₂-PO3 at pH 7.4 (10.30%), but it also gives a low DOX release amount (58.82%) at pH 5.0. However, insufficient PO linker would induce much leakage of DOX-Fe₃O₄@mSiO₂-PO1 at pH 7.4 (24.14%). As shown in Fig. 5A, DOX-Fe₃O₄@mSiO₂-PO2 is considered to be the right sample for exhibiting the appropriately controlled DOX release performance.

Besides, the magnetic targeting of Fe₃O₄ NPs, they can also be used as NIR photoabsorbers for PTT. So the photothermal effect of Fe₃O₄@mSiO₂ NPs under 808 nm NIR with different photoirradiation power was investigated and the results are shown in Fig. 5C. In marked contrast to pure water, the Fe₃O₄@mSiO₂ solution show a rapid increase of temperature from 36.9 to 43.2 ± 0.4 °C (2 W cm⁻²) at 8 min, and then the temperature tends to steady to 46.8 ± 0.2 °C at 14 min. Whereas the temperature just increases by 1.4 °C for pure water at the same times. It is believed that the elevation of temperature is because of the photothermal effect of Fe₃O₄. Additionally, by raising the photoirradiation power, the heating rate increases, and the final temperature can increase to 51.0 ± 0.4 °C (4 W cm⁻²) and 52.3 ± 0.2 °C (6 W cm⁻²) at 14 min. With the fast photothermal effect, these NPs can be used to carry out PTT for anti-cancer treatment. Furthermore, the influence of the photothermal effect on the drug release was also studied. From Fig. 5D, it can be see that under NIR irradiation the release rate obviously increases at pH 5.0 with the final release amount close to that obtained without NIR irradiation. However, there is not a particular difference between the release behavior at pH 7.4 with and without NIR irradiation. In view of the results shown in Fig. 5D, it can be seen that the increased temperature would improve the diffusion of DOX molecules (the release rate) but that it cannot induce the degradation of the PO linker to enhance the amount released.

To observe the cell uptake and subsequent localization of the samples, DOX-Fe₃O₄@mSiO₂-PO and DOX-Fe₃O₄@mSiO₂-PO-FA were incubated with HeLa cells for 3 h and 0.5, 1, and 3 h, and the fluorescence images were recorded. The blue fluorescence from the DAPI was used to mark the nucleus and the red emission from DOX was for tracking the carrier. From Fig. 6, for the results obtained with incubation for 0.5 h, some NPs can be observed in the cytoplasm through endocytosis or macropinocytosis. The fast uptake by HeLa cells is because of the small nanoparticle size (about 50 nm) and the excess expression of the FA receptor on the tumor cell.²¹ Without FA modification, DOX-Fe₃O₄@mSiO₂-PO exhibits decreased uptake by HeLa cells. By extending the incubation time to 3 h, more and more red fluorescence is seen in the nucleus, and this is ascribed to the acid enhanced DOX release in cancer cells which is helpful in improving the drug efficacy.

Fig. 6 Fluorescence images of HeLa cells after incubation for 0.5, 1, 3 h with DOX-Fe₃O₄@mSiO₂-PO-FA and for 3 h with DOX-Fe₃O₄@mSiO₂-PO. For each panel, the images from left to right show the DAPI labeled cell nucleus (blue), DOX fluorescence (red), and then a merged image.

The cell cytotoxicity of these samples against HeLa cells via MTT assay was verified in vitro. From the results shown in Fig. 7A, Fe₃O₄@mSiO₂-FA exhibits negligible cytotoxicity, and even when the concentration increases to 125 μg mL⁻¹, the cell viability can remain above 91.20 ± 0.73%, indicating the potential application of Fe₃O₄@mSiO₂-FA NPs as a nanocarrier in biopharmacy applications. In addition, DOX-Fe₃O₄@mSiO₂-PO-FA demonstrates a decreased cell viability (54.10 ± 1.10%), which is ascribed to the acid enhanced intracellular DOX release. The cell viability using free DOX was also determined as a comparison. From Fig. 7B, it is shown that DOX-Fe₃O₄@mSiO₂-PO-FA reveals the attenuated cell viability in compared with that of free DOX (with equivalent amount), ascribed to the high uptake of the nanocomposite by HeLa cells because of endocytosis, followed by the sensitive release of DOX inside the endosomal compartment. Furthermore, when NIR (808 nm, 2.0 W cm⁻²) irradiation is introduced, the cell viability further drops to 45.70 ± 1.23% with irradiation for 10 min. With prolonged irradiation time of 30 min, the cell viability further decreases to 19.20 ± 1.47%, and the enhanced cell cytotoxicity is derived from the synergistic action of chemotherapy and PTT.

Fig. 7 (A) Cell viability of HeLa cells incubated with different amounts of Fe₃O₄@mSiO₂-FA and DOX-Fe₃O₄@mSiO₂-PO-FA for 24 h. (B) Inhibition of HeLa cell growth in the presence of DOX-Fe₃O₄@mSiO₂-PO-FA, free DOX (with an equivalent amount) and irradiated with NIR (808 nm, 2.0 W cm⁻²) for different times.

Conclusion

In this research, a pH sensitive drug release and an NIR-induced PTT nanocomposite based on Fe₃O₄@mSiO₂ NPs has been successfully developed. Studies with HeLa cells revealed the rapid uptake of the nanocomposite of the host. Furthermore, the MTT assay shows that Fe₃O₄@mSiO₂-PO has good biocompatibility, and will not be harmful to the human body. Furthermore, the system can use for PTT. Therefore, the system has potential for targeted anti-cancer and drug delivery in cancer therapy on the basis of this investigation.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (21471041, 21571045), and College Youth Innovation Talents Training Program UNPYSCT-2015053.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra23292b

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