NIR Controlled Interaction of Transformable

Apr 4, 2017 - Then, this nanoparticle is transformed into “attack” state under NIR irradiation, achieving the supersensitive DOX release from the ...
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Letter pubs.acs.org/NanoLett

Tumor Acidity/NIR Controlled Interaction of Transformable Nanoparticle with Biological Systems for Cancer Therapy Dongdong Li,†,‡ Yinchu Ma,‡ Jinzhi Du,† Wei Tao,‡ Xiaojiao Du,† Xianzhu Yang,*,†,‡ and Jun Wang*,† †

Institutes for Life Sciences, School of Medicine and National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou, Guandong 510006, P. R. China ‡ School of Medical Engineering, Hefei University of Technology, Hefei, Anhui, 230009, P. R. China S Supporting Information *

ABSTRACT: Precisely controlling the interaction of nanoparticles with biological systems (nanobio interactions) from the injection site to biological targets shows great potential for biomedical applications. Inspired by the ability of nanoparticles to alter their physicochemical properties according to different stimuli, we explored the tumor acidity and near-infrared (NIR) light activated transformable nanoparticle DATAT-NPIR&DOX. This nanoparticle consists of a tumor acidity-activated TAT [the TAT lysine residues’ amines was modified with 2,3-dimethylmaleic anhydride (DA)], a flexible chain polyphosphoester core coencapsulated a NIR dye IR-780, and DOX (doxorubicin). The physicochemical properties of the nanoparticle can be controlled in a stepwise fashion using tumor acidity and NIR light, resulting in adjustable nanobio interactions. The resulting transformable nanoparticle DATAT-NPIR&DOX efficiently avoids the interaction with mononuclear phagocyte system (MPS) (“stealth” state) due to the masking of the TAT peptide during blood circulation. Once it has accumulated in the tumor tissues, DA TAT-NPIR&DOX is reactivated by tumor acidity and transformed into the “recognize” state in order to promote interaction with tumor cells and enhance cellular internalization. Then, this nanoparticle is transformed into “attack” state under NIR irradiation, achieving the supersensitive DOX release from the flexible chain polyphosphoester core in order to increase the DOX−DNA interaction. This concept provides new avenues for the creation of transformable drug delivery systems that have the ability to control nanobio interactions. KEYWORDS: Nanobio interaction, tumor acidity-responsive, NIR-responsive, activated TAT targeting, cancer therapy

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Recent fundamental studies have demonstrated that the physicochemical properties of nanoparticles (such as size, shape, surface charge, and targeting ligand) play a crucial role in determining their interactions with biological systems during the delivery process.23−25 Meantime, with the development of a sensitive polymer, nanoparticles can alter their physicochemical properties in response to endogenous and exogenous stimuli.26−30 Thus, the stimuli-responsive nanoparticles have exhibited the potential to mediate nanobio interactions in the living body.31−34 For example, a tumor acidity-responsive charge conversional nanoparticle, the zeta potential of which transformed from a negatively charged form into a positively charged

anomaterials have shown enormous potential as delivery systems for diagnostic and therapeutic agents in cancer therapy.1−4 To deliver the encapsulated cargo to its biological targets, a nanoparticle needs to interact with complex biological systems (such as biomolecules, cells, body fluids, tissues, and organs).5,6 An ideal nanoparticle is capable of mediating its interactions with biological systems (nanobio interactions) within the living body.7−13 For example, during circulation, a nanoparticle should minimize its interaction with the mononuclear phagocyte system (MPS) to avoid the rapid clearance by the reticuloendothelial system (RES).14−16 In the tumor tissue, its interaction with cancer cells needs to be enhanced to improve cellular uptake;17−19 within tumor cells, the encapsulated cargo requires rapid release to achieve the interaction with biological targets.20−22 Although we are well aware of the optimal design principles, currently available nanoparticles have difficulties fulfilling all of these design requirements. © 2017 American Chemical Society

Received: December 29, 2016 Revised: March 6, 2017 Published: April 4, 2017 2871

DOI: 10.1021/acs.nanolett.6b05396 Nano Lett. 2017, 17, 2871−2878

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Figure 1. Preparation and characterization of the dynamic nanoparticle DATAT-NPIR&DOX. (A) Schematic illustration of tumor acidity/NIR controlled transformable nanoparticle DATAT-NPIR&DOX for mediating nanobio interaction from the injection site to biological targets. (B) UV−vis−NIR absorption spectra of these IR-780/DOX co encapsulated nanoparticles. (C, D, E) Particle size distribution (C), TEM images (D), and zeta potential (E) of the transformable nanoparticle DATAT-NPIR&DOX and the control nanoparticles (TAT-NPIR&DOX and SATAT-NPIR&DOX). DA

form, has been developed to enhance its interaction with cells in the tumor tissue.35 In addition, Wang et al. have worked with a sheddable nanoparticle that deshielded its polyethylene glycol (PEG) shell with the aid of tumor acidity or overexpressed enzymes.36,37 They have succeeded in preparing the nanoparticle such that it avoids interaction with macrophages but improves the interaction with tumor cells. To increase the interaction of the encapsulated drug and biological targets, researchers have developed the redox, pH, and other external stimuli responsive nanoparticle for rapid intracellular release.28,38−40 Altering the physicochemical properties of nanoparticles for mediating nanobio interactions has shown potential for improving the efficacy of therapeutics. Despite significant progress, it is still challenging to precisely and systemically control the physicochemical properties of nanoparticles from the injection site to biological targets for mediating nanobio interactions. Herein, we explored a transformable nanoparticle (DATATNPIR&DOX) with tumor acidity-activated TAT (transactivator of transcription) and a flexible chain polyphosphoester core. This nanoparticle’s physicochemical properties can be controlled in a stepwise fashion by tumor acidity and NIR light, resulting in the mediation of nanobio interactions. As shown in Figure 1A,

TAT-NPIR&DOX (“stealth” state) can minimize its interaction with MPS cells in the blood circulation to avoid rapid clearance by RES. This is because its TAT function is inactivated by modifying the TAT lysine residues’ amines with DA. Once accumulated in the tumor tissues, the masked TAT peptide was reactivated by the tumor acidity, and DATAT-NPIR&DOX transformed into “recognize” state to markedly promote its interaction with tumor cells for enhanced cellular internalization. After internalization into the tumor cells, DATAT-NPIR&DOX transformed into “attack” state, and the NIR irradiation significantly increased the DOX−DNA interaction. The flexible chain polymer polyphosphoester, which formed the nanoparticular core to encapsulate DOX and photothermal agent IR-780, was capable of flowing at body temperature and exhibited supersensitive drug release under photothermal conversion according to our recently reported result.41 We systematically and comprehensively evaluated the effect of this transformable nanoparticle on its interaction with biological systems and on its overall anticancer efficacy. This study provides new avenues for the fabrication of the next generation of cancer drug delivery systems, treatments that will have superior therapeutic effects to those currently available. 2872

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Figure 2. (A) Plasma DOX concentration versus time after intravenous injection of IR&DOX, these IR-780- and DOX-loaded nanoparticles. (B) Flow cytometric analyses of the cellular uptake of DATAT-NPIR&DOX, SATAT-NPIR&DOX, and TAT-NPIR&DOX by macrophages for 2 h.

Figure 3. (A, B) Analysis of DOX content in tumor tissue by a Xenogen IVIS Lumina system (A) and UPLC (B). Tumors were excised 24 h after intravenous injection. (C) Flow cytometric analyses of MDA-MB-231 cells after incubation with DATAT-NPIR&DOX, SATAT-NPIR&DOX, and TATNPIR&DOX for 2 h at pH 6.5. (D, E) Confocal laser scanning microscope (CLSM) images (D) and UPLC quantitative analyses of DOX concentration (E) of MDA-MB-231 cells after incubation with these IR-780- and DOX-loaded nanoparticles at pH 7.4 or pH 6.5 for 2 h. DAPI (blue) and Alexa Fluor488 phalloidin (green) were used to stain cell nuclei and F-actin, respectively. The scale bar is 10 μm.

To obtain the DATAT-NPIR&DOX, the TAT peptide sequence “YGRKKRRQRRRC” containing a thiol group was first tethered

to the diblock copolymer of poly(ethylene glycol)-block-poly(2hexoxy-2-oxo-1,3,2-dioxaphospholane) with the maleimide end 2873

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signals were determined by flow cytometric analysis (Figure 2B). We observed that the macrophage raw 264.7 cells incubated with DA TAT-NPIR&DOX or SATAT-NPIR&DOX exhibited much weaker intracellular fluorescence of DOX compared to those incubated with TAT-NPIR&DOX, indicating that masking the TAT targeting function by DA or SA efficiently reduced its interaction with MPS cells. After circulating into the tumor tissue, DATAT-NPIR&DOX entered into the “recognize” state in order to promote its interaction with tumor cells and enhance cellular internalization. To verify this, we evaluated the DOX accumulation in the tumor tissue. After 24 h postinjection, the mice were sacrificed, and the tumor tissue was collected for Xenogen IVIS Lumina system analysis. As shown in Figure 3A and Figure S3, the highest fluorescent signal of DOX was visualized at tumor sites in DA TAT-NPIR&DOX compared with the mice administered SATATNPIR&DOX or TAT-NPIR&DOX. Furthermore, Figure 3B showed that the DOX accumulation in the tumor tissue after administration of DATAT-NPIR&DOX was 4.75-fold and 1.76fold higher than that of TAT-NPIR&DOX and SATAT-NPIR&DOX, respectively. The DATAT-NPIR&DOX and SATAT-NPIR&DOX exhibited similar pharmacokinetic properties (Figure 2A), ensuring that both nanoparticles were equally circulated into the tumor interstitium. However, the DATAT-NPIR&DOX group showed significantly improved DOX accumulation in tumor tissue as compared with those in the SATAT-NPIR&DOX group. To investigate this, we incubated the blank nanoparticle DATAT-NP and SATAT-NP in phosphate-buffered saline for 30 min and then lyophilized for 1H NMR analysis. As shown in Figure S4, the methyl proton peaks of DA of the DATAT-NP (Ha, 1.85 ppm) had almost disappeared at pH 6.5 within 30 min. In contrast, the proton resonance of the methylene hydrogen atoms adjacent to the amide (Ha′, 2.59 ppm) was not lessened for SATAT-NP after incubating at pH 6.5 for 30 min (Figure S5). In addition, the zeta potential of DATATNPIR&DOX gradually increased from −13.7 mV to 2.6 mV within 20 min at pH 6.5 (Figure S6), reaching a similar zeta potential to that of TAT-NPIR&DOX (Figure 1E). On the contrary, the zeta potential of DATAT-NPIR&DOX only slightly and slowly increased at pH 7.4. Nevertheless, the zeta potential of SATAT-NPIR&DOX exhibited only a negligible change at either pH 7.4 or pH 6.5, retaining a negative zeta potential. Additionally, to determine the degradation rate of the amide bonds formed between TAT lysine residues’ amines and DA or SA, the nanoparticles DATAT-NP and SATAT-NP were incubated at pH 7.4 and 6.5 conditions, and then the fluorescamine was used as a sensor to detect the resulting amine group. As shown in Figure S7, approximately 96% amide bonds of DATAT-NP was hydrolyzed and converted to amine bonds after incubation 4 h at pH 6.5, while only 37% amide bonds was degraded at pH 7.4. On the contrary, lower than 10% amide bonds of SATAT-NP was converted to amine bonds at either pH 7.4 or pH 6.5. These results demonstrated that the tumor’s slight acidity was capable of triggering the rapid degradation of amide bonds formed between TAT lysine residues’ amines and DA, and regenerating TAT peptide. It is possible for the regeneration of the TAT peptide with tumor acidity to accompany the enhanced interaction of nanoparticle and tumor cell. Thus, the cellular internalization of these nanoparticles at tumor acidity was investigated. After incubation with MDA-MB-231 cells at pH 6.5 for 2 h, the intracellular DOX fluorescent was determined by flow cytometric analysis (Figure 3C). The cells incubated with DA TAT-NPIR&DOX at pH 6.5 had a much stronger intracellular

group (Mal-PEG-b-PHEP) via a facile thiol−maleimide coupling reaction (Scheme S1). The successful synthesis was verified by 1 H NMR spectroscopy, indicating that the double bond of the maleimide (6.90 ppm, a, Figure S1) had almost disappeared; 83.5% of the maleimide end group was successfully conjugated with the TAT peptide using Ellman’s reagent 5,5′-dithiobis(2nitrobenzoic acid) as described in the Supporting Information. Subsequently, IR-780 and DOX were encapsulated into TATPEG-b-PHEP based polymeric micelles (TAT-NPIR&DOX) by a dialysis method, and then the TAT lysine residues’ amines of the obtained TAT-NPIR&DOX were modified with DA to obtain DA TAT-NPIR&DOX. The TAT lysine residues’ amines modified with succinic anhydride (SA) were used as a control (SATATNPIR&DOX). The extent of DA or SA conjugation to two lysine residues’ amines of TAT-PEG-b-PHEP was approximately 100% according to the 1H NMR analyses. For these DOX and IR-780loaded nanoparticles, the loading content of IR-780 and DOX was ca. 2.18 ± 0.23% and 4.36 ± 0.17%, respectively. The UV−vis−NIR spectra of DATAT-NPIR&DOX, SATATNPIR&DOX, and TAT-NPIR&DOX are shown in Figure 1B. All three showed strong absorbance at 480 and 790 nm, indicating the efficient coencapsulation of DOX and IR-780 for the three nanoparticles. The size distribution of these nanoparticle measured by dynamic light scattering (DLS, Figure 1C) was approximately 100 nm. The morphologies of DATAT-NPIR&DOX, SA TAT-NPIR&DOX, and TAT-NPIR&DOX observed by the transmission electronic microscopic (TEM) exhibited a compact and spherical morphology with a diameter of 80 nm (Figure 1D). The zeta potentials of these nanoparticles were also determined. As shown in Figure 1E, DATAT-NPIR&DOX and SATAT-NPIR&DOX showed a negative surface property with a zeta potential of ca. −13.7 mV, while TAT-NPIR&DOX showed a slightly positive zeta potential (3.8 mV). Additionally, these nanoparticles maintained their diameters for over 7 days (Figure S2) after incubation culture medium containing 10% fetal bovine serum (FBS) at 37 °C, which could be due to the PEGylation. As described above, this nanoparticle should minimize its interaction with MPS cells in circulation (“stealth” state) and avoid rapid clearance by the RES. It is well-known that the TAT peptide possesses the ability to quickly translocate into almost any live cells,42 which is also accompanied by the rapid clearance from circulation.43,44 Thus, for the DATAT-NPIR&DOX, the lysine residue amines of TAT peptide were amidized by DA to reduce its interaction with MPS cells and avoid the rapid clearance.45 To demonstrate this, we evaluated the pharmacokinetics of these nanoparticles. Figure 2A shows the percentage of injected DOX in plasma versus time curves; the pharmacokinetic parameters are presented in Table S1 using a noncompartment model. As expected, masking the DATAT-NPIR&DOX TAT peptide showed a prolonged half-life in the blood circulation compared with the TAT-functionalized nanoparticle TAT-NPIR&DOX, which could also be observed for SATAT-NPIR&DOX. The Cmax of DATATNPIR&DOX and SATAT-NPIR&DOX in the plasma were 2.33-fold and 2.23-fold of TAT-NPIR&DOX, respectively. Also note that the blood clearance of TAT-NPIR&DOX (64.5 L/h) was much faster than that of DATAT-NPIR&DOX (14.2 L/h) and SATAT-NPIR&DOX (13.0 L/h). It is possible that the prolonged circulation achieved by masking the TAT targeting function is due to the reduced interaction of DATAT-NPIR&DOX and MPS cells. To test this, the macrophage raw 264.7 cells were incubated with these nanoparticles, and then the intracellular DOX fluorescence 2874

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Figure 4. (A, B) Temperature evolution on tumors (A) and IR thermal images (B) of MDA-MB-231 tumor-bearing mice after intravenous administration of PBS, free IR&DOX, and IR-780- and DOX-loaded nanoparticles upon NIR irradiation (808 nm, 1.0 W/cm2, 10 min). (C) DOX release from DATAT-NPIR&DOX and DATAT-NPIR/DATAT-NPDOX with or without NIR irradiation (808 nm, 1.0 W/cm2, 5 min). (D) Change in average DOX fluorescence lifetime (ps) of these formulations ([IR-780] = 2.0 μg/mL], [DOX] = 4.0 μg/mL) under NIR irradiation (808 nm, 1.0 W/cm2, 10 min). (E, F) Flow cytometric analyses (E) and CLSM images (F) of MDA-MB-231 cells after NIR irradiation (808 nm, 1.0 W/cm2, 5 min) and further incubation for 4 h. The MDA-MB-231 cells were precultured with DATAT-NPIR&DOX for 1 h. The scale bar is 10 μm.

fluorescence when compared with those of SATAT-NPIR&DOX, reaching a similar mean fluorescence intensity to the cells treated with TAT-targeted nanoparticle TAT-NPIR&DOX. However, cells incubated with DATAT-NPIR&DOX and SATAT-NPIR&DOX exhibited similar intracellular DOX fluorescence at pH 7.4 (Figure S8), which is significantly lower than cells treated with TATNPIR&DOX. This is because the TAT peptide modified by DA can only be regenerated at the level of tumor acidity as described above. On the other hand, the cellular uptake at different pH levels was further corroborated by confocal laser scanning microscopic (CLSM) observations. After incubation with DATAT-NPIR&DOX, SA TAT-NPIR&DOX, or TAT-NPIR&DOX at pH 7.4 or 6.5 for 2 h, the cytoskeleton F-actin and the cell nuclei were counterstained with Alexa Fluor 488 phalloidin (green) and 6-diamidino-2-phenylindole (DAPI, blue), respectively. As shown in Figure 3D, the cellular uptake of TAT-NPIR&DOX and SATAT-NPIR&DOX were not affected by the pH value, while a much stronger cellular fluorescence for DATAT-NPIR&DOX at pH 6.5 was detected

compared to cells treated with DATAT-NPIR&DOX at pH 7.4. Moreover, the intracellular DOX contents were quantitatively determined. As shown in Figure 3E, the amount of intracellular DOX following incubation with DATAT-NPIR&DOX at pH 6.5 was comparable to that of TAT-NPIR&DOX at pH 7.4 or 6.5, which were ca. 1.75 times higher than the cells treated with DATATNPIR&DOX at pH 7.4 or SATAT-NPIR&DOX at both pH. Thus, we demonstrated that a TAT peptide masked by DA can be regenerated in the tumor’s slightly acidic microenvironment, resulting in the transformation of DATAT-NPIR&DOX into the “recognize” state. Therefore, the interaction of DATAT-NPIR&DOX and tumor cells was enhanced with tumor acidity, which accompanied by the promoted cellular uptake by tumor cells and improved drug accumulation in the tumor tissue. After the encapsulated cargo has entered the tumor cell, its rapid release and interaction with biological targets plays a crucial role in the final anticancer efficacy. For the DATAT-NPIR&DOX, hydrophobic flexible chain PHEP formed the nanoparticular core, which was capable of flowing at the physiological 2875

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Figure 5. (A, B) MDA-MB-231 tumor growth curves (A) and tumor growth rate (B) of various groups after intravenous administration of PBS, IR&DOX, and IR-780- and DOX-loaded nanoparticles with different treatments. (C) The weight of the tumor was assessed after the last determination. Mean ± SD, n = 5; *p < 0.05; **p < 0.01.

reaching 70%. Similar NIR-activated DOX release profiles were observed from the TAT-NPIR&DOX and SATAT-NPIR&DOX nanoparticles (Figure S9). This is because comparable localized heating was generated under NIR irradiation (Figure S10), which was capable of triggering the supersensitive DOX release from their hydrophobic PHEP flowable core. However, it is interesting that this NIR-activated drug release effect disappeared when the DOX and IR-780 were separately encapsulated into the nanoparticles (DATAT-NPIR/DATAT-NPDOX). It is likely that the increased temperature of the DATAT-NPIR solution under NIR irradiation is insufficient to trigger DOX release from the separate core of DATAT-NPDOX. To verify our assumption, we incubated the DATAT-NPIR/DATAT-NPDOX at 45 °C for 5 min (the same temperature and irradiation time under NIR), then we examined the influence of temperature on DOX release from TAT-NPIR&DOX (Figure S11). As expected, the DOX release rate was only slightly enhanced after preincubation at 45 °C. Furthermore, to demonstrate the reason for the supersensitive release, we monitored the temperature of the nanoparticle core by assessing the fluorescence lifetime with the time-correlated single photon counting (TCSPC) technique. We first calculated a calibration curve relating DOX lifetime of these nanoparticles and temperature using a TCSPC technique. As shown in Figure S12, the excited-state lifetime of DOX decreased with increasing temperature following a negative linear relationship with a calculated sensitivity of −8.5 ps/°C. Furthermore, these nanoparticles were exposed to an NIR laser (808 nm, 1.0 W/ cm2) at an IR-780 concentration of 2.0 μg/mL for 5 min as described above, and then the fluorescence lifetimes were determined. As shown in Figure 4D, the DOX fluorescence

temperature and exhibited supersensitive drug release under photothermal conversion.41 Thus, the NIR irradiation could trigger the supersensitive DOX release from the DATATNPIR&DOX and then increase the DOX-DNA interaction in tumor cells. Therefore, the photothermal conversion of these nanoparticles in the tumor tissue was a prerequisite for the triggered DOX release. After 24 h postadministration, the tumor site was irradiated by 808 nm NIR laser (1.0 W/cm2, 10 min), and the local temperature change in the tumor tissue was recorded. Note that the IR-780 and the DOX were separately encapsulated into DATAT-PEG-b-PHEP based nanoparticles (DATAT-NPIR/DATAT-NPDOX) for subsequent comparison. DA TAT-NPIR/DATAT-NPDOX exhibited similar circulation, biodistribution, tumor accumulation, and cellular uptake to the DA TAT-NPIR&DOX (data not shown). As shown in Figure 4A and B, the tumor temperature of the DATAT-NPIR&DOX and DATATNPIR/DATAT-NPDOX groups increased to ca. 48.0 °C within 5 min. In contrast, the tumor temperature of the SATAT-NPIR&DOX, TAT-NPIR&DOX and IR&DOX groups were not obviously evaluated, reaching 43.5 °C, 41.4 °C, and 39.3 °C, respectively. Based on our design, the abruptly raised intraparticle temperature of this transformable nanoparticle under NIR irradiation should suddenly trigger the DOX release from its flowable core. To demonstrate this, we exposed these nanoparticles to an IR-780 concentration of 2.0 μg/mL to an NIR laser (808 nm, 1.0 W/cm2) for 5 min, and then the release profile was monitored. As shown in Figure 4C, without NIR irradiation, only 25% of the DOX released from DATAT-NPIR&DOX in 8 h (slight drug leakage during blood circulation), while the DOX release was significantly accelerated after NIR irradiation, 2876

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monitored. As illustrated in Figure 5A, treatment with DATATNPIR&DOX or DATAT-NPIR/DATAT-NPDOX but no NIR irradiation showed comparable inhibition of tumor growth, while treatment with DATAT-NPIR&DOX plus NIR irradiation (DATATNPIR&DOX+NIR) exhibited the highest tumor growth inhibition among all of these formulations. In contrast, the anticancer efficacy of DATAT-NPIR/DATAT-NPDOX plus NIR irradiation (DATAT-NPIR/DATAT-NPDOX+NIR) was moderately improved after irradiation, which could be due to the photothermal ablation to the tumor. In comparison to the DATATNPIR&DOX+NIR group, the tumor growth was mildly inhibited by the intravenous injection of SATAT-NPIR&DOX and TATNPIR&DOX plus NIR irradiation. In addition, the inspection of the tumor growth rates among all of these formulations also indicated that the antitumor efficiency of the DATAT-NPIR&DOX+NIR group improved markedly (Figure 5B); the tumor weight in Figure 5C and tumor picture in Figure S16 further supported the above result. It is worth noting that there was not significant body weight loss in any of the groups (Figure S17), and the histological analyses by hematoxylin and eosin (H&E) staining of the main organs after treatment also did not show obvious biological toxicity (Figure S18), demonstrating that the formulations at these DOX doses were not noticeably toxic to the mice. Finally, the in vivo therapeutic efficacy and mechanism were analyzed by immunohistochemical staining after the treatment (Figure S19). It clearly demonstrated that the administration of DA TAT-NPIR&DOX plus NIR irradiation was the most effective formulation in reducing the percentage of increasing TUNELpositive tumor cells, indicating the optimal efficiency of the DA TAT-NPIR&DOX by tumor acidity/NIR mediated nanobio interaction. In summary, we prepared the DATAT-NPIR&DOX in order to mediate nanobio interactions for satisfactory anticancer efficacy; indeed, its properties can be precisely and systemically controlled from the injection site to biological targets. The TAT targeting function of DATAT-NPIR&DOX was masked by DA, which minimized its interaction with MPS cells (“stealth” state). Thus, the circulation time in the blood was prolonged. After circulating into tumor tissues, the masked TAT peptide was reactivated by the tumor acidity, and this DATAT-NPIR&DOX transformed into the “recognize” state in order to markedly promote its interaction with tumor cells for enhanced cellular internalization. Finally, after internalization into the tumor cells, the local heating generated in the nanoparticle flowable core suddenly triggered the supersensitive DOX release. Then, this transformable nanoparticle transformed into “attack” state in order to significantly increase the DOX-DNA interaction, thus exhibiting superior therapeutic effects in cancer treatment. We provide proof of the principle that systemically mediating nanobio interactions can be achieved by the rational design of nanoparticular delivery systems. More importantly, such a design strategy provides a promising avenue to fabricate the next generation of drug delivery systems with superior therapeutic effects in cancer treatment.

lifetime decreased by ca. 300 ps for the simultaneous encapsulation formulation, while it decreased by 90 ps for DA TAT-NPIR/DATAT-NPDOX. According to the calibration curve in Figure S12, the average temperature of the nanoparticle core for TAT-NPIR&DOX, SATAT-NPIR&DOX, DATAT-NPIR&DOX, and DA TAT-NPDOX increased by 34.1 °C, 33.9 °C, 34.1 °C, and 10.3 °C, respectively. We found that the internal temperature increase of the nanoparticle core for TAT-NPIR&DOX, SATAT-NPIR&DOX, and DATAT-NPIR&DOX under NIR irradiation was greater compared to the temperature increase of the nanoparticle core of DATAT-NPDOX for the DATAT-NPIR/DATAT-NPDOX formulation. It appears that the sudden temperature increase of the nanoparticle core was the primary trigger for the DOX release from the flowable core. Thus, when DOX and IR-780 were simultaneously encapsulated, the NIR-activated drug release could be achieved. In contrast, for separate encapsulation formulation, the generated localized heat within the nanoparticle DA TAT-NPIR cannot efficiently accelerate the DOX release from DA TAT-NPDOX (Figure S13). To examine whether this NIR-activated DOX release effect could be achieved within tumor cells, we incubated the cells with DA TAT-NPIR&DOX or DATAT-NPIR/DATAT-NPDOX ([DOX] = 4.0 μg/mL, [IR-780] = 2.0 μg/mL) at pH 6.5 for 1 h, and the cells were subsequently exposed them to a NIR laser (1.0 W/cm2, 5 min). After further incubation for 4 h, the intracellular DOX fluorescence was analyzed by flow cytometric analysis and CLSM. As shown in Figure 4E, without NIR irradiation, intracellular DOX fluorescence of DATAT-NPIR&DOX only slightly increased for the DATAT-NPIR&DOX group, whereas the NIR irradiation significantly enhanced intracellular DOX fluorescence. The DOX fluorescence was partially quenched after encapsulation into the nanoparticle (Figure S14). Thereby, the enhanced DOX fluorescence was due to the NIR-activated supersensitive DOX release from the nanoparticle DATATNPIR&DOX as described in Figure 4C. In contrast, such NIRenhanced intracellular DOX fluorescence almost disappeared when cells were treated with DATAT-NPIR/DATAT-NPDOX (Figure S15A). Additionally, the DOX distribution detected by CLSM demonstrated that stronger red DOX fluorescent signals were observed in cell nuclei when cells were treated with DATATNPIR&DOX plus NIR irradiation (Figure 4F). On the contrary, DOX was mainly localized in the cytoplasm when cells were treated with DATAT-NPIR/DATAT-NPDOX, regardless of the NIR irradiation (Figure S15B), indicating that DOX was still encapsulated in the nanoparticle. These results were consistent with the NIR-activated release profile (Figure 4C). Our findings demonstrate that, following accumulation in the tumor tissue, the NIR irradiation suddenly elevated the temperature of the nanoparticle’s core due to the photothermal effect of the encapsulated IR-780. The resultant temperature increase of the NP core triggered the supersensitive surrounding DOX release from the flowable core for DATAT-NPIR&DOX and then significantly increased the DOX-DNA interaction in the tumor cells. To indicate the advantage of the tumor acidity/NIR controlled transformable nanoparticle for in vivo cancer therapy, the antitumor effects of the various formulations were examined. Mice bearing the MDA-MB-231 tumors were treated with the above formulations. After 24 h postadministration, the tumor tissue was irradiated by 808 nm NIR laser at a power density of 1.0 W/cm2 for 10 min, and then the tumor growth was

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.6b05396. Experimental methods and supporting figures (PDF) 2877

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (X. Yang). *E-mail: [email protected] (J. Wang). ORCID

Xianzhu Yang: 0000-0002-1006-0950 Author Contributions

D. Li and Y. Ma contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51473043, 51390482, and 51633008), National Basic Research Program of China (2013CB933900, and 2015CB932100), the Natural Science Foundation for Distinguished Young Scholars of Guangdong Province, and the Fundamental Research Funds for the Central Universities.

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DOI: 10.1021/acs.nanolett.6b05396 Nano Lett. 2017, 17, 2871−2878