MnO2-Based Nanoplatform Serves as Drug ... - ACS Publications

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Letter

A MnO2-based Nanoplatform Serves as Drug Vehicle and MRI Contrast Agent for Cancer Theranostics Mei Zhang, Lei Xing, Hengte Ke, Yu-Jing He, Peng-Fei Cui, Yong Zhu, Ge Jiang, Jian-Bin Qiao, Na Lu, Huabing Chen, and Hulin Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15247 • Publication Date (Web): 14 Mar 2017 Downloaded from http://pubs.acs.org on March 16, 2017

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ACS Applied Materials & Interfaces

A MnO2-based Nanoplatform Serves as Drug Vehicle and MRI Contrast Agent for Cancer Theranostics Mei Zhang,+abc Lei Xing,+abc Hengte Ke,+ de Yu-Jing He,a Peng-Fei Cui,a Yong Zhu,a Ge Jiang,f Jian-Bin Qiao,a Na Lu,a Huabing Chen,*de and Hu-Lin Jiang,*abc

a.

State Key Laboratory of Natural Medicines, China Pharmaceutical University,

Nanjing 210009, China b.

Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University,

Nanjing 210009, China c.

Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China

Pharmaceutical University, Nanjing 210009, China d.

Jiangsu

Key

Laboratory

of

Translational

Research

and

Therapy

for

Neuro-Psycho-Diseases, and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu 215123, China e.

School for Radiological & Interdisciplinary Sciences (RAD-X), Collaborative

Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, and School of Radiation Medicine and Protection, Soochow University, Suzhou, Jiangsu 215123, China f. College of Life Sciences and Biotechnology, Dalian university, Dalian, China + These authors contributed equally to this work.

ACS Paragon Plus Environment


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*

Correspondence to:

Professor H. L. Jiang, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China. Tel: +86-25-83271027; Fax: +86-25-83271027; E-mail: [email protected] Professor H. B. Chen, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu 215123, China Tel: +86-512-65883370; Fax: +86-512-65883370; E-mail: [email protected]


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Abstract: Multidrug resistance (MDR) greatly impedes the therapeutic efficacy of chemotherapeutic agents. Overexpression of ATP-binding cassette (ABC) transporters, such as P-gp, on the surface of tumor cells is a major mechanism in MDR. In this study, we fabricated manganese dioxide (MnO2)/doxorubicin (DOX)-loaded albumin nanoparticles (BMDN) for magnetic resonance imaging and reversing MDR in resistant tumor. BMDN facilitated the delivery of DOX into MDR tumor cells through their MDR reversal effects including enhanced cellular uptake, reduced drug efflux, and decreased hypoxic tumor microenvironment. BMDN also acted as an effective MRI contrast agent, thereby causing good in vitro and in vivo T1-weighted imaging. Keywords: multifunction nanoparticles, manganese dioxide, multidrug resistance, doxorubicin, theranostics



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Chemotherapy has long been applied in the treatment of various types of cancers. However, the emergence of multidrug resistance (MDR) severely hinders the efficacy of chemotherapy. P-glycoprotein (P-gp), a membrane bound ATP-binding cassette (ABC) transporter encoded by MDR1, was one of the major mechanisms entangled in MDR.1,

2

Currently, the nanocarriers have emerged as a potential approach to

overcome MDR in tumors by either evading P-gp or inhibiting its drug efflux effect. Albumin has widely been explored as a candidate drug vehicle in the construction of nanocarrier owing to its excellent biocompatibility and low immunogenicity.3-5 Besides, the interaction between albumin and albumin receptors overexpressed on cancer cells such as albumin receptor gp60 or secreted protein acidic and rich in cysteine (SPARC), could be utilized as a targeting strategy to achieve preferential tumor accumulation and cellular uptake of drugs, thereby accounting for enhanced therapeutic effect and minimized adverse side effect.6-8 On the other hand, hypoxia is a major feature in most solid tumors owing to the severe imbalance between the supply and consumption of oxygen. Studies have demonstrated that hypoxic tumor microenvironment played a dominant role in the invasiveness, metastasis, and drug resistance of tumors.9-11 Cellular adaptive responses to hypoxia are mainly modulated through hypoxia inducible factor 1 (HIF-1), an oxygen-regulated transcript factor. Activated HIF-1 generally binds to hypoxic response elements, and thus leads to the transcription of various gene including multidrug resistance gene 1 (MDR1), thereby promoting the malignancy of tumors. In addition, HIF-1 also induces anaerobic metabolism in tumor cells for


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preserving energy, which leads to augments in the intracellular levels of lactic acid and reactive oxygen species (ROS), resulting in the aggravation of tumor and treat failures.12,13 Therefore, interfering with HIF-1 might act as a potential therapeutic strategy to overcome MDR in tumor. Metallic oxides, especially manganese dioxide, have been reported to modulate hypoxic tumor microenvironment and subsequently attenuate the expression of HIF-1α.14,15 Moreover, Mn-based contrast agents (CAs) have also shown prospective potential in magnetic resonance imaging (MRI) as alternatives of gadolinium (Gd)-based CAs for better biocompatibility. However, Mn-based CAs frequently encountered both poor delivery stability of Mn2+ and insufficient imaging performances.16-19 Therefore, it is highly desired to explore an effective Mn-based CA for achieving both regulation of hypoxia tumor microenvironment and improved MRI performance. In this study, as shown in Figure 1, we fabricated BSA-MnO2-DOX nanoparticles (BMDN) as a theranostic agent for cancer imaging and simultaneous chemotherapy of resistant tumor. BMDN facilitated drug internalization and inhibited the efflux effect of P-gp, both of which contributed to the remarkable reversal effect towards drug resistance in tumor cells. The sensitivity of BMDN towards mild acid promoted the responsive release of Mn2+, which resulted in enhanced T1-weighted imaging in tumor sites. We also investigated the anti-tumor effect and imaging performance of BMDN both in vitro and in vivo, validating that BMDN could serve as a potential nanoplatform for cancer theranostics. MnO2 colloid was synthesized by a simple redox reaction between KMnO4 and



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excessive Na2S2O3. The as-prepared MnO2 colloid in pH 7.2 water manifested suitable particle size (45.5 ± 0.3 nm) and zeta potential (- 37.19 ± 1.53mV) for intracellular drug delivery, yet it would agglomerate irreversibly with the existence of ions. Therefore, we coated BSA on the surface of MnO2 colloid to improve its stability for further application. As in Figure 2A, the protein corona of BSA on the surface of MnO2 helped to prevent agglomeration through steric repulsive force and electrostatic repulsive force. DOX was loaded onto BSA-MnO2 via electrostatic interaction and coordinate bond between Mn elements, anthraquinone and -NH2 in DOX,20 and the desolvation crosslinking method was subsequently utilized to prepare bovine albumin nanoparticles (BSA-MnO2-DOX nanoparticles, BMDNs). The successful loading of DOX and MnO2 in BMDN was confirmed by UV-vis spectroscopy and XPS respectively, with the maximum absorbance peak of DOX observed at 479 nm and peaks assigned to Mn (2p1/2) and Mn (2p3/2) at the binding energy of 653.9 and 642.7 eV (Figure 2B and 2C), respectively.21,22 The as-prepared BMDN exhibited favorable drug envelope efficiency (73.95 %), and their drug loading capacity was 7.40 % (each 100 mg of BMDN contains about 7.4 mg DOX). TEM imaging shows that BMDN had the uniform morphology and residual uncrosslinked BSA, the average diameter of BMDN was 77.9 ± 4.8 nm (Figure 2A), which was smaller than the hydrodynamic particle size (165.5 ± 3.0 nm) measured by DLS (Figure 2D), owing to the hydration layer of BSA. As shown in Figure 2E, BMDN still maintained good stability in RPMI 1640 medium with negligible change in hydrodynamic size, and the zeta potential increases from about -30 to -15 mV due


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to the buffer solution in RPMI 1640 medium. Additionally, BMDN exhibited a slight change in hydrodynamic size after 3 weeks in serum-free RPMI 1640 medium (data not shown), suggesting that BMDN manifested satisfactory stability for drug delivery. As shown in Figure 2F, the cumulative release of DOX was significantly increased in mild acidic buffer solution (pH = 5.0, 84.62%), when compared with that at neutral buffer solution (28.03 %) within 72 h. Different pH brought reversible changes to the surface charge and structure of BSA, and the crosslinking bonds among BMDN could be disintegrated in mild acid, thus a pH-responsive release of DOX could be observed when BMDN was treated with releasing medium at different pH.23 The pH-responsive release of DOX from BMDN could also contributed to the alleviation of side effects in normal tissues to the utmost extent, which presented a potential approach for the safe and efficient therapy of cancer. Results

of

MTT

assay

showed

that

BMDN

exhibited

time-

and

concentration-dependent proliferative inhibition effect against all investigated cell lines. BMDN manifested notable drug resistance reversal effect in MCF-7/ADR cells, with obviously higher cytotoxicity than free DOX at all tested concentrations (Figure 3A and 3C). The IC50 of BMDN toward MCF-7/ADR cells for 48 h was 36.82 µg/ml, which is comparable to [email protected] In consistence, BMDN induced remarkable apoptosis in MCF-7/ADR cells, while equivalent free DOX showed far less influence as elucidated in Figure 3E, 3F and 3G. The morphological features of BMDN contributed to the bypass of P-gp efflux pump, resulting in its excellent MDR reversal effect. Though the cytotoxicity of BMDN towards MCF-7 cells during 24 h was



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slightly milder than free DOX, yet its 48 h cytotoxicity was rather stronger than free DOX (Figure 3B and 3D). The lower cytotoxicity of BMDN during 24 h may be attributed to the time course during which DOX was released from BMDN and barely excretion of free DOX in MCF-7 cell with expressing less P-gp than MCF-7/ADR.24 Yet the prolonged incubation led to the full release of DOX, which consequently conduced to an enhanced cytotoxicity. Figure S1 also showed that BMDN and the drug-free BSA-MnO2 exhibited milder cytotoxicity in HUVEC (normal cells) than free DOX, which validated the prospect of BMDN to serve as a safe theranostic platform. Both genomic abnormalities and lack of oxygen contributed to MDR in hypoxic tumor microenvironment, which was mainly caused by proliferation alteration, DNA damage inhibition, cellular metabolism reprogramming, and enhanced drug efflux.25 As shown in Figure 3H and 3I, MCF-7 cells exhibited attenuated drug sensitivity towards free DOX at all treated concentrations with exposure to hypoxia for 24 h, which was consistent with the former studies. However, the proliferation inhibition effect of BMDN barely changed, suggesting that the hypoxia-induced MDR was effectively reversed by BMDN. Therefore, BMDN were capable of reversing MDR in tumor cells, whether it was induced by hypoxia or not, further confirming the potential of BMDN as an effective drug delivery platform in the treatment of malignant tumor. The mechanism behind the MDR reversal effect of BMDN was investigated through different methods. First, we investigated the cellular uptake pathways of


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BMDN in MCF-7 and MCF-7/ADR cell lines. BMDN entered cells through an energy-dependent process, which was proved by the inhibited endocytosis at 4 °C incubation (Figure 4A and 4B). The existence of albumin remarkably attenuated the uptake of BMDN, suggesting that albumin receptors dominantly participated in the internalization of BMDN. Albumin receptors overexpressed on cancer cells played a key role in the facilitated cellular internalization of BMDN through albumin receptor-mediated endocytosis. Noteworthy, the inhibition effect of BSA and HSA was more evident in MCF-7/ADR cells, further suggesting the prominent role of receptor-mediated internalization during the cellular uptake of BMDN in MCF-7/ADR cells. In addition, CMZ also inhibited the cellular uptake of BMDN, while

amiloride

and

nystatin

showed

no

influence,

demonstrating

that

clathrin-mediated endocytosis, instead of caveolae-mediated endocytosis, was mainly involved in the cellular uptake of BMDN. Facilitating drug accumulation was one of the main strategies to reverse drug resistance in tumor cells,26 thus the drug accumulation in MCF-7/ADR cells treated with BMDN or free DOX was compared intuitively by the fluorescent signal intensity of DOX under a CLSM at different time points. As in Figure 4C, the fluorescence of drug in cells treated with BMDN increased as the incubation time prolonged. The fluorescence was first located in endosomes (1 h), and then spread to the whole cytoplasm, representing the release of DOX (2-4 h). The nuclear entry of DOX was clearly observed after incubation for 6 h. As the incubation time prolonged (8-12 h), the morphologic changes of nuclear and cells were subsequently observed, indicating



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that DOX in BMDN finally reached its functional site, damaged nuclear, and then killed tumor cells. In contrast, there was scarcely any entry of DOX or morphological changes in cells treated with free DOX at all time points (Figure S3). This comparison indicated that BMDN could remarkably facilitate the cellular uptake of DOX. Apart from ameliorated cellular uptake, inhibition of drug efflux mediated by P-gp also led to higher drug accumulation.27,28 As elucidated in Figure 4D, in the BMDN-treated group, a large majority of DOX was still remained in cells after efflux assay, yet the cells in free DOX-treated group showed drastically decreased intracellular drug accumulation after endocytosis for the same time period. The structure features of BMDN enabled it to evade the efflux effect of P-gp, resulting in extended retention time as well as increased accumulation of DOX in cells. As a result, the drug resistance reversal effect of BMDN was achieved by both promoted cellular uptake and reduced efflux, thereby contributing to better therapeutic efficacy. To further investigate the molecular mechanism of the reversal effect of BMDN towards hypoxia-induced drug resistance in cells, we compared the expression levels of HIF-1α and MDR1 in MCF-7/ADR cells after treated with BMDN and free DOX under hypoxia. As elucidated in Figure 4E, the expression of MDR1 and HIF-1α was decreased in the cells treated with BMDN compared with control group. HIF-1α is the active subunit of HIF-1, a key mediator in the cellular adaptive response to hypoxia. Active HIF-1 translocates into nuclear and binds with hypoxia responsive element, promoting the expression of various downstream genes including MDR1.29 The reduction of HIF-1α in cells treated with BMDN initially led to the decrease of MDR1,


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which resulted in the inhibited drug efflux and subsequently facilitated cellular DOX accumulation. The DOX accumulated in cells then exerted its function and damaged nuclear, thus interfering HIF-1’s nuclear translocation and its promoting effect on the expression of MDR1. As a result, a drastically decrease of MDR1 was observed in cells treated with BMDN, according to the reversal effect toward hypoxia-induced MDR. On the contrary, the expression of HIF-1α and MDR1 in cells treated with free DOX was scarcely different from control group, which was consistent with the insufficient efficacy of free DOX. Therefore, the superiority of BMDN over free DOX for reversing hypoxia-induced MDR was validated at molecular level, further suggesting that BMDN hold the perspective to serve as a valid drug delivery system in tumor therapy. The imaging performance of BMDN was assessed using a 1.5 T clinical MRI scanner. As shown in Figure 5A and 5B, both of BMDN and BSA-MnO2 exhibited Mn concentration-dependent T1-weighted MRI performance, with BMDN possessing a much higher r1 value (11.794 mM-1s-1) than BSA-MnO2 (4.762 mM-1s-1). The enhanced relaxation of BMDN was possibly attributed to the large surface area to mass ratio of BMDN, where the chances of Mn exposed on the surface as well as the accessibility of surrounding water to Mn were both facilitated. The in vitro MRI capacity of BMDN was then evaluated in MCF-7/ADR cells (Figure 5C). In consistence with results in Figure 5A and 5B, images of cells treated with BMDN were brighter than cells treated with BSA-MnO2. However, the contrast in the images of BMDN was slightly lower than BMDN dispersion, resulting from the ions leakage



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caused by the cytotoxicity of BMDN. Moreover, the in vivo MRI capacity of BMDN was investigated in MCF-7/ADR tumor xenografted mice model. As elucidated in Figure 5D and 5E, the contrast enhancement led by BMDN and BSA-MnO2 was observed gradually over time, with a better contrast than the in vitro T1-weighted images. This enhanced contrast in the in vivo images originated from the full exposure of Mn, resulting from the acidic microenvironment as well as the abundant proteases in tumor tissue.30 Noticeably, the contrast enhancement of BMDN remained a longer imaging window (4 h) in tumor than that of BSA-MnO2 (2 h), as a result of the slow release profile of albumin nanoparticles. Consequently, these results confirmed that BMDN could act well as a theranostic nanoplatform in tumor treatment. The in vivo therapeutic efficacy of BMDN and free DOX was then investigated on mice with xenografted MCF-7/ADR tumors. As elucidated in Figure 5F, best inhibition effect towards tumor growth was observed in BMDN-treated mice, with scarcely any increase in tumor volume during experimental period. By contrast, the size of tumors in DOX-treated mice doubled at the end of the treatment. Tumors in mice treated with BSA-MnO2 showed no difference with control group, further indicating the excellent biocompatibility of this delivery platform. At the end of treating period, the size of tumor in mice treated with BMDN was the smallest among the four treating groups. This result was in consistence with TUNEL staining (Figure 5G), where massive apoptotic tumor cells were observed in BMDN-treated group. In contrast, mice treated with DOX exhibited far less apoptosis in tumor tissue. These results from in vivo study validated that BMDN possessed the potential as a safe and


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efficient delivery platform for the theranostics of malignant tumors. In this study, we established a MnO2-based nanoplatform for cancer theranostics. The BMDN possessed sustained release profiles of DOX and Mn2+, which respectively contributed to improved therapeutic efficacy, accompanying with better MRI performance in the mild acidic microenvironment of tumor. Through the enhancement of cellular uptake, inhibition of cellular efflux, as well as the intervention of HIF-1α, BMDN reversed basal drug resistance as well as hypoxia-induced drug resistance. Moreover, BMDN exhibited excellent T1-weighted imaging performance both in vitro and in vivo. Therefore, BMDN was validated to hold the potential as a theranostic nanoplatform in tumor therapy. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Cytotoxicity of BMDNs in HUVEC cells (Figure S1), cytotoxicity of BMDNs under hypoxia with ROS scavenger (Figure S2) and cellular uptake of DOX in MCF-7/ADR in different time periods (Figure S3). AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] Notes



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The authors have declared no conflict of interest.

ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (81573369 and 51473109), and the Natural Science Foundation of Jiangsu Province (Grant No. BK20140659). This work was also supported by the Project Program of State Key Laboratory

of

Natural

Medicines,

China

Pharmaceutical

University

(No.

SKLNMZZJQ201601) and National High Technology Research and Development Program of China (863 Program, No. 2015AA020314). This work was also supported by the Outstanding Youth Fund of Jiangsu Province of China (Grant No. BK20160031).


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Figure 1. Schematic illustration of BMDN as an MDR-reversal vector and T1-weighted contrast agent for MRI and simultaneous chemotherapy on resistant tumor .



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Figure 2. Physicochemical characterization of BMDNs. (A) Morphology of BSA-MnO2 and BMDNs under TEM. (B) UV-vis absorption spectra of BMDNs, free DOX and BSA-MnO2. (C) X-ray photoelectron spectroscopy of BMDNs, peaks assigned to Mn (2p1/2) and Mn (2p3/2) were detected at the binding energy of 653.9 and 642.7 eV. (D) Size distribution of BMDNs. (E) Stability of BMDNs upon dilution by RPMI 1640 medium. (F) Cumulative release of DOX from BMDNs under different pH condition (pH = 5.0 and 7.4) in 72 h.


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Figure 3. Cytotoxicity of BMDNs towards MCF-7/ADR (A, 24 h; C, 48 h), and MCF-7 (B, 24 h; D, 48 h) under normoxia；Apoptosis induced by (F) BMDNs or (G) DOX in MCF-7/ADR cells, (E) was blank control. Cytotoxicity of BMDNs under hypoxia towards MCF-7/ADR (H), and MCF-7 (I).



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Figure 4. Intracellular uptake of BMDNs in (A) MCF-7/ADR cells, and (B) MCF-7 cells with various endocytosis inhibitors (DOX: 60 µg/ml). (C) Cellular uptake of BMDNs in MCF-7/ADR during different time periods (DOX: 20 µg/ml). (D) Accumulation of DOX in MCF-7/ADR cells after treated with BMDNs and free DOX for different time periods (DOX: 20 µg/ml). (E) Expression of MDR1 and HIF-α in MCF-7/ADR cells after treated with BMDNs under hypoxia for 12 h (DOX: 60 µg/ml).


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Figure 5. (A) Plot of T1-1 versus Mn concentration of BSA-MnO2 and BMDNs dispersion, the slope of the regression equation was T1 relaxation rate. (B) T1-weighted results obtained from BSA-MnO2 and BMDNs dispersion at different Mn concentrations. (C) T1-weighted results obtained from BSA-MnO2 and BMDNs in MCF-7/ADR cells at different Mn concentrations. (D) T1-weighted results obtained from BMDNs and (E) BSA-MnO2 on MCF-7/ADR tumor-bearing nude mice before (Di, Ei) and after (Dii-Dvi, Eii-Evi) intratumoral administration of BMDNs and BSA-MnO2 respectively. The T1-weighted images were obtained at 0, 5, 30, 60, 120, and 240 min post-injection. (F) Tumor growth inhibition effect of BMDN. Treatment started after the tumor volume reached 100 mm3. The volume of tumor was measure after each administration. (G) Representative TUNEL staining in continuous sections from tumors in different treating groups.



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Table of Contents Graphic and Synopsis


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MnO2-Based Nanoplatform Serves as Drug ... - ACS Publications

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