A “submunition” dual-drug system based on smart hollow NaYF₄/apoferritin nanocage for upconversion imaging

Abstract

Synergetic therapy has exhibited important potential for the treatment of cancers, especially for drug-resistant cancers. In this report, bifunctional nanomaterials based on doxorubicin (DOX)-loaded NaYF₄ and verapamil (Vp)-loaded apoferritin nanocage dual-drug system (DOX/NaYF₄-Vp/AFn-FA) were synthesized for in vivo upconversion imaging and enhanced chemotherapy in breast cancers. Moreover, folic acid (FA) targeting promoted the cellular uptake, and accelerated the release of DOX in drug-sensitive MCF-7. This system is a multifunctional drug delivery system with significant tumor-targeting efficacy and is also the first time preparation of NaYF₄–AFn-FA. The dual-drug system enabled a temporal release of two drugs: Vp was released rapidly to inhibit the activity of P-gp and restore cell apoptotic signaling pathways, while DOX was released in a more sustained manner and highly accumulated in drug resistant cells to exert a therapeutic effect, due to the inactivation of P-gp by Vp. Toxicity assessment in vitro and in vivo revealed the good biocompatibility of the as-prepared DOX/NaYF₄-Vp/AFn-FA nanocomposites. In addition, NaYF₄–AFn-FA uptaken by cells and mouse by intravenous injection showed bright green emission without background noise under 980 nm infrared laser excitation. Thus, NaYF₄–AFn-FA has the potential for simultaneous targeted anti-cancer drug delivery or imaging, which suggested a new multi-mechanism pathway for tumor treatment.

---

1. Introduction

Recently, luminescent hollow-structured materials have attracted considerable attention owing to their unique properties and potential applications in biomedical fields.¹ Hollow/mesoporous structured nanomaterials not only have high specific surface² and cavity volumes but also possess luminescence properties, which means they can be commonly used as ideal carriers for drug delivery,^3–7 luminescent probes for biological imaging, or as tracers for drug-release monitoring.⁸ They have many advantages over conventional organic dye markers and quantum dots, such as high physicochemical stability, low toxicity, reduced photodamage to living organisms, high signal-to-noise ratio, and strong tissue penetration ability.⁹

Optical imaging has been considered as the most promising imaging modality. It has many advantages, such as high sensitivity, excellent temporal and spatial resolution for imaging in vivo and in vitro, relatively low cost and easy access. In addition, it has the capability to provide cellular- or molecular-level information.^10,11 Lanthanide (Ln)-doped upconversion (UC) luminescent nanomaterials, which can convert low-energy (near-infrared (NIR) photon) excitation to higher-energy (UV-visible light) emission by means of multiple absorption or energy transfer,^12–15 have been increasingly used for optical imaging. Use of hollow NaYF₄, which shows UC luminescent property, it expected to be a promising material in biological applications.

So far, there were many methods used to fabricate UC nanoparticle-based composites with hollow structure. Generally, a template-assisted strategy is commonly used, which involves the coating of templates with nanocrystals (precursors or targeted products). However, this procedure has disadvantages, such as tedious synthetic procedures, low yields and poor water solubility, which has largely impeded upscaled production and applications. Zhang et al. reported a facile solution-phase synthesis of NaYF₄:Yb/Er (Tm) UC hollow spheres based on nanoscale growth induced by the Kirkendall effect.¹⁶ In this paper, we report an efficient, easy operation, one-pot fabrication of hollow UC structures utilizing the well-known classical physical phenomenon of Ostwald ripening. In this reaction system, we used polyethylenimine (PEI) as the structure-stabilizing agent, which can efficiently control the nucleation and growth of NaYF₄ crystals in order to achieve hollow mesoporous structured nanospheres (HMNSs). The nanoscaled HNSs had high water solubility and good biocompatibility, which also showed intense multicolor UCL under 980-nm NIR laser excitation.

Apoferritin nanocages have been proposed to be a promising and versatile system.¹⁷ Ferritins comprise a family of proteins in different forms found in most living organisms. Each ferritin nanocage is made of 24 subunits, which self-assemble to form a cage-like nanostructure, with external and internal diameters of 12 and 8 nm, respectively.¹⁸ This unique architecture provides two interfaces, one outside and one inside, for possible functional loading.¹⁹ Moreover, since the subunits can be disassembled at acidic pH and reassembled at neutral pH in a shape-memory fashion, apoferritin can be exploited for the encapsulation of various organic molecules, thus representing an interesting scaffold for the development of biocompatible drug delivery systems.²⁰ Since ferritin is specifically cross-recognized in humans by the receptor of transferrin 1, it is found to be overexpressed in many types of tumor cells but not in normal cells and healthy tissues.²¹ Based on the above considerations, ferritin is a green biomaterial for loading drugs.

Breast cancer is the most common form of cancer in women, while also having the highest mortality rate worldwide.²² In this work, this is the first time to connect hollow NaYF₄ and ferritin nanocage, a dual-drug system. P-glycoprotein (P-gp) is one of the ATP-binding cassette superfamily proteins which participates in the transport of a wide variety of substrates. When over-expressed by tumors, P-gp-mediated efflux of drugs with resultant multidrug resistance (MDR) tumor cells is a major obstacle to successful clinical cancer chemotherapy. Therefore, in order to circumvent this problem, two drugs were loaded, namely DOX and Vp, because Vp is a first generation P-glycoprotein (P-gp) inhibitor and the two drugs have a synergistic effect, which can increase the intake of DOX in order to kill more tumor cells, thereby enhancing the therapeutic index. DOX is one of the most widely used chemo-therapeutics in the treatment of solid tumors, although the development of resistance and the occurrence of severe side effects, including cardiotoxicity and myelosuppression, caused by high dosages, limit its efficacy in clinical practice.²³ Additionally DOX presents poor solubility and is readily metabolised.²⁴ DOX is transported by diffusion into cancer cells which drastically limits the uptake of the compound.²⁵ The reason we use Vp, the first-generation P-gp inhibitor, along with the chemotherapy drugs, is that it can increase drug uptake in tumor cells that overexpress P-gp. The dual-drug system enabled a temporal release of two drugs: Vp was released rapidly to inhibit the activity of P-gp and restore cell apoptotic signaling pathways, while DOX was released in a sustained manner and is highly accumulated in drug resistant cells to exert a therapeutic effect. In DOX/NaYF₄-Vp/AFn-FA two different carriers are used to load the two drugs which should avoid mutual interference of the two drugs with each other. The drug-loading and release properties, cytotoxicity, therapeutic effects, and in vitro upconversion luminescence imaging of the as-prepared NaYF₄–AFn-FA were investigated in detail.

2. Materials and methods

2.1. Materials

Hydrated rare earth chloride (RECl₃·6H₂O, RE = Y, Yb, Er, 99.99%), sodium chloride (NaCl, purity ≥ 99.5%), sodium fluoroborate (NaBF₄, ≥99.0%), branched polyethylenimine (PEI, Aldrich, MW ∼ 25 000), folic acid (FA), AFn (apoferritin from equine spleen, product of USA), and diethylene glycol (DEG, Acros, 99+%) were used as received. DOX (99.9%) was purchased from Huafeng United Technology Co. (Beijing, China), Vp (catalog no. V4629) were purchased from Sigma-Aldrich (St. Louis, Missouri), dimethyl sulfoxide (DMSO), 1-ethyl-3-(3′-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Co. Ltd. (Shanghai, China). All the chemicals used were analytical reagents. The water used was deionized.

2.2. Synthesis of hollow and mesoporous structured NaYF₄:20%Yb³⁺,2%Er³⁺ NPs

Hollow and mesoporous structured NaYF₄:Yb³⁺,Er³⁺ nanoparticles were synthesized by a one-step hydrothermal process without any sacrificial templates. In a typical process, 10 ml RECl₃ (0.1 M, RE = 78%Y + 20%Yb + 2%Er) were dispersed into 30 ml of diethylene glycol (DEG) containing 0.8 g of polyethyleneimine (PEI) by vigorous stirring to obtain a transparent solution. Afterwards, 1 mmol NaCl was poured into the mixture solution with vigorous stirring for 0.5 h. Then, 5 ml NaBF₄ was added and the pH maintained about 8. After additional agitation for 0.5 h, the solution was transferred into a 40 ml Teflon autoclave, which was tightly sealed and maintained at 200 °C for 6 h. As the autoclave was cooled to the room temperature naturally, the as-prepared precipitates were separated via centrifugation, washed several times with ethanol and deionized water, and then the final products were obtained by freeze-drying. Hollow, mesoporous structured NaYF₄:20%Yb³⁺,2%Er³⁺ (labeled as NaYF₄) HMNPs were obtained.

2.3. In vitro NaYF₄:20%Yb³⁺,2%Er³⁺ loading with doxorubicin and release

DOX, was selected as a model drug to investigate the drug loading and release behaviors. First, 5 mg DOX were dissolved in 5 ml deionized water with stirring at room temperature until it dissolved. Subsequently, 10 mg of NaYF₄ HMNPs were added and stirred overnight in the dark at room temperature. Then the solution was centrifuged to collect the DOX-loaded NaYF₄ HMNPs (labeled as DOX/NaYF₄). The supernatant solutions were collected, and the content of DOX was determined by UV-vis spectral measurement at 480 nm. DOX-loaded NaYF₄ HMNP samples were immersed in 40 ml of phosphate buffer saline (PBS) (pH = 7.4 or 5.0) at 37 °C. At predetermined time points (5, 10, 30 min, 1, 2, 4, 8, 12, 24, 36, 48 h), buffer solution was taken and replaced with fresh buffer solution. The amount of DOX released was quantified by UV-vis spectrophotometry.

2.4. In vitro apoferritin loading with verapamil and release

AFn loading with Vp was prepared using the disassembly/reassembly method. Vp (4.9 mg ml⁻¹) was added to a AFn solution (3.7 mg ml⁻¹ in 0.15 M NaCl) and the mixture was adjusted to pH 2.0 by 0.1 M HCl. The pH was maintained for about 15 min when the dissociation of AFn was completed, then the pH value was increased up to 7.5 using 0.1 M NaOH. The resulting solution was stirred at room temperature for 2 h (labeled as Vp/AFn).²⁶ The supernatant solutions were collected, and the content of Vp was determined by UV-vis spectral measurement at 278 nm. Vp/AFn samples were immersed in 40 ml of phosphate buffer saline (PBS) (pH = 7.4 or 5.0) at 37 °C. At predetermined time points (5, 10, 30 min, 1, 2, 4, 8, 12, 24, 36, 48 h), buffer solution was taken and replaced with fresh buffer solution. The amount of Vp released was quantified by UV-vis spectrophotometry.

2.5. Synthesis of DOX/NaYF₄-Vp/AFn-FA NPs

Typically, a solution of 200 μl of Vp/AFn, 10 ml of 0.2 mg ml⁻¹ NHS, 10 ml of 0.6 mg ml⁻¹ EDC and 5 ml of 0.2 mg ml⁻¹ FA were mixed and stirred for 2 h at 37 °C. Subsequently, 20 mg of DOX/NaYF₄ were added and allowed to react with stirring overnight in dark, at room temperature. The materials were recovered by centrifugation and washed with twice with deionized water. The as-prepared products were labeled as DOX/NaYF₄-Vp/AFn-FA NPs. The same method was used to prepare NaYF₄–AFn-FA NPs.

2.6. Characterization

X-Ray power diffraction (XRD) was performed on a D8 Focus diffractometer (Bruker) with Cu-Kα radiation (λ = 0.15405 nm). Fourier-transform IR spectra were recorded on a Nicolet iS10 IR spectrophotometer using the KBr pellet technique. Transmission electron microscopy (TEM) was performed using FEI Tecnai G² S-Twin with a field emission gun operating at 200 kV. Images were acquired digitally on a Gatan multiple CCD camera. The UC emission spectra were obtained by using a 980-nm laser diode as the excitation source and the emission spectra were dispersed by a monochromator of the Acton SpectraPro-2758 equipped with R928 PMT, and the data were recorded from 400 to 700 nm. Fluorescence microscopy images were observed by fluorescence microscopy (Olympus, FV 1000).

2.7. In vitro cytotoxicity of DOX/NaYF₄-Vp/AFn-FA NPs

In vitro cytotoxicity of hollow mesoporous structured materials was assayed against MCF-7 cells. MCF-7 cells were seeded in a 96-well plate at a density of 8 × 10³ cells per well and cultured in 5% CO₂ at 37 °C for 24 h. Then free DOX, free Vp, DOX/NaYF₄, Vp/AFn, DOX/NaYF₄-Vp/AFn-FA and NaYF₄–AFn-FA were added to the medium, and the cells were incubated in 5% CO₂ at 37 °C for 24 h, respectively. The concentrations of DOX were 0.75, 1.5, 3.0, 6.25, 12.5, 25 or 50 μg ml⁻¹. At the end of the incubation, the media containing the nanospheres was removed, and 20 μl of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) solution (diluted in a culture media with a final concentration of 5.0 mg ml⁻¹) was added into each cell and incubated for another 4 h. The supernatant in each well was aspirated. 150 ml of dimethyl sulfoxide (DMSO) was added to each well before the plate was examined using a microplate reader at the wavelength of 490 nm.

2.8. Cellular uptake

Cellular uptake by MCF-7 cells was examined using UCL microscopy. For UCL, the MCF-7 cells were seeded in 6-well culture plates and grown overnight as a monolayer and were incubated with DOX/NaYF₄-Vp/AFn-FA and NaYF₄–AFn-FA HMNPs (DOX: 20 μg ml⁻¹) at 37 °C for 30 min, 1, 2 or 4 h. Thereafter, the cells were rinsed with PBS several times, and then 500 μl of PBS was added.

2.9. Anti-tumor effect of DOX/NaYF₄-Vp/AFn-FA NPs in vivo

2.9.1. Xenograft tumor mouse model. All animal experiments were performed under a protocol approved by Henan laboratory animal center. The S180 tumor models were generated by subcutaneous injection of 2 × 10⁶ cells in 0.1 ml saline into the right shoulder of female KM mice (18–22 g). Food and water were provided ad libitum. The mice were used for in vivo anti-tumor experiments while the tumor volume reached ∼100 mm³ (about 7 days after tumor inoculation).

2.9.2. In vivo anti-tumor efficacy. For the in vivo anti-tumor experiments, the tumor-bearing mice were divided into five groups (six mice per group to minimize the differences of weights and tumor sizes in each group). The mice were administered with (1) saline, (2) DOX, (3) Vp, (4) NaYF₄–AFn-FA and (5) DOX/NaYF₄-Vp/AFn-FA by tail vein injection every 2 days for 14 days, respectively [(1)–(4) dose: 10 mg kg⁻¹, (5) dose: 50 mg kg⁻¹]. Throughout the study, mice were weighed and tumors were measured with calipers every two days. Tumor volume (V) was calculated according to the formula: V = [length × (width)²]/2. At the end of experiment, the animals were sacrificed and tumor tissues were taken out and weighed. Then, six tumor tissues of each group were soaked in 10% formalin solution, embedded with paraffin for hematoxylin and eosin (H & E) staining. Morphological changes were observed under microscope (Eclipse 80i, Nikon, Japan).

2.9.3. Pharmacokinetics studies. The animals were randomly divided into experimental groups (varying from 3 to 6 rats per group) for treatment with saline and formulations of free DOX, free verapamil and DOX/NaYF₄-Vp/AFn-FA. Saline and formulations at doses of DOX (10 mg kg⁻¹) and Vp (2 mg kg⁻¹) were administered via the tail vein.

After injection, blood was sampled from retro orbital sinus at 10, 30 min, 1, 2, 4, 6, 8, 12 and 24 h. Blood samples (300 μl) were collected in heparinized tubes, and then centrifuged at 3000 rpm for 10 min at 4 °C to separate the plasma and stored at −20 °C until assayed for DOX.

2.9.4. HPLC analysis for DOX. The HPLC assay of Andersen et al.¹⁹ was used to analyze DOX with minor modification. Briefly, 1 ml acetonitrile was added to each 200 μl sample and centrifuged after mixing by vortex to precipitate proteins and extract DOX. The supernatant was entirely taken and dried under nitrogen, and reconstituted in 100 μl mobile phase prior to injection into a Symmetry® C18 column (150 mm × 4.6 mm, 5.0 mm, Waters e2695, USA). Detection was via DOX intrinsic fluorescence at 582 nm on the HPLC system with a fluorescence detector; mobile phase: methanol/0.01 M phosphate 55% : 45%; column temperature 30 °C; flow rate 0.8 ml min⁻¹ and injection volume 10 μl. The limit of quantitation (LOQ) was 1.5 ng ml⁻¹. In plasma, coefficient of variation (CV) of precision and accuracy was <10% and recovery was >90%.

3. Results and discussion

3.1. Preparation and characterization of NaYF₄ HMNPs

NaYF₄:Yb³⁺,Er³⁺ nanoparticles (labeled as NaYF₄ HMNPs) with a mesoporous shell and hollow interior structure were synthesized hydrothermally without sacrificial templates for the first time. The procedure for the formation of mesoporous NaYF₄–AFn-FA HMNPs is presented in Scheme 1. The composition and phase purity of the products were first examined by XRD. Fig. 1 shows XRD patterns of β-NaYF₄:20%Yb³⁺,2%Er³⁺ HMNPs. It was obvious that all the diffraction peaks could be clearly indexed to pure β-NaYF₄, which agreed well with the data reported in the JCPDS standard card (JCPDS no. 16–0334). No other phase can be detected in the XRD pattern, indicating the formation of pure β-NaYF₄, which is beneficial for obtaining bright luminescence.

Scheme 1 Schematic illustration for the synthesis and application of NaYF₄–AFn-FA composite HMNPs.

Fig. 1 XRD patterns of β-NaYF₄:20%Yb³⁺, 2%Er³⁺ HMNPs, and the calculated line pattern for β-NaYF₄.

The morphology and size distribution of the HMNPs were investigated using scanning electron microscopy and transmission electron microscopy. As can be seen in Fig. 2A and B, uniform nanospheres with a size of 100 ± 10 nm are observed with the presence of a characteristic cavity in hollow structures. Moreover, the PEI layer can be seen clearly and the thickness of PEI layer was estimated to be around 8 nm, as also confirmed by the TEM image presented in Fig. 2C.

Fig. 2 SEM images (A), TEM images (B and C) with different magnifications of water-dispersible hexagonal-phase NaYF₄:20%Yb,2%Er nanocrystals prepared without methanol.

The N₂ adsorption–desorption isotherm in Fig. 3 was analyzed by a surface area analyzer. The Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH) method were utilized to calculate the BET specific surface area and the pore size distributions, respectively. Results show that the isotherm is type II and reveals the existence of mesoporosity in the sample. The BET surface area and pore volume were calculated to be 61.362 m² g⁻¹ and 0.30 cm³ g⁻¹, respectively, and the pore-size distribution showed a narrow apex centered at 10.5 nm (inset in Fig. 3). The high pore volume and BET surface area indicated that HMNPs could be used as hosts for storing drug molecules.

Fig. 3 N₂ adsorption/desorption isotherms and pore size distribution curves (inset) of the hollow mesoporous NaYF₄:Yb³⁺,Er³⁺ nanoparticles.

In order to further confirm the composition and surface structure of the products, FT-IR spectroscopy was conducted for the final products. The FT-IR of AFn is shown in Fig. 4a and amide bonds are observed at 1653 and 1405 cm⁻¹. In Fig. 4b, the bands at 2920 and 2847 cm⁻¹ were attributed to the stretching vibrations of the –CH₂ group, respectively. In addition, the observations of unique absorption peaks from internal vibration of the amide bonds (1636 and 1403 cm⁻¹) and the peak at 1532 cm⁻¹, which is a characteristic asymmetric vibration of the –NH₂ group, demonstrates the existence of the PEI polymer.²⁷ Owing to the presence of hydrophilic PEI on the surface, these nanospheres could be readily dispersed in deionized water, and form a nearly transparent aqueous solution (Fig. 4, inset).

Fig. 4 FTIR spectra of (a) AFn, (b) NaYF₄ and (c) NaYF₄–AFn-FA HMNPs.

After conjugation with FA, a well-resolved vibrational peak at 1610 cm⁻¹ assigned to the N–H bending vibration of the CONH group and another obvious peak at 1694 cm⁻¹ attributed to the C=O amide stretching of the α-carboxyl group from the FA molecules were present in Fig. 4c.^28–33 These results further confirmed that FA ligands have been successfully grafted onto the NaYF₄ nanospheres.

3.2. Upconversion luminescence property

The up-conversion emission spectra were obtained by using 980-nm IR laser excitation, and the corresponding up-conversion emission spectrum of Yb³⁺ and Er³⁺ co-doped NaYF₄ nanospheres are shown in Fig. 5. The obtained spectrum containing three emission peaks at around 522, 542 and 656 nm can be assigned to ²H_11/2 → ⁴I_15/2, ⁴S_3/2 → ⁴I_15/2 and ⁴H_9/2 → ⁴I_15/2 electronic transitions of the Er³⁺ ion, respectively.^34,35

Fig. 5 Upconversion luminescence spectra of NaYF₄:20%Yb,2%Er.

3.3. In vitro cytotoxicity of NaYF₄–AFn-FA

Cytotoxicity is an essential concern when it comes to the development of nanomaterials for biomedical imaging and cancer-therapy applications.^36–39 The standard MTT cell assay was performed on MCF-7 cell lines to test the cytotoxicity of NaYF₄–AFn-FA. Fig. 6A showed the in vitro cell viability of MCF-7 cells incubated with NaYF₄–AFn-FA with different concentrations ranging from 6.25 to 200 μg ml⁻¹ for 24 and 48 h, respectively. This result showed that >90% of cell viabilities were observed even at a high-dose concentration (200 μg ml⁻¹) of the NaYF₄–AFn-FA composites after incubation for 24 and 48 h, respectively. These MTT assay results demonstrated that these NaYF₄–AFn-FA are almost nontoxic to living cells and could be potentially used as bioprobes for in vitro cell imaging or drug carriers for drug delivery.

Fig. 6 Cytotoxicity test and UCL cell imaging. (A) Cell viabilities of MCF-7 cells incubated with NaYF₄–AFn-FA at different concentrations for 24 and 48 h, respectively. (B and C) In vitro UCL images of MCF-7 cells using NaYF₄–AFn and NaYF₄–AFn-FA as luminescent probes under excitation with a 980-nm laser, respectively.

The cellular uptake process by MCF-7 cells was examined by UCL microscopy. MCF-7 cells were incubated with NaYF₄–AFn and NaYF₄–AFn-FA (100 μg ml⁻¹) for 2 h and then washed with PBS solution to remove the unreacted nanomaterials. The luminescence images were recorded using an inverted fluorescence microscope equipped with an external 980-nm laser as the excitation source. As shown in Fig. 6B and C, it can be seen that the cells incubated with NaYF₄–AFn-FA UC HNSs show a strong green luminescence signal under 980-nm laser excitation (Fig. 6B and C, middle), with no luminescence from cells incubated with NaYF₄–AFn, and no autofluorescence background from the MCF-7 cells was observed. The overlay of the bright-field and UCL image in Fig. 6B and C (right) further indicated that the green emission was evident and part of the UC HNSs were internalized into the cells.^40–43 The above results demonstrated that the FA has targeting ability and the as-prepared NaYF₄–AFn-FA HNSs can be used as a good luminescent probe for cell imaging.

3.4. Drug loading and release properties

DOX was chosen as a model drug to evaluate the loading and controlled release behaviors of the hollow NaYF₄. The content of DOX in the composites could be determined by the characteristic absorption peak centered at 480 nm, as shown in Fig. 7a, and the successful loading of DOX onto the NaYF₄ could be readily confirmed by a clear color change from white to red (Fig. 6A). From Fig. 7a and drug loading formula we can calculate the loading amount is 10.58%. In addition, the drug release content and rate of DOX increased with a decrease of pH. The in vitro cumulative DOX release profiles from hollow NaYF₄ HMNPs in PBS solutions at two different pH values (7.4 and 5.0) at 37 °C are shown in Fig. 7b, and clearly show a pH-dependent trend. This is because the deprotonation of –NH₂ groups of DOX molecules and dendrigraft cationic polymer PEI coated on the HMNPs at higher pH values can enhance the intermolecular interaction between DOX and hollow PEI HMNPs⁴⁴ and more DOX molecules could be adsorbed and stored in the inner cavity at higher pH values. From Fig. 7b, we observe only 40% of DOX was released from the nanocarriers, even after 48 h in PBS solution at pH 7.4. In contrast, when the pH value was decreased to 5.0, the system showed a fast drug release rate, which reached 48.8% within 1 h and more than 70% of DOX was released after 48 h. This is because under acidic conditions the protonation of the amino group in the DOX molecule would weaken its binding to the PEI polymer on the HMNPs surface and speed up the detachment of DOX from DOX/NaYF₄.³⁸ Generally, the extracellular pH of many solid tumors is lower than that of normal tissues and presents an acidic environment.^45,46 This suggest that the nanocarriers could potentially serve as a probe or drug carriers for monitoring the drug-release efficiency during cancer therapy and thus be beneficial to the targeting of cancerous tissues.^47,48

Fig. 7 Drug loading/release behaviors. (a) A comparison of UV/vis absorbance spectra of the NaYF₄, free DOX, and DOX/NaYF₄ at a loading pH of 7.0; (b) quantification of DOX release capacity at different pH values. (c) Quantification of Vp release capacity at different pH values. Higher drug release quantity was achieved at higher pH value.

Vp was chosen as a model drug to evaluate the loading and controlled release behaviors of AFn. The content of Vp in AFn could be determined by the characteristic absorption peak centered at 278 nm. We use the same method to calculate the loading amount as 3.79%. From Fig. 7c, we observe both at pH 5.0 or 7.4, that the system showed a fast drug release rate, with Vp released rapidly to inhibit the activity of P-gp and restore cell apoptotic signaling pathways, while DOX was released in a sustained manner, so the dual-drug system enabled a temporal release in order to exert a better therapeutic effect.

3.5. In vitro cytotoxicity effect on cancer cells

Since folate receptors are often overexpressed on the surface of human cancer cells, we often use folic acid (FA) as an attractive ligand for targeted anti-cancer drug delivery. Thus the FA conjugated nanomaterials were uptaken by cancer cells by receptor-mediated endocytosis.^49,50 In this work, we use MTT assay to test the pharmacological activity of DOX/NaYF₄-Vp/AFn on MCF-7 cells for 48 h incubation. In order to test the pharmacological activity of the DOX/NaYF₄-Vp/AFn and DOX/NaYF₄-Vp/AFn-FA composites on MCF-7 cells this was evaluated in vitro by MTT assay, and the results were compared with free DOX, DOX/NaYF₄ and Vp/AFn as shown in Fig. 8. The results indicated that DOX/NaYF₄ and Vp/AFn had low cytotoxic effect on MCF-7 cells within the tested concentration range. However, DOX/NaYF₄-Vp/AFn showed an increasing inhibition against MCF-7 cells compared to DOX/NaYF₄ and Vp/AFn. Moreover, DOX/NaYF₄-Vp/AFn-FA had an even higher cytotoxicity than DOX/NaYF₄-Vp/AFn under identical conditions, this may be attributed to the fact that the DOX/NaYF₄-Vp/AFn-FA can be taken up via receptor-mediated endocytosis by FA receptor-expressing MCF-7 cells and release DOX inside so as to induce cell death. It was noteworthy when the concentration of DOX was over 6.25 μg ml⁻¹, the cell-killing ability of DOX/NaYF₄-Vp/AFn-FA was close to that of free DOX. It should be noted that free DOX can diffuse into cells rapidly whereas the DOX-loaded nanocarriers have to be endocytosed to enter the cells. This suggested that FA-conjugated nanospheres can enhance the cell uptake efficiency for targeted drug delivery. The results also indicate that NaYF₄–AFn-FA are promising for use as a targeted anti-cancer drug delivery system and enhance the therapeutic efficacy.

Fig. 8 In vitro cytotoxicity of MCF-7 cells after incubation 48 h with DOX/NaYF₄, Vp/AFn, DOX/NaYF₄-Vp/AFn, DOX/NaYF₄-Vp/AFn-FA, and free DOX at different concentrations.

3.6. Cellular uptake

Cellular uptake process of as-prepared nanomaterials was examined by using fluorescence microscopy. As shown in Fig. 9, after incubation of MCF-7 cells with DOX/NaYF₄-Vp/AFn-FA for different times, the samples were taken up by MCF-7 cells, and by comparing to control cells, the cell uptake of samples can be measured. The results demonstrate a strong red luminescence signal from DOX molecules (Fig. 9), thereby confirming that the DOX/NaYF₄-Vp/AFn-FA composite could be internalized by the MCF-7 cells and then release DOX inside cells.^51,52 We also can see that the uptake amount of DOX/NaYF₄-Vp/AFn-FA endocytosed by MCF-7 cells was reached after 2 h. In order to verify the effect of DOX/NaYF₄-Vp/AFn-FA, we evaluated the cell uptake degree of free DOX using fluorescence microscopy analysis, as shown in Fig. 10a. The result showed that the uptake amount of DOX/NaYF₄-Vp/AFn-FA was much higher than that of free DOX under similar condition at 2 h. To further confirm the targeting capability of FA, we also evaluated the cell uptake degree of DOX/NaYF₄-Vp/AFn using fluorescence microscopy analysis, as shown in Fig. 10b. These results indicated that the uptake amount of DOX/NaYF₄-Vp/AFn-FA is much higher than that of DOX/NaYF₄-Vp/AFn at 2 h. Thus FA conjugated nanocarriers can enhance the cell uptake efficiency for targeted drug delivery, and therefore, the as-prepared HMNPs are promising candidates for image-guided drug delivery.

Fig. 9 Fluorescence microscopy images of MCF-7 cells incubated with DOX/NaYF₄-Vp/AFn-FA for 0.5, 1, 2 and 4 h at 37 °C, respectively.

Fig. 10 Fluorescence microscopy images of MCF-7 cells incubated with DOX (a), DOX/NaYF₄-Vp/AFn (b) and DOX/NaYF₄-Vp/AFn-FA (c) ([DOX] = 20 μg ml⁻¹) for 2 h at 37 °C, respectively.

3.7. Anti-tumor effect of DOX/NaYF₄-Vp/AFn-FA in vivo

3.7.1. Anti-tumor efficacy in vivo. To investigate the anti-tumor efficacy of DOX/NaYF₄-Vp/AFn-FA in vivo, comparative efficacy studies were conducted. Tumor-bearing mice were divided into five groups and tumor volume (V) was calculated according to the formula: V = [length × (width)²]/2. The changes of relative tumor volume as a function of time were plotted (Fig. 11a). After 14 days treatment, the control group showed a relative tumor volume (V/V₀) of 10.85 ± 0.34 and the NaYF₄–AFn-FA drug carrier group showed (V/V₀) of 10.33 ± 0.41, without significant difference, implying that NaYF₄–AFn-FA drug carrier does not affect the tumor growth. Free DOX, free Vp and DOX/NaYF₄-Vp/AFn-FA groups showed (V/V₀) of 7.67 ± 0.23, 10.02 ± 0.26 and 5.55 ± 0.32, respectively, suggesting that DOX/NaYF₄-Vp/AFn-FA was more effective than the other two therapeutic groups (P < 0.05).

Fig. 11 In vivo anti-tumor treatments: (a) tumor growth trend chart of mice in different treatment groups within 14 days; (b) changes of body weight of mice in different groups during treatment.

Furthermore, the changes of body weight as a function of time were plotted during the treatments (Fig. 11b) as high toxicity usually leads to weight loss. No weight loss was observed (Fig. 11b), indicating that toxicity of the treatments were not obvious.

In addition, on monitoring the heart, liver, spleen, lung, kidney, except for cardiac toxicity of the DOX group, almost no toxicity was observed (Fig. 12). In tumor tissues, tumor cells of saline control (Fig. 12a) and NaYF₄–AFn-FA (Fig. 12d) groups were in good condition of rapid proliferation and cells closely spaced with a big cell size. But it could be clearly observed in DOX (Fig. 12b) and Vp (Fig. 12c) that tumor cells began to shrink, implying DOX was toxic to tumor cells and the state of cell growth had begun to deteriorate. Furthermore, in the DOX/NaYF₄-Vp/AFn-FA group (Fig. 12e) typical symptoms such as obvious necrosis, karyotheca dissolving, and nucleolus disappearing all appeared, suggesting a good anti-tumor effect in vivo.

Fig. 12 H & E stained tumor tissues harvested from the mice with different treatments. (a) Blank; (b) DOX; (c) Vp; (d) NaYF₄–AFn-FA; (e) DOX/NaYF₄-Vp/AFn-FA. Data were presented as mean ± standard deviation (n = 3).

3.7.2. Pharmacokinetics studies. In order to investigate the pharmacokinetics of various drug complexes, blood samples of mice after injection of free DOX, free DOX and free Vp, or DOX/NaYF₄-Vp/AFn-FA at different times were determined by HPLC. Fig. 13 illustrates the plasma DOX concentration–time profiles of free DOX, free DOX and free Vp, and DOX/NaYF₄-Vp/AFn-FA. When DOX/NaYF₄-Vp/AFn-FA was administered, the plasma concentration–time profiles were different from those of free DOX, as shown in Fig. 13. Also, the addition of free Vp significantly (P < 0.01) altered some pharmacokinetic parameters of DOX when compared to free DOX without Vp (Table 1) where Vp increased significantly the T_1/2 and AUC_0–t and reduced CL of DOX. Compared with free DOX and free Vp treatment, a C_max of 68.90 ± 4.62 μg ml⁻¹ and an AUC_0–t 965.00 ± 79.21 μg h ml⁻¹ obtained from the DOX/NaYF₄-Vp/AFn-FA treatment represented a very significant 6.3-fold increase in AUC, confirming slower DOX removal from DOX/NaYF₄-Vp/AFn-FA. An elimination T_1/2 of 28.62 ± 2.71 was 4.3-fold higher and a CL of 0.01 ± 0.003 L mg⁻¹ h⁻¹ was 7-fold slower when compared to free DOX and free Vp.

Fig. 13 Plasma DOX concentration–time curve in mice of free DOX, free DOX and free Vp, and DOX/NaYF₄-Vp/AFn-FA.

Table 1 Summary of the pharmacokinetic parameters (mean ± SD, n = 3/group) for free DOX, free DOX and free Vp, and DOX/NaYF₄-Vp/AFn-FA at DOX i.v. dose of 10 mg kg⁻¹ in the absence and presence of verapamil (i.v. dose of 2 mg kg⁻¹)

Parameter	Tmax/h	AUC0–t/μg h ml^−1	MRT/h	Cmax/μg ml^−1	T1/2/h	Vss/ml	CL/l mg^−1 h^−1
Free DOX	0.1667	126.73 ± 10.11	6.20 ± 0.45	35.06 ± 5.12	2.62 ± 0.25	0.49 ± 0.02	0.08 ± 0.02
Free DOX and free Vp	0.1667	152.74 ± 12.72	8.71 ± 0.72	36.10 ± 5.37	6.61 ± 0.62	0.57 ± 0.03	0.07 ± 0.01
DOX/NaYF4-Vp/AFn-FA	0.1667	965.00 ± 79.21	36.88 ± 3.12	68.90 ± 4.62	28.62 ± 2.71	0.93 ± 0.03	0.01 ± 0.003

---

3.8. In vivo luminescence bioimaging

To reveal the optical bioimaging of the NaYF₄–AFn-FA, in vivo luminescence bioimaging of a Kunming mouse that was subcutaneously injected with NaYF₄–AFn-FA was tested under 980-nm laser excitation. From Fig. 14, we observe that intense UC signals were detected in tumor sites and varied in brightness with time, being most intense at 2 h. In order to further illustrate the results of bioimaging, we compare the bioimaging of NaYF₄–AFn-FA at 2 h and blank in vivo. From Fig. 15, the results suggested that NaYF₄–AFn-FA has good imaging capability, so the as-prepared NaYF₄–AFn-FA can act as luminescent nanoprobes for in vivo bioimaging.

Fig. 14 In vivo UC luminescence bioimaging of a Kunming mouse: NaYF₄–AFn-FA. The panels ranging from left to right correspond to overlay images in different times.

Fig. 15 In vivo UC luminescence bioimaging of a Kunming mouse: (a) blank and (b) subcutaneous injection of the NaYF₄–AFn-FA after 2 h. The panels ranging from left to right correspond to bright field images, UC images, and overlay images, respectively.

4. Conclusion

In summary, we developed hollow NaYF₄-based multi-functional nanoparticles and dual-drug NaYF₄–AFn-FA, which could not only specially target cancer cells but also transfer DOX and Vp into tumor tissue. The nanoscale formulation showed neglectable toxicity, and could serve as an active-targeting drug delivery carrier and in in vitro upconversion luminescence imaging. DOX is one of the most widely used chemotherapeutics in the treatment of solid tumors and Vp, as a first-generation P-gp inhibitor, can be effectively loaded on NaYF₄–AFn-FA to form a drug delivery system for cancer treatment or cell and vivo imaging. The dual-drug system enabled a temporal release of two drugs: Vp was released rapidly, while DOX was released in a more sustained manner. NaYF₄–AFn-FA was prepared for the first time and DOX/NaYF₄-Vp/AFn-FA showed a strong anti-tumor efficacy compared to the free drugs (DOX and Vp) and DOX/NaYF₄-Vp/AFn, suggesting that this is a viable new multi-mechanism strategy for tumor treatment employing NaYF₄–AFn-FA as drug carriers with DOX and Vp.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant no. 81402893).

References

1. W. J. Huang, M. Y. Ding, H. M. Huang, C. F. Jiang, Y. Song, Y. R. Ni, C. H. Lu and Z. Z. Xu, Mater. Res. Bull., 2013, 48, 300–304.
2. L. D. Carlos, R. A. S. Ferreira, V. D. Bermudez, B. Julian-Lopez and P. Escribano, Chem. Soc. Rev., 2011, 40, 536–549.
3. P. P. Yang, S. L. Gai and J. Lin, Chem. Soc. Rev., 2012, 41, 3679–3698.
4. K. Cheng and S. H. Sun, Nano Today, 2010, 5, 183–196.
5. H. J. Fan, M. Knez, R. Scholz, K. Nielsch, E. Pippel and D. Hesse, Nat. Mater., 2006, 5, 627–631.
6. E. Y. Yan, Y. L. Fu, X. Wang, Y. Ding, H. Q. Qian and C. H. Wang, J. Mater. Chem., 2011, 21, 3147–3155.
7. C. C. Huang, W. Huang and C. S. Yeh, Biomaterials, 2011, 32, 556–564.
8. Q. L. Wang, X. X. Huang, Y. J. Long, X. L. Wang, H. J. Zhang, R. Zhu, L. P. Liang, P. Teng and H. Z. Zheng, Carbon, 2013, 59, 192–199.
9. J. Zhou, Z. Liu and F. Y. Li, Chem. Soc. Rev., 2012, 41, 1323–1349.
10. R. Kumar, M. Nyk, T. Y. Ohulchanskyy, C. A. Flask and P. N. Prasad, Adv. Funct. Mater., 2009, 19, 853–859.
11. Z. L. Wang, J. H. Hao, H. L. W. Chan, G. L. Law, W. T. Wong and K. L. Wong, Nanoscale, 2011, 3, 2175–2181.
12. F. Wang and X. G. Liu, Chem. Soc. Rev., 2009, 38, 976–989.
13. L. Cheng, C. Wang and Z. Liu, Nanoscale, 2013, 5, 23–37.
14. Z. J. Gu, L. Yan, G. Tian, S. J. Li, Z. F. Chai and Y. L. Zhao, Adv. Mater., 2013, 25, 375–379.
15. F. Chen, W. B. Bu, S. J. Zhang, X. H. Liu, J. N. Liu, H. Y. Xing, Q. F. Xiao, L. P. Zhou, W. J. Peng, L. Z. Wang and J. L. Shi, Adv. Funct. Mater., 2011, 21, 4285–4294.
16. F. Zhang, Y. F. Shi, X. H. Sun, D. Y. Zhao and G. D. Stucky, Chem. Mater., 2009, 21, 5237–5243.
17. Z. P. Zhen, W. Tang, H. M. Chen, X. Lin, T. Todd, G. Wang, T. Cowger, X. Y. Chen and J. Xie, ACS Nano, 2013, 7, 4830–4837.
18. A. MaHam, Z. W. Tang, H. Wu, J. Wang and Y. H. Lin, Small, 2009, 5, 1706–1721.
19. M. Uchida, M. L. Flenniken, M. Allen, D. A. Willits, B. E. Crowley, S. Brumfield, A. F. Willis, L. Jackiw, M. Jutila, M. J. Young and T. Douglas, J. Am. Chem. Soc., 2006, 128, 16626–16633.
20. K. L. Fan, C. Q. Cao, Y. X. Pan, D. Lu, D. L. Yang, J. Feng, L. N. Song, M. M. Liang and X. Y. Yan, Nat. Nanotechnol., 2012, 7, 459–464.
21. L. Li, C. J. Fang, J. C. Ryan, E. C. Niemi, J. A. Lebron, P. J. Bjorkman, H. Arase, F. M. Torti, S. V. Torti, M. C. Nakamura and W. E. Seaman, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 3505–3510.
22. C. DeSantis, R. Siegel, P. Bandi and A. Jemal, Ca-Cancer J. Clin., 2011, 61, 409–418.
23. C. DeSantis, R. Siegel, P. Bandi and A. Jemal, Ca-Cancer J. Clin., 2011, 61, 409–418.
24. A. Mordente, G. Minotti, G. E. Martorana, A. Silvestrini, B. Giardina and E. Meucci, Biochem. Pharmacol., 2003, 66, 989–998.
25. R. Misra and S. K. Sahoo, Eur. J. Pharm. Sci., 2010, 39, 152–163.
26. K. M. Laginha, S. Verwoert, G. J. R. Charrois and T. M. Allen, Clin. Cancer Res., 2005, 11, 6944–6949.
27. F. Wang, D. K. Chatterjee, Z. Q. Li, Y. Zhang, X. P. Fan and M. Q. Wang, Nanotechnology, 2006, 17, 5786–5791.
28. S. Setua, D. Menon, A. Asok, S. Nair and M. Koyakutty, Biomaterials, 2010, 31, 714–729.
29. H. X. Wu, G. Liu, S. J. Zhang, J. L. Shi, L. X. Zhang, Y. Chen, F. Chen and H. R. Chen, J. Mater. Chem., 2011, 21, 3037–3045.
30. Z. H. Xu, P. A. Ma, C. X. Li, Z. Y. Hou, X. F. Zhai, S. S. Huang and J. Lin, Biomaterials, 2011, 32, 4161–4173.
31. I. F. Li, C. H. Su, H. S. Sheu, H. C. Chiu, Y. W. Lo, W. T. Lin, J. H. Chen and C. S. Yeh, Adv. Funct. Mater., 2008, 18, 766–776.
32. F. Wang, D. K. Chatterjee, Z. Q. Li, Y. Zhang, X. P. Fan and M. Q. Wang, Nanotechnology, 2006, 17, 5786–5791.
33. Y. F. Zhu, Y. Fang and S. Kaskel, J. Phys. Chem. C, 2010, 114, 16382–16388.
34. X. Wang, J. Zhuang, Q. Peng and Y. D. Li, Nature, 2005, 437, 121–124.
35. W. Q. Luo, C. Y. Fu, R. F. Li, Y. S. Liu, H. M. Zhu and X. Y. Chen, Small, 2011, 7, 3046–3056.
36. C. Wang, L. A. Cheng and Z. A. Liu, Biomaterials, 2011, 32, 1110–1120.
37. Z. X. Zhao, Y. N. Han, C. H. Lin, D. Hu, F. Wang, X. L. Chen, Z. Chen and N. F. Zheng, Chem.–Asian J., 2012, 7, 830–837.
38. G. Tian, W. L. Ren, L. Yan, S. Jian, Z. J. Gu, L. J. Zhou, S. Jin, W. Y. Yin, S. J. Li and Y. L. Zhao, Small, 2013, 9, 1929–1938.
39. F. Chen, W. B. Bu, S. J. Zhang, J. N. Liu, W. P. Fan, L. P. Zhou, W. J. Peng and J. L. Shi, Adv. Funct. Mater., 2013, 23, 298–307.
40. D. K. Chatteriee, A. J. Rufalhah and Y. Zhang, Biomaterials, 2008, 29, 937–943.
41. M. Wang, C. C. Mi, W. X. Wang, C. H. Liu, Y. F. Wu, Z. R. Xu, C. B. Mao and S. K. Xu, ACS Nano, 2009, 3, 1580–1586.
42. J. F. Jin, Y. J. Gu, C. W. Y. Man, J. P. Cheng, Z. H. Xu, Y. Zhang, H. S. Wang, V. H. Y. Lee, S. H. Cheng and W. T. Wong, ACS Nano, 2011, 5, 7838–7847.
43. Y. L. Dai, X. J. Kang, D. M. Yang, X. J. Li, X. Zhang, C. X. Li, Z. Y. Hou, Z. Y. Cheng, P. A. Ma and J. Lin, Adv. Healthcare Mater., 2013, 2, 562–567.
44. D. M. Yang, X. J. Kang, P. A. Ma, Y. L. Dai, Z. Y. Hou, Z. Y. Cheng, C. X. Li and J. Lin, Biomaterials, 2013, 34, 1601–1612.
45. D. C. Drummond, M. Zignani and J. C. Leroux, Prog. Lipid Res., 2000, 39, 409–460.
46. Z. M. Bhujwalla, D. Artemov, P. Ballesteros, S. Cerdan, R. J. Gillies and M. Solaiyappan, NMR Biomed., 2002, 15, 114–119.
47. Y. Zhao, L. N. Lin, Y. Lu, S. F. Chen, L. Dong and S. H. Yu, Adv. Mater., 2010, 22, 5255–5259.
48. W. Wei, G. H. Ma, G. Hu, D. Yu, T. Mcleish, Z. G. Su and Z. Y. Shen, J. Am. Chem. Soc., 2008, 130, 15808–15810.
49. L. L. Li, R. B. Zhang, L. L. Yin, K. Z. Zheng, W. P. Qin, P. R. Selvin and Y. Lu, Angew. Chem., Int. Ed., 2012, 51, 6121–6125.
50. J. Sudimack and R. J. Lee, Adv. Drug Delivery Rev., 2000, 41, 147–162.
51. H. A. Meng, M. Liong, T. A. Xia, Z. X. Li, Z. X. Ji, J. I. Zink and A. E. Nel, ACS Nano, 2010, 4, 4539–4550.
52. J. A. Liu, W. B. Bu, L. M. Pan and J. L. Shi, Angew. Chem., Int. Ed., 2013, 52, 4375–4379.

---