NIR-responsive copolymer upconversion nanocomposites for

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NIR-responsive copolymer upconversion nanocomposites for triggered drug release in vitro and in vivo Yeye Zhang, Guangzhao Lu, Yuan Yu, He Zhang, Jie Gao, Zhiguo Sun, Ying Lu, and Hao Zou ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00681 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on January 2, 2019

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ACS Applied Bio Materials

NIR-responsive copolymer upconversion nanocomposites for triggered drug release in vitro and in vivo Yeye Zhang1,2,#, Guangzhao Lu1,#, Yuan Yu1,#, He Zhang1, Jie Gao1, Zhiguo Sun1, Ying Lu1,*, Hao Zou1,*. 1Department

of Pharmaceutical Sciences, School of Pharmacy, Second

Military Medical University, Shanghai, China. 2Department

of Pharmacy, Zhongshan Hospital, Fudan University,

Shanghai, China.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] Author Contributions #Y.

Zhang, G. Lu, and Y. Yu contributed equally to this work.

Notes The authors declare no competing financial interest.

1

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ABSTRACT Light has several advantages as the stimulus for triggered drug release. Currently, the applications of phototriggered drug-release devices (PDDs) are largely limited by two factors: the limited tissue penetration and detrimental effects caused by excitation light (ultraviolet or visible light). To address this disadvantage, this study developed nanocomposites based on upconversion nanoparticles (UC), which could convert near-infrared light to ultraviolet-visible light and trigger drug release. By loading UC and doxorubicin (DOX) into photo-responsive copolymer PEG-NMABPLA

(PNP),

near-infrared

responsive

copolymer

upconversion

nanocomposites (PNP-DOX-UC) was constructed. We proved that PNPDOX-UC showed the fast release and strong cytotoxicity under near infrared irradiation in vitro. The therapeutic efficacy study indicated that PNP-DOX-UC+hv had the enhanced antitumor efficiency. In the study, UC becoming an internal ultraviolet-visible light source for near infrared excitation developes an applicable and efficient approach to meet the requirements for UV/Vis excitation, which is a major disadvantage in photosensitive materials developed for pharmaceutical and biomedical applications. KEY WORDS: Near-infrared light; Photo-responsive; Copolymer; Upconversion Nanoparticle; Nanocomposites; Triggered drug release 2


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INTRODUCTION Many smarter nanoscale therapeutic modalities have been developed for efficient and safe cancer therapy 1-3. Despite the significant progresses in nanocarriers such as tumor-targeted nanomedicines, the single "targeting" or "enrichment" cannot solve the problem of premature or slow release of drugs. In this aspect, stimuli-sensitive nanocarriers, such as responsive nanocarriers to pH

4, 5,

temperature 6, reduction gradients 7,

exhibit the enhanced therapeutic efficacy in vitro and in vivo 8-12. Because of the high spatiotemporal resolution in non-invasive manipulation, the phototriggering technology shows a significant advantage in the control of drug release 13, 14. To date, many types of phototriggered drug-release devices (PDDs) have been designed, including block copolymer micelles 15-17, hydrogels 18, 19,

and silica nanoparticles20-23.

These PDDs were designed based on

various phototriggers such as 2-nitrobenzyl azobenzene

29.

24-26,

coumarin

27, 28,

and

However, all photo-reactions of PDDs need high energy

ultraviolet-visible light, which is harmful to living tissues and owns poor penetration depth 30, 31. Thus, NIR light is more applicable and efficient in pharmaceutical and biomedical applications for the deeper penetration into tissues without harm to healthy cells photon techniques 35. Zhu

36

32-34.

NIR can be utilized by two-

and Guardado-Alvarez

37

reported a kind of

mesoporous silica nanoparticles (MSN), which could generate two NIR 3



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photons (λ=800 nm) to trigger disruption, unlike the one-UV-photon process. Although these systems accelerated the drug release, the irradiation could not work without femtosecond pulsar lasers, which were expensive and suffered certain harms. Moreover, only partial phototriggers possessing good focalization cross-section could be excited by two photons, and the slow and inefficient photoreactions suggested that the irradiation time was too long in practice. Recently, rare earth ion based upconversion nanoparticles (UCNPs) provided a novel method for PDDs triggered by NIR light. UCNPs own unique character which relatively low energy photons (NIR light) are absorbed by them and converted into photons with relatively higher energy in the ultraviolet-visible regions required in most photoreactions in an antiStokes process. In addition, UCNPs has higher conversion efficiency than quantum dots and NIR-phototriggers which are nonlinear multiphoton absorption. Compared with two-photon techniques, the upconversion process allows for much lower phonon energy and a much cheaper tunable near-infrared laser can be used as the light source. In addition, their narrow emission bandwidth, long luminescence lifetime, high chemical stability, single-photon emission, and low toxicity make UCNPs serve as bioimaging biolabels38-42. Therefore, UCNPs have been coupled with polymer materials (PEG, chitosan, polypeptide, MSN) used in many in applications such as biological imaging and cancer therapy 4


41, 43-45.

Zhao et al. first

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demonstrated controlled release of a drug under 980 nm NIR irradiation based on UCNPs 46. Then Chang-Ming Dong et al. used a PNBC-b-PEO copolymer loaded with doxorubicin (DOX) and coated on the UC nanoparticles to realize an effective NIR light responsive drug release. Although these approaches have been proved to be able to improve the response speed and shorten the exposure time, photoreactions in these approaches require high-energy NIR light (power density = 5 W/cm2) 43. In this work, we designed novel 980 nm NIR-triggered nanocomposites PEG-NMAB-PLA-UCNPs (PNP-UC) to realize DOX photo-controllable release for cancer therapy (Figures 1A-B). With 2nitrobenzyl as the phototrigger and PEG-PLA as the basic skeleton, we synthesized a phototriggered block copolymer PEG-NMDB-PLA (PNP), into which UCNPs and drugs were encapsulated. NIR light was converted into UV light owing to UCNPs coated in the nanocomposites. And NIRtriggered properties of the nanocomposites PNP-UC loaded with Nile Red (NR) were proved by the changes of fluorescence intensity. Nanocomposites PNP-UC loaded with DOX were prepared to realize the high encapsulation efficiency and drug loading. Under different irradiation time and power values, PNP-DOX-UC showed the fast drug release and strong cytotoxicity. In vivo study, the higher anti-tumor efficiency were showed in tumor xenografted mice treated with PNP-DOX-UC and NIR.

5



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EXPERIMENTAL SECTION Materials. trifluoroacetic

Vanillin, acid,

dimethylaminopyridine,

1,2-Dibromoethane, sodium

azide,

potassium

sodium

succinic

iodide,

cuprous

(Benzotriazol-1-yloxy)tripyrrolidinophosphonium

borohydride, anhydride, iodide,

4and

hexafluorophosphate

(PyBOP) were all purchased from Aladdin Regent (Shanghai, China).Linear monofunctional PEG amine NH2, PEG-NH2 , MW=2kDa), alkyne-functional polylactic acid (PLA2000-C≡C, MW=2kDa), and Polyethylene glycol-block-polylactic acid (PEG-PLA, PEG average Mn 2000, PLA average Mn 2000) were purchased from Biomatrik Inc. (Zhejiang, China). Doxorubicin hydrochloride (DOX · HCL), Nile red (NR) and triethylamine were obtained from Sigma-Aldrich Company. The core–shell NaYF4:0.3%Tm@CaF2 upconversion nanoparticles (UC) were prepared in Fuyou Li’s laboratory (Fudan University, Shanghai, P. R. China) according to previous protocol

47

and were dispersed in

tetrahydrofuran. Pancreatin, DMEM, FBS, trypsin-EDTA, penicillin and streptomycin were purchased from GIBCO (USA). MCF-7 cell line was gifted from Beijing University.

Synthesis of phototriggered block copolymer PEG-NMDB-PLA (PNP)

6


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Synthesis of the photo-trigger S-(o-nitro-m-methoxy-p-azide alkoxy benzyl ester)( NMAB-ester). According to the synthetic routine of PNP (Figure S1), photo-trigger NMAB-ester was synthesized in five steps. Firstly, vanillin (3.0 g, 19.7 mmol), 1,2-dibromoethane (37.0 g, 197.0 mmol) and potassium bicarbonate (8.2 g, 59.1 mmol) were added in 100 mL of acetonitrile, followed by the 3-h reaction at 90 °C. Secondly, the obtained compound was structurally modified via nitration reaction, reduction reaction and azido reaction to yield the compound 3. Thirdly, compound 3 (4.0 g, 7.5 mmol) and succinic anhydride (1.5 g, 15.0 mmol) were added in pyridine at 50 °C and were stirred for 24 h for complete reaction. The progress of reaction was monitored by thin-layer chromatography. The reaction mixture was then evaporated to dryness. The crude products were purified by silica-gel column chromatography. The purified products were characterized by 1HNMR and MS (Figures S2A-D).

Synthesis of photo-triggered block copolymer PNP.

The

phototrigger (NMAB-ester) synthesized above was used to connect PEGNH2 with PLA-C≡CH to prepare PEG-NMAB-PLA (PNP) (Figure S1). We developed the Cuprous-catalyzed intermolecular azide-alkyne cyclization to synthesize NMAB-PLA-ester : Preferably NMAB-ester （18.4mg，0.05mmol），PLA2000-C≡C（150mg，720.075mmol）and 7



Cuprous iodide（2.4mg，0.01mmol）were dissolved in tetrahydrofuran and a small quantity of triethylamine was added dropwise. The solution were heated continuously at 60 ℃ and monitored by a simple thin-layer chromatography (TLC). After the reaction，the system were rinsed with a mixture of water and methylene chloride. Further purification by Silica gel Column Chromatogram was used to get NMAB-PLA-ester (Figure S2E). Yield: 46.7%. 1H NMR (400 MHz, CDCl3): δ=7.94 (s, 1H), 7.66 (s, 1H), 7.04 (s, 1H), 5.54 (s, 2H), 5.19-5.11 (q, 1H, hydrogen atoms in tertiary carbon), 4.81 (t, 2H), 4.43 (t, 2H), 3.97 (s, 3H), 2.75 (s, 1H), 2.71 (t, 2H), 2.70 (s, 2H), 2.08 (m, 2H), 1.59-1.54 (d, J=7.20Hz, 2H, hydrogen atoms in methyl). The activated-NH2 group of PEG-NH2 (MW=1 kDa, 2 kDa, and 5 kDa) reacted with -COOH group of NMAB-PLA-ester to form the final product PNP (Figure 1C). Here is the detailed instruction: We dissolved PEG2000-NH2 （ PEG1000-NH2 or PEG5000-NH2 ）、 NMAB-PLA-ester and PyBop in methylene chloride with a drop of triethylamine. The synthetic process was followed by TLC. After reaction finished, rinsed with the mixture of ice diethyl ether and acetone, then dissolved in water and finally freeze-dried to obtain PNP. The desired product was confirmed by 1H NMR and FT-IR (Figures 1C-D).

8


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Preparation of the drug-loaded micelles (PNP-NR and PNP-DOX) and upconversion nanocomposites (PNP-UC, PNP-NR-UC, and PNPDOX-UC).

The micelles and nanocommposites were prepared by thin

film hydration. Firstly, 1 mg Dox-HCl salt with three molar equivalents of triethylamine was dissolved and stirred in chloroform for 2 h under roomtemperature to get DOX base. DOX base and 10 mg of PNP were codissolved in chloroform, evaporated to form thin film at 30 °C, and hydrated with water to yield the PNP-DOX. The blank micelles PNP were also prepared as above without the addition of DOX. Then Nile red tetrahydrofuran solution (10 μL) was added into the blank micelles (1 mL) to gain the concentration of 10-6 moL/L and loaded into PNP with sonication for 30 min. PNP (20 mg) and UC (40 mg) were dissolved in chloroform, respectively. Then the UC in chloroform was cautiously added into the copolymer solution, dropwise with vigorously stirring. Then a thin film was formed when the solvent was completely evaporated. The thin film was rehydrated in an aqueous solution and sonicated to yield the nanocomposite PNP-UC. For PNP-DOX-UC and PNP-NR-UC, the similar procedure was performed and then the obtained solution was centrifuged. Non-photo-responsive composites PP-NR-UC and PP-DOX-UC were also prepared for comparison in a similar method except that PEG-PLA was used to replace PNP. The resultant micelles and nanocomposite were kept at 4 °C for further characterization. 9



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The following designations are used to define our prepared nanomicelles or nanocomposites: blank PEG-NMAB-PLA nanomicelles (PNP), nile red-loaded PEG-NMAB-PLA nanomicelles (PNP-NR), doxorubicin-loaded

PEG-NMAB-PLA

nanomicelles

(PNP-DOX),

upconversion nanoparticles-loaded PEG-NMAB-PLA nanocomposites (PNP-UC), nile red and upconversion nanoparticles-loaded PEG-NMABPLA nanocomposites (PNP-NR-UC), doxorubicin and upconversion nanoparticles-loaded PEG-NMAB-PLA nanocomposites (PNP-DOX-UC), upconversion nanoparticles-loaded PEG-PLA nanocomposites (PP-UC), nile red and upconversion nanoparticles-loaded PEG-PLA nanocomposites (PP-NR-UC), doxorubicin and upconversion nanoparticles-loaded PEGPLA nanocomposites (PP-DOX-UC). Physicochemical characteristics of PNP-DOX, PNP-UC and PNPDOX-UC were identified using Malvern Zetasizer (Nano ZS, UK). The morphology was measured using JEM-1400 transmission electron microscope (TEM). And the encapsulation efficiency (EE%) and drug loading content (DL%) of DOX in the nanocomposites were calculated from the following formula: EE% =

Weight of DOX in nanocomposites Weight of totally added drug

× 100%

Weight of DOX in nanocomposites

DL% = Weight of totally added drug and polymers × 100%

10


(1) (2)

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Optical properties Absorption spectrum and emission spectrum. The emission spectrum of UC and nanocomposite PNP-UC solution placed under 980 nm NIR laser was analyzed by a fluorescence spectrophotometer. The absorption spectrum of PNP was obtained by UV-vis spectroscopy in the range of 300nm-500 nm. Photo-dissociation reactions. For the photoreaction, the nanocomposite PNP-UC solution was vertically irradiated with a 980 nm near-Infrared laser for given periods at 1 W cm−2. The time-resolved ultraviolet-visible spectra of the irradiated solutions were dertermined by UV-vis spectroscopy. Photo-triggered model drug release. For photo-triggered drug release, the nanocomposite PNP-NR-UC solution was vertically irradiated with a 980 nm near-Infrared for different periods at 1 W cm−2. The time-resolved changes of fluorescence intensity of the irradiated solutions were dertermined using a fluorescence spectrometer (Ex=550 nm, Em=633 nm). For comparison, the group without UC (PNP-NR+hv), the group without phototrigger (PP-NR-UC+hv), and the group without NIR exposure (PNPNR-UC) were tested under the same conditions.

Morphological changes after illumination. A certain concentrations of blank micelles PNP and nanocomposite PNP-UC were respectively 11



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irradiated with the 365 nm UV laser (15 mW cm-2) for 15 min or 980 nm near-Infrared laser (power density 1 W cm-2) for 180 min. The solutions were then shaken at 37 °C under 200 rpm for 24 h. The morphological changes were analyzed by TEM with or without staining.

NIR phototriggered drug release in vitro

The drug release profiles

from PNP-UC nanocomposite were studied using a dialysis method. PNPDOX, PNP-UC or PP-UC solution were dialyzed (MWCO 3500) with phosphate buffered saline at 37 °C with or without 980 nm NIR light (1 W cm-2) in different periods (30 min, 60 min). The dialysate buffer was collected at pre-determined time. The same volume of fresh PBS were added to the dialysate buffer. The amount of the released doxorubicin was analyzed by HPLC. To develop a new UCNPs-based PDD in living tumor tissues, the release profiles were tested under the light irradiation of 50 mW cm−2 according to the following steps. The phototriggered release was studied by irradiating the PNP-DOX-UC nanocomposite with 980 nm NIR laser (50 mW cm−2) for 48 h. Aliquots of 300 μL nanocomposite solution were withdrawn and the free DOX in the solution was collected by ultrafiltration and centrifugation for further HPLC analysis.

12


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In vitro cell uptake of PNP-UC nanocomposite under NIR laser irradiation. Due to their narrow emission bandwidth, long luminescence lifetime, high chemical stability, UCNPs have the function of imaging. Cell uptake of PNP-DOX-UC nanocomposite were imaged without other fluorescent dye. Briefly, MCF-7 cells were cultured overnight and attached in confocal dishes. Then, the cells were treated with 200 μL of PNP-UC solution for 2 h. After washing with phosphate buffered saline, the fluorescence was visualized using a laser confocal microscopy with NIR (980 nm) as the laser source.

In Vitro Cell Viability Assay. To assess the cytotoxic effect of PNP-DOXUC under NIR irradiation, CCK-8 assay was conducted according to the previously reported results 48. MCF-7 cells with the concentration of 3 × 103 cells per well were seeded in 96-well cell-culture plate. Then the cells were incubated at 37 °C and 100% humidity overnight under a 5% CO2 atmosphere. After 12 h, the cells were treated with 200 μL of fresh medium containing different kinds of nanocomposites (PNP-UC, DOX, PNP-DOX, PP-DOX-UC, and PNP-DOX-UC) at the desired concentration. The cells were incubated for 3 h with the nanocomposites to evaluate the endocytosis process. For the experiment groups with NIR irradiation, the MCF-7 cells was kept on the thermostatic culture plates and NIR 980 nm laser at a power density 1 W cm−2 were exposed from the top of the plate. To prevent 13



overheating, near-Infrared laser irradiation each time lasted 5 min followed by 5 min interval until the cumulative exposure time reached 30 min or 60 min. Then the cells were incubated for another 24 h or 48 h at 37 °C under a 5% CO2 atmosphere. The cell viabilities were determined according to the CCK-8 method described above. Besides, the cytotoxicity of 980 nm NIR with different power values was also tested.

In vivo toxicity of NIR-triggered PNP-DOX-UC. Animal experiments conformed to the guidelines of university laboratory animals regulations. MCF-7 cells at a density of 2×106 were inoculated into the left mammary glands of 24 female nude Balb/c mice to establish xenograft breast cancer model. As the volume of tumors grew up to 100 mm3, the mice were assigned in four groups randomly (n=6) and 0.20 mL of normal saline, DOX (5 mg/kg)+hv, PNP-DOX-UC or PNP-DOX-UC+hv were intravenously injected to each group twice a week. The phototriggered drug release in vivo was evaluated by irradiating the tumors region with continuous-wave 980 nm laser. The power density was set as 50 mW cm−2 and the exposure time last 60 min. The irradiating treatments were repeated every day in 26 days. The therapeutic efficacy of PNP-DOX-UC was assessed by changes of the tumor size every other day as well as tumor inhibition ratio in each group. And the body weight was also were 14


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determined

for

each

mouse.

Tumor

size

was

estimated

as

0.5×length×(width)2. At the end of the test after 26-day observation, all mice were sacrificed and the tumors mass were weighted in 26 days. Inhibition ratio of tumor (IRT%) was calculated using the following formula: IRT (%) =

𝑊𝑐 ― 𝑊𝑡 𝑊𝑐

(3)

× 100%

where wc and wt are respectively the average tumor mass weight of controls and treatment group.

RESULTS AND DISCUSSION Synthesis of phototriggered block copolymer. As described in experimental section, we prepared the phototrigger NMAB-ester and phototriggered block copolymer PNP, which were then characterized by IR, 1HNMR, MS (Supporting Information). As shown in Fig. 1C-D for PNP, the signals of 1H NMR spectrum are assigned as follows: δ=7.91 (Ha), 7.67 (Hb), 7.11 (Hc), 5.52 (Hd), 4.80 (He), 4.42 (Hf), 4.34 (Hg), 4.01 (Hh), 3.36 (Hi), 2.75 (Hj), 2.55 (Hk), and 2.18 (Hl). The chemical shifts from 3.83 to 3.06 were attributed to the methylene of PEG skeleton. δ= 5.35~5.07 and δ= 1.75~1.20 were respectively assigned to hydrogen atoms on tertiary carbon atoms and methyl groups of PLA skeleton. The signals at 7.67 ppm were assigned to the 1,2,3-1H-Triazole , indicating the new 15



modified PLA in main chains through click reactions. The results confirmed the presence of photo-trigger NMAB-ester, PEG and PLA in the PNP. In the FTIR spectrum of PNP, new peaks at 1625 cm−1, 1513 cm−1, and 875 cm−1 were respectively related to PEG and PLA skeletons. These peaks are usually caused by 1,2,3-1H-Triazole, Benzene and nitro, respectively. Besides, the peak at 3030 cm−1 disappeared due to alkynyl on PLA skeleton testified the successful synthesis of 1,2,3-1H-Triazole. All the results of 1H NMR and FTIR showed the presence of photo-trigger NMAB-ester, PEG, and PLA in the PNP.

Figure 1. (A) Schematic illustration of NIR-responsive copolymer UCNPs nanocomposites for triggered drug release; (B) NIR-responsive photoreaction of PNP polymer; (C) 1H NMR spectrum of PEG-NMAB-PLA in CDCl3 (Inset: Structure of PEG-NMAB-PLA); (D) FT-IR spectra of PEG-NH2, PLA-C≡CH, and PEG-NMAB-PLA. 16


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Characterization of the drug-loaded micelles (PNP-NR and PNP-DOX) and upconversion nanocomposite (PNP-UC, PNP-NR-UC, and PNPDOX-UC). The critical micelle concentrations (CMC) of the drug-loaded and upconversion nanocomposite were determined by the fluorescence method with NR as a probe. The observed critical micelle concentration was 33 μg/mL. In our study, we adopted the thin-film hydration method to prepare all the nanosystems for it showed high DL% and EE% compared to the dialysis method (Table S1). The detailed characterization results of all nanosystems are summarized in Table 1. After loading with DOX or UC, the composite size increased and the zeta potential was changed from negative to positive. When loaded with DOX and UC simultaneously, the size of PNP-DOX-UC increased to 350 nm with good poly-dispersity indexes. The trapping efficiency (62.85%) and drug-loading rate (6.07%) of PNP-UC-DOX were decreased compared to PNP-DOX. UC owned a smaller particle diameter of about 20 nm. As shown in the TEM images of PNP-DOX-UC (Figure 2A), UC was clearly loaded inside almost all nanocomposite and most of nanocomposites contain 3~7 UC nanoparticles (the inset in Figure 2A). In regard to all the nanocomposites, the loaded UC nanocomposites showed irregular morphology, because the number of encapsulated UC varies from nanocomposite to nanocomposite. Our prepared nanocomposites showed the larger loading capacity and better entrapment compared to the previous report 46. 17



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Table 1 Characterization results of PNP-DOX, PNP-UC, and PNP-UCDOX (n=3). Nanocomposite

Size (nm)

PDI

PNP

124.53±2.91

0.253±0.004

zeta potential (mV) -8.89±0.48

PNP-DOX

192.46±6.60

0.215±0.051

PNP-UC

200.2±4.62

PNP-DOX -UC

350.1±8.37

EE%

DL%

\

\

3.79±0.14

64.47±2.19

10.85±0.40

0.269±0.125

-4.27±0.91

\

\

0.288±0.036

5.50±0.21

62.85±9.90

6.07±0.85

Optical properties. To confirm the NIR-responsive composite dissociation and evaluate whether NIR laser can trigger the release of hydrophobic drug, a series of optical experiments were performed. Figure 2B shows the upconversion emission spectra and ultraviolet-visible absorption spectra of UC and PNP-UC nanocomposite solutions. When the NIR laser beam passes through the UC or PNP-UC solution, we can clearly observed the photoluminescence of visible light. The naked UC solution and PNP-UC solution had the same emission spectrum upon the exposure to 980 nm NIR laser. The mission spectrum emitted from the UC was around 350 nm.

The UV emission spectrum was matched well with the

absorption spectrum of the polymer PNP with o-nitrobenzyl groups as the phototrigger, indicating that PNP-UC had the application potential for the dissociation of NIR-triggered composites. The evolution of the ultravioletvisible absorption spectrum or fluorescence emission spectrum of PNP-UC or PNP-NR-UC under irradiation at 980 nm (1 W/cm-2) was illustrated 18


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(Figures S3A-B). After the exposure to NIR laser, absorption spectrums of PNP-UC or PNP-NR-UC with the phototrigger o-nitrobenzyl groups in the 300~350 nm were obtained and the absorbance of PNP-UC or PNP-NRUC solution increased upon irradiation due to a continuous photochemical reaction of photocleaved molecules on PNP polymer upon 980 nm NIR light excitation. Before NIR light exposure, the strong fluorescence of PNP-NR-UC under 550 nm excitation were found. Since quenching of the fluorescence of NR in water, the high fluorescence intensity confirmed that NR had been loaded in the composite. During the NIR exposure, the fluorescence intensity of NR continuously decreased, which indicated a continuous diffusion of NR hydrophobic dye from the nanocomposites. For PNP-NR+hv, PP-NR-UC+hv group, the result was completely different. After 6-h NIR laser exposure, the fluorescence spectrums did not change, indicating that the PNP copolymer was stable and NR remained to be loaded into the nanocomposites (Figure S3C). Without aid of UC and phototriggers, 980 nm NIR laser could not induce the photochemical reaction of NMAB or the phototriggered release of NR from PNP-UC nanocomposites. The TEM images (Figure 2C) showed morphological changes of the PNP-UC nanocomposites before and after NIR laser irradiation. Before the irradiation, UC was encapsulated inside PNP polymer micelles clearly. After 980 nm NIR laser exposure, the complete disintegration of composites was observed. As a result, UC was released 19



from the composites and then gathered. The photocleavaged hydrophilic and hydrophobic segments formed a layer of dark organic materials. These results indicated that CW 980 nm laser triggered the dissociation of PNPUC.

Figure 2. (A) Morphology of PNP-DOX-UC (scale bar = 0.5 μm; Inset is the magnified image, scale bar = 200 nm); (B) The upconversion emission and ultraviolet-visible absorption spectra (Black line and red line respectively represent the emission spectra of UC and PNP-UC; Blue represents the absorption spectrum of PNP). Inset: Upconversion emission photos of the UC (1) and PNP-UC (2); (C) The morphological changes of PNP and PNP-UC after irradiation.

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NIR phototriggered DOX release in vitro. As shown in Figure 3A, irradiation the DOX- encapsulated nanocomposites with 980 nm NIR laser can effectively change the cumulative DOX release profile. The higher DOX release ammount was found with longer NIR irradiation time, which means that accelerated drug release occurred under the stimulus of NIR light. Every group showed the two-phase release: fast release before 12 h and slow release after 12 h. After periodic irradiation with 980 nm laser, the DOX released from PNP-DOX-UC reached 60.6% (30 min) and 48.9% (60 min) within 12 h. The proportion of released DOX in the non-irradiated sample was 37.3% and the proportion of released DOX in the groups without UC or phototrigger were respectively 40.1% or 34.1%. Besides, PNP-UC-DOX under NIR exposure for 60 min showed a maximum release of about 68.8% compared to other groups (61.5%, 47.4%, 53.9%, and 51.2%). All these results could be attributed to NIR light-induced nanocomposite dissociation and the increased proportion of released DOX. More importantly, our nanocomposites also exhibited apparent lighttriggering properties with the lower power density NIR light (50 mW cm−2) (Figure S4).

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Figure 3 (A) In vitro cumulative DOX release profiles of PNP-DOX-UC and control groups under irradiation at 980 nm (1 W/cm2) in PBS Buffer (pH=7.4) (n=3); (B) Cell uptake of PNPUC towards MCF-7 cells (treated for 4 h, 1: Bright field; 2: Fluorescence; 3: Overlap of 1 and 2); In vitro cytotoxicity and IC50 values (μg/mL) of different groups under 1 W/cm2, 980 nm NIR light: (C) Cell viability of MCF-7 cells for 24 h by MTT assay; (D) IC50 values of MCF-7 cells for 24 h (n=3); (E) Cell viability of MCF-7 cells for 48 h by MTT assay; (F) IC50 values of MCF-7 cells for 48 h (n=3).

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Comments: (a)n.s.p>0.05 v.s. DOX; a,bp< 0.05 v.s DOX; c,fp< 0.001 v.s DOX; d,gp< 0.01 v.s PP-DOX-UC+hv 60 min; e,hp< 0.05 v.s PNP-DOX+hv 60 min or PNP-DOX-UC; ip> 0.05 v.s PNP-DOX-UC+hv 60 min (n=3). (b)n.s.p>0.05 v.s. DOX; ap< 0.05 v.s DOX; bp>0.05 v.s DOX or PP-DOX-UC+hv 60 min; cp< 0.01 v.s PNP-DOX+hv 60 min; dp< 0.05 v.s PNP-DOXUC; ep< 0.05 v.s PNP-DOX+hv 60 min; fp>0.05 v.s PNP-DOX-UC or PNP-DOX-UC+hv 60 min (n=3).

In vitro cell uptake and cytotoxicity tests of NIR-triggered PNP-DOXUC. We used the modified confocal microscopy to realize cell imaging without any auto-fluorescence (Figure 3B). Fluorescence images indicated that PNP-UC entered the cell cytoplasm, and upon NIR laser irradiation, strong photoluminescence was observed in MCF-7 cells without the interference of background fluorescence. The good biocompatibility of NIR light and UCNPs had been verified in vitro (Figures S5A-B). The cytotoxicity of the PNP-UC at different concentrations were studied in MCF-7 cells. The cell survival was more than 95% when the concentration of polymer was below 0.33 mg/mL. More than 50% of the cells survive even when the concentration of polymer was 1 mg/mL. Additionally, after the 980 nm NIR laser irradiation for different exposure time (15, 30 or 60 min) at different laser power (0.5 W cm−2, 0.75 W cm−2, and 1 W cm−2), the cell survival was still above

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95%. This result indicated that the near-infrared irradiation are not toxic for cells. To demonstrate the effectiveness of PDDs, PNP-DOX-UC+hv (30 min/60 min), the cytotoxicity were tested in MCF-7 lines for 24 h or 48 h, and DOX solution, PNP-DOX-UC, PNP-DOX+hv (60 min), PP-DOXUC+hv (30 min) was used as control. The higher DOX concentration and the longer incubation period mean the higher killing efficiency (Figures 3C-F; Table 2). The cytotoxicity of PNP-DOX-UC+hv (30 min or 60 min) against MCF-7 incubated for 24 h was plotted in Figures 3C-D. The PNPDOX-UC+hv (30 min/60 min) showed a dose-dependent cytotoxicity and the lowest IC50 compared to the control groups. Notably, the IC50 value of PNP-DOX-UC+hv (30 min/60 min) was more than 4 times lower than that of the control formulations of PNP-DOX-UC and PNP-DOX+hv (60min). Figures 3E-F shows the cell-killing activity incubated for 48 h. The PNP-DOX-UC+hv (30 min/60 min) remained the best cell-killing effect in all groups. The significant difference between PNP-DOX-UC+hv (30 min/60 min) and the control formulations in enhancing the cytotoxicity was obviously caused by the NIR combined with UCNPs. Furthermore, no significant statistical differences were found between the cytotoxicity of PNP-DOX-UC+hv (30 min) and PNP-DOX-UC+hv (60 min), indicating that our nanocomposites could realize NIR-triggered drug release within 30 min and increase the damaging effect on tumors. These results 24


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convincingly suggested that the DOX-loaded PNP-UC nanocomposites exhibited the NIR-responsive cytotoxicity, facilitating the development of stimuli-sensitive delivery system for cancer therapy. Table 2 IC50 values of MCF-7 treated for 24 h or 48 h (n=3). Groups

24h-IC50 (μg/mL)

48h-IC50 (μg/mL)

DOX

2.097±0.805

0.068±0.010

PP-DOX-UC+hv 60min

1.561±0.394

0.081±0.021

PNP-DOX+hv 60min

1.100±0.410

0.133±0.055

PNP-DOX-UC

1.176±0.262

0.092±0.014

PNP-DOX-UC+hv 30min

0.273±0.208

0.064±0.009

PNP-DOX-UC+hv 60min

0.247±0.211

0.057±0.008

Effects of NIR-triggered PNP-DOX-UC in vivo. The therapeutic efficacy of PNP-DOX-UC was evaluated in MCF-7 tumor xenografts. The tumors were irradiated with a near-infrared light. The power density was set as 50 mW cm−2 in order to minimize the effect of overheating generated by NIR laser on therapeutic efficacy results. The group treated with saline without near-infrared laser exposure was set as control. The PNP-DOX-UC+hv demonstrated the better inhibitory effect on tumor growth than PNP-DOXUC (P < 0.01) (Figure 4A). The tumors were isolated and weighed at the end point after the treatment (Figure 4B). The weight of tumors treated with the PNP-DOX-UC+hv was significantly lower than that of those 25



treated with PNP-DOX-UC (Figure 4C) (P < 0.01). Besides, PNP-DOXUC+hv showed the higher tumor inhibition ratio (46%) than PNP-DOXUC (27%) (Table 3). In our in vivo research, the results showed that lower antitumor efficacy for PNP-DOX-UC+hv compared to DOX-hv. There are many reports that the delivery efficiency of nanomedicine depend on physicochemical properties, including particle size and surface charge. A major explaination for this phenomenon in vivo is that the low accumulation at the tumor because of PNP-DOX-UC has a large particle size (350.1±8.37nm) which resulted in weaker passive targeting, but a much lower toxicity as desired and higher tumor inhibition ratio than nonirradiated group. Nanocomposites with different sizes will be investigated in future studies. Although the DOX solution had the best anti-tumor effect, the body weight was reduced at the most rapid rate compared to other groups (Table 3) (Figure 4D). The control group treated with free DOX resulted in 15% of weight decrease with poor physiological conditions, because of the systemic toxicity of free doxorubicin. Moreover, the group treated with PNP-DOX-UC+hv showed no significant weight loss compared to those of PNP-DOX-UC, indicating the comparable safety of the PNP-UC and NIR. These results demonstrated that PNP-DOX-UC+hv realized NIR-triggered drug release in living tissues and had major advantages for deep-seated tumor treatment.

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Figure 4 In vivo antitumor activity (n=6): (A) The tumor growth curve; (B) Images of excised tumors after 26-day, scale bar = 2 cm; (C) Tumor weight at the end of experiment after 26-day observation; (othersp 0.05 v.s DOX or PP-DOX-UC+hv 60 min; cp< 0.01 v.s PNP-DOX+hv 60 min; dp< 0.05 v.s PNP-DOX-UC; ep< 0.05 v.s PNP-DOX+hv 60 min; fp>0.05 v.s PNPDOX-UC or PNP-DOX-UC+hv 60 min (n=3).


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Fig. 4 In vivo antitumor activity (n=6): (A) Tumor growth curves; (B) Images of excised tumor, scale bar = 2 cm; (C) Tumor weight at the end of the test after 28-day observation; (othersp

NIR-responsive copolymer upconversion nanocomposites for

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