NIR-sensitive UCNP@mSiO₂ nanovehicles for on-demand drug release and photodynamic therapy

Abstract

Nanocomposites have attracted the most attention for antitumor treatment. Here, we present a near-infrared (NIR) sensitive nanovehicle to reveal synergistic chemotherapy and photodynamic therapy (PDT). Upconverting nanoparticles (UCNP) NaYF₄:Yb, Tm@NaYF₄ were adopted as the core using a one-step coprecipitation method to realize the coating of the mesoporous silica shell and the doping of photosensitizer Hypocrellin A (HA). Furthermore, a UV light-cleavable 4-(2-carboxy-ethylsulfanylmethyl)-3-nitro-benzoic acid linker (CNBA) was synthesized and grafted outside as a “gate” to insure the encapsulation of the anticancer drug doxorubicin (DOX). Upon NIR irradiation, the UV light emission (derived from UCNP) can induce the break of the CNBA linker to make the “gate” open and cause drug release. Besides, the blue emission (450–470 nm) can excite HA to generate reactive oxygen (ROS) to achieve PDT. Owing to the nanoscale particle size (75 nm) and targeting transferrin (Tf) modification, these nanocomposites possess fast uptake by cancer cells (HeLa and MCF-7) and the enhanced cytotoxicity is derived from the synergistic effect of chemotherapy and PDT that would easily be controlled by the acting strength and time of NIR irradiation. Hence, the NIR light-sensitive nanocomposites are expected to be the promising and flexible platform for cancer treatment.

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Introduction

During the past 50 years, significant effort has been devoted to find effective means for antitumor treatment. Today the most common approach to treat cancer is chemotherapy, but it is well known to have several severe and general problems such as toxic side effects and drug resistance. Nanotechnology has emerged in the field of medicine to overcome many limitations of classic chemotherapy and is considered as the best potential strategy for anti-cancer therapy. Recently, liposomal, albumin, chitosan, ceramic, and metal-based nanoparticles are engineered to load antitumor drug to induce the drug molecules to be transported into the location of diseases directly and accurately, insuring the target anti-cancer therapy. Mesoporous silica nanoparticles (MSNs) have become a potential delivery vehicle owing to their advantageous structural features, such as high internal surface area/pore volume, tunable pore sizes, stability, and modifiable surface property, which enable the high drug loading and various functionalization.^1–5 Additionally, many external (e.g. temperature, light, electronic, magnetic) and internal (e.g. pH, biomolecules and enzymes) stimuli have been employed to construct stimuli-responsive release MSNs system.^6–20

Among those available stimuli means, the light is viewed as one of the most possible methods to achieve the microenvironment target release as the position, strength, and time of the action can be handled conveniently. Based on the previous reports, the light-sensitive release always requires light with high energy, such as UV and visible light, to induce the bond breaking or isomerism. However, the toxic of the UV light and the deficient tissue penetration of visible light restrict the application of the light on biomedicine, especially on the deep tissue treatment. The lanthanide upconversion nanoparticles (UCNPs) are able to absorb near-infrared (NIR) light and convert it into UV-vis emission. With much less damage and deeper tissue penetration of NIR light, UCNPs are widely used on NIR acting bioimaging. UCNPs as core and mesoporous silica as shell, the UCNP@mSiO₂ based-nanocomposites possess the advantage of both PDT and always adopted in stimuli-responsive release.^21–23 For instance, Retama and co-workers designed a drug delivery system that based on UCNP@mSiO₂ nanoparticles and functionalized with ortho-nitrobenzylalcohol derivate molecules to release cargo upon NIR light illumination.²⁴ Chen and co-workers designed and fabricated a UV light-responsive UCNP@mSiO₂ release system by controlling the wetting behavior of the surface of mesoporous silica.²⁵

Furthermore, besides the sensitive drug release, the light also can promote the photodynamic therapy (PDT). When the photosensitizer is excited by a certain light, the generation reactive oxygen (ROS) would cause the irreversible damage to cancer tissue to achieve PDT. Based on the previous reports, there are some disadvantages for the development of PDT on anticancer treatment, including the proper irradiation, the poor chemical stability/water-solubility and lack of discrimination between the normal and tumor site of photosensitizers. Currently, some carriers, mainly polymers, micelles and graphene, were used to help them to gather into the focal site. Later, some photosensitizers also were covalently linked to silica nanoparticles. Wang et al. developed a heavy atom (I) containing dipyrromethene boron difluoride doped nanoparticles by covalent incorporation method. It can enhance the generation efficiency of ROS and reduce dye leakage and subsequently unwanted side effects.²⁶

Currently, many anti-cancer investigations focus on the either chemotherapy or PDT. To further enhance therapy efficacy, the combination of them would be expected to be an effective method. In this study, we present a functional UCNP@mSiO₂ nanocomposite to show the NIR-sensitive PDT and drug release synchronously as shown in Scheme 1. Here, NaYF₄:Yb, Tm@NaYF₄ was utilized as core which can produce the UV and visible light emission under 980 nm NIR irradiation. Firstly, Hypocrellin A (HA) was chose as photosensitizer, because the blue light emission (450–470 nm) can excite HA to generate ROS to achieve PDT. Herein, a coprecipitation method was used to obtain HA doped mesoporous silica shell (UCNP@mSiO₂/HA) on one-step. It is a possible way to overcome some of the above defects to improve PDT. Considering the novel UV emission, a UV cleavable o-nitrobenzyl derivative 4-(2-carboxy-ethylsulfanylmethyl)-3-nitro-benzoic acid (CNBA) was designed and synthesized (Scheme S1 and Fig. S1†), and the novel structure feature makes it can be connected outside UCNP@mSiO₂ by a simple silane coupling reaction to seal the doxorubicin (DOX, the model anticancer drug). Under the NIR irradiation, the UV light emission can induce the breaking of the CNBA linker to make the “gate” open and drug release. It is the single light source to induce the chemotherapy and PDT in the meantime. Meanwhile, to promote receptor-mediated endocytosis,²⁷ transferrin (Tf) have been chosen as the ideal active targeting agent because their receptors (TfR) were found to be more widely distributed on the tumor tissues than the normal tissues. In addition, transferrin is nonimmunogenic and can be conjugated without losing its biological activity.²⁸ And the detail cell experiments further reveal by used using HeLa and MCF-7 as typical cancer cells and 293T as model normal cells. The enhanced specific cytotoxicity is ascribing to the improved uptake by cancer cells and the synergistic action of chemotherapy and PDT that makes its potential application on anticancer therapy.

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

Materials and methods

Materials

Unless specified otherwise, all of the chemicals used were of analytical grade and used without further purification. Yttrium(III) chloride hexahydrate (YCl₃·6H₂O), ytterbium(III) chloride hexahydrate (YbCl₃·6H₂O), thulium(III) chloride hexahydrate (TmCl₃·6H₂O), ammonium fluoride, oleic acid, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), tetraethylorthosilicate (TEOS), (3-aminopropyl)triethoxysilane (APTES), 3-(triethoxysilyl)propyl isocyanate, 3-mercaptopropionic acid and 4-bromomethyl-3-nitrobenzoic acid were all purchased from Aladdin. 1,3-Diphenylisobenzofuran was taken on J&K. Hypocrellin A was purchased from Chengdu Biopurify Phytochemicals Ltd. 1-Octadecene and transferrin were obtained from Sigma-Aldrich.

Instruments

Samples were characterized by transmission electron microscopy (TEM Hitachi H-8100) at an accelerating voltage of 20 kV. X-ray diffraction (XRD) data is characterized using a Siemens D5005 diffractometer with Cu Ka radiation at 40 kV and 30 mA. The fluorescence spectra were surveyed by HORIBA FL-3. A Fourier transform infrared (FT-IR) spectroscopy spectrometer (JASCOFT/IR-420) was used to record the infrared spectra of mesoporous materials. Zeta potential was carried out on the Brookhaven. Ultraviolet-visible (UV-vis) spectra were taken on a Lambda 45 spectrophotometer. The isotherms of N₂ adsorption/desorption were measured at the temperature of liquid nitrogen using a Micromeritics ASAP 2010M system. The pore size distributions were calculated from the adsorption branches of the N₂ adsorption isotherms using the Barrett–Joyner–Halenda model.

Synthesis of NaYF₄:Yb, Tm and NaYF₄:Yb, Tm@NaYF₄ nanocrystals (UCNP)

In a typical synthesis of monodisperse NaYF₄:Yb, Tm (20/0.2 mol%), YCl₃·6H₂O (1.59 mmol), YbCl₃·6H₂O (0.40 mmol), and TmCl₃·6H₂O (0.01 mmol) in deionized water were added to a 100 mL flask containing 15 mL oleic acid and 30 mL 1-octadecene. The solution was stirred at room temperature for 1 h. Then the mixture was slowly heated to 120 °C to get rid of water under argon atmosphere, and maintained at 156 °C for about 1 h until a homogeneous transparent yellow solution was obtained. The system was then cooled down to room temperature with the flowing of argon. Then 10 mL methanol solution of NH₄F (296.3 mg, 8 mmol) and NaOH (200 mg, 5 mmol) was added and the solution was stirred at room temperature for 2 h. After methanol evaporated, the solution was heated to 290 °C and kept for 1.5 h before it was cooled down to room temperature. The mixture were first precipitated by the addition of 20 mL ethanol, and collected by centrifugation at 10 000 rpm for 10 min. Product was re-dispersed with 5 mL cyclohexane and precipitated by adding 15 mL ethanol, then collected by the same centrifugation. After four times' washing, the final product was re-dispersed in 20 mL cyclohexane.

For the synthesis of NaYF₄:Yb, Tm@NaYF₄ nanocrystal, about 1.0 mmol NaYF₄:Yb, Tm was firstly prepared using the similar procedures as mentioned above. Then, 800 μmol YCl₃·6H₂O in water solution was added in to a 100 mL flask containing 15 mL oleic acid and 30 mL 1-octadecene. The solution was stirred at room temperature for 1 h. Then the mixture was slowly heated to 120 °C to get rid of water under argon atmosphere, and maintained at 156 °C for about 1 h until a homogeneous transparent yellow solution was obtained. The system was then cooled down to room temperature with the flowing of argon. Then, 5 mL pre-prepared NaYF₄:Yb, Tm core (dispersed in cyclohexane) was added and kept for another 30 min before heated to 80 °C to remove cyclohexane. Then, 10 mL methanol solution of NH₄F (1 mmol) and NaOH (1.685 mmol) was added and the solution was stirred at room temperature for 2 h. After methanol evaporated, the solution was heated to 270–280 °C and kept for 1.5 h before it was cooled down to room temperature. The same washing steps were followed and sample was re-dispersed in 5 mL cyclohexane.

Synthesis of HA linker and UCNP@mSiO₂/HA

Briefly, 54.65 mg (0.1 mmol) of hypocrellin A were dissolved in 2 mL tetrahydrofuran at room temperature. Then, 50 μL (0.2 mmol) of 3-(triethoxysilyl)propyl isocyanate (TPI) were added to the solution, the reaction mixture was heated to 50 °C with the flowing of argon and stirred for 12 h.

As-synthesized OA coated UCNPs were transferred to an aqueous phase using cationic surfactant CTAB. A typical formation process of mesoporous silica coating was performed by self-assembly of the CTAB and silica precursor TEOS in basic solution. In a typical procedure, 0.5 mL of the prepared UCNPs in chloroform was poured into 5 mL of aqueous CTAB solution (0.2 M), and the resulting turbid oil-in-water microemulsion was stirred and sonicated vigorously for 1 h. Then, the mixture was heated up to 70 °C and aged for 30 min under stirring to evaporate the chloroform, resulting in a transparent solution. When the temperature was stable, the resulting solution was added to a mixture of water (5 mL), NaOH solution (150 μL, 0.1 M), 150 μL of 20% TEOS in ethanol and different amounts of 30, 45, 60 μL HA-linker solution (0.05 M) solution were added at 30 minute intervals and stirred for 24 h. It is noteworthy that TEOS should be added dropwise and the mixture must be stirred heavily. After the reaction was completed, the as-synthesized UCNP@mSiO₂/HA nanoparticles were washed 2 times with ethanol to remove the unreacted species. Then, the collected products were extracted for 3 h with a 1 wt% solution of sodium chloride (NaCl) in methanol at room temperature to remove the template CTAB.

Drug loading and Tf conjugation

UCNP@mSiO₂/HA (60 mg) and DOX (3 mg) were added to the methanol solution (10 mL) and stirred at 25 °C for 12 h. And then 20, 40 and 60 μL supernatant fluid of CNBA-APTES was added to the mixed solution. The obtained solid (named as UCNP@mSiO₂/HA-DOX@CNBA1, UCNP@mSiO₂/HA-DOX@CNBA2, and UCNP@mSiO₂/HA-DOX@CNBA3, respectively) was centrifuged, and washed several times with ethanol solution. The loading amount of DOX was determined by the UV/vis spectroscope at 480 nm. The loading efficiency (LE wt%) of DOX can be calculated by using the formula (1). The experiment repeated three times.

Then, the nanocomposites were activated by carboxyl firstly. In a typical reaction, 100 mg UCNP@mSiO₂/HA-DOX@CNBA was dispersed in 20 mL of toluene and 50 μL of APTES added to the solution. The solution was heated to 50 °C and stirred for 4 h with the flowing of argon. Then, we can get the amino-functional nanocomparticles. Next, 50 mg of as-prepared product was dissolved in 10 mL of DMSO. 15 mg of succinic anhydride and 15 mg of triethylamine were added to the solution. The reaction mixture was reacted for 48 h under stirring at 40 °C. After the reaction was completed, the nanoparticles were washed 2 times with ethanol to remove the unreacted species.

30 mg as-synthesized nanoparticles, 19.17 mg of EDC, 11.51 mg of NHS and 15 mg of Tf were added to 2 mL of dimethyl sulfoxide, the reaction mixture was reacted for 24 h under stirring at room temperature. At last, the obtained UCNP@mSiO₂/HA-DOX@CNBA nanocomposites were washed 2 times with ethanol to remove the unreacted species.

Release in vitro

Photo-triggered release of DOX in solution was done by mixing 5 mg UCNP@mSiO₂/HA-DOX@CNBAs with 5 mL PBS buffer solution and the solution was irradiated with a 980 nm NIR laser for different time duration at different power density. Control experiment was done without any NIR irradiation.

Cell culture

HeLa cells (cervical cancer cell line) were grown in monolayer in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Tianhang Bioreagent Co., Zhejiang) and penicillin/streptomycin (100 U mL⁻¹ and 100 μg mL⁻¹, respectively, Gibco) in a humidified 5% CO₂ atmosphere at 37 °C.

Cell cytotoxicities assay

In vitro cytotoxicity was assessed by the standard MTT assay. HeLa cells were seeded into 96-well plates at a density of 1 × 10⁵ per well in 100 μL of media and cultured in 5% CO₂ at 37 °C for overnight. Then, different NPs were added to the medium. For materials without NIR irradiation, the cells were incubated in 5% CO₂ at 37 °C for 24 h. For the materials with NIR irradiation, the NPs were removed after 6 h of incubation, followed by exposing to different light dosage. Then, the cells were further incubated for 24 h in the dark. Following this incubation, cells were incubated in the media containing 500 μg mL⁻¹ of MTT for 4 h. The medium was then replaced with 150 μL of dimethyl sulfoxide (DMSO) per well and the absorbance was monitored using a microplate reader (WD-2102A) at the wavelength of 490 nm. The cytotoxicity was expressed as the percentage of cell viability compared to untreated control cells. All samples were modified by Tf targeting before cell experiments.

Cellular uptake

To check cellular uptake and DOX release under NIR exposure, HeLa cells were cultured in 6-well plates in incubation medium (DMEM) for 24 h and then treated with FITC modified UCNP@mSiO₂/HA-DOX@CNBA and Tf-UCNP@mSiO₂/HA-DOX@CNBA at the same final concentration of 500 μg mL⁻¹. After the incubation for 6 h, the media were removed, and the cells were then washed twice with PBS. Then, the cells were subjected to various NIR light dosage (different time and different power). After the incubation, the cells were softly washed 3 times with PBS. The fluorescence imaging experiments were carried out on the Leica DFC450C Microsystems Ltd. using a 40× objective.

Flow cytometry

For flow cytometry studies, HeLa cells (1 × 10⁶) were seeded in 6-well culture plates and grown overnight. The cells were then treated with FITC-modified UCNP@mSiO₂/HA-DOX@CNBA and Tf-UCNP@mSiO₂/HA-DOX@CNBA at 37 °C for 6 h. A single cell suspension was prepared consecutively by trypsinization, washing with PBS, and filtration through nylon mesh (300 mesh). Thereafter, the cells were analyzed using a flow cytometer (BD C6) for FITC.

Results and discussion

In this paper, well-dispersed NaYF₄:Yb, Tm (18 nm) and NaYF₄:Yb, Tm@NaYF₄ (22 nm) nanocrystals were prepared (Fig. 1(A) and (B)). The high-resolution TEM (HRTEM) image and XRD patterns (Fig. 1(D)) of these nanocrystals reveal their high crystallinity, and the d-spacing value of 0.51 nm as shown in the inset of Fig. 1(A) is ascribed to d₁₀₀ of hexagonal-phase NaYF₄. Then, the coprecipitation strategy was used to coat these UCNPs with HA doped mesoporous silica shell (UCNP@mSiO₂/HA). From Fig. 1(C), the core–shell nanoparticle is around 74 nm in diameter with 25 nm thickness of shell can be found, obviously. The mesoporous structure of them is further surveyed by small angle X-ray diffraction. As present in Fig. S2,† the diffraction peak at about 2θ = 2.26 from the pattern suggests the mesoporous structure derived from surfactant CTAB.

Fig. 1 TEM images of (A) NaYF₄:Yb, Tm, (B) NaYF₄:Yb, Tm@NaYF₄ (UCNP), (C) UCNP@mSiO₂/HA. (D) Powder X-ray diffraction (XRD) pattern for the NaYF₄:Yb, Tm and NaYF₄:Yb, Tm@NaYF₄ (UCNP) and the calculated line pattern for the hexagonal NaYF₄ phase. Inset: HRTEM image of NaYF₄:Yb, Tm shows distinct lattice fringes with interplanar spacing of 0.51 nm ascribed to the (100) plane of hexagonal NaYF₄.

Fig. 2 shows the fluorescence spectra of pure NaYF₄:Yb, Tm nanocrystals, NaYF₄:Yb, Tm@NaYF₄ (UCNP) and UCNP@mSiO₂ core–shell nanoparticles with the excitation of 980 nm NIR (0.5 W cm⁻²). All of the fluorescence spectra show emission peaks at about 360, 452, 474, and 800 nm, which are assigned to the ¹D₂ → ³H₆, ¹D₂ → ³F₄, ¹G₄ → ³H₆ and ³H₄ → ³H₆ transition from Tm³⁺, respectively. After the growth of NaYF₄ shell, NaYF₄:Yb, Er@NaYF₄ exhibits enhanced fluorescence emission.^29–31 And the decreased fluorescence of UCNP@mSiO₂ due to the light-scattering effect on both emission and incident light by the silica layer.³² The digital photos further clearly display the detectable luminescence, suggesting the potential biomedical applications such as imaging, detection, and sensing of these nanoparticles.^33,34 In addition, with the increase of the irradiation power, the emission is also increased (Fig. S3†).

Fig. 2 Emission spectra of (a) pure NaYF₄:Yb, Tm nanocrystals, (b) NaYF₄:Yb, Tm@NaYF₄ (UCNP) and (c) UCNP@mSiO₂ core–shell nanoparticles. Samples were dispersed in aqueous solution at the same concentration. Inset: the corresponding digital photos of (a), (b) and (c).

Owing to the special luminescence of UCNPs, Hypocrellin A (HA) was adopted as the PS agent because the blue light emission can excite HA to generate singlet oxygen (¹O₂) and to achieve PDT. The singlet oxygen (¹O₂) was assessed in vitro by monitoring the time-dependent photodegradation of diphenylisobenzofuran (DPBF), a widely used singlet oxygen detector. From Fig. 3(A), the typical absorption peak of DPBF at 407 nm significantly decreases as the function of irradiation time because of the efficient generation of ¹O₂ from UCNP@mSiO₂/HA. However, even under the same laser irradiation, DPBF, UCNP@mSiO₂ and mSiO₂/HA can not induce the degradation of DPBF. Based on the investigation, the combination of upconversion fluorescent and HA makes sure the generation of ROS (Fig. 3(B)).

Fig. 3 (A) UV-vis spectra of DPBF after irradiation of the UCNP@mSiO₂/HA (60 μL) 980 nm light (0.5 W cm⁻²) for 0 to 60 min. (B) Decay curves of DPBF absorption at 407 nm in different solutions as a function of irradiation time.

To verify the organic modification and the drug loading, FT-IR spectroscopy was used to study these components of samples. The corresponding FT-IR spectra of (a) UCNP@mSiO₂/HA, (b) UCNP@mSiO₂/HA-DOX, (c) UCNP@mSiO₂/HA-DOX@CNBA, (d) Tf-UCNP@mSiO₂/HA-DOX@CNBA are illustrated in Fig. 4(A). From Fig. 4(A), a broad band in the region of 3435 cm⁻¹ is assigned to the symmetric stretching of Si–OH. Besides, the characteristic peaks at 1087 and 806 cm⁻¹ further confirm the silica network of the samples. The 1720 and 1630 cm⁻¹ peak is ascribed to carboxylate and C=O stretching vibration from HA linker (Fig. 4(A)(a)). After the drug loading, the peaks at 840 and 1460 cm⁻¹ is owing to =C–H bending vibration and C=C stretching vibration of the benzene skeleton in DOX (Fig. 4(A)(b)). As shown in Fig. 4(A)(c), the characteristic absorption peaks of NO₂ at 1525 and 1345 cm⁻¹ approve that CNBA has been introduced successfully. Moreover, the UV-vis absorption spectra of them are recorded as displayed in Fig. 4(B). The typical adsorption peaks of HA (467 nm) and DOX (477 nm) can be found, obviously. Moreover, the change of color and fluorescence as displayed in Fig. S4† further confirms the HA graft and drug loading. In addition, the zeta-potential was further used to monitor the surface charge. From Table 1, the zeta-potential of UCNP@mSiO₂/HA is −19.12 ± 0.52 mV that is derived from the negative charge of the surface Si–OH. And that increases to 8.38 ± 1.92 mV of UCNP@mSiO₂/HA-DOX@CNBA1, 11.66 ± 1.46 mV of UCNP@mSiO₂/HA-DOX@CNBA2 and 14.84 ± 0.68 mV of UCNP@mSiO₂/HA-DOX@CNBA3 due to the decrease of surface Si–OH substituted by CNBA linker. With the increase of the amount of CNBA, the higher value of zeta potential is present.

Fig. 4 (A) FTIR spectra of (a) UCNP@mSiO₂, (b) UCNP@mSiO₂/HA, (c) UCNP@mSiO₂/HA-DOX and (d) UCNP@mSiO₂/HA-DOX@CNBA. (B) The UV/vis spectrum of UCNP, UCNP@mSiO₂/HA, UCNP@mSiO₂/HA-DOX and UCNP@mSiO₂/HA-DOX@CNBA.

Table 1 Zeta potential of UCNP@mSiO₂/HA, UCNP@mSiO₂/HA-DOX@CNBA1, UCNP@mSiO₂/HA-DOX@CNBA2 and UCNP@mSiO₂/HA-DOX@CNBA3 under different conditions

Zeta potential test (mV)	Before NIR irradiation	After NIR irradiation
UCNP@mSiO2/HA	−19.12 ± 0.52	—
UCNP@mSiO2/HA-DOX@CNBA1	8.38 ± 1.92	−13.77 ± 2.08
UCNP@mSiO2/HA-DOX@CNBA2	11.66 ± 1.46	−8.61 ± 0.94
UCNP@mSiO2/HA-DOX@CNBA3	14.84 ± 0.68	−3.83 ± 2.87

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Nitrogen adsorption–desorption analyses were carried out to reveal the pore structure and relative textural properties of UCNP@mSiO₂/HA and UCNP@mSiO₂/HA-DOX@CNBAs. As depicted in Fig. 5(A), all samples possess the typical IV isotherm curves with H1 hysteresis loop, suggesting the cylindrical channel structure of the nanoparticles. Nitrogen adsorption amount of UCNP@mSiO₂/HA-DOX@CNBAs decrease compared with UCNP@mSiO₂/HA, implying that the drug loading and organic modification block some pore of the samples. And the corresponding BET surface area and pore volume also decrease to 203 m² g⁻¹ and 0.34 cm³ g⁻¹, 164 m² g⁻¹ and 0.23 cm³ g⁻¹, 124 m² g⁻¹ and 0.11 cm³ g⁻¹ smaller than the one measured on UCNP@mSiO₂/HA, which is 324 m² g⁻¹ and 0.42 cm³ g⁻¹ (Table 2). Fig. 5(B) reveals the pore size distribution curves of the three samples. As shown in Fig. 5(B), UCNP@mSiO₂/HA-DOX@CNBAs also exhibits the reduced pore size (2.47–2.23 nm), while that of UCNP@mSiO₂/HA centers at 2.60 nm.

Fig. 5 (A) Nitrogen adsorption–desorption isotherms and (B) pore size distribution for (a) UCNP@mSiO₂/HA, (b) UCNP@mSiO₂/HA-DOX@CNBA1, (c) UCNP@mSiO₂/HA-DOX@CNBA2, and (d) UCNP@mSiO₂/HA-DOX@CNBA3.

Table 2 Pore parameters and loading efficiency of the samples

Sample	Surface area (m^2 g^−1)	Pore volume (cm^3 g^−1)	Pore diameter (nm)	LE (wt%)
UCNP@mSiO2/HA	324	0.42	2.60	—
UCNP@mSiO2/HA-DOX@CNBA1	203	0.34	2.47	4.07 ± 0.5
UCNP@mSiO2/HA-DOX@CNBA2	164	0.23	2.29	4.68 ± 0.3
UCNP@mSiO2/HA-DOX@CNBA3	124	0.11	2.23	5.04 ± 0.3

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To demonstrate the photoinduced controlled release behavior of these nanocomposites, UCNP@mSiO₂/HA-DOX@CNBAs were exposed to NIR light irradiation at 0.5 W cm⁻² and the in vitro release profiles were recorded in Fig. 6(A). In dark condition, UCNP@mSiO₂/HA-DOX@CNBA1 releases little cargo (about 18.6%), and the increased “CNBA gate” make sure the fewer prematurity (below 8.1% of UCNP@mSiO₂/HA-DOX@CNBA2 and UCNP@mSiO₂/HA-DOX@CNBA3). However, the release is enhanced obviously upon the NIR light irradiation. From Fig. 6(A), it takes 2 h to reach 54.8%, 48.9%, and 39.3% and about 6 h to reach the maximal amount 61.8%, 57.4%, and 47.9% of UCNP@mSiO₂/HA-DOX@CNBA1, UCNP@mSiO₂/HA-DOX@CNBA2, and UCNP@mSiO₂/HA-DOX@CNBA3, respectively. The photoinduced drug release is ascribed to the UV cleavable CNBA linkers, which are grafted on to the surface of the nanocomposites as the “gate” to insure the drug loading. Upon NIR illumination, UV emission disrupts the CNBA linker to cause “gate” open and drug release. However, without NIR CNBA groups also block the pores and remain cargo inside, inducing the few immaturity and high pharmacological efficacy as well. Meanwhile, the behavior of NIR triggered-linker broken also was monitored by zeta-potential analysis. Review to Table 1, the zeta potential decrease to −13.77 ± 2.08, −8.61 ± 0.94 and −3.83 ± 2.87 mV after NIR light irradiating owing to the chain broken, leaving –SH on the surface.

Fig. 6 (A) Release profiles and (B) Higuchi plot for the release of DOX from UCNP@mSiO₂/HA-DOX@CNBA in pH 6.8 (a, c and e) with and (b, d and f) without NIR irradiation (980 nm, 0.5 W cm⁻²). Red: UCNP@mSiO₂/HA-DOX@CNBA1, green: UCNP@mSiO₂/HA-DOX@CNBA2, and blue: UCNP@mSiO₂/HA-DOX@CNBA3. (C) Release profiles of DOX from mSiO₂/HA-DOX@CNBA2 (a) with and (b) without NIR irradiation, and UCNP@mSiO₂/HA-DOX (c) with and (d) without NIR irradiation (980 nm, 0.5 W cm⁻²). (D) Release profiles of DOX from UCNP@mSiO₂/HA-DOX@CNBA2 under alternative NIR light irradiation/dark for every 30 min.

To further study the release behavior, the release data are analyzed by Higuchi model.^35,36 As we known, drug release kinetics from an insoluble, porous carrier matrix are frequently described by the Higuchi model, and the release rate can be described by the follow equation:

Q = kt^1/2

where Q is the quantity of drug released from the materials, t denotes time, and k is the Higuchi dissolution constant. According to the model, for a purely diffusion-controlled process, the linear relationship is valid for the release of relatively small molecules distributed uniformly throughout the carrier. As can be seen in Fig. 6(B), with NIR irradiation, all UCNP@mSiO₂/HA-DOX@CNBAs exhibit a two-step release (0–120 min and 120–360 min) based upon the Higuchi model. In the first 120 min, UCNP@mSiO₂/HA-DOX@CNBA1 possesses the highest dissolution constant k (the slope of the fitting line), followed with UCNP@mSiO₂/HA-DOX@ CNBA2 and UCNP@mSiO₂/HA-DOX@CNBA3. The reason is that the fewest CNBA linker is degraded most quickly, making the highest dissolution constant k as well as the fastest release rate of UCNP@mSiO₂/HA-DOX@CNBA1. At the same time, UCNP@mSiO₂/HA-DOX@CNBA3 with the most amount of CNBA graft reveals the lowest dissolution constant k before 120 min. In the second release step (120–360 min), the release rates of UCNP@mSiO₂/HA-DOX@CNBAs decrease and tend to smooth and accordance. As present in Fig. 6(A), it is believed that most drug molecules have been released after “gatekeepers” were broken in the first release step. In other words, the first release step depends mainly upon the degradation of “CNBA gate” and the second release step is determined just by the mesoporous structure of the host. All in all, the amount of “gate” can be used to regulate the release performance of the drug delivery system. From here we see that UCNP@mSiO₂/HA-DOX@CNBA2 is the best candidate.

Further to explore the NIR light trigger release performance, mSiO₂/HA-DOX@CNBA2 and UCNP@mSiO₂/HA-DOX with or without NIR irradiation were shown in Fig. 6(C). Regardless of whether NIR irradiation or not, mSiO₂/HA-DOX@CNBA2 displays few DOX release (below 8.7%), indicating that without up-conversion luminescence the “gate” would keep blocking the pore and inhibiting the drug release. However, without the “gate” UCNP@mSiO₂/HA-DOX exhibits the free release, and NIR-triggered DOX release trend can be surveyed from Fig. 6(C). And the inconsiderable increased DOX release of UCNP@mSiO₂/HA-DOX under NIR irradiation would be ascribed to the partial absorption of 980 nm NIR of water to induce the heat.³⁷ In addition, the influences of irradiation intensity and time on drug release also were delicately studied. Fig. 6(D) displays the release profiles of UCNP@mSiO₂/HA-DOX@CNBA2 under alternative NIR light irradiation/dark for every 30 min. When the working power density of the 980 nm laser is increased from 0.5 to 1.5 W cm⁻², the release rate and the final release amount increase as well. As shown in Fig. 6(D), these release behaviors reveal the on–off pattern of the excitation source, indicating that the release process could be fine-tuned by remote control of the NIR irradiation and the amount of the released drug is highly dependent on the duration and intensity of NIR exposure.

And then, the in vitro therapeutic performance of the proposed platform nanocomposites has been surveyed by incubated with HeLa cells for 24 h and evaluated by the MTT assay. As shown in Fig. 7(A), there is a negligible reduced cell viability of UCNP@mSiO₂/HA-DOX@CNBA (94.5%) and mSiO₂/HA-DOX@CNBA (95.7%) even with the high incubated concentration of 500 μg mL⁻¹, suggesting the excellent biocompatibility of the nanoplatforms under dark condition. Yet, the cell viabilities of UCNP@mSiO₂/HA-DOX decreases to 48.4% owing to the leakage of DOX with out the “CNBA gate”. After incubated with UCNP@mSiO₂/HA and UCNP@mSiO₂/HA-DOX@CNBA (6 h in the dark), the cells were exposed to various 980 nm excitation (0.05, 0.1, and 0.15 W cm⁻²) and the NIR-triggered cytotoxicity can be found in Fig. 7(B) obviously. From Fig. 7(B), under NIR excitation, UCNP@mSiO₂/HA reveals the plain cytotoxicity (62.5%, 0.15 W cm⁻²) due to the PDT derived from the generation of reactive oxygen (Fig. 3). Compared with UCNP@mSiO₂/HA, the enhanced cytotoxicity of UCNP@mSiO₂/HA-DOX@CNBA is ascribed from the NIR-sensitive reactive oxygen and drug release. With the increase of irradiation intensity, the remarkable cytotoxicity can be found (62.8%, 38.3%, and 24.7% of 0.05, 0.1, and 0.15 W cm⁻²) because of the enhanced up-conversion luminescence.

Fig. 7 (A) MTT cell viability assay of UCNP@mSiO₂/HA-DOX@CNBA, mSiO₂/HA-DOX@CNBA, UCNP@mSiO₂/HA-DOX on HeLa cells for 24 h incubation. (B) UCNP@mSiO₂/HA and UCNP@mSiO₂/HA-DOX@CNBA under 980 nm irradiation 20 min with different intensities (0–0.15 W cm⁻²) after 6 h incubation.

Besides, more cell examines were carried out to further illuminate the NIR-sensitive cytotoxicity. The viability of HeLa cells incubated with 980 nm (0.15 W cm⁻²), mSiO₂/HA, mSiO₂/HA-DOX@CNBA, UCNP@mSiO₂/HA, UCNP@mSiO₂-DOX@CNBA, UCNP@mSiO₂/HA-DOX and UCNP@mSiO₂/HA-DOX@CNBA without or with NIR irradiation (0.15 W cm⁻²) are summarized in Fig. 8. From Fig. 8, the mere NIR and nano-vehicle reveal above 94.2% viability of HeLa cells and the cytotoxicity derived from the NIR-triggered drug release and reactive oxygen. Furthermore, the synergistic effect of PDT and chemotherapy induces the improved cytotoxicity to HeLa cells rather than the simple plus effect of the two. Beside the irradiation strength, the higher irradiation time, the more cytotoxicity can be found (44.2% 10 min, 24.7% 20 min). Because the extended irradiation would excite extended vis and UV-emission that induces more drug release and the generation of more reactive oxygen ¹O₂ to bring the irreversible damage to cancer cell. The detail cell examine further reflect the vitro investigation: NIR-triggered PDT and chemotherapy induces the improved cytotoxicity to cancer that is associated with the convenient operation to bring up a novel potential nanomedicine. To further survey the possible application, 293T (normal epithelial cell) and MCF-7 (breast cancer cell) also were used as the model cells. As can be seen in Fig. S5A,† the cell viability of 293T and MCF-7 still retain 95.7% and 94.2% after incubated with UCNP@mSiO₂/HA@CNBA, implying the negligible cytotoxicity of the nanocarrier. Under 980 nm (0.15 W cm⁻²) irradiation for 20 min, the obvious cytotoxicity of MCF-7 cells (25.4%) is ascribed to the DOX release and ROS generation.

Fig. 8 Cell viability of HeLa cells incubated with 980 nm (0.15 W cm⁻²), (A) mSiO₂/HA, (B) mSiO₂/HA-DOX@CNBA, (C) UCNP@mSiO₂/HA, (D) UCNP@mSiO₂-DOX@CNBA, (E) UCNP@mSiO₂/HA-DOX and (F) UCNP@mSiO₂/HA-DOX@CNBA without or with 980 nm irradiation (0.15 W cm⁻²).

To observe the targeting uptake and subsequent localization of the nanocomposites, Fig. 9 shows the UCNP@mSiO₂/HA-DOX@CNBA (with and without Tf) pre-incubated with the HeLa cells for 3 h in the dark and then 980 nm irradiation for 5 and 20 min. As can be seen from Fig. 9(A), green (FITC, 520 nm) and red (DOX, 480 nm) emission can be found in cytoplasm, implying these nanocomposites have been internalized by HeLa cells through endocytosis or macropinocytosis owing to nano-size particles. Furthermore, compared with the emission as shown in Fig. 9(A) and (B) exhibits the enhanced green and red color, ascribing to the enhanced uptake of Tf-UCNP@mSiO₂/HA-DOX@CNBA into HeLa. And the increasing red light intensity in cell nucleus over time indicates a continuous released DOX molecules gather into nucleus to improve the chemotherapy. To quantitative analysis the cellular uptake, the flow cytometry analysis was actualized as present in Fig. 10. From Fig. 10(A), the more Tf-UCNP@mSiO₂/HA-DOX@CNBA nanocomposites gather into HeLa cells than UCNP@mSiO₂/HA-DOX@CNBA. Moreover, MCF-7 cells also reveal the enhanced uptake amount compared with 293T cells (Fig. 10(B)), owing to the higher expression of TfR in tumor cells (HeLa and MCF-7 cells) than normal cells (293T cells).³⁸ The targeting associated with the sensitive chemotherapy/PDT further insures the specific cytotoxicity to the focal sites.

Fig. 9 Fluorescent images of HeLa cells incubated with UCNP@mSiO₂/HA-DOX@CNBA without and with Tf modification for 3 h pre-incubation and then that were exposed to the 980 nm light (0.15 W cm⁻²) for 5 and 20 min before observations. All images share the same scale bar (20 μm). For each panel, the images from left to right show nanoparticles stained by FITC (green), DOX fluorescence in cells (red), bright field and the merge of the three images.

Fig. 10 (A) Flow cytometry analysis of the HeLa cells incubated with FITC modified UCNP@mSiO₂/HA-DOX@CNBA (black) and Tf-UCNP@mSiO₂/HA-DOX@CNBA (red) for 6 h. (B) Flow cytometry analysis of the 293T cells (red) and MCF-7 (green) cells incubated with FITC modified Tf-UCNP@mSiO₂/HA-DOX@CNBA for 6 h.

Conclusion

In summary, a novel NIR-triggered nanocomposite based on UCNP@mSiO₂ core–shell nanostructure has been prepared. To make the best use of the upconversion fluorescence (visible and UV emission), the light-triggered drug release and photodynamic therapy were constructed in to one-nanoplatform. First, an effective photosensitizer Hypocrellin A (HA) was covalently incorporated into mesoporous silica framework on one-step to avoid the leakage. And then, a UV light-cleavable linker (CNBA) was synthesized and to keep DOX storing in the mesopore. Upon NIR illumination, the UV light emission can induce the break of CNBA linker to make the “gate” open and drug release. Besides, the blue light emission can excite HA to generate reactive oxygen (ROS) to achieve PDT. Both nanoscale particle size (75 nm) and targeting Tf graft improve the specific phagocytosis by cancer cells. The synergistic effect of PDT and chemotherapy induces the enhanced cytotoxicity which would be conveniently adjusted by varying the intensity and/or time duration of NIR light irradiation. Therefore, this novel NIR-triggered nanoplatform should have a great potential for anticancer treatment.

Acknowledgements

Financial support for this study was provided 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: The synthetic approach and NMR spectrum of the as-synthesized linker; small angle XRD; additional results. See DOI: 10.1039/c6ra03186b

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