Multifunctional near infrared-emitting long-persistence luminescent nanoprobes for drug delivery and targeted tumor imaging

Biomaterials xxx (2014) 1e11

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Multifunctional near infrared-emitting long-persistence luminescent nanoprobes for drug delivery and targeted tumor imaging Junpeng Shi, Xia Sun, Jinlei Li, Huizi Man, Jiangshan Shen, Yanke Yu, Hongwu Zhang* Key Lab of Urban Pollutant Conversion, Institute of Urban Environment, Chinese Academy of Sciences, Jimei Road 1799, Xiamen 361021, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 August 2014 Accepted 2 October 2014 Available online xxx

In this paper, near infrared-emitting long-persistence luminescent porous Zn1.1Ga1.8Ge0.1O4:Cr3þ, Eu3þ @SiO2 nanoprobes have been prepared using mesoporous silica nanospheres both as morphologycontrolling templates and as vessels. These nanoprobes possessed an excellent capacity for drug delivery and allowed for real-time monitoring of the delivery routes of the drug carriers in vivo. The nanoprobes demonstrated a typical mesoporous structure, a brighter NIR emission (696 nm) and a long afterglow luminescence that persisted for 15 d. Furthermore, after surface modification with folic acid (FA), a tumor-targeting group, these nanoprobes exhibited an excellent ability to target tumors with high sensitivity in vitro and in vivo. Importantly, these modified nanoprobes could accurately diagnose tumors and allow for long-term tumor monitoring via in situ and in vivo re-excitation by a red LED lamp. Furthermore, the drug release data demonstrated that the modified nanoprobes could be loaded with a large amount of doxorubicin hydrochloride (DOX) and showed sustained release behavior. Together, the results of this study indicate that these nanoprobes can accurately diagnose tumors, allow for long-term in vivo and in situ monitoring and release DOX in situ to cure tumors. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Persistent luminescence Mesoporous Drug delivery Real-time monitoring Tumor targeting

1. Introduction Mesoporous silica materials have attracted increasing attention for drug delivery in recent decades due to their unique properties, which include a stable mesoporous structure, high specific surface area, easy surface modification and excellent biocompatibility [1e4]. However, because of the complicated environment in vivo, drug carriers entering into biological host systems are difficult to monitor in real time and also may not be able to effectively locate the targeted site (e.g., a tumor), which would decrease the pharmacological function and potentially result in serious adverse reactions and side effects. Thus, it is critical to realize real-time monitoring of the delivery routes of drug carriers in vivo [5]. Fluorescent labeling has been proven to be a simple and effective way of monitoring the route of drug carriers in vivo [6]. Mesoporous silica materials functionalized to have fluorescent properties have attracted much interest because of their potential application in tracking drug carriers [7,8]. To date, a large number of silica-based fluorescence labeling materials, such as organic dyes, upconverting nanoparticles and semiconductor quantum dots (QDs), have been applied for drug delivery [9e12]. However, there

* Corresponding author. Tel./fax: þ86 592 6190773. E-mail addresses: [email protected], [email protected] (H. Zhang).

are still some critical limitations for using these fluorescent probes to monitor drug delivery in vivo. Due to serious photo-bleaching and poor stability, organic dyes are only suitable for observation over short time periods [13]. Although fluorescent nanoprobes, such as semiconductor nanocrystals and upconverting nanocrystals, can effectively overcome the problems of photo-bleaching and instability associated with traditional organic fluorophores [14e16], these photoluminescent nanoprobes still have some shortcomings for practical applications. For example, low photoluminescent signals and strong auto-fluorescence from biological tissues, can drastically decrease the signal-to-noise ratio (SNR) and can even result in false diagnosis [17,18]. Such intrinsic disadvantages would affect their utility for tracking drug delivery in vivo. Recently, there has been increasing interest in employing longpersistence luminescent nanoparticles (LPLNPs) for in vivo imaging. Because the long afterglow of these nanoparticles can last for several hours after they are excited in vitro, real-time in vivo imaging can be achieved after injection without requiring any external illumination source. Thus, the SNR can be significantly improved by removing the background noise originating from in situ excitation [19,20]. Moreover, the afterglow luminescence of near infrared (NIR)-emitting long-persistence luminescent nanoparticles (NLPLNPs) (the afterglow wavelength varies from 650 nm to 900 nm) falls within the tissue transparency window, where light attenuation is largely due to scattering rather than absorption, which is advantageous for long-

http://dx.doi.org/10.1016/j.biomaterials.2014.10.033 0142-9612/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Shi J, et al., Multifunctional near infrared-emitting long-persistence luminescent nanoprobes for drug delivery and targeted tumor imaging, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.033

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Fig. 1. Characterization of NLPLNPs@MSNs. (A) TEM image of NLPLNPs@MSNs. (B) N2 adsorption/desorption isotherm and pore size distributions (inset) of NLPLNPs@MSNs. (C) XRD patterns of NLPLNPs@MSNs. (D) EDS of NLPLNPs@MSNs. Scale bar ¼ 50 nm.

term in vivo imaging with deep penetration and a high SNR [21]. Scherman and co-workers designed NLPLNPs (CaMgSi2O6: Eu, Pr, Mn) to realize in vivo imaging for more than 1 h [22]. Moreover, Yan et al. have utilized NLPLNPs (Zn2.94Ga1.96Ge2O10:Cr3þ, Pr3þ) to diagnose tumors and realize long-term monitoring of tumors [23]. More recently, new and non-toxic NLPLNPs (ZnGa1.995Cr0.005O4) with a narrow size distribution were used to realize tumor targeting and efficient cell tagging to track the biological fate of cells in vivo, without any acute toxicity, in healthy mice [24]. Although the materials mentioned above have exhibited great advantages for longterm in vivo imaging with a high SNR, they were still unsuitable for use as drug carriers because of their lack of a porous structure and the non-uniform particle sizes and morphologies, all of which have hindered the application of NLPLNPs in tracking drug delivery in vivo. Therefore, it is necessary to develop novel monodisperse NLPLNPs with a porous structure to realize not only tumor diagnosis but also the drug delivery. Here, we utilized mesoporous silica nanospheres (MSNs) both as drug carriers and as morphology-controlling templates to design porous NLPLNPs. Furthermore, based on a recent report, the long afterglow performance of Zn1.1Ga1.8Ge0.1O4:Cr3þ was the best among those of all germanium substituted ZnGa2O4 (Zn1þxGa22xGexO4:Cr3þ), including ZnGa1.995Cr0.005O4 and Zn2.94Ga1.96Ge2O10:Cr3þ [25]. Thus, in this paper, we loaded Zn1.1Ga1.8Ge0.1O4:Cr3þ, Eu3þ into the pores of MSNs to prepare a novel trackable drug carrier (Zn1.1Ga1.8Ge0.1O4:Cr3þ, Eu3þ @SiO2) (NLPLNPs@MSNs). Due to the MSN templates, the obtained samples showed a porous structure, spherical morphology, narrow size

distribution, lager specific surface area and large pore capacity, indicating excellent drug carrier properties. Moreover, the NLPLNPs@MSNs demonstrated a brighter NIR emission (696 nm), and the long afterglow luminescence persisted for 15 d. In particular, the NLPLNPs@MSNs could be excited by a red LED lamp in vivo, indicating the capacity for real-time tracking and imaging continuously in vivo for long periods of time. Furthermore, after surface modification with folic acid (FA), a tumor-targeting group, the NLPLNPs@MSNs exhibited excellent tumor targeting ability with high sensitivity both in vitro and in vivo. All the results indicate that NLPLNPs@MSNs-FA can accurately diagnose tumors, be used for long-term in vivo and in situ monitoring and release a drug in situ to cure tumors. 2. Material and methods 2.1. Materials Zn(NO3)2$6H2O, Ga(NO3)3$6H2O, Cr(NO3)3$9H2O, Eu(NO3)3$6H2O, and GeO2 were purchased from Aladdin (Shanghai, China). Tetraethoxysilane (TEOS), 3-aminopropyl triethoxysilane (APTES), hexadecyl trimethyl ammonium bromide (CTAB), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethanol, dichloromethane and diethanolamine were purchased from Shanghai Chemical Reagent Company (Shanghai, China). N-(3Dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 4-dimethylaminopyridine (DMAP), folic acid (FA), DOX and methyl thiazolyl tetrazolium (MTT) were

Please cite this article in press as: Shi J, et al., Multifunctional near infrared-emitting long-persistence luminescent nanoprobes for drug delivery and targeted tumor imaging, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.033

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purchased from SigmaeAldrich (Sigma, USA). RPMI 1640 medium, MEM medium, and fetal bovine serum (FBS) were purchased from Life Technologies (Gibco, USA). Hepatic carcinoma cells (HepG2), normal liver cells (L02) and human umbilical vein endothelial cells (HUVECs) were obtained from the Cell Resource Center of the Shanghai Institutes for Biological Sciences (SIBS, China). Kunming normal mice and H22 tumor-bearing mice were purchased from the Shanghai lab animal research center (Shanghai, China). 2.2. Synthesis of NLPLNPs@MSNs The MSNs were synthesized according to a previous method with modifications [26]. In a typical procedure, 0.2 g of CTAB, 25 mL of water, 5 mL of ethanol, and 50 mL of diethanolamine were mixed together and stirred in a water bath at 60  C for 30 min. Then, 2 mL of TEOS was added into the mixture dropwise over 2 min while stirring. An additional 2 h of stirring was employed. The solution was then cooled to room temperature. Subsequently, the MSNs were collected by centrifugation, and the precipitates were dried at 80  C. The dried sample was calcined at 550  C for 5 h to remove the CTAB templates. The NLPLNPs@MSNs were synthesized by a mesoporous silica nanoparticles template method with some modifications [19]. Briefly, 1 mL of Zn2þ (1 M), 3.28 mL of Ga3þ (0.5 M), 0.9 mL of Ge4þ (0.1 M), 0.82 mL of Cr3þ (0.01 M) and 0.82 mL of Eu3þ (0.01 M) were prepared by dissolving Zn(NO3)2, Ga(NO3)3, GeO2, Cr(NO3)3, and Eu(NO3)3 according to the chemical formula of Zn1.1Ga1.8Ge0.1O4:0.5% Cr3þ, 0.5% Eu3þ. Subsequently, 0.2 g of MSNs was added into the above mixed ionic solution under slow stirring, and the reaction was kept at 45  C for 24 h. Then, the sample was dried at 80  C for 3 h and 110  C for 4 h. Finally, the dried sample was calcined in air at 600  C for 2 h.

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(Edinburgh, UK). The FT-IR spectra were recorded on a Nicolet is10 spectrometer (Thermo Scientific, USA). The UVevis spectrum of DOX was obtained using an ultraviolet and visible spectrophotometer (Thermo Scientific, USA). The N2 adsorption/desorption isotherms were measured at liquid nitrogen temperature (77 K) using a Micromeritics ASAP 2020 instrument. Zeta potential was measured on a Zetasizer Nano-ZS (Malvern, UK). 2.5. Cytotoxicity assay To determine the cytotoxicity of NLPLNPs@MSNs-NH2, the MTT assay and a cell apoptosis assay were performed. Briefly, cells

2.3. Surface functionalization of NLPLNPs@MSNs The modification of the NLPLNPs@MSNs was conducted according to a previous method with slight modification [23,24]. Briefly, NLPLNPs@MSNs were dispersed in a 5 mM NaOH solution by sonication for 5 min. Subsequently, the NLPLNPs@MSNs solution was vigorously stirred for 24 h. The NLPLNPs@MSNs solution was centrifuged at 10,000 rpm for 10 min to collect the precipitates. The obtained sample was dried at 60  C and denoted as NLPLNPs@MSNs-OH. To obtain NLPLNPs@MSNs-NH2, 0.1 g of NLPLNPs@MSNs-OH was redispersed in 40 mL of DMF, and 400 mL of APTES was added under constant stirring. The suspension was then stirred at room temperature for 24 h. The resulting NLPLNPs@MSNs-NH2 was collected by centrifugation and washed with DMF to remove excess APTES. To conjugate FA to NLPLNPs@MSNs-NH2, 10 mg of NLPLNPs@MSNs-NH2 was dispersed in 10 mL of dichloromethane by sonication, and then 10 mL of DMSO and 8 mg of FA were added. Subsequently, 10 mg of EDC, 20 mg of NHS and 10 mg of DMAP were added into the mixture. The mixture was gently stirred for 48 h in the dark at room temperature. Finally, the resulting NLPLNPs@MSNs-FA were obtained by centrifugation. 2.4. Characterization The morphology of the samples were characterized by transmission electron microscopy (TEM, Hitachi, Japan). XRD patterns were recorded on a X'Pert Pro instrument (PANalytical, Netherlands). The actual composition of NLPLNPs was determined on Axios-MAX X-ray fluorescence spectrometer (PANalytical, Netherlands). The excitation and emission spectra and the NIR afterglow decay were determined on an FLS920 spectrometer

Fig. 2. Optical properties of NLPLNPs@MSNs. (A) Excitation and emission spectra of NLPLNPs@MSNs. (B) Afterglow decay curve of NLPLNPs@MSNs after 5 min of irradiation with a 254 nm UV lamp and a red LED lamp (the inset shows digital photos of NLPLNPs@MSNs under 254 nm UV excitation). (C) NIR afterglow decay images detected by a CCD camera at different time points after stopping UV irradiation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Shi J, et al., Multifunctional near infrared-emitting long-persistence luminescent nanoprobes for drug delivery and targeted tumor imaging, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.033

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Fig. 3. Schematic diagram for the synthesis, surface modification and application of NLPLNPs@MSNs.

Fig. 4. Surface modification of NLPLNPs@MSNs. (A) FT-IR spectra. (B) Zeta potentials.

Fig. 5. Characterization of NLPLNPs@MSNs-FA. (A) Excitation and emission spectra of the NLPLNPs@MSNs-FA in PBS. (B) Afterglow decay curves of NLPLNPs@MSNs-FA after 5 min of irradiation with a 254 nm UV lamp and a red LED lamp (the inset shows digital photos of NLPLNPs@MSNs-FA in PBS under 254 nm UV excitation). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Shi J, et al., Multifunctional near infrared-emitting long-persistence luminescent nanoprobes for drug delivery and targeted tumor imaging, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.033

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(HUVECs, L02 and HepG2) were seeded into 96-well plates and cultured in a CO2 incubator (Heracell, Germany) for 12 h. Then, the old medium was removed and replaced with fresh medium containing various concentrations of NLPLNPs@MSNs-NH2. Subsequently, the 96-well plates were placed in a CO2 incubator for 24 h. To measure the toxicity, MTT (0.5 mg/mL) was added to each well for 4 h at 37  C. Then, DMSO was added to each well, and the plate was agitated on a plate shaker for 20 min. The absorbance was then measured at 490 nm using a microplate reader (Molecular Devices, USA). Cell apoptosis was measured using the Annexin-V-Fluos and propidium iodide (PI) apoptosis detection kit (Roche, Germany). Briefly, cells (HUVECs, L02 and HepG2) were exposed to NLPLNPs@MSNs-NH2 for 24 h. Then, the cells were collected and re-suspended in 100 mL of Annexin-V-Fluos labeling solution (containing the Annexin-V-Fluos labeling reagent and PI). The cells were incubated with the labeling solution at 25  C for 15 min, transferred to 96-well plates, and then analyzed using a flow cytometer (Beckman Coulter, USA). 2.6. In vitro imaging To evaluate the potential for tumor cell targeting with NLPLNPs@MSNs-FA in vitro, HepG2 and L02 cells were cultured in MEM and RPMI 1640 with 10% FBS. The same number cells were seeded in 35 mm culture dishes for 2 h in a CO2 incubator. After adhesion, the two different cell types were treated with fresh medium containing the nanoprobes (50 mg/mL) for 2 h at 37  C. Subsequently, the cell medium was removed, and the cells were

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washed with PBS several times. The cell luminescence imaging was then collected on a laser scanning confocal microscope (LSCM) (Zeiss, Germany). In addition, afterglow imaging was collected on an IVIS Lumina II imaging system (Xenogen, USA) after excitation with 254 nm for 60 s. The exposure time was set to 60 s. To evaluate the tumor cell detection sensitivity in vitro, HepG2 cells were cultured with 50 mg/mL of NLPLNPs@MSNs-FA for 2 h at 37  C. The cells were then washed and resuspended in PBS, and the cell number was counted using a hemocytometer and microscope (Zeiss, Germany). Lastly, the HepG2 cells labeled with NLPLNPs@MSNs-FA in 100 mL PBS were seeded into a 96-well plate by 1:2 serial dilutions from 50,000 cells to 98 cells. The plate was imaged using an IVIS Lumina II imaging system after excitation with 254 nm for 30 s. The exposure time was set to 60 s. 2.7. Drug loading and release The drug loading and release was characterized according to a previous method with slight modification [10,27]. Briefly, 10 mg of NLPLNPs@MSNs-FA was dispersed in 4 mL of DOX aqueous solution (1 mg/mL). After stirring for 12 h in the dark condition, the DOXloaded sample was collected by centrifugation and denoted as DOX-NLPLNPs@MSNs-FA. The DOX-NLPLNPs@MSNs-FA were then transferred to a dialysis tube and immersed in 2 mL of pH ¼ 7.4 phosphoric acidic buffer (PBS) at 37  C. At selected time points, the samples were centrifuged, and PBS was removed and replaced with an equal volume of fresh PBS. The amount of released DOX in the supernatant solutions was measured based on the absorbance at 480 nm using a UVevis spectrophotometer. To calculate the DOX-

Fig. 6. Cytotoxicity of NLPLNPs@MSNs-NH2. (A) Cell viability assay (MTT). (B, C) Cell apoptosis assay. The data represents the mean ± SD (n ¼ 3).

Please cite this article in press as: Shi J, et al., Multifunctional near infrared-emitting long-persistence luminescent nanoprobes for drug delivery and targeted tumor imaging, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.033

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loading efficiency, the residual DOX content (RDOx) and the original DOX content (ODOx) were determined by UVevis measurements at 480 nm. The loading efficiency of DOX was then calculated as follows: [(ODOx  RDOx)/ODOx]  100%.

variance (ANOVA) followed by Dunnett's multiple comparison tests. A value of P < 0.05 was considered significantly different. All analyses were performed using the statistical package for the social science program (SPSS 16.0).

2.8. In vivo imaging

3. Results and discussion

To evaluate the potential for tumor imaging with NLPLNPs@MSNs-FA in vivo, different numbers of HepG2 cells (1500, 3000 and 6000) labeled with NLPLNPs@MSNs-FA were subcutaneously injected into the abdomen of a mouse after excitation at 254 nm for 60 s. Then, analysis on an IVIS Lumina II imaging system was used to determine the correlation between cell number and luminescence in vivo. Male Kunming (KM) mice implanted with murine hepatoma H22 tumors overexpressing folate receptors were used to investigate tumor targeting with DOX-NLPLNPs@MSNs-FA in vivo [28,29]. The DOX-NLPLNPs@MSNs-FA was dispersed in PBS solution (1 mg/ mL), excited for 60 s with a 254 nm UV lamp, and then injected through the tail vein into a normal and H22 tumor-bearing mice. The luminescence signals were subsequently collected on an IVIS Lumina II imaging system. The exposure time was set to 60 s.

3.1. Synthesis and characterization of NLPLNPs@MSNs

2.9. Statistical analysis Results are expressed as the mean ± SD from three independent experiments, the data were analyzed by a one-way analysis of

The strategy for preparing multifunctional NLPLNPs@MSNs first involved the synthesis of MSNs as morphology-controlling templates, followed by the formation of Zn1.1Ga1.8Ge0.1O4:Cr3þ, Eu3þ nanoclusters inside their pores. Transmission electron microscopy (TEM) showed that the MSN templates possessed a highly regular spherical morphology and a narrow size distribution centered at approximately 50 nm (Fig. S1A). And the XRD pattern of MSNs showed a typical mesoporous silica nanoparticles broad peak at low angles (Fig. S1B). Furthermore, it could be observed that the synthesized NLPLNPs@MSNs maintained their original spherical morphology and free-standing nature (Fig. 1A). The N2 adsorption/ desorption isotherms of the NLPLNPs@MSNs exhibited a typical type IV isotherm for mesoporous materials (Fig. 1B). The BrunauerEmmett-Teller (BET) surface area and average pore size were calculated to be 158.3 m2/g and 2.3 nm (Fig. 1B, inset), respectively. The combination of a uniform mesoporous pore size and small particle size is highly advantageous and favorable for drug delivery applications. In addition, the XRD pattern of the NLPLNPs@MSNs

Fig. 7. Tumor targeting in vitro. (A) Luminescence imaging of L02 and HepG2 cells incubated with NLPLNPs@MSNs-FA for 2 h (B) LSCM images of L02 and HepG2 cells incubated with NLPLNPs@MSNs-FA for 2 h. Scale bar ¼ 20 mm.

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showed that the typical spinel phases of ZnGa2O4 and Zn2GeO4 were formed by the heat treatment at 600  C, and the MSN templates remained as amorphous silicon dioxide phase (Fig. 1C). Energy dispersive spectroscopy (EDS) results indicated the existence of Si, Zn and Ga (Fig. 1D), which further confirmed the formation of mesoporous NLPLNPs@MSNs. The actual composition of NLPLNPs was determined to be Zn1.1Ga1.8Ge0.1O4:Cr3þ0.009, Eu3þ0.009 by X-ray fluorescence spectrometry. Collectively, these results indicated that Zn1.1Ga1.8Ge0.1O4:Cr3þ0.009, Eu3þ0.009@SiO2 with a highly regular spherical morphology, narrow size distribution and mesoporous structure were successfully synthesized. The excitation and emission spectra of the NLPLNPs@MSNs are shown in Fig. 2. Fig. 2A showed that a narrow-band emission peak was at 696 nm after excitation at 254 nm. This NIR emission is characteristic of Cr3þ ions and can be attributed to the spinforbidden 2E / 4A2 transition [30]. The excitation spectrum of the NLPLNPs@MSNs consisted of four main excitation bands with peaks at 270 nm, 412 nm, 580 nm and 664 nm, which were attributed to the charge transfer of CrOþ / O2, 4A2 / 4T1, 4 A2 / 4T2 and 4A2 / E transition of Cr3þ, respectively [23,30]. Furthermore, the NLPLNPs@MSNs also exhibited excellent superlong-lasting NIR luminescence. As shown in Fig. 2B, the persistent luminescence of the NLPLNPs@MSNs at 696 nm showed a slow decay after irradiation with 254 nm light for 5 min. The NIR afterglow could still be detected by a CCD camera after 15 days without any illumination (Fig. 2C), indicating that the NLPLNPs@MSNs have excellent optical properties for bioimaging in vivo. It should be noted that the red light (600e700 nm) in the tissue transparency window can also induce the long-lasting luminescence (Fig. 2B), which suggests that the afterglow image signal of the NLPLNPs@MSNs in tissue may be recovered and can persist further via in situ and in vivo re-excitation by red light [31]. All these result

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indicated that the NLPLNPs@MSNs could provide real-time monitoring for drug delivery in vivo. 3.2. Surface functionalization of NLPLNPs@MSNs To improve their water-solubility, biocompatibility and tumortargeting ability, FA, a tumor-targeting group, was grafted onto the surface of the NLPLNPs@MSNs through a three-step reaction scheme, as illustrated in Fig. 3. First, a large number of hydroxyl groups were produced on the surfaces of the NLPLNPs@MSNs via treatment with NaOH. Subsequently, amino groups were linked with the hydroxyl groups to form NLPLNPs@MSNs-NH2. Conjugation of NLPLNPs@MSNs-NH2 with FA was then carried out to produce the final NLPLNPs@MSNs-FA nanoprobes for drug loading and tumor targeting (Fig. 3). Surface functionalization of the NLPLNPs@MSNs was confirmed by Fourier transform infrared spectrometry (FTIR) and Zeta potential analysis. The FTIR spectra showed a strong absorption band at 3441 cm1 (OeH), indicating successful surface modification with hydroxyl groups (Fig. 4A). After amination, the appearance of absorption bands at 1390 cm1 (CeN stretching bands), 1496 cm1 (primary amine bands), 2933 cm1 (CH2 stretching bands) and 3400 cm1 (NeH stretching bands) suggested the successful modification with APTES (Fig. 4A). In the FTIR spectrum of NLPLNPs@MSNs-FA, the absorption bands at 1508 cm1 (amide II band) and 1633 cm1 (stretching vibration of C]O) suggested that FA was successfully grafted onto the surface of the NLPLNPs@MSNs (Fig. 4A) [32]. In addition, the change in the zeta potential from negative (24 mV) to positive (þ12 mV) after the amination process also supported the success of the amination modification (Fig. 4B). The zeta potential changed from þ12 mV to 35 mV after FA modification (Fig. 4B), further supporting the formation of the tumor-targeting NLPLNPs@MSNs-FA nanoprobes.

Fig. 8. Detection sensitivity of HepG2 labeled with NLPLNPs@MSNs-FA in vitro. (A) Luminescence imaging of different numbers of HepG2 cells labeled with NLPLNPs@MSNs-FA. (B) Correlation between cell number and luminescence signal.

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After functionalization, the NLPLNPs@MSNs-FA nanoprobes were very easily dispersed in water to form a luminescence solution. Compared with the NLPLNPs@MSNs, the emission spectrum of the NLPLNPs@MSNs-FA solution was unchanged and also exhibited an NIR emission peak at 696 nm after excitation at 254 nm. The surface modification induced some changes in the excitation spectrum of the NLPLNPs@MSNs-FA solution. The two bands at 254 and 580 nm became much stronger, while the intensity of the bands at 475 and 664 nm was obviously decreased (Fig. 5A). More importantly, the NLPLNPs@MSNs-FA solution also exhibited excellent long-lasting NIR luminescence after irradiation with 254 nm light for 5 min, and the afterglow signal could be recovered and persisted for a long time after re-excitation by red light (Fig. 5B), suggesting that these tumor-targeting nanoprobes can be utilized to realize real-time monitoring of the drug carrier in vivo.

vein endothelial cells (HUVECs) and hepatic carcinoma cells (HepG2) to evaluate the cytotoxicity of the NNLPLNPs@MSNs-NH2. As shown in Fig. 6A, no significant toxicity to the three different cell types were observed after exposure to 10e100 mg/L of NNLPLNPs@MSNs-NH2 for 24 h. Even when the sample concentration was 100 mg/L, the cell viability remained at 94.3%, 93.5% and 95.3% for the L02, HepG2 and HUVEC cells, respectively, suggesting low toxicity to the cells. The apoptosis assay further confirmed this result. As shown in Fig. 6B and C, when the concentration of NNLPLNPs@MSNs-NH2 reached 100 mg/L, the percent of viable cells was 98.8%, 97.0% and 93.9% for the L02, HepG2 and HUVEC cells, respectively. Together, these results suggest that this drug delivery system has low cytotoxicity, which was essential for their subsequent application in vivo imaging and drug delivery. 3.4. Tumor imaging in vitro and in vivo

3.3. Cytotoxicity assay Because an excessive amount of Cr3þ is toxic to humans, toxicity testing of the NLPLNPs@MSNs is a critical requirement for future clinical applications. Thus, in this experiment, MTT and apoptosis assays were performed on normal liver cells (L02), human umbilical

To evaluate the targeting ability of the NNLPLNPs@MSNs-FA to tumor cells, HepG2 cells, which overexpress folate receptors, and L02 cells, which have low folate receptor expression, were chosen as model cells [33]. As shown in Fig. 7A, the afterglow luminescence signal of the HepG2 cells was much higher than that of the L02 cells

Fig. 9. Detection of HepG2 cells labeled with NLPLNPs@MSNs-FA in vivo. (A) In vivo luminescence images of a normal mouse after subcutaneous injection of HepG2 cells labeled with NLPLNPs@MSNs-FA (60 s of irradiation with a 254 nm UV lamp before injection). (B) In vivo luminescence images of secondary excitation with a red LED lamp for 2 min. The images were taken 2 h after subcutaneous injection. (a) Correlation between cell number and in vivo luminescence signal after subcutaneous injection. (b) Correlation between cell number and in vivo luminescence signal after secondary excitation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Shi J, et al., Multifunctional near infrared-emitting long-persistence luminescent nanoprobes for drug delivery and targeted tumor imaging, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.033

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after 5 min. Though the afterglow luminescence signal gradually decreased over time, the afterglow luminescence signal of the HepG2 cells was still strong enough to be precisely measured after 30 min, while the afterglow luminescence signal of the L02 cells was very weak. These results suggested that the NNLPLNPs@MSNs-FA possessed a stronger ability to target HepG2 tumor cells compared to normal L02 cells. In addition, confocal microscopy was used to further study the interactions between the NLPLNPs@MSNs-FA and cells. As shown in Fig. 7B, there was a large difference between the L02 and HepG2 cells in terms of the intracellular uptake of NLPLNPs@MSNs-FA. The uptake of NLPLNPs@MSNs-FA in the HepG2 cells was much higher than in the L02 cells, and this enhanced efficiency resulted from receptor-mediated uptake of the NLPLNPs@MSNs-FA by their corresponding receptors, which are known to be overexpressed on the surface of cancer cells [34]. These results suggested that the NLPLNPs@MSNs-FA possessed an excellent targeting capacity for tumor cells in vitro. It is very important to detect tumor cells with high sensitivity and a good correlation between signal and tumor cell number. As shown in Fig. 8A, there was a strong afterglow luminescence signal when the cell number was 50,000. As the cell number decreased, the luminescence signal gradually decreased. However, the luminescence of as few as 98 cells was still detectable (SNR ¼ 5.5), and a good correlation (R2 ¼ 0.9956) between the cell number and luminescence signal was also observed (Fig. 8B). These results indicated that NLPLNPs@MSNsFA could be employed to detect cancer cells in vitro with good sensitivity and a correlation between signal and cell number. To investigate the potential of NLPLNPs@MSNs-FA for tumor imaging in vivo, different numbers of HepG2 cells labeled with NLPLNPs@MSNs-FA were subcutaneously injected into the abdomen of a mouse after 254 nm excitation for 60 s. As shown in Fig. 9A, the luminescence signal could be clearly detected in the abdomen of the mouse. Similar to the in vitro results, a good correlation (R2 ¼ 0.9884) between cell number and luminescence signal was also obtained (Fig. 9A), suggesting the potential utility of NLPLNPs@MSNs-FA for tumor cell detection in vivo. Furthermore, even after 20 min, the excellent correlation between cell number and afterglow luminescence signal was maintained, indicating that NLPLNPs@MSNs-FA can be utilized to realize precise tumortargeting detection for long times. Although the super-long afterglow of NLPLNPs@MSNs-FA can afford long detection times in vivo, decreases in their afterglow luminescence intensity over time are unavoidable and resulted in decreased luminescence signal and then low detection sensitivity. Thus, we adopted an in vivo and in situ repeatable excitation method to obtain continuous, real-time and high SNR imaging in vivo. Fig. 9B showed the results for the repeated excitation of the NLPLNPs@MSNs-FA with a red LED lamp (600e700 nm) for 2 min after 2 h of emission decay in vivo. Importantly, the luminescence signal could be recovered at the HepG2 site. Furthermore, a good correlation between cell number and luminescence signal could still be obtained after repeated in vivo excitation (Fig. 9B), indicating that the excellent tumor-targeting detection of NLPLNPs@MSNs-FA can persist for any required amount of time. Moreover, red light sources (especially 650 nm) have been applied widely in medical diagnosis because they have a strong capacity for tissue penetration and cause minimal tissue injury [31]. Therefore, our results revealed that NLPLNPs@MSNs-FA demonstrated not only an excellent capacity for continuous, real-time and high SNR imaging in vivo but also realized continuous in vivo and in situ monitoring of drug delivery.

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commonly used chemotherapeutic drug for cancer therapy [35,36], was selected as a model drug to evaluate the loading and controlled releasing behavior of this system. The amount of loaded DOX was calculated to be 4.5% by weight, as determined by the UVevis absorbance of DOX at 480 nm. Cumulative drug release profiles for the DOX-NLPLNPs@MSNsFA in vitro (PBS, pH ¼ 7.4, normal physiological environment) are shown in Fig. 10A. A fast release from the DOX-NLPLNPs@MSNs-FA was observed within 15 h, followed by slow release. Approximately 94% of the DOX was released within 50 h. Furthermore, almost all of the DOX (99%) was released from the DOX-NLPLNPs@MSNs-FA within 60 h. This drug carrier system obviously demonstrated sustained release behavior. In addition, Fig. 10B shows the UVevis spectra of pure and released DOX. It can be observed that the maximum absorption wavelength did not change, and no new bands appeared. Therefore, the DOX was released in its original form, and no detectable impurities were created in this system. These results indicated that NLPLNPs@MSNs-FA could act as a drug storage/release carrier. 3.6. Tumor targeting and drug tracking in vivo To investigate the potential application of NLPLNPs@MSNs-FA as a trackable drug carrier for real-time monitoring and tumor targeting in vivo, DOX-NLPLNPs@MSNs-FA (0.2 mg) was injected via the tail vein into H22 tumor-bearing mice and normal mice. As

3.5. Drug release in vitro To further investigate the drug loading and release abilities of NLPLNPs@MSNs-FA, Doxorubicin hydrochloride (DOX), a

Fig. 10. Drug release. (A) Drug release profiles for DOX-NLPLNPs@MSNs-FA. (B) UVevis spectrum of DOX.

Please cite this article in press as: Shi J, et al., Multifunctional near infrared-emitting long-persistence luminescent nanoprobes for drug delivery and targeted tumor imaging, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.033

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J. Shi et al. / Biomaterials xxx (2014) 1e11

shown in Fig. 11A, the afterglow luminescence signal of the DOXNLPLNPs@MSNs-FA could be detected in the whole body within 10 min, and a strong luminescence signal was observed at the tumor site, indicating the excellent tumor-targeting capacity of DOXNLPLNPs@MSNs-FA. Furthermore, the tumor image with high SNR persisted for more than 15 min due to the long time NIR afterglow. Even after 20 min, the SNR at the tumor site remained at 2.85 (Fig. S2), which was strong enough to distinguish the tumor. To further enhance the SNR and the resolution of the tumor image, the DOX-NLPLNPs@MSNs-FA was re-excited after 2 h in vivo using irradiation from a red LED lamp for 2 min. It can be noted that the

clear luminescence signal at the tumor site recovered (Fig. 11B, C). More importantly, the tumor site showed a stronger SNR (5.7) after being re-irradiated (Fig. S3), which suggested that more DOXNLPLNPs@MSNs-FA accumulated at the tumor site with blood circulation. This result proved that DOX-NLPLNPs@MSNs-FA possessed an excellent capacity for continuous, real-time and high SNR imaging in vivo. Furthermore, we evaluated the luminescence signal from major organs and a tumor after 6 h of emission decay. Almost no luminescence signal could be observed in the heart and kidney. In contrast, strong luminescence signals were observed in the liver and spleen, and clear signals could also be

Fig. 11. In vivo luminescence images of H22 tumor-bearing mice. (A) H22 tumor-bearing mice after intravenous injection of DOX-NLPLNPs@MSNs-FA. (B, C) Secondary excitation with a red LED lamp for 2 min, performed 2 h after intravenous injection of DOX-NLPLNPs-FA. (D) Luminescence images of the following isolated organs and a tumor from a H22 tumor-bearing mouse taken 6 h after injecting DOX-NLPLNPs@MSNs-FA: (1) liver, (2) spleen, (3) kidney, (4) heart, (5) lung, and (6) tumor. (E) Normal mouse after intravenous injection of DOX-NLPLNPs@MSNs-FA. (F) Secondary excitation with a red LED lamp for 2 min, performed 2 h after intravenous injection of DOX-NLPLNPs@MSNs-FA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Shi J, et al., Multifunctional near infrared-emitting long-persistence luminescent nanoprobes for drug delivery and targeted tumor imaging, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.033

J. Shi et al. / Biomaterials xxx (2014) 1e11

found in the lung and tumor (Fig. 11D). These results were very consistent with the results for tumor imaging in vivo and further confirmed the excellent tumor targeting ability of NLPLNPs@MSNsFA. In addition, for comparison, we performed similar experiments on normal mice, but no luminescence signal could be observed at the same site of the normal mice (Fig. 11E). We also did not observe an enhanced luminescence signal in the same site of normal mice after repeat irradiation with a red LED lamp (Fig. 11F), which provided additional evidence for the excellent tumor targeting ability of NLPLNPs@MSNs-FA. Collectively, these results indicated that DOX-NLPLNPs@MSNs-FA could accurately diagnose tumors and be used for continuous and real-time monitoring of drug delivery in vivo. 4. Conclusions In summary, mesoporous silica nanospheres were used both as morphology-controlling templates and as vessels for the synthesis of multifunctional NIR long-persistence luminescence mesoporous nanoprobes. After surface modification with the tumor targeting agent FA, NLPLNPs@MSNs-FA not only maintained an excellent long afterglow NIR luminescence but also possessed a strong tumortargeting capacity. Tumor targeting assays in vitro and in vivo confirmed that NNLPLNPs@MSNs-FA possessed a stronger targeting capacity for HepG2 tumor cells than for normal L02 cells, and a good correlation between cell number and luminescence signal was observed in vitro and in vivo. Furthermore, our results indicated that DOX-NLPLNPs@MSNs-FA could accurately target tumors and be used for continuous real-time monitoring of drug delivery in vivo. We expect this study to provide a new perspective on the design of multifunctional structures for trackable drug delivery based on NLPLNPs. Acknowledgments This work was supported by the Natural Science Foundation of Ningbo (Grant No. 2013A610127), the National High-Tech R&D Program of China (863 Program, 2012AA062607), ShuangBai plan of Fujian province, the National Key Technology Support Program (2012BAC25B04) and the Science and Technology Project in Xiamen (3502Z20132012). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.10.033. References [1] Yang P, Quan Z, Hou Z, Li C, Kang X, Cheng Z, et al. A magnetic, luminescent and mesoporous coreeshell structured composite material as drug carrier. Biomaterials 2009;30:4786e95. [2] Vallet-Regi M, Balas F, Arcos D. Mesoporous materials for drug delivery. Angew Chem Int Ed 2007;46:7548e58. [3] Slowing II , Trewyn BG, Giri S, Lin VY. Mesoporous silica nanoparticles for drug delivery and biosensing applications. Adv Funct Mater 2007;17:1225e36. [4] Li ZJ, Zhang YJ, Zhang HW, Fu HX. Long-lasting phosphorescence functionalization of mesoporous silica nanospheres by CaTiO3:Pr3þ for drug delivery. Microporous Mesoporous Mater 2013;176:48e54. [5] Lei J, Wang L, Zhang J. Superbright multifluorescent core-shell mesoporous nanospheres as trackable transport carrier for drug. ACS Nano 2011;5: 3447e55. [6] Qiu F, Wang D, Zhu Q, Zhu L, Tong G, Lu Y, et al. Real-time monitoring of anticancer drug release with highly fluorescent star-conjugated copolymer as a drug carrier. Biomacromolecules 2014;15:1355e64. [7] Yang P, Gai S, Lin J. Functionalized mesoporous silica materials for controlled drug delivery. Chem Soc Rev 2012;41:3679e98. [8] Lu J, Liong M, Li Z, Zink JI, Tamanoi F. Biocompatibility, biodistribution, and drug-delivery efficiency of mesoporous silica nanoparticles for cancer therapy in animals. Small 2010;6:1794e805.

11

[9] He Q, Shi J. Mesoporous silica nanoparticle based nano drug delivery systems: synthesis, controlled drug release and delivery, pharmacokinetics and biocompatibility. J Mater Chem 2011;21:5845e55. [10] Dai Y, Pa Ma, Cheng Z, Kang X, Zhang X, Hou Z, et al. Up-conversion cell imaging and pH-induced thermally controlled drug release from NaYF4: Yb3þ/ Er3þ@ hydrogel coreeshell hybrid microspheres. ACS Nano 2012;6:3327e38. [11] Kim J, Lee JE, Lee J, Yu JH, Kim BC, An K, et al. Magnetic fluorescent delivery vehicle using uniform mesoporous silica spheres embedded with monodisperse magnetic and semiconductor nanocrystals. J Am Chem Soc 2006;128: 688e9. [12] Gai S, Yang P, Li C, Wang W, Dai Y, Niu N, et al. Synthesis of magnetic, upconversion luminescent, and mesoporous core-shell-structured nanocomposites as drug carriers. Adv Funct Mater 2010;20:1166e72. [13] Resch-Genger U, Grabolle M, Cavaliere-Jaricot S, Nitschke R, Nann T. Quantum dots versus organic dyes as fluorescent labels. Nat Methods 2008;5:763e75. [14] Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, et al. Quantum dots for live cells, in vivo imaging, and diagnostics. Science 2005;307:538e44. [15] Wang ZL, Hao J, Chan HL, Law GL, Wong WT, Wong KL, et al. Simultaneous synthesis and functionalization of water-soluble up-conversion nanoparticles for in-vitro cell and nude mouse imaging. Nanoscale 2011;3:2175e81. [16] Chatterjee DK, Rufaihah AJ, Zhang Y. Upconversion fluorescence imaging of cells and small animals using lanthanide doped nanocrystals. Biomaterials 2008;29:937e43. [17] Maldiney T, Kaikkonen MU, Seguin J, le Masne de Chermont Q, Bessodes M, Airenne KJ, et al. In vitro targeting of avidin-expressing glioma cells with biotinylated persistent luminescence nanoparticles. Bioconjug Chem 2012;23: 472e8. [18] Maldiney T, Richard C, Seguin J, Wattier N, Bessodes M, Scherman D. Effect of core diameter, surface coating, and PEG chain length on the biodistribution of persistent luminescence nanoparticles in mice. ACS Nano 2011;5:854e62. [19] Li ZJ, Zhang HW, Sun M, Shen JS, Fu HX. A facile and effective method to prepare long-persistent phosphorescent nanospheres and its potential application for in vivo imaging. J Mater Chem 2012;22:24713e20. [20] Wu BY, Wang HF, Chen JT, Yan XP. Fluorescence resonance energy transfer inhibition assay for a-fetoprotein excreted during cancer cell growth using functionalized persistent luminescence nanoparticles. J Am Chem Soc 2010;133:686e8. [21] Liu F, Yan W, Chuang YJ, Zhen Z, Xie J, Pan Z. Photostimulated near-infrared persistent luminescence as a new optical read-out from Cr3þ-doped LiGa5O8. Sci Rep 2013;3. ac C, Seguin J, Pelle  F, Maîtrejean S, Jolivet JP, et al. [22] Chermont QlM De, Chane Nanoprobes with near-infrared persistent luminescence for in vivo imaging. Proc Natl Acad Sci 2007;104:9266e71. [23] Abdukayum A, Chen JT, Zhao Q, Yan XP. Functional near infrared-emitting Cr3þ/Pr3þ co-doped zinc gallogermanate persistent luminescent nanoparticles with superlong afterglow for in vivo targeted bioimaging. J Am Chem Soc 2013;135:14125e33. re A, Seguin J, Teston E, Sharma SK, Viana B, et al. The [24] Maldiney T, Bessie in vivo activation of persistent nanophosphors for optical imaging of vascularization, tumours and grafted cells. Nat Mater 2014;13:418e26. [25] Allix M, Sb Chenu, Veron E, Poumeyrol T, Kouadri-Boudjelthia EA, Alahrache S, et al. Considerable improvement of long-persistent luminescence in germanium and tin substituted ZnGa2O4. Chem Mater 2013;25:1600e6. [26] Qiao ZA, Zhang L, Guo M, Liu Y, Huo Q. Synthesis of mesoporous silica nanoparticles via controlled hydrolysis and condensation of silicon alkoxide. Chem Mater 2009;21:3823e9. [27] Hou Z, Li C, Pa Ma, Cheng Z, Li X, Zhang X, et al. Up-conversion luminescent and porous NaYF4: Yb3þ, Er3þ@ SiO2 nanocomposite fibers for anti-cancer drug delivery and cell imaging. Adv Funct Mater 2012;22:2713e22. [28] Geng J, Li K, Ding D, Zhang X, Qin W, Liu J, et al. Lipid-PEG-folate encapsulated nanoparticles with aggregation induced emission characteristics: cellular uptake mechanism and two-photon fluorescence imaging. Small 2012;8: 3655e63. [29] Li K, Ding D, Huo D, Pu KY, Thao NNP, Hu Y, et al. Conjugated polymer based nanoparticles as dual-modal probes for targeted in vivo fluorescence and magnetic resonance imaging. Adv Funct Mater 2012;22:3107e15. [30] Pan Z, Lu YY, Liu F. Sunlight-activated long-persistent luminescence in the near-infrared from Cr3þ-doped zinc gallogermanates. Nat Mater 2012;11: 58e63. [31] Cheong WF, Prahl SA, Welch AJ. A review of the optical properties of biological tissues. IEEE J Quantum Elect 1990;26:2166e85. [32] Mallik R, Wa C, Hage DS. Development of sulfhydryl-reactive silica for protein immobilization in high-performance affinity chromatography. Anal Chem 2007;79:1411e24. [33] Xu Y, Jin X, Ping Q, Cheng J, Sun M, Cao F, et al. A novel lipoprotein-mimic nanocarrier composed of the modified protein and lipid for tumor cell taŕ geting delivery. J Control Release 2010;146:299e308. [34] Sudimack J, Lee RJ. Targeted drug delivery via the folate receptor. Adv Drug Deliv Rev 2000;41:147e62. [35] Luo Z, Hu Y, Cai K, Ding X, Zhang Q, Li M, et al. Intracellular redox-activated anticancer drug delivery by functionalized hollow mesoporous silica nanoreservoirs with tumor specificity. Biomaterials 2014;35:7951e62. [36] Li X, He Q, Shi J. Global gene expression analysis of cellular death mechanisms induced by mesoporous silica nanoparticle-based drug delivery system. ACS Nano 2014;8:1309e20.

Please cite this article in press as: Shi J, et al., Multifunctional near infrared-emitting long-persistence luminescent nanoprobes for drug delivery and targeted tumor imaging, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.033