NIR-to-Red Upconversion Nanoparticles with Minimized Heating

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Biological and Medical Applications of Materials and Interfaces

NIR-to-Red Upconversion Nanoparticles with Minimized Heating Effect for Synchronous Multidrug Resistance Tumor Imaging and Therapy Xiaoqin Chen, Yajun Tang, Amin Liu, Yuda Zhu, Dong Gao, You Yang, Jing Sun, Hongsong Fan, and Xingdong Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00409 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

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ACS Applied Materials & Interfaces

NIR-to-Red

Upconversion

Nanoparticles

with

Minimized Heating Effect for Synchronous Multidrug Resistance Tumor Imaging and Therapy Xiaoqin Chen, Yajun Tang, Amin Liu, Yuda Zhu, Dong Gao, You Yang, Jing Sun, Hongsong Fan*, Xingdong Zhang National Engineering Research Center for Biomaterials, Sichuan University, Sichuan, Chengdu 610065, P. R. China KEYWORDS: NIR-to-Red emission, theranostic agent, nanomaterials, chemotherapy, overheating effect, multi-drug resistance ABSTRACT: Lanthanide-doped upconversion nanoparticles (UCNPs), especially the 808 nm activated UCNPs, are promising imaging agents for biological applications due to their minimal tissue overheating effects and low autofluorescence background. Optimizing the emission peaks located in the “biological window (600 nm-1100 nm)” is of vital importance to obtain maximum penetration depth and intense deep tissue imaging. On the other hand, because of the widely existed multi-drug resistance (MDR) of tumor cells, traditional tumor chemotherapy often fails to achieve the desired effect. Herein, a new type of 808 nm excited pure red luminescence core– shell Nd3+-sensitized NaY(Mn)F4:Yb/Er@NaYbF4:Nd UCNPs (CSUCNPs) were designed and synthesized for deep tissue imaging and MDR tumor diagnosis with minimized heating effect. In

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the meanwhile, D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) coating was introduced to endow CSUCNPs with capabilities of drug loading and overcoming MDR. In vitro cytotoxicity test revealed that CSUCNPs-TPGS-DOX (D-CSUCT) had excellent MDR cancer cell killing efficacy. In vivo test showed D-CSUCT can target tumor site by enhanced retention effect (EPR), and the intense luminescent signal from tumor site in the deep tissue were detected. Generally, this work shows D-CSUCT can overcome MDR effect, diagnosis tumor, inhibit tumor growth and induce tumor cells necrosis and apoptosis, without causing damage to major organs and other side-effects. Overall, the study demonstrates the conjugating of red-emitted UCNPs with minimized heating effect and anti-MDR carrier is high promising for developing multi-functional theranostic system with effective simultaneous diagnosis and multi-drug resistant tumors treatment.


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Introduction In recent years, nanomaterials and nanotechnology has been attractive strategies for biomedical application.1-8 Among various kinds of nanomaterials, lanthanide-doped upconversion nanoparticles (UCNPs) have been emerging as imaging agents for their unique optical properties.9-14 In comparison with conventional down-conversion fluorescence labels, UCNPs have higher penetration depth and lower background light.15-18 Till now, the most popular UCNPs are doped with ytterbium (Yb3+) ions as sensitizers with 980 nm irritation excitation.19-21 However, the light absorption band of 980 nm has a large overlap with the absorption of water in the biological tissue sample, resulting dramatically weakened deep tissue imaging.22-24 Moreover, the energy absorbed by biological sample may also cause thermal damage to cells and tissues.25 To solve this problem, it is urgent to find a more suitable near infrared (NIR) excitation wavelength from the “optical window in biological tissue”. Therefore, in recent researches, UCNPs with 808 nm excitation have been introduced to prevent energy loss and overheating effect, and improve the tissue penetration depth.26-28 Unfortunately, when the Yb3+-sensitized UCNPs are excited by 808 nm, the low absorption coefficient of Yb3+ ions makes it difficult to detect upconversion luminescence (UCL). To improve the UCL, Nd3+/Yb3+/activator-based UCNPs are designed and fabricated.29 In this configuration, Nd3+ features a strong absorption band around 808 nm, and there is an effective energy transfer between Yb3+ and Nd3+.30-31 Therefore, Nd3+/Yb3+/activator can effectively generate UCL through energy transfer between Nd3+→Yb3+→activator. Another key parameter for in vivo deep tissue imaging is to develop UCNPs with emission bands in the red to NIR spectral range (600-1100 nm), which minimizes scattering, absorption and autofluorescence backgrounds. Although most of Nd3+-sensitized


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UCNPs

such

as

NaYF4:Yb/Nd/Er@NaYF4:Nd,

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NaYF4:Yb/Er/Nd@NaYF4,

NaYF4:Yb/Tm/Nd@NaYF4:Nd are able to generate light in 600-1100 nm region under 808 nm excitation, this configuration also usually exhibits intensive blue, green or yellow emission, resulting decreased deep tissue penetration.28-29,

32-33

Therefore, a great challenge remains to

fabricate intensive pure red emission UNCPs in an 808 nm excited Nd3+/Yb3+ co-doped system. In this paper, we prepared co-doping Nd3+/Yb3+/Mn2+/activator in a core–shell structured nanoparticles NaY(Mn)F4:Yb/Er@NaYbF4:Nd (CSUCNPs) through a facile liquid-solid-solution (LSS) strategy with oleic acid (OA) as surfactant.34-35 These CSUCNPs can be excited by NIR 808 nm laser and emit pure red luminescence at 660 nm, and realize NIR-to-Red deep tissue imaging with minimal tissue overheating. To reduce cross-relaxation between the Nd3+ ions and the activator ions, a core−shell structure involving the Yb3+, Nd3+ in the shell layer and Yb3+, activator ions in the core was designed, which was able to simultaneously enhance energy transfer from Nd3+ to Yb3+ and minimize the cross-relaxation between the Nd3+ and the activator ions (Scheme 1). Meanwhile, because of the widely existed multi-drug resistance (MDR) of tumor cells, traditional tumor chemotherapy often fails to reach the desired effect. It is necessary to introduce anti-MDR modifier into the anti-tumor conjugates. D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) is a biocompatibility FDA approved pharmaceutical adjuvant, which has been reported to have the ability to overcome MDR effect by inhibiting the Pglycoprotein (P-gp) mediated drug efflux.36-38 Besides, TPGS has an amphiphilic structure (Figure S1). Its lipophilic alkyl tail binds to the OA-coated CSUCNPs through hydrophobic interactions leading to the formation of a hydrophobic layer which favors doxorubicin (DOX) loading. Meanwhile, its hydrophilic polar head endows the resulting composite with good hydrophilicity. Therefore, a novel theranostic agent (D-CSUCT) based on CSUCNPs with NIR-


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to-Red upconversion luminescence and high drug loading capacity could be realized to overcome MDR cancer and achieve in vivo deep tissue imaging.

RESULTS AND DISCUSSION Preparation and Characterization of CSUCNPs, CSUCT and D-CSUCT NanoConjugates. The synthetic route for CSUCNPs is shown in Scheme 1A. The original Mn2+doped UCNPs core was synthesized via a LSS method,34-35,

39

followed by growing a

NaYb(Nd)F4 shell on the inner core.39 The phase composition of prepared UCNPs core and CSUCNPs were analyzed by X-ray powder diffraction (XRD). As shown in Figure 1A, the XRD patterns of UCNPs and CSUCNPs could be well indexed as pure cubic phase (JCPDS: 06-0342), and no other phases or impurities were found. The size distribution of CSUCNPs were measured by dynamic light scattering (DLS) and showed in Figure 1B, the mean hydrodynamic diameter of the CSUCNPs is in the range of 20 to 40 nm..The typical transmission electron microscopy (TEM) images shown in Figure 1C suggest that CSUCNPs have a core-shell structure with the size in the range of 20 to 30 nm. Clearly, the DLS size is slightly larger than the TEM size. The composition of CSUCNPs was analyzed by energy dispersive x-ray spectroscopy (EDS) as shown in Figure 1D, confirming the existence of Na, Y, Yb, Er, Nd and Mn elements. The surface property of CSUCNPs was further investigated by Fourier transforming infrared spectroscopy (FT-IR) (Figure 1E). The stretching vibrations of the -CH=CH- at 2982 cm-1 and – COOH group at 1612 cm-1 suggest the presence of OA ligands on the surface of CSUCNPs. Attractively, as Figure 1F shows, these CSUCNPs reveal a single intense emission peak at around 660 nm under 808 nm excitation. The energy transfer of CSUCNPs under 808 nm excitation (Scheme 1B) suggests that the red emission (652 nm) comes from Er3+ with the 4F9/2→ 4

I15/2 transition. We argue that for the Nd3+ ions which located in the shell, they served as


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Scheme 1. (A) The synthetic process for D-CSUCT nanoparticles. (B) The energy transfer mechanism of CSUCNPs for photon upconversion under 808 nm excitation. (C) Schematic illustration of the imaging and therapy procedure of D-CSUCT nanoparticles in vivo. sensitizers to absorb and transfer the energy to the 2F5/2 state of nearby Yb3+ ions in the shell. Then, the energy was transferred to the Yb3+ ions in the core to active the Er3+ ions in the core. Subsequently, the non-radiative energy transfer took place, which was from the 4S3/2 and 2H9/2


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Figure 1. Characterizations of CSUCNPs. (A) XRD patterns of UCNPs and CSUCNPs. (B) Size distribution of CSUCNPs. (C) TEM images of CSUCNPs. (D) EDS analysis of CSUCNPs. (E) FT-IR spectra of CSUCNPs. (F) Upconversion emission spectra of CSUCNPs under 808 nm laser. Inset: corresponding photograph of CSUCNPs dispersed in cyclohexane. levels of Er3+ to the 4T1 level of Mn2+and back to the 4F9/2 level of Er3+. Finally, the bright red emission (652 nm) was observed. The slight red-shift of emission peak from 652 nm to 660 nm may be caused by the parcel of the NaYb(Nd)F4 shell. The above results demonstrate the successful preparation of core-shell CSUCNPs with monodispersed nanoscale spheres. With the surface OA ligands, the CSUCNPs can be easily modified and functionalized by lipophilic and/or amphiphilic molecules through hydrophobic interactions. In addition, this intense red up-conversion emission makes the material ideal for constructing multifunctional vectors suitable for deep tissue imaging and tumor diagnosis.


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To realize synchronous tumor diagnosis and treatment, the fabricated CSUCNPs should have high drug loading capacity and overcome MDR ability. For this purpose, TPGS was first used to functionalize the OA encapsulated CSUCNPs to obtain biocompatible CSUCNPs-TPGS (CSUCT). As shown in Scheme 1A, the hydrophobic tail of TPGS is tightly bound to the surface OA ligand of CSUCNPs via hydrophobic-hydrophobic interaction; meanwhile, the hydrophilic head which is directed outward improves the hydrophilicity of the CSUCNPs. Then doxorubicin (DOX) was loaded into the hydrophobic layer which was formed by the hydrophobic end of TPGS and OA chain of CSUCNPs to obtain D-CSUCT. Figure 2A presents the UCL spectra of CSUCNPs, CSUCT and D-CSUCT after surface modification and drug loading. Under 808 nm excitation, the three emission spectra exhibited similar curves, except for the slight drop of luminescence intensity. However, the relative high photoluminescence reveals that D-CSUCT still has high NIR-to-Red efficiency and is very promising for deep tissue imaging. The FT-IR spectra are shown in Figure 2B, and the stretching vibrations of the -CH=CH- at 2982 cm-1 and COOH group at 1612 cm-1 suggest the presence of OA chain on CSUCNPs. After TPGS modification on the CSUCT surface, the peaks appearing at 1100-1500 cm-1 and 3610 cm-1 corresponding to the stretching vibrations of C-O and -OH groups, which indicate the successful modification of TPGS on CSUCNPs. When DOX is loaded, the stretching vibration peaks of NH2 appear in the range of 1000 cm-1-1100 cm-1, indicating that DOX is successfully loaded in the D-CSUCT. Besides, the zeta potential of the materials shown in Table S1 indicate good colloidal stability of these nanoparticles.


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Figure 2. (A) Upconversion emission spectra of CSUCNPs, CSUCT and D-CSUCT. (B) FT-IR spectra of CSUCNPs, CSUCT and D-CSUCT. (C) UV-vis absorption spectra of DOX, DCSUCT and CSUCT. (D) DOX release profile from D-CSUCT at different pH during 168 h. Drug Loading and pH-Sensitive Release. As shown in Figure 2C, DOX has obvious characteristic absorption peak at 480 nm. In comparison, CSUCT drug carrier has no absorption at 480 nm. Hence, the identical peak at 480 nm in D-CSUCT indicates the successful loading of DOX on CSUCT. The drug loading capacity was 28%, which is relatively high as compared to most of other nanocarriers reported.5, 40 The high capacity of drug loading is attributed to the small size of the nanoparticles with large specific surface area, providing a large hydrophobic layer between TPGS and OA. Figure 2D shows the pH-sensitive drug release profiles from DCSUCT over a period of 168 h. We found that lower pH can promote the DOX release from our


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D-CSUCT. Under simulated physiological conditions (pH = 7.4), only 15% and 26% of DOX was released respectively during 24 and 168 h. Interestingly, for our simulated cancer environment (pH = 5.0), the DOX release reached respectively about 65% and 94%, during the periods of 24 and 168 h. This pH-induced release profile could be attributed to the increase of hydrogen ions (H+) at lower pH values, and DOX became more water-soluble in acidic environment. In addition, the acidic environment induced the protonation of the amino group of DOX (Figure S1); the resulting positive charge weaken the hydrophobic interaction and increased the release of DOX. The above-mentioned pH-induced drug release characteristic is important for the clinical cancer treatment, due to the acidic microenvironment of the tumor tissue. In Vitro Cell Labeling and In Vivo Imaging of D-CSUCT. Cell labeling of D-CSUCT was investigated with two-photon confocal laser scanning microscopy (TCLSM) under 808 nm excitation. As Figure 3A-B show, the red UCL of CSUCNPs was observed in both human breast adenocarcinoma DOX-resistant (MCF-7/ADR) cells and DOX-sensitive MCF-7 cells, and no background fluorescence was observed. These observations suggest that D-CSUCT could be easily taken by cells, and thus successfully label cells for bioimaging application. For deep tissue imaging, D-CSUCT (25 µL, 5 mg/mL) solution was subcutaneously or intramuscularly injected in the back or lower limb of anesthetized BALB/C mice. The UCL image was observed under 808 nm excitation. As Figure 3C shows, strong red UCL can be observed at the injection site even through deep intramuscular injection (about 1 cm depth). Worth to note that, the mouse hair did not cause interference to the UCL, indicating the excellent penetrating ability of D-CSUCT for NIR-to-Red imaging. For in vivo tumor diagnosis,


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Figure 3. In vitro cell labeling and in vivo tumor imaging. (A) Live cell imaging of MCF-7 cell and (B) MCF-7/ADR cell co-cultured with D-CSUCT for 1 h was observed by TCLSM. (C) UCL image of Balb/c mouse after injection of D-CSUCT (5 mg/mL, 25 µL). Images of MCF7/ADR tumor bearing nude mouse after the injection of D-CSUCT (5 mg/mL, 200 µL). (D) UCL imaging of tumor tissue observed by TCLSM. Tumor tissues were separated from nude mice after D-CSUCT treatment during 14 days. (EX=808 nm). D-CSUCT was administered by caudal vein injection into MDR tumor bearing nude mice. Figure 3C shows the UCL image obtained under 808 nm laser irradiation. Interestingly, red UCL was successfully captured at the tumor site. We speculate this tumor accumulation is due to the


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good hydrophilicity and proper size of D-CSUCT nanoparticles, which can be passively targeted to the tumor site of nude mice by EPR effect.41 To further investigate the material accumulation in the targeted tumor site for imaging and therapy, we observed the UCL of sliced tumor tissue by TCLSM after intravenous injection of D-CSUCT. As shown in Figure 3D and S2, tumor slices emit strong red UCL at 808 nm excitation. Under the same excitation, the luminescence intensity appears weaker at the denser tissue areas, relatively stronger at the looser tissue areas with vacuoles. Areas with stronger luminescence should have high concentration of D-CSUCT, leading to severe tumor necrosis, forming more vacuoles. Therefore, it can be concluded that the UCL was quite stable and can be detected for a long time after cancer treatment, without quenching in the complex biological environment. Cellular Uptake Efficiency to Overcome MDR. MDR is the main obstacle of cancer chemotherapy, which leads to the failure of the cancer treatment in clinical.42-43 The overexpression of P-gp protein as drug efflux pumps plays the main role for MDR.44 The introduction of TPGS in D-CSUCT is expected to inhibit P-gp protein expression.36-37 and successfully deliver DOX to MCF-7/ADR cells without being pumped out. The red fluorescence (shown in Figure 4) represents the intracellular DOX signal and the blue fluorescence indicates the nuclei of cells. The DOX fluorescence was mainly in the cytoplasm around the nuclei. DOX fluorescence intensities in DOX or CSUCNPs-DOX treated groups were relatively weaker, comparing with TPGS contained groups (D-CSUCT or TPGS-DOX). This demonstrates the important role of TPGS for enhancing drug delivery. The inhibition of P-gp expression by TPGS leads to the inability of P-gp pump to excrete the drug and increase the aggregation of the drug in the cells (Figure 4B), thus detecting the highest DOX fluorescence intensity of D-CSUCT group,


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Figure 4. MDR overcoming study. (A) Intracellular DOX fluorescence (red) imaged by confocal laser scanning microscopy (CLSM). The nuclei were stained by DAPI (blue). (B) P-gp expression of cells incubated with different materials. (C) Cellular uptake characterized by flow cytometry (FCM) for MCF-7/ADR cells after 2 h incubation with different solutions. (D) Mean Intracellular DOX fluorescence intensity after different treatments. n=3, *p

NIR-to-Red Upconversion Nanoparticles with Minimized Heating

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