Construction of Hierarchical Polymer Brushes on Upconversion

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Construction of Hierarchical Polymer Brushes on Upconversion Nanoparticles via NIR-Light-Initiated RAFT Polymerization Zhongxi Xie, Xiaoran Deng, Bei Liu, shanshan Huang, Ping'an Ma, Zhiyao Hou, Ziyong Cheng, Jun Lin, and Shifang Luan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09124 • Publication Date (Web): 23 Aug 2017 Downloaded from http://pubs.acs.org on August 25, 2017

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ACS Applied Materials & Interfaces

Construction of Hierarchical Polymer Brushes on Upconversion

Nanoparticles

via

NIR-Light-

Initiated RAFT Polymerization Zhongxi Xie,a,b Xiaoran Deng,a Bei Liu,a Shanshan Huang,a Pingan Ma,a Zhiyao Hou,a Prof. Ziyong Cheng,*a Prof. Jun Lin,*a Prof. Shifang Luan*c a. State Key Laboratory of Rare Earth Resource Utilization Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China. b. University of Science and Technology of China No.96, JinZhai Road BaoheDistrict, Hefei, Anhui, 230026, P. R. China. E-mail: [email protected]; [email protected] c. State Key Laboratory of Polymer and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, People’s Republic of China E-mail: [email protected]

KEYWORDS Surface RAFT polymerization, upconversion nanoparticles, NIR light initiation, drug delivery system, cancer therapy

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ABSTRACT

Photoinduced

RAFT

(reversible

addition–fragmentation

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chain

transfer)

polymerization generally adopts high energy ultra violet (UV) or blue light. In combination with photoredox catalyst, the excitation light wavelength was extended to visible and even near infrared (NIR) region for electron transfer RAFT (PET-RAFT) polymerization. In this report, we introduce for the first time a surface NIR-light-initiated RAFT polymerization on upconversion nanoparticles (UCNPs) without adding any photocatalystand construct a functional inorganic core/polymer shell nanohybridforthe application in cancer theranostics. The multilayer core-shell UCNPs (NaYF4:Yb/Tm@NaYbF4:Gd@NaNdF4:Yb@NaYF4) with surface anchoring of chain transfer agent (CTA) can serve as efficient NIR-to-UV light transducer to initiate the RAFT polymerization. A hierarchical double block copolymer brush, consisting of poly(acrylic acid) (PAA) and poly(oligo(ethylene oxide)methacrylate-co-2-(2-methoxy-ethoxy)ethyl methacrylate) (PEG for short), was grafted from the surface in sequence. The targeting arginine-glycineaspartic peptide (RGD) was modifiedon the end of the copolymer through the trithiolcarbonate end group. After loading of doxorubicin (DOX), the UCNPs@PAA-b-PEG-RGD exhibited the enhanced U87MG cancer cell uptake efficiency and cytotoxicity. Besides, the unique upconversion luminescence of the nanohybridswas used as the autofluoresence-free cell imaging and labeling. Therefore, our strategy verified that UCNPs could efficiently activate RAFT polymerization by NIR photoirradiation and construct the complex nanohybrids, exhibiting the prospective biomedical applications due to the low phototoxicity and deep penetration of NIR light.


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INTRODUCTION As a kind of controlled/“living” radical polymerization, RAFT polymerization provides a powerful tool for synthesizing polymers with low polydispersity index (PDI) and complex architectures, such as multiblock copolymers and star-like polymers.1-4 Compared with atom transfer radical polymerization (ATRP) and nitroxide-mediated polymerization (NMP), RAFT process has some inherent merits such as no heavy metal demand, mild reaction conditions, the compatibility for numerous monomers and ease of end group functionalization. In recent years, many efforts have been devoted to combine RAFT polymerization with surface modification of inorganic nanoparticles, such as silica/silicon5-7, TiO28 and Au9-12. According to the pathways for preparation of polymer brushes on nanoparticles, the polymerization strategies could be divided into “grafting to”, “grafting through” and “grafting from” methods.13 The last one that implemented through surface CTA modification, is more appropriate for the maximum grafting density and high degree control of the thickness of polymer brushes.14 CTA is usually anchored on nanoparticles through its Z-group or R-group.15,16 For Z-group approach, the stabilizing group of CTA is covalently attached to the surface and therefore almost no trithiolcarbonate group will be lost during polymerization. However, with the polymer brushes length increasing, the free radical near the surface will be shielded, which results in the termination of polymer chains. On the contrary, R-group strategy could not only prevent such side effect, but also facilitate end group modification of polymer brushes. Trithiolcarbonate group at the end of polymer chains could be reduced into active thiol by hydrolysis17,18, aminolysis19,20 or NaBH421,22. Thus, further surface functionalities for distinct requirements such as linking of target molecules or fluorescent markers would be easily conducted.


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Traditional photoinduced RAFT polymerization adopts high energy UV or blue light as irradiation source, which may cause unavoidable side reactions like self-initiation of monomers and degradation of compounds.23 To solve those problems, Boyer and co-workers recently proposed a photo-induced electron transfer RAFT (PET-RAFT) polymerization that could be initiated by a wide visible spectrum and even NIR light through introducing appropriate organic photoredox catalyst.24-27 Apart from organic dyes, rare earth doped UCNPs also attracted much attention for the ability to convert long wavelength photons into short ones though anti-Stokes processes. UCNPs have merits of the low background fluorescence interference, high chemical and physical stability and less toxicity,28-30 which make them good candidates in many areas such as multi-modal imaging and labeling31-34, drug delivery systems (DDS)

35-38

and chemical

bond cleavage reaction.39-41 Besides, UCNPs have been applied to activate free radical polymerization by adding photosensitizers like Eosin Y et al. for preparation of polymerencapsulated nanocomposites.42,43 However, to the best of our knowledge, the in situ NIRintroduced RAFT polymerization on the surface of UCNPs has never been reported. In this article, we developed a new methodology to introduce homogeneous diblock copolymer brushes onto UCNPs through upconversion luminescence (UCL) initiated RAFT polymerization and further explored its application as an intelligent DDS. Herein CTA modified UCNPs (NaYF4:25%Yb/0.5%Tm@NaYbF4:50%Gd@NaNdF4:10%Yb@NaYF4) were employed as the NIR-to-UV light transducer to successfully construct diblock copolymer brushes that composed of poly(acylic acid) (PAA) and PEG (copolymer of DEGMA and PEGMA) onto UCNPs in sequence under mild 808 nm laser radiation without adding any additional initiators or sensitizer. This strategy demonstrates that the UV light coming from the upconversion process can efficiently cleave the trithiolcarbonate group and initiate the RAFT polymerization. Then, the


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trithiolcarbonate group at polymer chain end was aminolysised to form thiol group and further modified with RGD target peptide. The final nanohybrids carrying with the payload (DOX) demonstrated the pH-sensitive drug release behavior and enhanced therapeutic effect for U87MG cancer cell treatment. The synthesis procedure was illustrated in scheme 1.

RESULTS AND DISCUSSIONS Rare earth doped UCNPs could absorb low energy photons and transform them into high energies from NIR to visible and UV light via successive energy transfer (ET) processes. Among them, β-NaYF4:Yb/Tm nanoparticles have been intensively studied for NIR-to-UV light conversion and widely used as the promising candidates for bioimaging and NIR-sensitive chemical bond cleavage materials.44 In this study, monodisperse β-NaYF4:Yb/Tm nanoparticles (note as core) with size of 20 nm were successfully synthesized from the traditional thermolysis route (Fig. 1a).45 However, the luminescence of this core-only structure may be seriously quenched by surface defects, ligands and the local chemical environment.46 To overcome this hurdle, hierarchical core-shell structure UCNPs is usually employed for improving luminescence efficacy. Herein, we fabricated multilayer-shell NaYF4:25%Yb/0.5%Tm@NaYbF4:50%Gd@ NaNdF4:10%Yb@NaYF4 by successive layer-by-layer ejection of shell precursor solutions. From the TEM observation, the average size of the core-shell nanoparticles increased to 45 nm after shell coating (Fig. 1b). The X-ray powder diffraction (XRD) pattern shows that the coreshelled UCNPs have coincided phase with the pure β-NaYF4 diffraction data (JCPDS 16-0334) (Fig. 2). The UCL of core-shell UCNPs demonstrated the remarkable improvement compared with the core-only UCNPs (Fig. 3). The insets in Fig. 3 are corresponding digital photos of nanoparticles dispersed in cyclohexane. Both of the UCL spectra include 290 (1I6→3H6), 350


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(1I6→3F4), 365 (1D2→3H6), 450 (1D2→3F4) and 475 (1G4→3H6) nm transition bands. Calculated by the integration of spectrum area, the light-emitting intensity between 200 nm and 600 nm improved 22 times compared with core-only UCNPs, while the increment of UV region (250400 nm) reached up to 44 times. The result suggested that the core-shell strategy is very effective in improving photon transition and UV light strength. The possible ET processes were described in Fig. 4. In brief, under NIR light excitation, the rare earth ion could absorb several photos and leap to higher energy levels. When they jump back to the ground state, high energy light like UV or visible light would be emitted. The high concentration of Nd3+ (90%) in shell-2 guarantees the efficient 808 nm NIR light harvesting. Yb3+ ions play the important role for relaying energy from Nd3+ to the internal layers and eventually to Tm3+ via dipole-dipole ET. Once the Tm3+ is excited, it will be pumped into higher excited states by multiphoton processes and giving out UV light through 1I6→3H6, 1I6→3F4 and 1

D2→3H6 ET, respectively. The spatial separation of Tm3+/Nd3+ into different layers will prevent

the back energy transfer from Tm3+ to Nd3+. Finally, the inert NaYF4 was taken as the outermost shell that can effectively depress surface defects and diminishes ET from interior to outer environment. Such design not only solved the photon quenching effect, but also maximizes the use of 808 nm exciting light and greatly improves the UCL. The as-synthesized UCNPs were completely hydrophobic due to the OA coating. Further hydrophilic conversion and functionalization is prerequisite for the biological applications. Due to the strong affinity of phosphate than carboxylic acid group to UCNPs, alendronate may quickly replace OA on UCNPs by ligand exchange process and concomitantly introduce NH2 group at the surface (note as UCNPs-Ale), which allowed for anchoring CTA molecules by amide conjugation (note as UCNPs-CTA). As shown in FT-IR spectrum (Fig. 5), in contrast to


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the UCNPs-OA, the spectrum of UCNPs-Ale shows an emerging peak at 1639 cm-1 (δN-H).The wide bands that settled between 1260-990 cm-1 is associated with the P-O and P-O-C stretching vibrational in alendronate.47,48 For UCNPs-CTA, the enhanced wide band around 1065 cm-1 (νC=S), the new peaks at 3297 cm-1 (νN-H) and 1642 cm-1 (νC=O) clearly shown the existence of CTA at UCNPs.49-51 In addition, the UCL spectra changed much after CTA anchoring (Fig. 6). The UV emission at 365 nm was dramatically eliminated in intensity, while the 290 and 350 nm emissions were completely quenched. This should be attributed to the strong absorption of trithiocarbonates of CTA in UV region and partial overlap with the UCL of UCNPs (Fig. 7), which leads to the ET from UCNPs to CTA. In order to verify the feasibility of NIR-triggered bond cleavage, UCNP-CTA sample was dispersed in 2 mL ethanol at ambient temperature with vigorous stirring to keep a suspension and irradiated in a quartz cuvette with 808 nm laser (7 W/cm2) in the presence of air. After a certain interval, the UCNPs and supernatant were collected separately by centrifugation for UV-Vis measurement. Simultaneously, the UCNPs sediments were re-dispersed in fresh ethanol and operated as before. As shown in Fig. 8, UCNP-CTA suspension presented a characteristic absorption band of trithiolcarbonate around 300 nm at the beginning time. Along with the NIR irradiation, the intensity of trithiolcarbonate absorption in UCNP-CTA solution was dropped rapidly and almost could not be detected after 40 min (Fig. 8a). In comparison, the supernatant solution demonstrated the trithiolcarbonate absorption and their intensity were obviously in corresponding to the variety of UCNP-CTA solution (Fig. 8b). This should be attributed to the low dissociation energy of the C-S bond near R group of CTA molecules that closely attached on UCNPs. Under 808 nm irradiation, the UCNPs served as the internal light source for generating UV emission and broke C-S bond. The produced free radicals may be quickly quenched by


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oxygen so that the re-capture of CTA by UCNPs was blocked and the S-C(=S)-S fragment remained in supernatant solution. It should be mentioned that the solution temperature was only slightly elevated from 25 to 28 oC under NIR laser irradiation, which excluded the possibility of thermal cleavage of C-S bond. NIR-introduced grafting-from RAFT polymerization was performed using acrylic acid (AA) as monomer and the as-prepared UCNPs-CTA as substrate. The Schlenk tube containing DMSO, UCNPs-CTA and AA was degassed strictly by freeze-evacuate-thaw cycles and filled with N2. The whole device was immersed into EtOH as cooling system to avoid photothermol effect. The reactive system was in equilibrium at 32 oC under 808 laser irradiation with power of 12 W/cm2. The homogeneous PAA layer could be seen in TEM image with the thickness of 3.5 nm (Fig. 1c). Moreover, the FT-IR spectrum (Fig. 5d) presented the new peaks around 1740 cm-1 which belong to the –C=O stretching vibration of PAA. The transmission band at 1165 cm-1 was assigned to the stretching vibration of C-O groups.52 The wide band around 3400 cm-1 reflected the strong O-H stretching vibration of the carboxyl groups in PAA layer. Note that the absorption band on 300 nm of UCNPs@PAA is still remained after polymerization (Fig. 9b), illustrating the existence of CTA molecules at the end of polymer chains. So, it offers the possibility of initiating the second block polymerization. Grafting-from polymerization was re-initiated by addition of two monomers, di(ethylene glycol) methyl ether methacrylate (DEGMA) and poly(ethylene glycol) methacrylate (PEGMA), to construct the second random copolymer layer. The PEG modification is important for nanodrug carrier. It can improve the biocompatibility of nanoparticles and prevent nanoparticles from nonspecific protein adsorption, which eliminates the blood clearance and increases the blood circulation time.53 Based on the TEM observation (Fig. 1d), the thickness of polymer shell further increased to 6.5 nm after PEG polymerization.


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Accordingly, two additional peaks at 1563 cm-1 (νC=O) and 1265 cm-1 (νC-O-C) were observed in FT-IR spectrum after PEGylation, indicating the successful polymerization of PEG layer (Fig. 5e).54,55 Moreover, X-ray photoelectron spectroscopy (XPS) was applied to investigate the surface modified UCNPs. Fig. 10 shows the high-resolution C 1s XPS spectra of UCNPs@CTA, UCNPs@PAA and UCNPs@PAA-b-PEG, respectively. Compared with UCNPs-CTA spectra (Fig. 10a), new peaks at 286.0 (C-C=O species) and 288.4 eV (O=C-O species) appear and the intensity of 284.5 eV (C-C species) increases after PAA polymerization (Fig. 10b). In addition, the peak at 285.0 eV (C-S species) decreases due to the inevitable loss of surface-linked CTA.56,57 For UCNPs@PAA-b-PEG (Fig. 10c), a significant enhancement of the peak intensity at 284.5 eV (C-C species) is observed than that in UCNPs@PAA which derives from the extended polymer backbone. The peak intensity of 285.8 (C-C=O/C-O species) and 288.4 (O=CO species) eV also increases after introducing oligo PEG side chains. In addition, the composition of the nanohybrids were determined by thermogravimetry (TG) analysis. In Fig. 11, as the temperature changing from 30 to 750 °C, the weight loss percent of UCNPs@PAA and UCNPs@PAA-b-PEG were respectively increased 5.10% and 4.50% in contrast to before polymerization, indicating the PAA and PEG contents in the nanocomposite. This result is consistent with dynamic light scattering (DLS) characterization. As shown in Table 1, the hydrodynamic size at 25 ºC were gradually increased from 71.2 nm of UCNPs-CTA to 160.6 nm of UCNPs@PAA-b-PEG. Moreover, it is known that copolymers containing oligo(ethylene glycol) side chains is temperature-responsive, which is inclined to shrinking of the polymer chains as temperature increasing.58 As the result, the particle size of UCNPs@PAA-b-PEG decreased from 166.7 nm at 20 ºC to 142.3 nm at 35 ºC by DLS measuring in PBS buffer. Moreover, the different surface functionalities of the UCNPs also resulted in the significant


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variety of the zeta potential, which is -32 mV for UCNPs@PAA and -16 mV of UCNPs@PAAb-PEG, respectively (Fig. 12). The anti-protein adsorption performance of different UCNPs were tested by fluorescence microscopy observation (Fig. 13). The FITC-labeled avidin, HBA and BSA were incubated with UCNPs@PAA and UCNPs@PAA-b-PEG, respectively. The green luminescence from FITCproteins is obviously weaker for UCNPs@PAA-b-PEG in comparison with the UCNPs@PAA, indicating that the PEGylation on nanoparticles play an active role of mitigating nonspecific protein adsorption. The trithiolcarbonate group is convenient to be reduced into active thiol group through aminolysis et al. treatment. Taking advantage of this property, targeted Arg-Gly-Asp (RGD) molecules was further conjugated onto the surface of UCNPs@PAA-b-PEG to promote the specific cancer cell treatment. NH2-contained RGD peptide was immobilized on UCNPs@PAAb-PEG sample via coupling agent. In the control group, NH2-PEG (Mw = 600) was employed to replace NH2-RGD for further in vitro cell investigation (UCNPs@PAA-b-PEG-PEG600 for short). After RGD modification, the UV-Vis spectrum (Fig. 9d) shows that the absorption bands centered on 300 nm completely disappeared, reflecting the conversion of trithiolcarbonate group. The RGD-containing peptides were widely used as the probes for cell recognition and adhesion. It has a high affinity and specificity for integrin receptors αVβ3 that is overexpressed on endothelial cells of tumor blood vessel as well as various tumor cells types such as human U87MG glioblastoma cell.59,60 To test the targeting effect of RGD peptide on hybrid nanoparticles, UCNPs@PAA-b-PEG-RGD was first incubated with U87MG cells with and without free RGD blocking, respectively. Fig. 14 shows the inverted fluorescence microscope images of U87MG cells after incubation for 2 h. Compared with the U87MG cells that were pre-


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treated with free RGD targeted peptide, the UCL increased sharply for the non-blocking cell group. The results indicate that the addition of excess free RGD can significantly retard the uptake of the UCNPs@PAA-b-PEG-RGD by U87MG cells. Then, UCNPs@PAA-b-PEGPEG600 and UCNPs@PAA-b-PEG-RGD were incubated with normal U87MG cells. The UCL microscopy images are shown in Fig. 15. The incubation time were controlled to be 10 min, 30 min, 1 h, 4 h and 6 h to examine the targeted effect of RGD peptide. With the progress of incubation time, it can be seen that the blue UCL derived from UCNPs were gradually enhanced in both groups. Whereas the UCNPs@PAA-b-PEG-RGD group exhibited the evidently stronger luminescence signals than that of UCNPs@PAA-b-PEG-PEG600 control group at each given time point. The results implied that that RGD peptide modification promoted the endocytosis of UCNPs and has potential to improve efficacy for cancer therapy. As a widely used chemotherapeutic drug, doxorubicin hydrochloride (DOX) was loaded on UCNPs nanohybrids for assessments of drug loading and cancer therapy abilities in vitro. UCNPs@PAA-b-PEG-RGD sample was shaken gently for 24 h in DOX solution and then the drug loading efficiency was calculated to be about 16% in weight by UV-Vis spectrophotometer. PAA component play an important role for loading DOX molecules through Coulomb interaction. The positive-charged DOX molecules were attracted by negative-charged PAA block, which greatly improve the drug loading efficiency. Fig. 16 shows the in vitro drug release profiles of the samples at 37 ºC in PBS buffer solutions with different pH values. At pH = 7.4, that is similar with the normal physiological environment, the drug release rate was relative slow that less than 10% percent DOX was released within 24 hours (Fig. 16a). In comparison, almost 60% of DOX was released with the pH value decreasing to 5.0 (Fig. 16b). This should be attributed to the protonation of carboxyl groups in PAA in acidic environment, which reduces the


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electrostatic interaction and accelerates the DOX release. Thus, we have successfully created a pH-responsive DDS that is favor of reducing DOX release in neutral blood circulation while promoting the drug release in mildly acidic tumor tissue, especially in more acidic endosome and lysosome after endocytosis. To evaluated the cytotoxicity of as-prepared DOX-UCNPs@PAA-b-PEG-RGD in cancer treatment, the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) analysis on U87MG cells was carried out. As shown in Fig. 17, the cell viability in UCNPs@PAA-b-PEG group had a slightly decline as concentration increased, suggesting that almost there is no harm of the synthesized hybrid materials. Free DOX treated group also has not presented the effective cytotoxicity, which may be generated by the efflux effect of U87MG cells after long time incubation.44,61 Howerer, both DOX-UCNPs@PAA-b-PEG-PEG600 and DOX-UCNPs@PAA-bPEG-RGD nanodrug demonstrated the remarkably improved ability for killing U87MG cell. We speculate that this is owing to the different internalization mechanism of nanoparticles compared with free DOX. Nanodrugs were internalized into cells through pinocytosis.62 After being captured by the endosome or lysosome, DOX absorbed in the PAA layer was facilitated for releasing due to the mildly acidic environment and then entered nucleus, resulting in cell death. The completely different pathway by cells uptake of nanodrug may enhance the DOX distribution in cytoplasm and overcome the defects of conventional free drug delivery mode.54 Importantly, DOX-UCNPs@PAA-b-PEG-RGD nanodrugs showed the highest cytotoxicity effect and clear superiority to the other three control groups. This enhanced cellular uptake indicates the RGD-mediated endocytosis in U87MG cells played the key role in U87MG cells therapy.63 The result shows high prospect of the synthesize hybrid nanodrugs in further cancer therapy applications.


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CONCLUSIONS In summary, we have exhibited a proof-of-concept experiment that realized 808-nm-NIRlight-activated RAFT polymerization by utilizing multi-layer UCNPs as NIR-to-UV photon transducer. This study confirmed the feasibility that the surface-anchored RAFT agent with trithiocarbonate groups on UCNPs can be directly activated by NIR light without adding any photoinitiator and realizes the grafting-from RAFT polymerization. Thus, provides a pathway to extend photo-regulated RAFT polymerization to NIR region that overcomes the phototoxcity of high-energy UV light and displays the promising applications in biomaterials. Additionally, this process is promising and interesting to be further expanded to the different NIR exciting wavelength and multi-color-emission UCNPs or combining with diverse photocatalysis to introduce RAFT polymerization. The as-prepared nanohybrid was used as a multifunctional drug carrier for cancer therapy, in which UCNPs, PAA and PEG play the role of probe for upconversion florescent imaging, absorption of anticancer drug DOX by Coulomb force and anti-nonspecific protein adsorption, respectively. Moreover, trithiocarbonate groups at the end of polymer brush were converted to thiol group for targeting RGD peptide linking. The nanomedicine presented the remarkable superiority to free DOX for U87MG cancer cell treatment in vitro. MATERIALS AND METHODS Materials Rare earth oxides and rare earth chlorides were purchased from Beijing HWRK Chem Co. Ltd. Oleic acid (OA), octadecylene (ODE), acrylic acid (AA), di(ethylene glycol) methyl ether methacrylate (DEGMA), poly(ethylene glycol) methacrylate (PEGMA), 2-bromoacetic acid and carbon disulfide and 3-succinimidyl-3-maleimido-propionate (SMP) were purchased from


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Aldrich.

1-(3-dimethylamino-propyl)-3-ethylcarbodiimide

hydrochloride

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(EDC)

and

N-

Hydroxysuccinimide (NHS) were purchased from Aladdin. Fluorescein isothiocyanate labeled peptide (FITC-Avidin, FITC-HBA, FITC-BSA) was obtained from Bioss (Beijing) Co., Ltd. Gly-Arg-Gly-Asp-Ser-NH2 (RGD) was purchased from GL Biochem (Shanghai) Co., Ltd. PEGNH2 (Mw = 600) was obtained from Beijing Kaizheng Biotech Development Co. Ltd. Other agents were purchased from Beijing Chemical Regent Co. Ltd. Characterization The morphology of nanoparticles was obtained using transmission electron microscope (TEM) with a field emission gun operating at 200 kV. The crystal structure was characterized by X-ray powder diffraction (XRD) performed on a D8 Focus diffractometer (Bruker). The upconversion luminescence (UCL) spectra were obtained with a F-7000 fluorescence spectrometer (Hitachi) with 980 and 808 nm laser as the excitation source, respectively. Fouriere-transform infrared spectra (FT-IR) were obtained by a Vertex Perkin-Elmer 580BIR spectrophotometer (Bruker). U-3310 spectrophotometer was used for UV-Vis spectra measurement. The X-ray photoelectron spectra (XPS) were obtained by ECSALAB 250. Thermogravimetry analyse (TGA) was characterized by a Netzsch Thermo analyzer STA 409 instrument in the protection of N2 with a heating rate of 10 °C min−1. Dynamic light scattering (DLS) and surface zeta potential were characterized by a Zatasizer Nano (Malvern). Methods Preparation of CTA. Chain transfer agent, 2-(((dodecylthio) carbonothioyl) thio) acetic acid, was synthesized as followed. KOH (5.6 g) were first dissolved into 50 mL of methanol. Then, 1dodecanethiol (25 mL) were dropwise added into the mixture at room temperature. After stirring for half an hour, CS2 (10 mL) were added into the mixture slowly and stirred at room


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temperature for 3 h. Then, 2-BrAcOH (6.49 g) dissolved in methanol were added into the flask dropwise. After 8 h reaction, excess DI water were added into the solution before the organic phase was extracted. The final solution was removed by rotary evaporation. For further purification, products were extract with chloroform and water for 3 times. Preparation of UCNPs. The β-NaYF4:30%Yb/0.5%Tm nanoparticles (note as core) were synthesized using a traditional thermal decomposition strategy according to the literature.45, 64 In brief, a water solution of YCl3·6H2O (0.65 mmol), YbCl3·6H2O (0.3 mmol), TmCl3·6H2O (0.005 mmol), OA (6 mL) and ODE (15 mL) were first added into a 100-mL three-neck round-bottom reaction vessel. After evaporating water, the temperature was increased to 156 ºC and kept for 30 min. Then the vessel was cooled down to room temperature naturally followed by the addition of a methanol solution containing NaOH (2.5 mmol) and NH4F (4 mmol). The mixture was kept at 50 ºC for at least 30 min to remove methanol and form the nanoparticle seed. Under N2 protection, the temperature was increased up to 300 ºC and aging for 1 h. The final product was purified with centrifugation in ETOH and cyclohexane alternatively and dispersed into 5 mL chloroform for further usage. Before shell coating, Ln(CF3COO)3 were first prepared with Ln2O3 following the wellestablished protocol reported by our group previously.65 Then, multilayers of 0.5 mmol NaYbF4:50%Gd, 1 mmol NaNdF4:10%Yb and 1.5 mmol NaYF4 were coated onto the core following the hot injection protocol. Briefly speaking, chloroform solution of core, OA (10 mL) and ODE (10 mL) were added into a 100-mL four-neck round-bottom reaction vessel and kept at 50 ºC under vacuum for 30 min to evaporate the chloroform. N2 was blown into the vessel at 120 ºC and maintained for 10 min. Then at 310 ºC, a hot solution containing CF3COONa (0.5 mmol), Yb(CF3COO)3 (0.25 mmol), Gd(CF3COO)3 (0.25 mmol), OA (0.5 mL) and ODE (0.5 mL) were


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injected into the vessel quickly. Reaction was kept for 1 h for shell coating. Other shells were built in the same way excepted for changing the mole ratio of each trifluoroacetic acid salts. The mixture was purified in the same way with the core and dissolved in 20 mL of chloroform for further use. Immobilization of CTA. For hydrophilic transforming of UCNPs, UCNPs and the water solution of alendronate (40 mL, 5 mg/mL) were added to a 100 mL beaker with 2 h ultrasound treatment. After stirring overnight. The hydrophilic UCNPs was obtained by centrifugation with water and dissolved in DMF. The RAFT agent, 2-(((dodecylthio)carbonothioyl)thio) acetic acid, were anchored onto the surface of amine-functionalized UCNPs by using EDC and NHS. Typically, the carboxylic group of CTA (50 mg) in DMF solution was first activated by EDC and NHS for about 30 min under vigorous stirring. Afterwards the as-prepared UCNPs were added into the mixture and stirred for 24 h at room temperature. Yellowish precipitate was acquired (note as UCNPs-CTA) after centrifugation three times in EtOH. RAFT polymerization on UCNPs. UCNPs-CTA were dispersed into DMF to produce 10 mg/mL solution. AA was added into the mixture at a volume ratio of 1:5 with the total solution volume and filled with nitrogen. Polymerization underwent 808 nm irradiation with strict magnetic stirring for desired time. The compound was purified by centrifugation (10000 rpm for 10 min) with EtOH and dissolved into DMF (note as UCNPs@PAA). Further co-block polymerization of PEGMA and DEGMA (note as UCNPs@PAA-b-PEG) was achieved in almost the same way except for replacing AA monomer with PEGMA and DEGMEMA at a desired mole ratio to adjusted the LCST.68 The final products were purified with centrifugation and dissolved in EtOH.


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Immobilization of RGD. RGD peptide with active primary amine were anchored at the polymer chains end in the following steps. First, 100 µL of hexylamine were added into 2 mL of UCNPs@PAA-b-PEG solution (5 mg/mL) under N2 protection and stirred for 1 h. After purification, SMP (3 mg) and trimethylamine (100 µL) were added into the solution and kept stirring for 3 h in N2 atmosphere. After centrifugation, RGD (3 mg) were mixed with UCNPs@PAA-b-PEG-succimide and stirred overnight to produce final UCNPs@PAA-b-PEGRGD nanocompound. For the counterpart group, PEG-NH2 (Mw = 600) were used replacing RGD to generate UCNPs@PAA-b-PEG-PEG600. Anti-protein adsorption. FITC labeled proteins with different isoelectric point were cultivated with UCNPs@PAA and UCNPs@PAA-b-PEG for 20 min in PBS buffer, respectively. After centrifugation, the precipitate was redispersed in PBS buffer and imaged on microslide. UCNPs@PAA-b-PEG-RGD imaging. Human U87MG glioblastoma cells were chosen as the targeted cancer cell in this article. Cells were seeded in 6-well plates with a density of 1.5×105 cells/well and growing overnight. For RGD shielding group, free RGD were added into the plates with the concentration of 0.1 mg/mL. Cells were then incubated for 2 h before the addition of UCNPs@PAA-b-PEG-RGD. After another 2 h incubation, cells were treated with 4% paraformaldehyde (1 mL/well) at 37 ºC for 1 h. Then, fresh PBS was used to replace paraformaldehyde solution. Fluorescence microscope was used to test cell imaging. Cell imaging. U87MG were seeded in 6-well plates with a density of 1.5×105 cells/well and growing overnight. UCNPs@PAA-b-PEG-PEG600 and UCNPs@PAA-b-PEG-RGD (0.1 mg/mL) were added into each well with the concentration of 50 µg/mL. Cells were incubated at 37 ºC for 10 min, 30 min, 1 h, 4 h and 6 h before paraformaldehyde treatment as before.


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Drug loading and release. DOX (1 mg/mL, 1mL) were mixed with the water solution of UCNPs@PAA-b-PEG (3 mg/mL, 2 mL) and stirred for 24 hours at room temperature. The supernatant was collected for DOX-loading efficiency characterization. For DOX release examination, DOX-UCNPs@PAA-b-PEG sample were immersed into the phosphate buffered saline solution (pH = 7.4 and 5.0) and incubated at 37 ºC for desire time. Then, the buffer solution was collected and replaced with fresh buffer solution at selected time. All supernatant’s absorbing spectrum were tested and recorded. Cytotoxicity Assay. Human U87MG glioblastoma cells were seeded in 96-well plate with a density of 1×104 cells/well and growing overnight. Free DOX, UCNPs@PAA-b-PEG-RGD, DOX-UCNPs@PAA-b-PEG-PEG600 and DOX-UCNPs @PAA-b-PEG-RGD were dispersed in MEM medium respectively. The concentrations of nanoparticles were 10, 20, 40 and 80 µg/mL. The concentrations of DOX were 1.5625, 3.125, 6.25 and 12.5 µg/mL, respectively. After incubation for 24 h, MTT solution (0.5 mg/mL, 20 µL) were added into each well and incubated for another 4 h in dark room. Then, DMSO (150 µL) were added into each well and cells were detected by a microplate reader.

ACKNOWLEDGMENT This project is financially supported by the National Natural Science Foundation of China (NSFC 51472231, 51572257, 51472233, 51372241, 51332008), the National Basic Research Program of China (2014CB643803). Projects for science and technology development plan of Jilin province (20170101187JC, 20170414003GH).

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FIGURES AND CAPTIONS

Scheme 1. Schematic illustration of the procedure for the synthesis of hierarchical block copolymer brushes on upconversion nanoparticles via NIR-light-initiated grafting-from RAFT polymerization and its further application for drug delivery.


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Figure 1. Transmission electron microscopy (TEM) images of monodispersed (a) NaYF4:30%Yb/0.5%Tm,

(b)

NaYF4:30%Yb/0.5%Tm@NaYbF4:50%Gd@NaNdF4:10%Yb

@NaYF4, (c) UCNPs@PAA and (d) UCNPs@PAA-b-PEG nanoparticles.


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Figure

2.

Wide-angle

XRD

pattern

of

synthesized

(a)

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β-NaYF4:Yb3+/Tm3+,

(b)

NaYF4:30%Yb/0.5%Tm@NaYbF4:50%Gd@NaNdF4:10%Yb@NaYF4 core-shell UCNPs and (c) standard data for β-NaYF4 (JCPDS No. 16-0334).


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Figure 3. Upconversion luminescence (UCL) spectra of NaYF4:Yb3+/Tm3+ core-only (excited with 980 laser) and NaYF4:30%Yb/0.5%Tm@NaYbF4:50%Gd@NaNdF4:10%Yb@NaYF4 coreshell UCNPs (excited with 808-nm laser, 7 W/cm2), respectively. Nanoparticles were dispersed in cyclohexane solution (0.05 mM). The excitation power was 7 W/cm2. The insets show the photos of (a) core-only and (b) core-shell UCNPs dispersed in cyclohexane solution (0.05 mM).


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Figure 4. The upconversion mechanisms of UCNPs under 808 nm irradiation.


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Figure 5. Fouriere-transform infrared spectra (FT-IR) of (a) UCNPs, (b) UCNPs-Ale, (c) UCNPs-CTA, (d) UCNPs@PAA, and (e) UCNPs@PAA-b-PEG.


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Figure 6. UCL spectra of (a) UCNPs in cyclohexane and (b) UCNPs-CTA in DMF excited with 808 nm laser, respectively. The concentration of each sample was 0.05 mM. The excitation power was 7 W/cm2.


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Figure 7. (a) The absorption spectrum of CTA dispersed in EtOH (0.5 mg/mL). (b) The emission spectra of NaYF4:30%Yb/0.5%Tm@NaYbF4:50%Gd@NaNdF4:10%Yb@NaYF4 core-shell UCNPs dispersed in cyclohexane (0.05 mM).


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Figure 8. UV-Vis absorption spectra of (a) UCNPs-CTA (0.05 mM in EtOH) and (b) supernatant collected at different irradiation time. The power of 808 nm laser was 7 W/cm2.


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Figure 9. UV-Vis absorption spectra of (a) UCNPs-CTA, (b) UCNPs@PAA, (c) UCNPs@PAAb-PEG and (d) UCNPs@PAA-b-PEG-RGD nanoparticles. All samples were dispersed in EtOH with 0.05 mM.


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Figure 10. High-resolution XPS C1s spectra and their peak fitting curves of the (a) UCNPsCTA, (b) UCNPs@PAA and (c) UCNPs@PAA-b-PEG.


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Figure 11. Thermogravimetric analysis of (a) UCNPs-CTA, (b) UCNPs@PAA, and (c) UCNPs@PAA-b-PEG under the protection of N2.


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Table 1.The particles size and hydrodynamic size distribution (PDI) of UCNPs-CTA, UCNPs@PAA, UCNPs@PAA-b-PEG, UCNPs@PAA-b-PEG-RGD and DOX- UCNPs@PAAb-PEG-RGD at different temperature.

Nanoparticles UCNPs-CTA UCNPs@PAA

Temp. (°C) Particle size (nm) 25 71.2 25 105 20 166.7 25 160.6 UCNPs@PAA-b-PEG 30 154.5 35 142.3 UCNPs@PAA-b-PEG-RGD 25 162.6 DOX-UCNPs@PAA-b-PEG-RGD 25 161.9

PDI 0.06 0.12 0.09 0.16 0.19 0.41 0.54 0.58


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Figure 12. Zeta potential of UCNPs-CTA, UCNPs@PAA, and UCNPs@PAA-b-PEG, respectively.


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Figure 13. Inverted fluorescence microscope images of FITC-Avidin, FITC-HBA and FITCBSA interaction with (a) UCNPs@PAA and (b) UCNPs@PAA-b-PEG. All scale bars are 20 µm.


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Figure 14. Inverted fluorescence microscope images of U87MG cells (a) with and (b) without free RGD blocking after incubation with 50 µg/mL UCNPs@PAA-b-PEG-RGD for 2 h. The scale bar is 50 µm.


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Figure 15. In vitro UCL images of U87MG cancer cell incubated at 37 ºC with (a)UCNPs@PAA-b-PEG-PEG600 and (b)UCNPs@PAA-b-PEG-RGD for 10 min, 30 min, 1 h, 4 h and 6 h, respectively. All scale bars are 50 µm.


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Figure 16. In vitro drug release of DOX-UCNPs@PAA-b-PEG-RGD at (a) pH = 7.4 and (b) pH = 5.0, respectively. The results were calculated from UV-Vis absorption spectra at 480 nm.


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Figure 17. In vitro cytotoxicity of free DOX (black bars), UCNPs@PAA-b-PEG-RGD (red bars), DOX-UCNP@PAA-b-PEG-PEG600 (blue bars) and DOX-UCNP@PAA-b-PEG-RGD (brown bars) on U87MG cancer cell after incubation for 24 h.


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Graphic for manuscript:


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Construction of Hierarchical Polymer Brushes on Upconversion

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