NaYF₄:Yb/Tm up-conversion nanoparticles (UCNPs) were purchased from Hefei Fluonano Biotech Co., Ltd. Anhydrous acetic acid, dimethyl sulfoxide (DMSO), chloroform, ethanol and cyclohexane were purchased from Beijing Chemical Reagent Company. Alfa Aesar Ltd. supplied N,N’-dicyclohexylcarbodiimide (DCC), 4,4’-trimethylene dipiperidine and 1,6-hexanediol diacrylate. JenKem Technology USA Inc. provided methoxy PEG amine (5K). iRGD (CRGDKGPDC) was purchased from GL Biochem (Shanghai) Ltd. Doxorubicin hydrochloride (DOX, 99.9%) was purchased from Adamas-beta. Aldrich Chemical Reagent Company provided sodium hydroxide (NaOH, 98%), sodium phosphate dibasic dodecahydrate, potassium phosphate monobasic, sodium chloride, potassium chloride, anhydrous sodium acetate and (4-carboxybutyl)triphenyl phosphonium bromide. N-hydroxysuccinimide (NHS) was purchased from Aladdin Chemical Co. DOX-resistant small-cell lung cancer cell line H69AR was kindly provided by Prof. Linglang Guo (Zhujiang Hospital, Southern Medical University). Fetal bovine serum (FBS) was purchased from PAN^TM Biotech, Germany. HyClone^TM offered 0.25% Trypsin-EDTA (1X) with phenol red, Roswell Park Memorial Institute (RPMI) 1640 cell-culture medium and antibiotics (penicillin 100 U/mL and streptomycin 100 µg/mL). Beyotime Institute of Biotechnology (Shanghai, China) supplied ATP assay kit and JC-1 mitochondrial membrane potential assay kit. Dojindo Molecular Technologies (Kumamoto Techno, Japan) offered CCK-8 kit. Mitotracker and Hoechst 33342 were purchased from Life Technologies (Shanghai, China). Lumiprobe Corporation (USA) supplied Cy5 NHS ester. Heparin sodium salt was supplied from J&K Scientific Ltd (Beijing, China). Chloral hydrate (98.5%) was purchased from Acros Organics, ThermoFisher Scientific (China) Co., Ltd. Anti-CD31 antibody (ab28364) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody were purchased from Abcam. All of the chemicals were analytical grade and applied with no further purification. The experiments adopted deionized water.

The collection of UV-Vis spectra was performed on a Varian Cary-50 UV-Vis spectrophotometer. The recording of fluorescence emission spectra was conducted on a Varian Cary Eclipse Fluorescence spectrophotometer. Up-conversion photoluminescence measurements were conducted on a PTI Quantamaster spectrofluorometer. A transmission electron microscope (TEM, Tecnai G2 20 S-TWIN, USA) was adopted to acquire morphologies of the samples. Zetasizer Nano ZS90 (Malvern Instruments Company, UK) was employed to perform dynamic light scatting (DLS) and zeta potential analysis. The Fourier transform Bruker EQUINOX55 spectrometer with the KBr pellet technique was employed to collect the Fourier transform infrared (FTIR) spectra. Confocal fluorescence microscopy (A1/LSM-Kit, Nikon) was employed to image the cells. The fluorescent images of mice were taken by PerkinElmer IVIS Lumina LT Series II Spectrum.

The light-cleavage polymer (HTMP) and iRGD-containing polymer (iPHT) were synthesized according to a reported protocol with minor modifications.^[

The polymer micelle (iPHM) containing HTMP and iPHT was prepared by using the solvent substitution method. Typically, HTMP (10 mg) and iPHT (4 mg) were added and dissolved in 2 mL of DMF (the amount of substance ratio of HTMP/iPHM was 1). Then, 10 mL of distilled water was added dropwise under vigorously stirring. The solution was then moved to a dialysis bag and underwent extensive dialysis against PBS buffer (pH=7.4) to form micellar nanoparticles. iPUN composited nanoparticles were synthesized as follows: the dispersion of oleic acid-altered UCNPs was performed in cyclohexane at 10 mg/mL, followed by 15-min ultrasonic dispersion. HTMP (10 mg) and iPHT (4 mg) were dispersed in 1 mL of chloroform, then, 500 μL of 10 mg/mL UCNPs mentioned above were added with vigorous stirring for 30 min. Next, 10 mL of distilled water was added dropwise to the above organic phase and the mixture was ultrasonicated for 20 min to be emulsified. The 12-h stirring of the emulsified solution was performed at room temperature for the evaporation of the organic solvent. The solution was then dialyzed for 24 h against distilled water to remove the residual organic solvent and impurities.

The NIR-responsive PEG detachment of iPUN was analyzed by DLS and ¹H-NMR. Typically, 1 mL of the aqueous solution with 0.5 mg/mL of iPUN was put into a vial and then irradiated with 980 nm NIR light (1 W/cm²). After 5 min, the hydrodynamic diameter and zeta potential of the particles were determined by DLS at 20 °C applying Zetasizer Nano S. To study the change of iPUN under NIR irradiation, 20 mg of lyophilized iPUN sample was dissolved in 600 µL of CDCl₃. After NIR irradiation (1 W/cm², 1 cm² faculous region) for different time, iPUN containing solution was centrifuged, and the supernatant was collected for ¹H-NMR characterization. Similarly, the acid-responsive size and zeta potential change of iPUN was also analyzed by DLS. The preparation of TEM samples was made below: a drop of the solution containing UCNPs or iPUN was dripped on a carbon-coated copper grid (400-mesh) and absorbed off the excess solution from the edge of the grid by filter paper after 1 min (repeated for 3 times). The measurement of the mean diameters of particles was performed from 10 particles in the TEM micrographs.

To be brief, 45 mg (0.10 mmol) of TPP was added to 10 mL of anhydrous N,N-dimethylformamide (DMF) in a 25 mL round-bottom flask and stirred to dissolve completely. Then, DCC (25 mg, 0.012 mmol) and NHS (14 mg, 0.12 mmol) were added into the above solution. After stirring for 3 h, the centrifugation of the mixture was conducted to eliminate dicyclohexylurea (DCU), and the collection of supernatant was conducted. Next, the purchased DOX (60 mg, 0.10 mmol) was desalted by using marginal excess of triethylamine. The activated TPP was then appended dropwise to the DOX solution. The solution was kept stirring for 12 h at room temperature in dark. Afterward, the crude product was obtained by precipitation in a great excessive amount of diethyl ether and centrifugation. Then, the precipitate was dissolved in chloroform and rinsed with saturated sodium chloride solution to remove other impurities from the product. The organic phase was then collected and dried in a vacuum. TDOX was characterized by FTIR (KBr pellet). Zetasizer Nano ZS90 (Malvern Instruments Company, UK) was adopted to perform Zeta potential analysis.

To prepare iPUTDN, a similar route for the preparation of iPUN was exploited. Briefly, oleic acid-coated UCNPs were dissolved in cyclohexane. HTMP (10 mg), iPHT (4 mg), and TDOX (1, 2 or 5 mg) were dissolved in chloroform. Firstly, the solutions were mixed, and distilled water was appended dropwise to the organic phase under vigorous stirring. After another 12 h stirring, the dialysis of the solution against water was conducted. iPUTDN nanoparticles were obtained by lyophilization.

To examine the TDOX release profile of iPUTDN under different conditions, four groups of samples were produced containing the same concentration of TDOX at 0.1 mg/mL. The conditions were set as solution pH at 7.4 or 5.0 and with or without NIR light irradiation. At predetermined time points, 3 mL of solution was collected from the vial containing 20 mL of solution for the measurement of TDOX absorbance at 480 nm, and 3 mL of fresh buffer was appended for keeping the volume in the vial. The calculation of the concentrations of TDOX was performed from a standard curve of TDOX absorbance at 480 nm.

DOX-resistant small-cell lung cancer H69AR cells were kindly given by Prof. Linglang Guo (Zhujiang Hospital, Southern Medical University). H69AR cells were used to examine the cell inhibition efficiency of iPUTDN. H69AR cells were cultured with RPMI supplemented with 20% FBS, 1.0×10⁵ U/L penicillin (Sigma), and 100 mg/L streptomycin at 37 °C in 5 % CO₂. H69AR cells were seeded into a 96-well cell culture plate (8000 cells per well) and incubated at 37 °C with 5% CO₂. A fresh culture medium was employed to replace the growth medium after 24 h. Afterward, iPUTDN or control samples (free TDOX and DOX) were added into wells (sample size=6). The irradiation of cells was performed with NIR laser (980 nm, 2 W/cm², 30 s) at 2 h and 12 h, and cultured for another 24 h. CCK-8 assays were adopted to determine cytotoxicity. An ELISA plate reader was adopted to measure the absorbance of every well at a test wavelength of 450 nm. The cell growth suppression of samples was calculated as follows:

where I_sample and I_control mean the intensity decided for cells handled with various samples and for control cells (untreated).

H69AR cells were seeded in confocal dishes at a density of 2×10⁴ cells per well for 24 h at 37 °C in 5 % CO₂. Afterward, DOX, TDOX or iPUTDN with or without NIR irradiation (1 W/cm², 2 min, maintenance concentration of DOX or TDOX at 2 μg/mL) were performed with the cells in RPMI 1640 at 37 °C in 5% CO₂ for 2 h and 24 h, respectively. Later, the cells were washed with PBS buffer three times. Then, the cells were stained with 5 μg/mL Hoechst 33342 at 37 °C for 10 min, then washed with cold PBS for three times and instantly observed with CLSM. The fluorescent intensity was measured by ImageJ. For the internalization pathways study, the incubation of cells was performed with various inhibitors such as nocodazole (10 μg/mL), chlorpromazine hydrochloride (1 μg/mL), amiloride hydrochloride hydrate (50 μmol/L), and genistein (100 μmol/L) in serum-free RPMI 1640 medium for 30 min before incubation with the iPUTDN or iPUTDN+NIR for another 4 h. To avoid the effect of NIR irradiation on cells, iPUTDN was irradiated with NIR (1 W/cm², 2 min) in an aqueous solution before adding to the cells. Later, 4% paraformaldehyde was adopted to fix the cells for 10 min and then washed with PBS for three times. 5 μg/mL Hoechst 33342 was employed to stain the cells at 37 °C for 10 min, then washed with PBS for three times. Confocal images were obtained by excitation of the samples at 561 and 405 nm. The fluorescent intensity was measured by ImageJ.

H69AR cells were seeded in culture dishes at an initial density of 1×10⁴ cells. After 24 h, TDOX, iPUTDN with or without NIR irradiation (1 W/cm², 2 min, equivalent to 2 μg/mL of TDOX) were appended and incubated with cells for another 24 h. After the incubation, the cells were washed with PBS solution and a fresh medium was added. 100 nmol/L Green fluorescent Mitotracker was added to dishes and incubated at 37 °C for 30 min. The nuclei were fluorescently visualized by staining with 5 μg/mL of Hoechst 33342. The cells were observed with CLSM.

JC-1 was employed to monitor the potential of the mitochondrial membrane. The probe will transform a monomer (red fluorescence) to a converged state (green fluorescence) in response to low mitochondrial membrane potential. Typically, H69AR cells were cultured in 6-well plates (2×10⁴ cells/well) for 24 h, then incubated with TDOX or iPUTDN with or without NIR for 2 h. iPUTDN was irradiated with NIR (1 W/cm², 2 min) in an aqueous solution before adding to the cells. JC-1 (500 μL, 10 μg/mL) was adopted to stain the cells for another 20 min. After rinsing twice, the examination of the cells was performed with a microplate reader (Infinite 200 Pro, Tecan, Zürich, Switzerland).

H69AR cells were cultured with 6-well plates (2×10⁴ cells per well) for 24 h at 37 °C, then incubated with TDOX and iPUTDN with or without NIR for 4 h. To avoid the effect of NIR irradiation on cells, iPUTDN was irradiated with NIR (1 W/cm², 2 min), in an aqueous solution before adding to the cells. The ATP assay kit was adopted to examine ATP levels, which were measured by a multi-mode microplate reader (Spark^TM 10M, Tecan, Zürich, Switzerland). The ATP levels were decided on basis of the ATP standard curve.

Beijing WeiTongLiHua Animal Co., Ltd. provided female BALB/c nude mice (4−6 weeks, 13±2 g). The preparation of DOX resistant subcutaneous model of small cell lung cancer was performed according to the procedure below. 4% Chloral hydrate at a dosage of 10 mL/kg was intraperitoneally injected to anesthetize female BALB/c nude mice, followed by a subcutaneous injection of 1×10⁷ H69AR cells. In the case of a palpable tumor at the position of the leg, the mice were subject to the experiments below. All the animal experiments were conducted by the guidelines approved by the Animal Care and Use Committee of CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety.

EDC/NHS coupling reaction was employed to synthesize iPUN-Cy5. To be brief, Cy5-NHS (Lumiprobe, USA) was appended to iPUN nanoparticles (mass ratio=1:1000) and then stirred for about 12 h. The product was obtained by lyophilization post extensive dialysis against distilled water for 24 h. Free Cy5 (200 μL) and iPUN-Cy5 (Cy5 concentration, 1 μg/mL) were intravenously administrated (n=3). For NIR group, the tumor was exposed to NIR irradiation (1 W/cm²) for 5 min after administration for 30 min, 4 h and 12 h. At the time points of 1 h, 6 h, 24 h post-injection, Lumina III in vivo imaging system (λ_Ex/λ_Em=640/680 nm) took the fluorescent images of the living mice. Then, the mice were sacrificed and the tumors were extracted to immunostaining with the anti-CD31 antibody and Alexa Fluor^®488 secondary antibody. Finally, the distribution of iPUN-Cy5 penetration from blood vessels was visualized by CLSM and the penetration depth and the fluorescence intensity of iPUN-Cy5 were analyzed by ImageJ. Similarly, free TDOX and iPUTDN (TDOX concentration, 8 mg/kg) were administrated into mice (1 W/cm² NIR irradiation was performed for 5 min at tumor area 30 min after i.v. injection). At 24 h after injection, the sampling and imaging of heart, liver, spleen, lung, kidney and tumor were conducted by an ex vivo imaging system with excitation wavelength 465/640 nm and emission wavelength 580/680 nm.

Ten days after inoculation of H69AR cells, the treatment was performed every other two days at a dose of 8 mg/kg for TDOX, lasting for three weeks (n=5 per group). The intravenous injection was performed through the tail vein. Later, the tumor area was exposed to NIR (980 nm, 1 W/cm², 10 min) at 1 and 12 h post-injection. The monitoring of body weight and the palpable tumor was performed every two days. After the treatments, all the mice were sacrificed and tumors were dissected and weighted. H&E staining was adopted to analyze histological sections of the main organs.

Whole blood (1 mL) taken from BALB/c nude mice was placed into a 4 mL centrifuge tube with 2.5 μL of 2% heparin sodium, the slow shaking of the mixture was performed. Later, after adding the same volume of normal saline, 10-min centrifugation was performed at 1500 r/min. The supernatant was removed, and the dispersion of the precipitate was performed in 10 mL of normal saline. The 12-min centrifugation was conducted at 1500 r/min, and the red blood cell suspension (RCS) was obtained by repeating the same procedures three times. Later, (I) 0.8 mL of PBS as a negative control, (II) 0.8 mL of deionized water as a positive control, and (III) 0.8 mL of an aqueous dispersion of iPUN at various concentrations in a scope of 0−250 μg/mL were mixed with 0.2 mL of RBC suspension, respectively. The centrifugation of mixtures was performed (12000 r/min, 5 min) after a 1-h incubation at 37 °C, and optical density (OD value) was determined with the supernatants in a microplate reader at a wavelength of 541 nm.

After the treatments, the mice were anesthetized by injecting 4% chloral hydrate into the peritoneal cavity, and blood was collected by removing the eyeballs. Whole blood (100 μL) was added into the EP tubes with 1 mL of anticoagulation (1.5 mg/mL EDTA dipotassium salt dihydrate, Macklin, Shanghai, China) and the samples were examined with routine blood tests at Animal Laboratory Testing Center, Peking University Health Science Center.

At the end of treatments, the mice were sacrificed and mouse tumors were fixed in 4% paraformaldehyde at 4 °C overnight. The samples were cut as 5 μm slices and stained with H&E and Ki-67. The images were recorded by an inverted fluorescence microscope (Olympus IX73).

Quantitative data are presented as mean±SD and analyzed by a two-tailed Student’s t-test. P<0.05 was accepted as a statistically significant difference.

The synthesis of nanoparticles was performed according to a reported protocol.^[

Fig 1  Fabrication and characterization of UCNPs and iPUNs. (A) TEM image of UCNPs. Scale bar=100 nm. (B) TEM image of iPUN. Scale bar=100 nm. (C) The hydrodynamic diameter of iPUN, iPUN+NIR (1 W/cm², 5 min) and iPUN in 100 mmol/L pH 5.0 acetate buffer. (D) Upconversion emission spectra of free UCNP in hexane and iPUN in water under 980 nm excitation and UV-Vis absorbance spectra of Nbz in DMSO. (E) ¹H-NMR spectra of HTMP after irradiated by NIR (980 nm, 1 W/cm²) over time. (F) The cumulative TDOX release profile of iPUTDN at different conditions (mean±SD, n=3). The green arrows indicate the time points of 980 nm laser on, and the black arrows mean laser off.

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TDOX was synthesized according to the reference.^[

Firstly, the cell uptake and intracellular distribution of TDOX were observed by confocal laser scanning microscopy (CLSM), using red emitted fluorescence of DOX at 630 nm when excited at 510 nm. After incubating H69AR cells with free DOX, TDOX, iPUTDNs with or without NIR at 2 h, the most intense red fluorescence was found in the TDOX and iPUTDN+NIR groups compared to other groups (Fig. 2A and Fig. S4 in ESI). After 24 h incubation, iPUTDN+NIR group retained the most intense fluorescence in four groups. Given that the H69AR cells are DOX resistant, it is reasonable to see the meager intracellular amount of free DOX at both time points. In stark contrast, iPUTDN plus NIR irradiation displayed a relatively slower but effective cell uptake because of the cascade behavior of PEG detachment and release of TDOX from particles.

Fig 2  Intracellular transport of iPUTDN in H69AR DOX resistance tumor cells. (A) Confocal microscopic images of H69AR cells incubated with DOX, TDOX and iPUTDN with or without NIR (980 nm, 1 W/cm², 2 min) for 2 h or 24 h. The nuclei were stained with Hoechst 33342 (blue). Scale bar=50 μm. (B) Colocalization images of TDOX (red) and mitochondria (green) in H69AR cells incubated with TDOX, iPUTDN or iPUTDN with NIR (980 nm, 1 W/cm², 2 min) for 24 h. Pearson’s correlation is 0.775 (TDOX), 0.726 (iPUTDN), 0.898 (iPUTDN with NIR). Pearson’s correlation coefficient for colocalization was obtained by colocalization analysis of NIS-Elements Viewer. Scale bar=10 µm.

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Next, the endocytosis pathway of iPUTDN was investigated in the H69AR cells by CLSM. Chlorpromazine (CPZ) can restrain clathrin to suppress clathrin-dependent endocytosis.^[

Mitochondrion targeting of DOX in the H69AR cell line under different conditions was examined by CLSM imaging. DOX shows a red fluorescence while mitochondria were labeled as green color. The co-localization of DOX with mitochondria displays merged yellow dots. As shown in Fig. 2(B), after incubating the cells with TDOX, iPUNTD, and iPUTDN + NIR for 24 h, iPUTDN + NIR group shows markedly more yellow dots than the other two groups and gives the highest Pearson’s correlation coefficients from the analysis by ImageJ. In line with the results of cell uptake and changes in particle physicochemical properties, it verified that an effective translocation of DOX towards mitochondria was attributed to a cascade transform of nanoparticles, including the PEG peel-off by NIR, iRGD exposure promoting cell endocytosis of the particles, accelerated particle dissociation to release TDOX and TPP-inducing mitochondrial enrichment.

To acquire an insightful understanding of mitochondria-targeted chemotherapy, we explored the mitochondrial membrane potential and ATP level variation in H69AR cells. The test of mitochondrial membrane potential was performed by using the mitochondria permeable dye JC-1. The decline in the ratio of JC-1 emitted red/green fluorescence means mitochondrial depolarization.^[

Fig 3  The mechanism of iPUTDN to overcome DOX resistance in H69AR tumor cells. (A) Confocal images of mitochondrial membrane potential were examined by JC-1 as a probe. Scale bar=20 μm. (B) The JC-1 red/green fluorescence ratio in Fig. 3(A) is measured by ImageJ. (C) The effect of various samples on intracellular ATP was shown. The concentration of TDOX was maintained as 2 µg/mL. (D) Cell viability of free TDOX, iPUTDN and iPUTDN with NIR (980 nm, 2 W/cm², 30 s) in H69AR cells (mean±SD, n=6). *, P<0.05; **,P<0.01; ***,P<0.001.

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In vitro cell inhibition efficacy of DOX, TDOX, iPUTDN, or iPUTDN plus NIR against H69AR cells was investigated by CCK-8 assay (Fig. 3D and Fig. S6 in ESI). The IC50 value of DOX in H69AR cells was 20.3 µg/mL, whereas the IC50 value of TDOX was reduced to 8.4 µg/mL. iPUTDN plus NIR irradiation exhibits a slightly higher cell inhibition ability (IC50=~7.5 µg/mL) in comparison to TDOX. The result implied that the cascade stimuli-responsive change of iPUTDN with the assistance of NIR ensured the rapid cell endocytosis and efficacious inhibition of drug efflux, which outperformed the other carriers.^[

To examine the distribution of nanoparticles in vivo, we established a BALB/c nude murine model bearing a subcutaneous H69AR tumor. Cy5 was modified onto iPUN or iPUTDN via EDC/NHS reaction between N-hydroxysuccinimide group on Cy5-NHS and amino groups on iPUN or iPUTDN. The fluorescent nanoparticles were conducive to being monitored in vivo. After intravenous injections of different samples, the live fluorescence of Cy5 in mice was monitored at 1, 6, and 24 h by Lumina III in vivo imaging system. According to Fig. 4(A), Cy5 fluorescence gradually elevated over time in mice injected with iPUN-Cy5. Upon NIR irradiation, iPUN-Cy5 exhibits a superior accumulation in tumors of mice by comparing with that of the no radiation group (Fig. S7A in ESI). Moreover, we next examined DOX distribution in mice by visualizing its ex vivo fluorescence in excised tissues of mice handled with TDOX, iPUTDN, iPUTDN + NIR after administration for 24 h. Similarly, DOX delivered by iPUTDN plus NIR shows more efficient tumor accumulation than the other two samples (Fig. 4B and Fig. S7B in ESI).

Fig 4  Biodistribution of iPUN in vivo. (A) Fluorescence imaging of live mice upon 1. free Cy5; 2. iPUN-Cy5; 3. iPUN-Cy5 with NIR (1 W/cm², 5 min). The white circles emphasize the areas of tumors; (B) Fluorescence imaging of excised tumors and organs of mice upon free TDOX, iPUTDN, and iPUTDN with NIR (1 W/cm², 5 min); (C) Analysis of Cy5-labeled nanoparticles distribution in tumors. The immunostaining of tumor blood vessels was performed with CD31 antibody. Scale bar=50 μm. For magnified images. Scale bar=10 μm. (D) The mean distance between Cy5 labeled nanoparticles and the blood vessels from (C); (E) Cy5 fluorescence of CD31-stained tumor tissues was quantified by ImageJ (mean±SD, n=4).

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To examine the vascular penetration of nanoparticles, the immunostaining of tumor tissues at 24 h after injection was performed with fluorescently remarked CD31 antibody to visualize blood vessels. According to Fig. 4(C), the extravasation of considerably more Cy5-labeled nanoparticles was conducted from the tumor blood vessels by NIR than no NIR regulation because of NIR-caused activation of iRGD and down-sizing of nanoparticles by NIR. There was even less Cy5 fluorescence around blood vessels in the free Cy5 group (Fig. 4E) because of the low cumulation of free Cy5 in tumors. Moreover, the average distance between nanoparticles and the tumor blood vessels was the longest in iPUN-Cy5/NIR group (Fig. 4D), suggesting improved penetration capability.

The biocompatibility of the nanoparticles in vivo was investigated in the subcutaneous H69AR tumor-bearing BALB/c nude mice. The i.v. injection of iPUN at the highest dosage (250 μg/mL) did not cause hemolysis of the red blood cells (Fig. S8 in ESI). After administering the samples and NIR irradiation, mice’s body weights kept similar between these groups for three weeks (Fig. 5A). There was no obvious variation in routine blood biochemical tests (Fig. S9 in ESI). These results attested that the administration of iPUTDN with or without NIR irradiation was biocompatible in mice. In the meantime, the analyses for hematoxylin and eosin (H&E) staining of healthy organs after administration of iPUTDN revealed no severe adverse effects on the liver and kidney (Fig. S10 in ESI).

Fig 5  Inhibitory effect of iPUTDN in DOX resistant H69AR tumor in BALB/c nude mice. (A) Average body weights of mice upon indicated treatments in three weeks (mean±SD, n=5). (B) Variations in tumor volumes during the treatment cycle (mean±SD, n=5). (C) Tumor dissection photographs from the mice through intravenous administration. I: PBS + NIR, II: TDOX + NIR, III: iPUTDN, IV: iPUTDN + NIR. (D) Average tumor weights in (C) (mean±SD, n=5). **, P<0.01; ***,P<0.001. (E) Representative pictures of H&E and Ki-67 staining of the excised tumors from each group after the treatments, respectively. Scale bar=20 μm.

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The examination of the in vivo anticancer therapeutic efficacy of the nanoparticles against a drug-resistant subcutaneous H69AR tumor in BALB/c nude mice was performed. By comparing with the controls, iPUTDN plus NIR irradiation potently restrained the tumor growth in mice for three weeks (Fig. 5B). The significant reduction in tumor weights after the treatments also proved the antitumor effects of iPUTDN with NIR irradiation (Figs. 5C and 5D). In addition, hematoxylin and eosin (H&E) staining of the tumor section at the end of the treatment indicated that the tumor cells exhibited less proliferation and the apparent propensity of apoptosis with the treatment of iPUTDN + NIR as compared to the other treatment groups (Fig. 5E). Similar results were found in the Ki-67 staining (Fig. 5E).

Subcellular organelle-targeted drug delivery has been reported as a promising way to improve disease diagnosis and therapy.^[

Numerous endogenous stimuli-responsive mitochondria-targeted nanosystems have been developed,^[