Versatile Octapod-Shaped Hollow Porous Manganese(II) Oxide

Jul 9, 2019 - Multifunctional nanoplatforms featuring promising properties including excellent loading efficiency, real-time monitoring, and improved ...
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Letter Cite This: Nano Lett. XXXX, XXX, XXX−XXX

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Versatile Octapod-Shaped Hollow Porous Manganese(II) Oxide Nanoplatform for Real-Time Visualization of Cargo Delivery Ruixue Wei,† Xuanqing Gong,† Hongyu Lin,† Ke Zhang,‡ Ao Li,† Kun Liu,† Hong Shan,‡ Xiaoyuan Chen,§ and Jinhao Gao*,†

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State Key Laboratory of Physical Chemistry of Solid Surfaces, The MOE Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China ‡ Guangdong Provincial Engineering Research Center of Molecular Imaging, Center for Interventional Medicine, The Fifth Affiliated Hospital, Sun Yat-Sen University, Zhuhai 519000, China § Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, United States S Supporting Information *

ABSTRACT: Multifunctional nanoplatforms featuring promising properties including excellent loading efficiency, real-time monitoring, and improved cargo bioavailability and bioselectivity are in great demand by the biomedical research community. During the development of such nanoplatforms, stimuli-responsive nanoparticles (NPs) as a smart nanoplatform have recently received extensive attention. Herein, we report small-sized octapod-shaped hollow porous manganese(II) oxide (HPMO) NPs as a stimuli-responsive T1activatable nanoplatform for tumor-specific cargo delivery and realtime monitoring. The HPMO NPs functionalized by zwitterionic dopamine sulfonate (ZDS) can act as a versatile platform to load organic dyes or chemotherapeutic drugs with high loading efficiency. The obtained Cargo@HPMO would decompose into paramagnetic Mn2+ ions and subsequently release cargoes in mild acidic conditions, especially in tumor microenvironment and lysosome. The released Mn2+ can enhance T1 magnetic resonance signal for real-time monitoring of the cargo delivery in vivo. This octapodshaped Cargo@HPMO can act as a smart and versatile nanoplatform with pH-responsive multimodal imaging and site-specific drug delivery for the development of accurate diagnosis and effective therapy for cancer. KEYWORDS: hollow porous MnO, pH-responsive, activatable T1 contrast agent, cancer therapy

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water-soluble Mn2+ ions in response to tumor microenvironment and enhance T1 magnetic resonance imaging (MRI) signal.15,28−34 Moreover, the resulting Mn2+ could be rapidly excreted by kidneys without potential long-term toxicity. Among them, hollow manganese oxide nanomaterials have attracted widespread attention because of their large cavities and excellent loading efficiency.6,35−37 However, the largesized (≥100 nm) manganese oxide NPs tend to accumulate in mononuclear phagocyte system (MPS), which undermines the advantage of targeting tumor through enhanced permeability and retention (EPR) effect.38,39 For these reasons, hollow manganese oxide NPs with small size, large cavity, and proper surface coating can act as a desired drug delivery nanoplatform. Herein, we report small-sized octapod-shaped hollow porous manganese(II) oxide (HPMO) NPs as an intelligent drug

he accurate diagnosis and prompt treatment of cancer are of great significance in the clinic.1−5 Therefore, many efforts have been dedicated to develop intelligent nanomaterials with combined diagnostic and therapeutic functions in response to tumor microenvironment (low pH values,6−9 hypoxia,10 or high glutathione levels11) over the past decades. 12−20 Inorganic-based (e.g., Au, Fe 3 O 4 , and FePt)21−26 and organic-based (e.g., human serum albumin, liposome, and poly(lactic-co-glycolic acid))10,27 nanomaterials were frequently used as nanocarriers to deliver therapeutic agents in biomedical applications. However, the long-term toxicity of inorganic-based nanoparticles (NPs) and the inability of organic-based NPs in imaging significantly restrict their utilization for accurate and safe diagnosis of diseases. Therefore, the development of multifunctional nanocarriers with integration of biocompatible character and imaging property is particularly critical. Manganese-based oxide NPs are frequently employed as bioresponsive nanoprobes because they could dissociate into © XXXX American Chemical Society

Received: May 8, 2019 Revised: June 24, 2019 Published: July 9, 2019 A

DOI: 10.1021/acs.nanolett.9b01900 Nano Lett. XXXX, XXX, XXX−XXX

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The transmission electron microscope (TEM) images (Figure 2a) of MnO NPs revealed the octapod-shaped morphology and narrow size distribution (about 35 nm in diameter). The X-ray diffraction (XRD) patterns and selected area electron diffraction (SAED) patterns indicated that the asprepared product was of a typical MnO structure (JCPDS NO. 07−0230, Supporting Information Figure S1b,c). The TEM images (Figure 2b) demonstrated that HPMO NPs retained high uniformity of the original size and octapod shape with unique hollow structure, which was not yet reported. The wall thickness was around 3 nm. The XRD and SAED data suggested the amorphous nature of HPMO NPs (Supporting Information Figure S1d,e). As shown in X-ray photoelectron spectroscopy (XPS) spectra (Supporting Information Figure S2), the peaks of 640.0 eV (Mn 2p3/2) and 652.2 eV (Mn 2p1/2) indicated the +2 oxidation state of Mn in HPMO NPs. The small-angle X-ray scattering (SAXS, Figure 2c) revealed that the surface-area-to-volume ratio and the pore size of HPMO NPs were approximately 0.86 nm−1 and 1.03 nm, respectively. We also performed the N2 adsorption analysis and found that the specific surface area (SBET) and pore diameter of HPMO NPs were 157 m2 g−1 and 2.5 nm, respectively (Supporting Information Figure S3). The SAXS and BET data revealed the hollow and porous structures of HPMO NPs, which allow it to be a potential candidate for cargo carriers. Dynamic light scattering (DLS) and UV−vis spectroscopy were used to detect the changes after cargo loading. DLS analysis revealed that the hydrodynamic diameter (HD) of HPMO NPs and Cargo@HPMO were ∼39 and ∼41 nm, respectively (Supporting Information Figure S4a). The UV− vis spectra and optical photographs of Cargo@HPMO (Supporting Information Figure S4b−d) suggesting the successful loading of cargoes. The loading capacity was evaluated by UV−vis spectroscopy (Supporting Information Figure S5). The amount of DOX, CPT, or Rh123 that 1 mg of HPMO could load was about 0.73, 0.03, or 0.18 mg, respectively, and the loading efficiency was proportional to the water solubility of the cargoes. Manganese oxide is known to be stable under neutral and basic conditions, but ruptures and decomposes into Mn2+ ions in mild acidic environment.43 Hence, we monitored the pHinduced degradation of HPMO NPs by TEM images (Figure 2d). After incubation in PBS buffer, the morphology of HPMO NPs showed time-dependent decomposition behavior at pH 5.4, but did not change at pH 7.4. Considering that the breakup of HPMO NPs is closely associated with the change of UV−vis absorbance, we also used UV−vis spectrometry to assess the decomposition behavior of HPMO NPs (Figure 2e and Supporting Information Figure S6). The change in UV− vis absorbance revealed the pH-responsive behavior of HPMO NPs. TEM images and UV−vis results suggested that HPMO NPs could be degraded in response to mild acidic environment. The relaxivity measurements of HPMO NPs at different pH values were carried out on a 0.5 T MRI scanner (Figure 2f and Supporting Information Figure S7). The results showed r1 values of 0.8, 5.2, and 8.3 mM−1 s−1 for pH 7.4, 6.5, and 5.4, respectively. The r1 value increased as the pH value decreased. However, there was no appreciable distinction in r1 value of Mn2+ at different pH values (about 8.7 mM−1 s−1 at pH 7.4 and about 8.9 mM−1 s−1 at pH 5.4, Supporting Information Figure S7). The amorphous nature of HPMO NPs (Supporting Information Figure S1d,e) suggests that there are few magnetic ions (i.e., Mn 2+ ) with unsaturated

delivery nanoplatform (Cargo@HPMO), which can realize activated T1-weighted MRI and real-time monitoring of cargo release in response to mild acidic environment. The octapodshaped HPMO NPs were synthesized with a two-step method (Supporting Information Figure S1a) and functionalized with zwitterionic dopamine sulfonate (ZDS) molecules to enhance the water solubility and reduce the nonabsorption of proteins in physiological environment.40,41 Chemotherapeutic drugs or organic dyes (e.g., doxorubicin (DOX), camptothecin (CPT), or Rhodamine123 (Rh123)) were further loaded into the HPMO nanocarriers. The released paramagnetic Mn2+ ions under low pH conditions could serve as an efficient T1 MRI contrast agent for real-time monitoring of cargo release in vivo. Furthermore, the fluorescence of released cargoes could further visualize the location of the cargoes (Figure 1). As a proof of

Figure 1. Scheme of pH-responsive small-sized octapod-shaped HPMO NPs as a T1-activatable nanocarrier with real-time monitoring of cargo delivery. Cargo@HPMO can release Mn2+ ions and cargoes simultaneously for T1-activatable MR imaging, drug delivery, and/or fluorescence imaging in response to low pH in tumor microenvironment and lysosomes.

concept, DOX@HPMO can realize both T1 MR and fluorescence signals turning “ON” in tumor regions for realtime monitoring of drug delivery. This responsive multimodal imaging is of great significance and impendency for accurate diagnosis of tumor. Moreover, compared to free DOX, DOX@ HPMO showed efficient tumor uptake through the EPR effect and remarkable ability to inhibit tumor growth with less side effects in vivo because of the reduced accumulation of anticancer drugs in normal tissues. This nanoplatform with pH-responsive multimodal imaging and site-specific chemotherapeutic drug release would find tremendous biomedical applications in accurate diagnosis and prompt chemotherapy for cancer. Results and Discussion. Characterization and pHActivated T1-Signal-Enhancement Behavior of HPMO. The octapod-shaped Cargo@HPMO nanoplatform was prepared as follows (Supporting Information Figure S1a): At the first stage, octapod-shaped MnO NPs were synthesized by thermal decomposition of manganese oleate complex in 1-octadecene in the presence of oleylamine and sodium oleate. The octapodshaped HPMO NPs were obtained by an acid etching method in 90% trioctylphosphine oxide (TOPO) under a nitrogen atmosphere.42 Subsequently, ZDS was used to coat HPMO NPs to enhance the solubility in water.40,41 Lastly, DOX, CPT, or Rh123 was loaded by vigorous stirring with HPMO@ZDS in phosphate-buffered saline (PBS buffer, pH 7.4) for 48 h. B

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Figure 2. Characterizations and pH-responsive degradation of small-sized octapod-shaped HPMO NPs. TEM images of octapod-shaped (a) MnO (inset, the geometric model of octapod) and (b) HPMO NPs (inset, the high-magnification TEM). (c) SAXS curve of HPMO NPs. (d) TEM images of HPMO NPs after incubation in PBS at different pH values (7.4 or 5.4). Scale bar: 100 nm for all images. (e) UV−vis absorbance at 380 nm of HPMO NPs incubated at different pH values (7.4, 6.5, or 5.4). (f) Relaxivity measurements of HPMO NPs at different pH values (7.4, 6.5, or 5.4), n = 3/group. (g) T1-weighted phantom images of HPMO NPs with increased Mn concentrations at different pH values (7.4, 6.5, or 5.4).

Figure 3. Cargo release and pH-activatable T1-weighted MR and fluorescence analysis. (a) Fluorescence spectra of free DOX and DOX@HPMO at different pH values. Inset: fluorescence images of free DOX and DOX@HPMO (DOX, 400 μg/mL) with excitation at 500 nm. (b) Fluorescence spectra of free CPT and CPT@HPMO at different pH values. Inset: optical photographs of free CPT and CPT@HPMO (CPT, 10 μg/mL) with excitation at 365 nm by a UV lamp. (c) Fluorescence spectra of free Rh123 and Rh123@HPMO at different pH values. Inset: fluorescence images of free Rh123 and Rh123@HPMO (Rh123, 10 μg/mL) with excitation at 500 nm. Kinetics of cargo release for (d) DOX@HPMO, (e) CPT@ HPMO, and (f) Rh123@HPMO at different pH values, n = 3/group. Insets in (d−f), T1-weighted phantom images of corresponding Cargo@ HPMO before incubation, at 5 and 24 h after incubation at pH 5.4.

HPMO NPs had brighter signals at pH 5.4 than at pH 6.5 than at pH 7.4 with Mn at the same concentration. These phenomena could be attributed to the decomposition of

coordination for water on the particle surface, which may result in extremely low r1 value of HPMO NPs at neutral solution. T1-weighted phantom images (Figure 2g) suggested that C

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Nano Letters HPMO NPs into Mn2+ ions in mild acidic environment. The generated Mn2+ ion has five unpaired 3d electrons and is a typical T1 contrast agent.6,44 These results indicated that HPMO NPs could act as an excellent candidate of pHresponsive T1-activatable nanocarriers. pH-Activated T1 MR and Fluorescence Signals Enhancement of Cargo@HPMO. To achieve accurate diagnosis in the clinic, multimodal imaging is usually employed. With the property of HPMO NPs that the T1 signal could be turned “ON” in acidic condition, T1-weighted MRI can serve as one imaging approach to monitor drug delivery. Fluorescence imaging can offer another imaging means since the signal of fluorescent probes is annihilated after loading due to the aggregation-caused quenching (ACQ)45−47 and restored when the nanocarrier collapses and frees the cargoes. Therefore, the combination of HPMO nanocarriers with fluorescent probes can undergo a turning “ON” process for both T1 MR and fluorescence imaging, which facilitates accurate imaging and diagnosis in vivo. The fluorescence spectra and imaging of Cargo@HPMO indicated that the fluorescence of cargo was significantly quenched after loading cargo inside HPMO NPs but recovered at pH 5.4 (Figure 3a−c). Cargo is in an aggregated state in the HPMO cavity, which suppress the fluorescence. The decomposition of HPMO NPs causes cargo to recover its free state and restore its fluorescence under acidic condition. The process of fluorescence quenching and recovery can further facilitate visual monitoring of the decomposition of Cargo@HPMO and the release of cargo. The pH-triggered drug release behaviors of Cargo@HPMO in various physiological environments (including blood pH 7.4, tumor microenvironment pH 6.5, and intracellular pH 5.4) were studied by UV−vis spectroscopy (Figure 3d−f). The release percentage of DOX was less than 5% at pH 7.4 over 24 h. However, the DOX release rates increased to 48% (pH 6.5) and 92% (pH 5.4), owing to the pH-triggered decomposition of HPMO NPs. The release behaviors of CPT@HPMO and Rh123@HPMO were similar to that of DOX@HPMO, i.e., the release percentage increased with decreasing pH values. Moreover, we also observed the T1-activatable phantom imaging during cargo release process (insets of Figure 3d−f), indicating that Cargo@HPMO decomposed into Mn2+ ions and released cargoes at low pH value. In other words, cargo release and fluorescence recovery could only occur after the decomposition of Cargo@HPMO, which further confirmed that cargoes were loaded into HPMO NPs rather than physically absorbed on the surface. The pH-induced T1 signal and fluorescence recovery behavior of Cargo@HPMO allows monitoring the release of cargoes in cells. The T1 MR signal of SMMC-7721 cells slightly increased after 2 h incubation, indicating that HPMO NPs slowly decomposed into Mn2+ ions, which led to the T1 signal enhancement (Figure 4a,b). The highest T1 signal was achieved at 4 h after incubation, suggesting that HPMO NPs was completely decomposed at this time point. Accordingly, we then conducted the confocal fluorescence imaging of SMMC-7721 cells after treatment with Rh123@HPMO (10 μM for Rh123) or DOX@HPMO (10 μM for DOX) for different incubation times to monitor the intracellular drug delivery (Figure 4c and Supporting Information Figure S8). The confocal fluorescence images showed that the fluorescence signal of cargoes was low after 2 h incubation, suggesting that the cargoes were still aggregated in HPMO NPs with fluorescence quenched. After 4 h, the fluorescence signal

Figure 4. Both T1 MRI and fluorescence signals are turned “ON” after cellular uptake. (a) T1-weighted MR images of SMMC-7721 cells after incubation with HPMO NPs (100 μM Mn) for different times. (b) Quantitative MR signals analysis for (a), n = 3/group. (c) Confocal fluorescence images of SMMC-7721 cells treated with Rh123@ HPMO (10 μM Rh123) for different times. Hoechst 33342 and LysoTracker Green were used to stain cell nuclei (blue) and lysosome (green), respectively.

increased, indicating the significant release of cargoes. The well overlap of cargo fluorescence signal and LysoTracker indicated that Cargo@HPMO were successfully internalized by endocytosis and proceeded to decomposition in lysosomes under acid conditions. We conducted flow cytometry to monitor the CPT release and cell response (Supporting Information Figure S9) and found that the fluorescence enhancement of CPT and cell apoptosis increased over time, indicating that pH-responsive cargo release could subsequently induce cell apoptosis. To further evaluate the pH-responsive behavior in cells, we used DOX@HPMO as an example and conducted the confocal fluorescence imaging in different culturing environments (serum starvation or inhibition). Serum starvation causes a decrease in lysosomal pH,48 whereas the lysosomal pH increases when the cells are incubated with inhibitors, such as chloroquine (CQ).49 The DOX fluorescence inside cell lysosomes was significantly enhanced during serum starvation but became invisible in the presence of CQ (Figure 5a and Supporting Information Figure S10). These results demonstrated that low pH value could dramatically improve the release rate of DOX, which was consistent with our aforementioned observations. However, the red fluorescence of SMMC-7721 cells after treated with free DOX indicated that free DOX could directly enter into the cell nucleus within 4 h (Figure 5a), probably by ATP-dependent transport.50 The endocytosis-mediated intracellular drug delivery of Cargo@ HPMO is beneficial for increasing the intracellular release of the anticancer drugs, thereby boosting the lethality to cancer cells. Based on these findings, it could be concluded that DOX@HPMO enter into the cells by endocytosis and release DOX in response to acidic environments. T1-weighted MR images of SMMC-7721 cells after 4 h incubation with DOX@HPMO (100 μM for Mn, 10 μM for DOX) under different conditions were also acquired on a 0.5 T MRI scanner (Figure 5b,c). The T1 signal of the cells during serum starvation was significantly enhanced because of the high released Mn2+ concentration. In contrast, a slightly enhanced T1 signal of the cells was observed in the presence of CQ, indicating the low released Mn2+ concentration. However, the total amount of Mn element in cells measured by inductively coupled plasma mass spectrometry (ICP-MS) D

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Figure 5. Activatable T1 MR and fluorescence imaging to monitor pH-responsive drug release in cells. (a) Confocal fluorescence images of SMMC7721 cells treated with DOX@HPMO, free DOX (100 μM for Mn, 10 μM for DOX) for 4 h. Serum starvation and CQ treatments represent strong acidic and low acidic lysosomes, respectively. Hoechst 33342 and LysoTracker Green were used to stain cell nuclei (blue) and lysosome (green), respectively. (b) T1-weighted MR images of SMMC-7721 cells after 4 h incubation with DOX@HPMO (100 μM Mn, 10 μM for DOX). (c) Quantitative MR signals analysis for (b), n = 3/group. (d) Total amount of Mn in SMMC-7721 cells in (b) measured by ICP-MS, n = 3/group. (e) Cell viability of SMMC-7721 cells treated with free DOX, and DOX@HPMO under different culturing conditions for 24 h, n = 5/group. (f) Comparison of IC50 values in (e), n = 5/group. The error bars represent the standard deviations (SD).

Figure 6. In vivo activatable T1 MR imaging and fluorescence imaging. (a) T1-weighted MR imaging of H22 tumor-bearing mice before and after local injection of DOX@HPMO (1 mg Mn per kg, 1 mg DOX per kg) into tumor and muscle tissues at 7.0 T. Yellow and white arrows indicate the tumor and muscle tissues, respectively. (b) Related SNR in (a), n = 3/group. (c) T1-weighted MR images of mice before and post i.v. injection of DOX@HPMO (at a dose of 2 mg Mn per kg, 2 mg DOX per kg). The yellow arrows, white dotted lines, and red circles indicate the location of tumor, liver, and bladder, respectively. (d) Quantitative analysis of SNR in (c), n = 3/group. (e) Ex vivo fluorescence imaging of major organs and tumors from the mice before and after i.v. injection of DOX@HPMO (at a dose of 4 mg Mn per kg, 4 mg DOX per kg). (f) Fluorescence intensity analysis of tumors in (e), n = 3/group. The error bars represent the standard deviations (SD).

were similar (Figure 5d). The apparent contradiction between T1 signals and ICP-MS results could be attributed to that the ICP-MS detects the total concentration of Mn in cells, while enhanced T1 signal is mainly caused by the released free Mn2+

ions. Therefore, in the case of cells undergoing serum starvation, most of Mn is released as free Mn2+ ions due to the low pH in lysosomes, accounting for the enhanced T1 signal; while a significant amount of HPMO is not E

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Figure 7. In vivo cancer therapy study using HPMO NPs as drug carriers. (a) Tumor growth curves and (c) body weight change curves of the mice during treatment with DOX@HPMO, DOX, HPMO NPs, or PBS. Arrows indicate the day for treatment (n = 3/group). (b) Average weights of tumors collected from the mice after 20 d treatment (n = 3/group). p values in (a) and (b) were calculated by Tukey’s posthoc test (***p < 0.001, **p < 0.01). (d) H&E histology images of the tumors and liver from mice after 20 d treatment. Black arrows indicate the typical necrotic cells in the tumors. Yellow arrows indicate the typical damaged cells in the liver. (e) Quantitative analysis of biochemistry indexes of the mice, including alanine aminotransferase (ALT), alkaline phosphatase (ALP), indirect Bilirubin (IBil), and total bilirubin (TBil), after 20 d treatment (n = 3/ group). The mauve region indicates the reference safety range. The error bars represent the standard deviations (SD).

In Vivo MR Imaging and Drug Release Monitoring in Mice. We employed BALB/c mice bearing H22 subcutaneous tumors to study the pH-responsive behavior of DOX@HPMO in vivo on a 7 T MRI scanner. We first locally injected DOX@ HPMO (1 mg Mn per kg, 1 mg DOX per kg) into the tumor and muscle tissues to quickly assess the feasibility (Figure 6a,b). As expected, the T1 signal in tumor region became brighter after 2 h, but no significant signal change in the muscle region, indicating the pH-responsive T1 contrast enhancement ability of DOX@HPMO for tumor MR imaging. MR imaging was subsequently performed on mice after intravenous (i.v.) injection with DOX@HPMO at a dose of 2 mg Mn per kg (Figure 6c and Supporting Information Figure S12). At 2 h post injection, the tumor, liver, and bladder showed slight T1 signal enhancement. At 4 h post injection, the T1 signal in tumor region increased and reached a peak level, revealing the high accumulation and significant decomposition of DOX@ HPMO in the tumor region. The dark T1 signal of tumor at 5 d post-injection suggested that Mn2+ ions have been metabolized. The bladder also exhibited time-dependent T1 signal enhancement and reached a maximum between 4 and 6 h postinjection, indicating the excretion of Mn2+ ions through urine. The bladder was still slightly brighter at 5 d post-injection than that before injection, which might be ascribed to the slow decomposition of DOX@HPMO and metabolism of Mn2+ ions in other organs. The minor change in the signal of liver suggested that DOX@HPMO did not significantly accumulate in liver probably due to the zwitterionic surface coating.37,38

decomposed due to the relatively high pH in lysosomes in the case of cells treated with CQ. These results further confirmed the ability of DOX@HPMO for intracellular pH-triggered T1weighted MR and fluorescence imaging. Considering the outstanding anticancer and fluorescent properties of DOX as well as the high loading efficiency, we took DOX@HPMO as an example for further study. Before assessing the cytotoxicity of DOX@HPMO, we first evaluated the cytotoxicity of HPMO NPs and Mn2+ ions to SMMC-7721 cells with methyl thiazolyl tetrazolium (MTT) assays and showed that they had no appreciable cytotoxicity after 72 h incubation even at a concentration of Mn up to 200 μM (Supporting Information Figure S11). After 24 h incubation, we observed a significant drop in viability for cells treated with DOX and DOX@HPMO as the concentration of DOX increased (Figure 5e). The corresponding half maximal inhibitory concentrations (IC50, Figure 5f) indicated that DOX@HPMO (IC50 = 1.52 ± 0.41 μM for DOX) showed higher cytotoxicity than free DOX (IC50 = 2.23 ± 0.46 μM for DOX). Considering that the pH-dependent DOX release might affect the cytotoxicity of DOX@HPMO, the cytotoxicity of DOX@HPMO to SMMC-7721 cells undergoing serum starvation and in the presence of CQ were also evaluated, and the IC50 values after 24 h incubation were 0.78 ± 0.14 and 4.65 ± 0.91 μM with respect to DOX, respectively (Figure 5e,f), indicating that DOX@HPMO showed the highest cytotoxicity under serum starvation conditions. F

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7a,b and Supporting Information Figure S16). The weight of mice treated with free DOX started to decrease at Day 6, while the weights of mice treated with DOX@HPMO, HPMO NPs, and PBS maintained consistent during the treatment period (Figure 7c). These results revealed that DOX@HPMO showed minimal side effects during treatment, while free DOX manifested great adverse effects in mice. Chemotherapy is widely used as a cancer treatment in clinic. However, the biodistribution of chemotherapeutic drugs (such as arsenic trioxide, DOX, and CPT) is not ideal in the body. One reason is that only 2−5% of the drugs reach the tumor site, and most of them are accumulated in normal tissues.51,52 Another issue is that free drugs could be pushed back into the blood relatively easily, causing the poor retention in tumor.53,54 Prolonging the blood circulation and reducing the uptake of the MPS are effective means to increase drug targeting to tumors.55 The leaky tumor vasculatures and the impaired lymphatic drainage of the tumor microenvironment are used to achieve passive accumulation of nanomedicine by the EPR effects.38,56 HPMO nanocarriers could prolong the blood circulation time of DOX and facilitate drug delivery through passive targeting, which is consistent with the greater lethality to tumor and less side effects of DOX@HPMO compared to free DOX. The overexposure to free Mn ions could induce deleterious effects on the central nervous system and cause parkinsonism-like syndrome,57,58 and the aqueous Mn ions have been found to impart neurotoxicity with a lethal dose (LD50) of 0.3 mmol/kg (16.5 mg/kg, intravenous injection) for mice.59 The injection dose in this work was 2 mg Mn/kg, which is much lower than the LD50 values. Safety assessment of the chemotherapy is of pivotal concern for further biomedical applications. We conducted hematoxylin and eosin (H&E) staining and main biochemistry index analysis to evaluate the systemic toxicity in the animals. The majority of tumor cells were severely damaged in the group of mice treated with DOX@HPMO after 20 d (Figure 7d). The tumor cells were slightly damaged after treatment with free DOX. The tumor cells treated with HPMO NPs or PBS showed no significant damage. All major organs did not exhibit appreciable microscopic lesions after treatment with DOX@ HPMO, HPMO NPs, and PBS (Supporting Information Figure S17). However, the liver of the mice treated with free DOX showed significant damage (Figure 7d). The biochemistry indexes (Figure 7e) were consistent with H&E staining results, suggesting the severe liver damage in free DOX treated group. These results demonstrated that DOX@HPMO could kill tumor cells effectively with relieved side effects in vivo. Conclusions. In summary, we have developed a novel octapod-shaped HPMO nanoplatform with efficient cargo loading and pH-activated T1 MR imaging. As a nanocarrier, HPMO NPs could decompose into Mn2+ ions and release cargoes in response to mild acid environment. The activated T1 MR and fluorescence signals could be used to visually monitor the delivery of cargoes. For example, DOX@HPMO passively targeted to tumor via the EPR effect, and efficiently released paramagnetic Mn2+ ions and DOX in responsive to acidic environments. The T1 MR signal changes enabled real-time monitoring of drug release in vivo. Particularly, compared to free DOX, DOX@HPMO reduced the side effects of DOX while effectively inhibited the tumor growth. This strategy that accomplishes real-time multimodal monitoring of drug release via pH-responsive T1 MR and fluorescence imaging is of significant importance and impendency for accurate diagnosis

We then calculated the signal-to-noise ratios (SNRs) of MR imaging to quantify the contrast enhancement post-injection (Figure 6d). The SNRpost/SNRpre values of tumor, liver, and bladder at 4 h post-injection were 1.9, 1.4, and 1.6, respectively, which confirmed that the tumor region showed the highest T1 contrast enhancement. The result indicated that DOX@HPMO could effectively target tumor via the EPR effect and release Mn2+ ions that specifically enhance T1 signal in tumor. Importantly, most of the generated Mn2+ ions could be cleared through urine within 5 d. It is noteworthy that the T1 signal increase in tumor also reflected the release of DOX, suggesting that DOX@HPMO could achieve real-time monitoring of drug release in tumor. Since the fluorescence of DOX coincides with the autofluorescence of mice (mainly from fur and blood), we performed ex vivo fluorescence imaging (Figure 6e,f) of mice to further monitor the DOX release after intravenous injection of DOX@HPMO (at dose of 4 mg DOX per kg). At 4 h post injection of DOX@HPMO, the enhanced fluorescent signal of tumor and the almost invisible fluorescent signals of other major organs demonstrated the efficient delivery and accumulation of DOX@ HPMO in tumor. The decreased fluorescent signal of tumor at 6 h after injection suggested that DOX had been partially metabolized or cleared. For biodistribution analysis of Mn in major organs and tumor (Supporting Information Figure S13), the ICP-MS results showed high tumor uptake of DOX@ HPMO at 4 h post-injection, which is consistent with the results of in vivo MR imaging. It is noted that the accumulation of DOX@HPMO in major organs (e.g., liver and spleen) at 4 h post-injection was apparent. However, there was almost no fluorescence signals in these organs (Figure 6e), which suggests that DOX@HPMO did not decompose to release DOX in these organs because of the neutral environment. As evidenced by the MR and fluorescence imaging, DOX@ HPMO could accumulate in tumor and release Mn2+ ions and DOX in situ, and simultaneously “turn on” and amplify the T1 MR and fluorescence signal. Therefore, the encapsulation of DOX into HPMO nanocarriers could not only increase the enrichment of DOX in tumor tissues but also accomplish visual monitoring of DOX delivery in vivo. We also investigated the metabolism of DOX via the fluorescence analysis of mice urine (Figure S14). The deep orange color and strong fluorescence signal of the mouse urine at 1 h after injection of free DOX indicated the fast renal clearance of free DOX. However, the renal clearance of DOX for mice injected with DOX@HPMO was not observed until 4 h after injection. This observation could be attributed to the fact that the HPMO nanocarriers could effectively avoid the fast clearance and therefore increase the blood circulation time, which enhances the passive targeting efficiency (Supporting Information Figure S15). In Vivo Cancer Therapy. Encouraged by the efficacy of DOX@HPMO in cells, we further evaluated the potency in inhibiting tumor growth in vivo. For in vivo experiments, to minimize the interference of potential immune response between different living subjects, we chose the murine hepatoma cell line H22 tumors, which have also been widely used as a liver tumor mouse model for therapeutic study. After intravenous injection of DOX@HPMO, free DOX, HPMO NPs, or PBS into mice (at a dose of 2 mg DOX per kg or 2 mg Mn per kg), both DOX@HPMO and free DOX inhibited the growth of solid tumor, while the tumors of mice treated with HPMO NPs and PBS grew rapidly during treatment (Figure G

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Nano Letters

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and precise therapy in biomedical applications. We believe that this versatile drug-carrier nanoplatform with controlled release and T1 signal amplification holds great potential for considerable applications in cancer management.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.9b01900. Materials and methods, TEM images, XRD, UV−vis, MTT, flow cytometry, confocal imaging, and H&E staining (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (86)-592-2180278. ORCID

Jinhao Gao: 0000-0003-3215-7013 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (21771148, 21602186, 21521004, and 81430041), IRT_17R66, the Natural Science Foundation of Fujian Province of China (2018J01011), and Fundamental Research Funds for the Central Universities (20720170020, 20720170088, and 20720180033).

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REFERENCES

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DOI: 10.1021/acs.nanolett.9b01900 Nano Lett. XXXX, XXX, XXX−XXX