Amorphous Manganese

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Unconventional Preparation to Polymer/Amorphous Manganese Oxide-Based Biodegradable Nanohybrids for Low Premature Release and Acid/Glutathione-Activated Magnetic Resonance Imaging Xubo Zhao, Yudian Qiu, Yalei Miao, Zhongyi Liu, Wenjing Yang, and Hongwei Hou ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00307 • Publication Date (Web): 23 May 2018 Downloaded from http://pubs.acs.org on May 23, 2018

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ACS Applied Nano Materials

Unconventional Preparation to Polymer/Amorphous Manganese Oxide-Based Biodegradable Nanohybrids for Low Premature Release and Acid/Glutathione-Activated Magnetic Resonance Imaging

Xubo Zhao*†, Yudian Qiu†, Yalei Miao†, Zhongyi Liu*†‡, Wenjing Yang§, Hongwei Hou † †

College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, 450001, China

‡

Zhengzhou Sino-Crystal Diamond Company Limited, Zhengzhou, 450001, China

§

Department of Anesthesiology, The First Affiliated Hospital, Zhengzhou University, Zhengzhou 450002, China.

* E-mail address: [email protected]

[email protected]

Abstract: A smart poly(methacrylic acid-co-N,N-bis(acryloyl)cystamine)/DOX/MnO2-2/polyethylene glycol theranostic nanohybrids (PMAABACy/DOX/MnO2-2/PEG TNs) were rationally fabricated using in-situ generation of amorphous MnO2 by taking advantage of the spatial confinement effect of PMAABACy nanohydrogels, and its PEGylation

was

accomplished

through

Mn-N

coordinate

bonding.

The

amorphous 2+

PMAABACy/DOX/MnO2-2/PEG TNs was synthesized through the chelation between Mn

MnO2

of

ions and carboxyl

groups of PMAA chains, which served as a gatekeeper to prevent the premature leakage of DOX during blood circulation. In the presence of intracellular acidic glutathione (GSH), the release of Mn2+ ions from amorphous MnO2 as a dual T1/T2 contrast agents endowed the nanohybrids with enhanced acid/GSH-activated magnetic resonance imaging (MRI). Meantime, the site-specific release of chemotherapeutic drug (DOX) was also realized because of the disintegration of both amorphous MnO2 and PMAABACy in response to the biological endogenous stimulus such as increased GSH level and slightly acidic pH in tumor cells. The newly as-synthesized nanohybrids exhibited some excellent characteristics: prevented premature release, enhanced the stability under physiological conditions, excellent T1/T2 MRI performances, remarkable biodegradability, and efficiently site-specific release of DOX. Our findings indicated that such polymer/amorphous manganese oxide-based biodegradable nanohybrids can facilitate the development of the drug delivery system (DDS) with a capacity of low premature release for real-time MRI-guided cancer therapy.

Key words: PMAA-based nanohydrogels; Amorphous MnO2; Biomineralization; Premature leakage; Reduction-sensitive property; cancer therapy

1.

Introduction

Until now, the treatments of cancer therapy have faced a lot of challenges as the ecosystem and environment being increasingly destroyed. As far as we know, surgery, radiotherapy, and chemotherapy serving as conventional cancer treatment options have become more and more important in cancer treatment. Recently, these conventional cancer treatments are struggling toward ever-increasing cancer patient. Particularly for chemotherapy, further

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development to this treatment remains a major challenge because of the low therapy efficiency, high recurrence rate, and systemic side effects of free anticancer drugs.1,2 With the development of nanotechnology, the DDS based on nanomaterials have attracted extensive attention for effectively delivering chemotherapy agent and avoiding the severe side effects in healthy tissues because of their outstanding performance and unique structure.3-5 In particular, through controlled release of anticancer drugs, the DDS have efficiently improved the chemotherapy efficiency to inhibit the solid tumor. Meanwhile, most of these reported multifunctional DDS have been explored as theranostic agents, implying that they possess therapeutic and diagnostic functions for cancer treatment. These multifunctional DDSs had same following functions: the solid tumor could be killed by loaded cargos and monitored by real-time contrast-enhanced imaging, which is of great significance for positive cancer therapy.6,7 Among numerous DDSs, PMAA-based platform has shown a great advantage as a smart and biocompatible DDS because of its abundant carboxyl groups and hydrophilic properties.8,9 Recently, by integration of both diagnostic and therapeutic functions into “all-in-one” multifunctional DDS has already became a powerful route for convenient real-time early detection of the therapeutic effects during the cancer therapy.3,10 In the early 1990's, molecular image has been attracted extensive attention because of non-invasively visualizes and understanding the mechanisms in biological tissues at a molecular level. As for cancer therapy, through the molecular imaging techniques, assessing metabolic changes, determining bio-distribution of drugs, and detecting progress of solid tumor now become more and more feasible in real time.11,12 With the rapid development of nanotechnology, representative contrast agents including photoacoustic imaging, ultrasound, optical microscopy, single photon emission computed tomography, positron emission tomography, X-ray computed tomography, and MRI have been explored for a wide range of imaging modalities.12,13 In particularly, some representative MRI modalities have been integrated into the DDS because of these following three main merits: high resolution of soft tissues, no tissue penetrating limit, and diversity of contrast agents.12 Drezek’s group designed a Fe3O4/Ag complexes for application of photothermal therapy and magnetic resonance imaging. They revealed the Fe3O4/Ag complexes were able to enhance the T2 magnetic resonance imaging and debulk tumors, further improve survival with PTT.14 Meade et al. synthesized a multimeric MRI-optical contrast agent based on macrocyclic Gd(III) chelates for multimodal imaging. Notably, this multimeric MRI-optical contrast agent possessed water solubility and high relaxivity.15 Meanwhile, Liu and coworkers prepared a polydisulfide MRI contrast agent by grafting diethylenetriaminepentaacetic to disulfide-containing poly(amido amine)s-graft-poly(ethylene glycol) followed by Gd(III) complexation. Owing to the introduction of Gd(III), the polydisulfide MRI contrast agent exhibited a high r1 value and served as the better MRI imaging with fewer side effects.16 Among the numerous contrast agents, MnO2-mediate MRI played a crucial role in traditional MRI modalities.17,18 Furthermore, the tumourigenesis can generate a apparently different intracellular microenvironments, which include oxidative medium in mitochondria with a high concentration of H2O2, reductive medium owing to the presence of high level GSH or cysteine in the cytoplasm and endosomes, slightly acidic microenvironments inside lysosomes and endosomes (pH 4.5–6.5), and various metabolites and biomolecules within cytoplasm.19-25 On the basis of these obvious difference, MnO2-based materials with ultrasensitivity in response to the reductive and slightly acidic medium are disintegrated and further release Mn2+ ions; therefore, the accessibility of paramagnetic centers [Mn2+ ions] to surrounding water molecules can be enhanced, leading enhanced T1 and T2-MRI performances.26 Most importantly, the as-released Mn2+ ions from the disintegration of MnO2-based materials can


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be easily metabolized by the kidneys. Owing to the remarkable biocompatibility, the MnO2-based materials would evade the intractable degradation issue of traditional inorganic such as well-known carbon, gold, and silica-based nanomaterials.7,27 Shi and coworkers reported a platform with ultrasensitive pH-triggered T1-weighted MRI and anticancer-drug releasing based on MnO2 nanosheets for intelligent cancer diagnosis and therapy. Notably, the fast disintegration of 2D MnO2 nanosheets in a mildly acidic environment can substantially enhance the T1-MRI performance.7 It is well-known that MnO2-based materials could be reduced to Mn2+ ions in the presence of high intracellular GSH level. Tan’s group synthesized a DNAzyme–MnO2 nanosystem via the physisorption of nucleobases on MnO2 nanosheets for efficient cancer therapy. Release Mn2+ ions upon intracellular GSH served as an activated MRI contrast agent in real time.28 Meanwhile, traditional MnO2 materials are crystal structure with long-range order to hinder the release rapidly of Mn2+ ions in response to endogenous stimuli of tumor microenvironment.29 We decide to fabricate the amorphous MnO2 materials, which are expected to be rapidly ionized in the reductive and slightly acidic medium for on-demand Mn2+ ions release and enable a subsequent localized enhanced acid/GSH-activated MRI for specific cancer therapy with visualization. These amorphous alloys and metals, which can be defined as metallic glasses (MGs) with a disordered atomic structure, have aroused wide concern because of their remarkable physicochemical properties in biomedical application.30 Discontentedly, the development of amorphous alloys and metals was restricted owing to especial synthesis conditions, such as the very high cooling rate (usually higher than 106 K s-1) to freeze the molten metal, and then hinder the nucleation of crystalline phases.31 Furthermore, the premature leakage of chemotherapeutic drug from DDS significantly reduces the therapy efficiency and causes the systemic side effects, thereby this disadvantageous leakage of chemotherapeutic drug should be evaded during the process of cancer therapy. To overcome these limitations, herein, we have designed and prepared a smart PMAABACy/DOX/MnO2-2/PEG TNs based on amorphous MnO2 and PMAABACy for avoiding leakage of the cargo under physiological conditions, simultaneously enhancing the intracellular pH/GSH dual-sensitive MRI, and realizing the site-specific release of DOX at tumor intracellular microenvironment for cancer therapy. As presented in Scheme 1, the PMAABACy nanohydrogels with disulfide cross-linked were prepared by a mild one-pot distillation-precipitation polymerization, which possessed abundant carboxyl group to load

chemotherapeutic

drug

(DOX)

and

chelate

the

Mn2+

ions.

Subsequently,

the

functional

PMAABACy/DOX/MnO2 containing amorphous MnO2 was crafted through a biomineralization process, which would induce the transformation of metal ions into amorphous metal oxide under mild temperature. Finally, through the PEGylation, the PMAABACy/DOX/MnO2-2/PEG nanohybrids were crafted. As shown in Scheme 1, Mn2+ ions released from amorphous MnO2 as a contrast endowed the DDS with the enhanced GSH and pH-activated MRI. This amorphization for MnO2 is expected to improve the biodegradability and magnetism-related properties of DDS because of the disordered atomic arrangement. Meantime, the chemotherapeutic

drug

(DOX)

is

also

rapidly

released

from

the

disintegration

of

functional

PMAABACy/DOX/MnO2-2/PEG TNs in response to both the high intracellular GSH concentrations and slightly acidic pH in tumor cells. This research presents a reasonable design to incorporate different functionalities into the single PMAABACy/DOX/MnO2-2/PEG TNs, which is promising for future cancer therapy.



NH2-PEG

Mn2+

DOX

OH-+O2

pH 7.4

Uptaking

Tumor Cell I MR 2+

M

n

Che mot hera py DO X

+

PMAA PMAABACy

GSH

H

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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PMAABACy/DOX

PMAABACy/DOX/MnO2

PMAABACy/DOX/MnO2-2/PEG

Scheme 1. Schematic illustration of the preparation of pH/GSH dual-sensitive PMAABACy/DOX/MnO2-2/PEG TNs, and the activation mechanism of PMAABACy/DOX/MnO2-2/PEG TNs by GSH and pH triggers, MRI mediated by Mn2+ ions, and DOX release.

2.

Experimental

2.1. Materials and Reagents. 2,2′-Azobisisobutyronitrile (AIBN), manganese chloride (MnCl2), analytical grade acetonitrile and acryloyl chloride were obtained from Tianjin Chemical Co. Ltd. Furthermore, the AIBN was recrystallized from methanol. Methacrylic acid (MAA) was provided by Aldrich and used after reduced pressure distillation. GSH was obtained from J & K Chemical Ltd. Doxorubicin hydrochloride (DOX) was purchased from Beijing Huafeng United Technology Co. Ltd. Monofunctional PEG (NH2-PEG1000) was obtained from Beijing Kaizheng Biological Engineering Development Co. Ltd. Throughout the testing process, ultrapure water was used.

2.2. Preparation of the reduction-responsive PMAABACy nanohydrogels. The previous procedure was utilized to synthesize the disulfide crosslinker BACy.32 The typical PMAABACy nanohydrogels were prepared via a facile and mild distillation precipitation copolymerization. In this copolymerization, the MAA was selected as a monomer, the BACy as a crosslinker, and the AIBN as an initiator, respectively. A typical procedure was as follows: MAA (405.1 mg) and BACy (131.6 mg) were dissolved in acetonitrile (80 mL) in a dried 100 mL of single round bottom flask. Subsequently, AIBN (10.5 mg) was added in the above mixtures. Then, the mixtures were heated to boiling within 30 min. After 30 mL of acetonitrile being distilled off within 2 h, the reaction was stopped. Then the suspension was centrifuged to obtain the


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reduction-responsive PMAABACy nanohydrogels. And the crude products of reduction-responsive PMAABACy nanohydrogels were washed by abundant deionized water to remove acetonitrile. Lastly, the resulting products were lyophilized and stored at 4 °C.

2.3. Drug Loading Drug-loading to the PMAABACy nanohydrogels were conducted according to the previous work.33 100.0 mg of PMAABACy nanohydrogels were dispersed in 50.0 mL of 0.6 mg mL−1 DOX solution at pH 7.4 under slightly magnetic stirring in the darkness for 24 h. Then, the dispersion was centrifuged and washed several times with abundant water to remove excess or unabsorbed chemotherapy drug DOX. In the end, the DOX-loaded PMAABACy nanohydrogels were lyophilized and stored at 4 °C.

2.4. Fabrication of PMAABACy/DOX/MnO2-2/PEG TNs Using the Mn2+ ions reduced from MnO2 as contrast endows the nanosystem with enhanced acid/GSH-activated MRI. To engineer the smart nanosystem, the functional PMAABACy/DOX/MnO2-2 TNs were fabricated through a biomineralization process, which would induce the transformation of metal ions into amorphous metal oxide within PMAABACy nanohydrogels under mild temperature. Briefly, 112 mg of DOX-loaded PMAABACy were dispersed in 80 mL of water under magnetic stirring with the aid of slight ultrasound. Then, the 10 mL of MnCl2 solution (188 mg) was slowly added under vigorous magnetic stirring. After the addition of MnCl2 solution, the pH value of mixtures was adjusted by NaOH (1.0 M) to pH 11. After reaction under vigorous stirring at 37 °C for 24 h, the functional PMAABACy/DOX/MnO2-2 TNs were washed by abundant deionized water to remove excess Mn2+. Furthermore, the PMAABACy/DOX/MnO2-2 TNs were lyophilized and stored at 4 °C. Meanwhile, the feeding ratios of MnCl2 (376 mg) were utilized to control the performance of PMAABACy/DOX/MnO2 complexes under similar above procedures to obtain PMAABACy/DOX/MnO2-1 TNs. Lastly, PMAABACy/MnO2-2 complexes were crafted via similar above methods to evaluate the related biocompatibility. For PEGylation, the 20 mg of PMAABACy/DOX/MnO2-2 or PMAABACy/MnO2-2 complexes were dispersed in 100 mL of water under the magnetic stirring by ultrasound treatment. Then, the 20 mg of NH2-PEG was added under vigorous magnetic stirring with the aid of slight ultrasound for 8 h, which was repeated for three times to insure the efficient PEGylation.

2.5. Triggered Release. Drug release from the stimuli-responsive PMAABACy/DOX/MnO2-2/PEG TNs were performed according to the previous work.33 Specifically, The typical condition (pH 5.0 in the presence of 10 mM GSH) was chosen to evaluate the DOX release from the PMAABACy/DOX/MnO2-2/PEG TNs simulating the tumor microenvironment. To

compare

with

the

tumor

microenvironment,

the

drug

release

performance

of

the

PMAABACy/DOX/MnO2-2/PEG TNs were investigated in PBS (pH 7.4 in the presence of 10 µM GSH) to simulate the normal physiological condition of human body, with the same procedure as the pH 5.0.



Page 6 of 21

2.6. Cell Toxicity Assays. The biocompatibility of the PMAABACy/MnO2-2/PEG TNs was evaluated via the general MTT assay using HeLa cells. Furthermore, the PMAABACy/DOX/MnO2-2/PEG TNs were utilized to investigate the ability of inhibition growth to HeLa cell. In the typical steps, the HeLa cells were transferred into 96-well plates at densities of 5×103 cells per well for 24 h. Subsequently, different concentrations of the PMAABACy/MnO2-2/PEG nanoparticles, free DOX, and PMAABACy/DOX/MnO2-2/PEG TNs were added and incubated for 24 h. Then, the cell viability of HeLa cells were determined by using the MTT assay.

2.7. Cellular Uptake of PMAABACy/DOX/MnO2-2/PEG TNs. The confocal laser scanning microscopy (CLSM) (LYMPUS FV-1000) was utilized to investigate the cellular uptake using HeLa cells after 24 h incubation according to the previous work.33 The location of intracellular fluorescence was determined by characteristic excitation wavelengths of 405 nm for Hoechst and 480 nm for DOX.

2.8. Analysis and Characterization. A JEM-1200 EX/S transmission electron microscope (TEM) and a JSM-6380 scanning electron microscope (SEM) were applied to evaluate the morphologies and sizes of the samples. For determination of the average hydrodynamic diameter (Dh) of PMAABACy, PMAABACy/DOX/MnO2-2, PMAABACy/DOX/MnO2-2/PEG TNs, and disintegration of PMAABACy/DOX/MnO2-2/PEG TNs by GSH and pH-triggers, DLS measurements were carried out with a Light Scattering System BI-200SM device (Brookhaven Instruments). The Fourier transform infrared (FT-IR) spectra of PMAABACy/DOX/MnO2-2 and PMAABACy/DOX/MnO2-2/PEG were detected in the range of 500-3900 cm-1. The X-ray powder diffraction (XRD) was utilized to evaluate the crystalline structure of PMAABACy/DOX/MnO2-1 and PMAABACy/DOX/MnO2-2 over the range of 5~60°. The

X-ray

photoelectron

spectroscopy

(XPS)

and

element

contents

of

these

PMAABACy

and

PMAABACy/DOX/MnO2-2 were performed with an Elementar Vario EL instrument (Elementar Analysensysteme GmbH, Munich, Germany). A clinical 3T MRI scanning (Siemens MAGNETOM Verio) was applied to detect the acid/GSH-activated MRI under different media. Different amounts of the PMAABACy/DOX/MnO2-2/PEG TNs were dispersed in the related buffer solutions (pH 7.4 in the presence of 10 µM GSH and pH 5.0 in the presence of 10 mM GSH) for 24 h to determine transverse relaxation rate (1/T2) and longitudinal relaxation rate (1/T1), respectively. A Lambda 35 UV−vis spectrometer was applied to detect the dual reduction-/pH-triggered release of DOX from the PMAABACy/DOX/MnO2-2/PEG TNs. The encapsulation efficiency and drug-loading capacity were evaluated by using equations as follows: DOX encapsulation efficiency (DEE) (%) =


× 100%.

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DOX-loading capacity (DLC) (%) =

3.

;

Results and Discussion

3.1. Synthesis and Characterization PMAABACy/DOX/MnO2-2/PEG TNs. The synthetic strategy to PMAABACy/DOX/MnO2-2/PEG nanohybrids was presented in Scheme 1. The PMAABACy nanohydrogels with reduction-property were prepared by a mild and facile one-pot distillation-precipitation polymerization, which possessed abundant carboxyl group to load chemotherapeutic drug (DOX) and adsorb the Mn2+ ions via electrostatic interaction and chelation, respectively. Subsequently, the functional PMAABACy/DOX/MnO2 nanohybrids containing amorphous MnO2 were fabricated through a biomineralized process, which would induce the transformation of metal ions into amorphous metal oxide within PMAABACy nanohydrogels under mild temperature.8,17 Through the PEGylation, the PMAABACy/DOX/MnO2-2/PEG nanohybrids were crafted. Most importantly, Mn2+ ions reduced from amorphous MnO2 as a contrast endowed the nanohybrids with enhanced acid/GSH-activated MRI as presented in Scheme 1. Owing to the introduction of disulfide crosslinking bonds into PMAA

nanohydrogels

and

pH/GSH

dual-sensitive

amorphous

MnO2,

the

decomposition

of

PMAABACy/DOX/MnO2-2 TNs were triggered in response to high intracellular GSH concentrations and slightly acidic pH in tumor cells to prompt the site-specific release of DOX.28

A

B

C

D

E

MnO2 (001) 0.24 nm

2 nm F

G

I J

H

2 nm Figure 1. The TEM images of PMAABACy (A), PMAABACy/DOX/MnO2-1 TNs (B), PMAABACy/DOX/MnO2-2 TNs (G) and PMAABACy/DOX/MnO2-2/PEG TNs (F); and the high magnification TEM images of PMAABACy/DOX/MnO2-1 TNs (C and D) and PMAABACy/DOX/MnO2-2 TNs (H and I); and the SEM images of PMAABACy/DOX/MnO2-1 TNs (E) and PMAABACy/DOX/MnO2-2 TNs (J), respectively. TEM images revealed that the as-prepared PMAABACy nanohydrogels had spherical structure with an average diameter of approximately 96 nm (Figure 1A). Meanwhile, the morphology and size of PMAABACy nanohydrogels kept consistent with the previous works.8,34 Subsequently, the chemotherapeutic drug DOX was introduced into the PMAABACy nanohydrogels via electrostatic interaction under neutral condition for 24 h. At last, through a biomineralized process of Mn2+ ions at basic environment under abundant oxygen atmospheres, which would induce the formation of amorphous metal oxide within PMAABACy/DOX complexes from abundant Mn2+ ions.



Page 8 of 21

During the biomineralization process, the PMAABACy/DOX complexes dispersed in deionized water was mixed with MnCl2 under vividly magnetic stirring. Then, the Mn2+ ions would be anchored onto carboxyl groups of PMAABACy/DOX complexes.17,35 Lastly, the NaOH aqueous solution (1 M) was slowly added to adjust the pH value of mixtures and trigger the formation of metal oxide within the PMAABACy/DOX complexes. Meanwhile, the biomineralization process was following reaction: 2MnCl2 + 4NaOH + O2 = 2MnO2 + 4NaCl + 2H2O.17,35 As shown in presented Figure 1B, C, and E, the introduction of metal oxide nanoclusters resulted in a rough surface. Meanwhile, the color of mixtures was changed from pink to brown (Figure 2E and F). Then, the enlarged TEM image (Figure 1C) of PMAABACy/DOX/MnO2-1 further presented the obvious MnO2 aggregation to confirm the generation of MnO2. The high resolution TEM (HRTEM) image taken from PMAABACy/DOX/MnO2-1 exhibited the well-defined lattice fringes with interplanar distance of 0.24 nm, implying the (100) plane of MnO2 sheet along with the [001] direction.2,36 In Figure 1E, the SEM images showed an irregular and rough surface to PMAABACy/DOX/MnO2-1. This undesirable phenomenon might be attributed to the excess of Mn2+ ions. On the basis of above analysis, equivalent Mn2+ ions with carboxyl groups of PMAABACy/DOX complexes resulted in a rough surface. To address the problem of rough surface, the experimental steps were optimized by the control of amounts of MnCl2. Therefore, the feeding ratios of MnCl2 (188 mg) were utilized to control the surface of PMAABACy/DOX/MnO2 nanoparticles to obtain the desired PMAABACy/DOX/MnO2-2. As shown in Figure 1G, H, and J, the obtained PMAABACy/DOX/MnO2-2 had unique spherical structure with an average diameter of approximately 102 nm. This average diameter of PMAABACy/DOX/MnO2-2 kept consistent with that of original PMAABACy nanohydrogels. Meanwhile,

the

PMAABACy/DOX/MnO2-2

possessed

a

smooth

surface

compared

with

that

of

PMAABACy/DOX/MnO2-1. The enlarged TEM and SEM images further revealed this smooth surface as shown in Figure 1H and J, respectively. Then, the HRTEM images didn’t show the characteristic lattice fringes, demonstrating that the generation of amorphous MnO2. Along with the concentration of Mn2+ ions being decreased, the precursor solution was restricted by the small space within PMAABACy nanohydrogels to further effectively hinder the long-range diffusion of Mn2+ ions, and the nucleation and the growth of crystalline phases were thus suppressed. The suppression of nucleation was attributed to the increasing of viscosity to decrease the concentration of Mn2+ ions, further restricted the generation of MnO2 aggregation. This phenomenon that could be also described a confinement effect by mediating of PMAABACy nanohydrogels. Meanwhile, PMAA chains also chelated partial Mn2+ ions to reduce the concentration of free Mn2+ ions, and further suppressed the nucleation and the growth of crystalline phases. Along with the increasing of oxidation time, the chelated Mn2+ ions would be gradually released to form the amorphous MnO2. On the basis of the above discussion, the decreasing of concentration of Mn2+ ions and the chelation of PMAA toward Mn2+ ions synergistically hindered the long-range diffusion of Mn2+ ions, and then suppressed the nucleation and the growth of crystalline phases. Bu’s groups also deeply studied that the citrate as a chelator to decrease the concentration of free iron ions, and further hindered nucleation and the growth of crystalline phases.30 Notably, these amorphous matters easily occurred disintegration in response to external stimulis (such as H+ or reducer).30 Furthermore, to explain the amorphous MnO2 in PMAABACy/DOX/MnO2, Raman spectra of PMAABACy/DOX/MnO2-1 and PMAABACy/DOX/MnO2-2 TNs were determined as shown in Figure 2B. The characteristic peaks in the range of 575-675 cm-1 could be attributed to the birnessite-type MnO2 within PMAABACy/DOX/MnO2-1.37 As compared with birnessite-type MnO2 to


Page 9 of 21

PMAABACy/DOX/MnO2-1, the typical peaks in the range of 575-675 cm-1 were hard to detect because of the introduction of amorphous MnO2 for PMAABACy/DOX/MnO2-2. Meanwhile, Figure 2C exhibited the XRD spectra to PMAABACy/DOX/MnO2-1 and PMAABACy/DOX/MnO2-2 TNs. It can be seen from the spectrum of PMAABACy/DOX/MnO2-1 that the (001) and (006) planes were attributed to birnessite-type MnO2,38 whereas these peaks were hard to find from the spectrum of PMAABACy/DOX/MnO2-2 TNs, implying that the existence of amorphous MnO2. These results are consistent with conclusion from TEM images as presented in Figure 1D and I.

A

Mn2p 1/2

B

Mn2p 2/3

PMAABACy/DOX/MnO2-1 PMAABACy/DOX/MnO2-2

C1s

O1s

N1s

PMAABACy S2s

PMAABACy/DOX/MnO2-2

800

700

600 500 400 300 Binding energy(eV)

200 200

400

600

800 1000 1200 1400 1600 1800

Raman Shift (cm-1)

C

D

E

F

006

001

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PMAABACy PMAABACy/DOX PMAABACy/DOX/MnO2-2 TNs

PMAABACy/DOX/MnO2-2 PMAABACy/DOX/MnO2-1

10

Figure

2.

XPS

20

30 40 2theta(degree)

spectra

of

50

PMAABACy

60

520

540

560

580

600

620

640

Emission wavelength (nm)

and

PMAABACy/DOX/MnO2-2

(A);

Raman

spectra

of

PMAABACy/DOX/MnO2-1 and PMAABACy/DOX/MnO2-2 (B); XRD spectra of the PMAABACy/DOX/MnO2-1 and PMAABACy/DOX/MnO2-2 (C); and Fluorescence spectra of the PMAABACy, PMAABACy/DOX, and PMAABACy/DOX/MnO2-2 in deionized water with concentration of 4.0 × 10−4 mg mL−1 (D), respectively. In addition, the inserts indicated that the color of mixtures was changed before (E) and after (F) the formation of MnO2 coating (F), respectively. Chemical state of characteristic element of PMAABACy nanohydrogels and PMAABACy/DOX/MnO2-2 were studied by X-ray photoelectron spectroscopy (XPS). As illustrated in Figure 2A, the band at 227.17 eV revealed the presence of S components in the surface of the PMAABACy nanohydrogels. Furthermore, the band at 399.75 eV revealed the presence of N components in the surface of the PMAABACy nanohydrogels. These information indicated that the PMAABACy nanohydrogels with reduction-sensitive property were successfully prepared via a mild and facile distillation–precipitation copolymerization. As revealed by X-ray photoelectron spectroscopy (XPS), our obtained PMAABACy/DOX/MnO2-2 showed two typical binding-energy peaks at 642 eV and 654 eV in



Figure 2A, which corresponded to Mn(IV)2p1/2 and Mn(IV)2p3/2 compared with XPS of PMAABACy. In addition, the MnO2 content of PMAABACy/DOX/MnO2-2 was 24.84 wt% by XPS. This phonemoneon implied that the manganese in the as-obtained PMAABACy/DOX/MnO2-2 was in the valence of IV and further confirmed the successful generation of MnO2.39 Compared with PMAABACy nanohydydrogels in deionized water, the significant fluorescence of PMAABACy/DOX was found, implying that the DOX was successfully loaded into the PMAABACy nanohydydrogels (Figure 2D). Subsequently, it is noted that the fluorescence quenching of DOX was caused by a biomineralized

process

of

MnO2.

The

clear

difference

in

PMAABACy,

PMAABACy/DOX,

and

PMAABACy/DOX/MnO2-2 demonstrated that DOX was successfully loaded into the PMAABACy nanohydydrogels and the formation of amorphous MnO2. In addition, the average hydrodynamic diameter (Dh) of the PMAABACy nanohydrogels and PMAABACy/DOX/MnO2-2 at pH 7.4 were tracked by using the DLS technique. As shown in Figure 3B, PMAABACy nanohydrogels and PMAABACy/DOX/MnO2-2 showed a narrow unimodal size distribution. Meantime, the Dh of PMAABACy nanohydrogels and PMAABACy/DOX/MnO2-2 were approximately 106 nm and 118 nm, respectively. This almost Dh between the PMAABACy nanohydrogels and the PMAABACy/DOX/MnO2-2 implied the MnO2 adequately dispersing into PMAABACy nanogydrogels. As a consequence, we believe that the amorphous MnO2 in PMAABACy/DOX/MnO2-2 was successfully synthesized by controlling the amounts of Mn2+ ions, revealing the new strategy for fabrication of amorphous MnO2. Importantly, along with the decreasing of proportion of MnO2 in preliminary experiment, the as-prepared PMAABACy/DOX/MnO2 complexes exhibited obvious premature release of DOX. In addition, with the increasing of proportion of MnO2, the corresponding complexes

possessed

crystalline

MnO2

(such

as

PMAABACy/DOX/MnO2-1).

And

thus

the

PMAABACy/DOX/MnO2-2 has been chosen as a desired vehicle.

a

b c

100

d

80

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 21

60 40 20

PMAABACy/DOX/MnO2-2 PMAABACy/DOX/MnO2-2/PEG

3500 3000 2500 2000 1500 1000 500

0 0

40

Wavenumber(cm-1)

80 120 Dh(nm)

160

Figure 3. FT-IR spectra of the PMAABACy/DOX/MnO2-2 and PMAABACy/DOX/MnO2-2/PEG (A), and the typical Dh distributions of the PMAABACy (B-b), PMAABACy/DOX/MnO2-2 (B-c), PMAABACy/DOX/MnO2-2/PEG TNs (B-d) and the disintegration of PMAABACy/DOX/MnO2-2/PEG TNs (B-a) at pH 5.0 in the presence of 10 mM GSH under magnetic stirring for 24 h, respectively. In addition, the insets were the digital photos of PMAABACy/DOX/MnO2-2/PEG TNs (C) and PMAABACy/DOX/MnO2-2 TNs (D) under physiological conditions for 60 min, respectively. To improve the stability and biocompatibility of the hybrids, thus, the PMAABACy/DOX/MnO2-2 were modified with NH2-PEG via Mn-N coordinate bonding. After the modification of PEG brushes, the characteristic peak of


Page 11 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


C-O-C stretching vibration at 1110 cm-1 appeared by compared to PMAABACy/DOX/MnO2-2 as shown in Figure 3A, further indicating the successful preparation of PMAABACy/DOX/MnO2-2/PEG TNs. In addition, as compared

with

the

original

PMAABACy/DOX/MnO2-2

(Figure

3D),

the

as-obtained

PMAABACy/DOX/MnO2-2/PEG TNs have favorable stability under physiological conditions as shown in Figure 3C. And that the obtained PMAABACy/DOX/MnO2-2/PEG TNs possessed a spherical structure and its average diameter

increased

to

around

124

nm

as

presented

in

Figure

1F.

Meanwhile,

the

Dh

of

PMAABACy/DOX/MnO2-2/PEG TNs increased to around 162 nm compared with that of PMAABACy/DOX/MnO2-2 (Figure 3B-d). Thus, the PEG brushes was successfully introduced on the surface of PMAABACy/DOX/MnO2-2 TNs. 3.2. Stability and disintegration of the PMAABACy/DOX/MnO2-2/PEG TNs. It is possible that various metal cations, GSH, cysteine (Cys), and hemocyanin (Hcy) exhibit potential selectivity to this nanocarriers. However, the prime objective of this work is not to delve into selectivity for various metal cations and Cys, Hcy of PMAABACy/DOX/MnO2-2/PEG TNs but to focus on the typical selectivity to GSH and pH of the nanohybrids, which can efficiently deliver the as-loaded drug at targeted cancer cells and at the same time perform MRI. It is well known that the reductive conditions of intracellular microenvironments of tumor tissues are drastically different from the normal tissues because of a high level of GSH and lower pH in the cytoplasm and lysosomes.22,40 On the basis of the intracellular microenvironments of tumor tissues, the only selectivity of PMAABACy/DOX/MnO2-2/PEG TNs to GSH and pH have been investigated in this work. To evaluate whether such a sensitive PMAABACy/DOX/MnO2-2/PEG TNs were suitable for disintegration by pH/GSH dual-sensitive triggers, a variety of characterization technologies (TEM and DLS) were conducted for further assessment of the related performance. As shown in Figure 4C, the PMAABACy/DOX/MnO2-2/PEG TNs were stable and dispersed well in PBS (mimicking the physiological medium) and DMEM (Dulbecco's Modified Eagle Medium) after 7 days, implying that the remarkable stability of the nanocarriers. Considering the reactivity of GSH and H+ toward MnO2, the disintegration behavior of the PMAABACy/DOX/MnO2-2/PEG TNs were carefully studied in typical medium. In brief, the PMAABACy/DOX/MnO2-2/PEG TNs were dispersed in acetate buffer solution ((pH 5.0 with 10 M GSH), mimicking the intracellular environment of tumor cell) at 37 ˚C for 12 h and 24 h, respectively. As shown in Figure 4A and B, the medium of pH 5.0 in the presence of 10 mM GSH would accelerate the decomposition of PMAABACy/DOX/MnO2-2/PEG TNs for 6 h, and the abundant nanorods were observed in Figure 4A. Meanwhile, TEM image (Figure 4A) revealed the as-generated nanorods from the PMAABACy/DOX/MnO2-2/PEG TNs complexes uniformly dispersed onto the surface of the complexes, demonstrating that the acidic and reductive conditions triggered the decomposition. Subsequently, the decomposition time of PMAABACy/DOX/MnO2-2/PEG TNs were lengthened to 24 h. As shown in Figure 4B, the previous nanorods of MnO2 disappeared in the field of vision, indicating the decomposition of MnO2 after reduction for 24 h. We therefore speculate that the decomposition of PMAABACy/DOX/MnO2-2/PEG TNs derive from transformation from MnO2 to Mn2+ ions in response to both elevated GSH level and slightly acidic condition. During reductive reaction, the MnO2 was reduced to Mn2+ ions, simultaneously the reductive agent GSH was oxidized to GSSG through the thiol-disulfide exchange by following reaction: MnO2 + 2GSH + 2H+ →Mn2+ + GSSG + 2H2O. The previous report also supported the reaction of thiol-disulfide exchange.6,18 Notably, for the PMAABACy nanohydrogels with reduction-sensitive property, swell of nanohydrogels were found in the field of vision upon the GSH for 12 h



compared with original PMAABACy nanohydrogels. This phenomenon indicated that PMAABACy nanohydrogels were

disturbed

via

GSH-trigger.

Meanwhile,

extending

the

reaction

time

to

24

h,

the

PMAABACy/DOX/MnO2-2/PEG TNs disappeared in the field of vision, implying the decomposition of PMAABACy/DOX/MnO2-2/PEG TNs in response to slightly acidic and reductive medium (Figure 4B). Importantly, the abundant small matters were detected under elevated GSH level and slightly acidic medium as shown in Figure 3B-a, further implying the decomposition of PMAABACy/DOX/MnO2-2/PEG TNs by GSH and pH triggers. On the basis of the above analysis, owing to the introduction of both disulfide bond and pH/GSH dual-sensitive amorphous MnO2, the PMAABACy/DOX/MnO2-2/PEG TNs possess reduction-sensitive property in the presence of GSH and slightly acidic medium.

100 80

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 21

pH 7.4 PBS 24 h pH 7.4 PBS 168 h DMEM 24 h DMEM 168 h

60 40 20 0 120

140

160 180 Dh(nm)

200

Figure 4. The TEM images to the disintegration of PMAABACy/DOX/MnO2-2/PEG TNs treated with 10 mM GSH and pH 5.0 for 12 h (A) and 24 h (B), respectively. In addition, the stability of PMAABACy/DOX/MnO2-2/PEG TNs in PBS or DMEM for different periods of time (C). 3.3. MRI Properties Measurements. The relevance of the disintegration process and corresponding enhanced T1 and T2-MRI through mediating of Mn2+ ions could be revealed by the clinical 3T MRI scanning. Different amounts of the PMAABACy/DOX/MnO2-2/PEG TNs were dispersed at the related buffer solutions (pH 7.4 in the presence of 10 µM GSH and pH 5.0 in the presence of 10 mM GSH) for 24 h. These two buffer solutions were chosen to imitate the normal blood circulation environment and intracellular environment of tumor tissues, respectively. The as-released Mn2+ ions from PMAABACy/DOX/MnO2-2/PEG TNs by GSH and pH-stimulus showed much stronger enhancement in both the longitudinal relaxivity r1 and transverse relaxivity r2 in comparison with the original the PMAABACy/DOX/MnO2-2/PEG TNs at pH 7.4 in the presence of 10 µM GSH as presented in Figure 5. Then, the longitudinal relaxivity r1 and transverse relaxivity r2 of the PMAABACy/DOX/MnO2-2/PEG TNs at pH 5.0 in the presence of 10 mM GSH, obtained by measuring the relaxation rate as a function of Mn concentration, presented 42- and 110-fold enhancement compared with those of the imitating normal blood circulation environment, respectively. This effect was clearly found in the T1- and T2-weighted MRI obtained at 3T magnetic field. Once amorphous MnO2 components of the PMAABACy/DOX/MnO2-2/PEG TNs were reduced to Mn2+ ions by GSH and pH-triggers, which corresponded to the largest enhancement in relaxivity.18 During the reductive and slightly acidic medium, numerous Mn2+ ions were generated, and then every Mn2+ ions could serve as a MRI contrast agent for positive T1-weighted MRI or negative T2-weighted MRI signal intensity enhancement. Comparatively, the PMAABACy/DOX/MnO2-2/PEG TNs treated with neutral buffer solution exhibited no significant signal


Page 13 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


changes along with the increasing of Mn concentration. As far as we know, Mn is shielded from the aqueous environment, decreasing their ability to enhance water proton relaxation.6,41 Such a remarkable MRI signal enhancement could be attributed to the disintegration of the PMAABACy/DOX/MnO2-2/PEG TNs in the presence of GSH at pH 5.0. As mentioned above, the PMAABACy/DOX/MnO2-2/PEG TNs not only act as a both positive and negative contrast on T1- and T2-weighted MRI, but also exhibit fast decomposition through a reduction-responsive trigger. Therefore, the smart PMAABACy/DOX/MnO2-2/PEG TNs with both MRI and reduction-responsive properties would serve as a desired DDS to deliver chemotherapeutic drug.

Figure 5. Plots of A) r1 and B) r2 versus [Mn] for PMAABACy/DOX/MnO2-2/PEG TNs solution (♦) and PMAABACy/DOX/MnO2-2/PEG TNs solution treated with GSH (■), respectively. C) T1-weighted and D) T2-weighted MRI results obtained from (A) and (B), respectively. Left to right: [Mn]=0, 0.2, 0.4, 0.8, and 1.6 mM. The top and bottom rows in (C) and (D) corresponded to PMAABACy/DOX/MnO2-2/PEG TNs in the presence and absence of GSH, respectively. 3.4. Drug Loading and Controlled Release. As a desired DDS, the loading capacity of chemotherapeutic agent plays an important role in cancer treatment. 100.0 mg of PMAABACy nannohydrogels were dispersed in 50.0 mL of 0.6 mg mL−1 DOX solution at pH 7.4 for loading of chemotherapy drug. Then, the dispersion was centrifuged and washed several times with abundant water to remove unabsorbed chemotherapy drug DOX, and further obtained PMAABACy/DOX. Subsequently, the 188 mg of MnCl2 and 100 mg of PMAABACy/DOX were dispersed in deionized water to form PMAABACy/DOX/MnO2-2 TNs, and then centrifuging and washing several times to remove leakage of DOX. Through UV−vis spectrometer, the loading capacity and encapsulation efficiency of DOX were determined, and these typical value are 26.3±1.8% and 87.67±6.0%, respectively. To evaluate the release behavior of DOX from the PMAABACy/DOX/MnO2-2/PEG TNs, the typical conditions were chosen to simulate the intracellular environment of tumor tissues and normal blood circulation environment. In brief, 10 mg of the PMAABACy/DOX/MnO2-2/PEG TNs were dispersed at the related buffer solutions (pH 7.4 in the presence of 10 µM GSH, pH 7.4 in the presence of 10 mM GSH, pH 5.0 in the absence of GSH, and pH 5.0 in the presence of 10 mM GSH). In addition, the corresponding Mn2+ release from PMAABACy/DOX/MnO2-2/PEG TNs was also performed at the typical buffer solutions (Figure 6B). The PMAABACy/DOX/MnO2-2/PEG TNs



didn’t exhibit the obviously leakage of DOX within 2 days under physiological conditions as presented in Figure 6A, which kept consistent with the Mn2+ release at pH 7.4 in the absence of GSH (Figure 6B). As far as we know, MnO2 coating had favorable stability in neutral environment. Meanwhile, the previous investigation revealed that weakly reducing bioagents such as glucose and fructose didn’t lead to the reduction of MnO2 coating.18 So these results suggested that the PMAABACy/DOX/MnO2-2/PEG TNs prevented the leakage of chemotherapy agent

pH 7.4 10 µM GSH pH 7.4 10 mM GSH

80 60 40

pH 5.0 pH 5.0 10 mM GSH

20 0 0

500

1000

1500

2000

Time (min)

2500

3000

Cumulative release of Mn2+(%)

during blood circulation, and then safely carried the chemotherapy agent to tumor tissues.

Cumulative release of DOX (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 21

100 80

pH 7.4 10 µM GSH pH 7.4 10 mM GSH pH 5.0 pH 5.0 10 mM GSH

60 40 20 0 0

200

400

600

800

Time(min)

Figure 6. Cumulative DOX release from the PMAABACy/DOX/MnO2-2/PEG TNs in simulated body fluids (A);

Cumulative Mn2+ release from the PMAABACy/DOX/MnO2-2/PEG TNs in simulated body fluids was revealed by the clinical 3T MRI scanning (B); The TEM images to the disintegration of PMAABACy/DOX/MnO2-2/PEG TNs by treatment with 10 mM GSH and pH 5.0 for 0 min (C), 720 min (D) and 1440 min (E), respectively. Furthermore, the color changes of PMAABACy/DOX/MnO2-2/PEG TNs were exhibited after dispersed in buffer solutions (pH 7.4 with 10 µM GSH (F) and pH 5.0 with 10 mM GSH (G)) for 24 h at 37 °C, respectively. Notably, along with the increasing of GSH concentration to 10 mM, the cumulative Mn2+ release from the PMAABACy/DOX/MnO2-2/PEG TNs was accelerated. This release of Mn2+ triggered the destruction of MnO2 coating to cause the release of DOX from the nanocarriers (Figure 6A). When the release condition was changed to pH 5.0, the cumulative Mn2+ release from the PMAABACy/DOX/MnO2-2/PEG TNs was found because of the destruction of MnO2 coating at typical acidic condition as shown in Figure 6B. Then, the release of DOX was also detected. As for a reductive and acidic medium ( pH 5.0 in the presence of 10 mM GSH) mimicking the intracellular environment of tumor tissues, the cumulative Mn2+ release from the PMAABACy/DOX/MnO2-2/PEG TNs was obviously enhanced. And the cumulative release ratio increased to 92.34 wt % at pH 5.0 in the presence of 10 mM GSH during the testing time (Figure 6A), indicating that the MnO2 coating was completely destructed. Most importantly, this obvious destruction is conductive to the release of DOX molecules from the nanocarriers. As shown in Figure 6A, PMAABACy/DOX/MnO2-2/PEG TNs also didn’t present the leakage of DOX within 5 h. As the fast disintegration of PMAABACy/DOX/MnO2-2/PEG TNs in an acidic and reductive environment, the


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substantial cumulative release of DOX exhibited obvious enhancement in the following time. During the testing time, cumulative release ratio increased to 79.13 wt % at pH 5.0 in the presence of 10 mM GSH. For comparison with physiological medium, the reductive and acidic medium not only disintegrated the structure of the DDS to fragments, but also enhanced the solubility of DOX to further lead the fast diffusion of DOX. The structure changes of PMAABACy/DOX/MnO2-2/PEG TNs were confirmed via insets of TEM images of different time (Figure 6C, D and E). As compared with the normal physiological medium (Figure 6F), the color of PMAABACy/DOX/MnO2-2/PEG TNs was obviously changed at pH 5.0 in the presence of 10 mM GSH for 24 h as shown in Figure 6G. Meanwhile, the structure changes of different time also keep consistent with the trends of cumulative release of Mn2+. As a desired DDS for delivering cytotoxic chemotherapy drug, they could effectively prevent the premature DOX leakage during blood circulation owing to their improved stability at pH 7.4. As in the intracellular environment of tumor tissues after reductive and acidic trigger for 10 h, most DOX could be quickly released and then in a sustained release model. Therefore, these results demonstrated that such pH/GSH dual-sensitive PMAABACy/DOX/MnO2-2/PEG TNs possess an ideal structure for delivering chemotherapy agent and realizing the MRI image with reduced side effects.

Figure 7. Cellular uptake to HeLa cells by treatmeant with the PMAABACy/DOX/MnO2-2/PEG TNs detected via

CLSM after the incubation of 12 h (A) and 24 h (B), respectively. Left to right: the bright field, blue Hoechst in cell nuclei, red DOX fluorescence in cells, and the merged one. In addition, the value of scale bar is 50 µm. 3.5. CLSM Analysis. The capability to intracellular drug delivery via PMAABACy/DOX/MnO2-2/PEG TNs at

different time was further systematically evaluated using HeLa cells by the CLSM technique as shown in Figure 7. After 12 h incubation, the tiny minority of DOX molecules with red fluorescence was detected as shown in Figure 7A. Along with the increasing of incubation time to 24 h, it is worth noting that strong red DOX fluorescence

appeared at the

cell nucleus as

shown in Figure

7A,

meaning the

efficient

uptake

of

the

PMAABACy/DOX/MnO2-2/PEG TNs and efficiently intracellular release of DOX at tumor cells. Meanwhile, the stronger

fluorescence

emerged

image

also

revealed

that

the

as-released

DOX

from

the

PMAABACy/DOX/MnO2-2/PEG TNs were accumulated mainly in the nucleus (Figure 7B). Most importantly, this result showed that the PMAABACy/DOX/MnO2-2/PEG TNs were disintegrated and chemotherapy drug was



released in correspondence with the intracellular reductive medium with high-level GSH in within endosomes and lysosomes.4,42 Subsequently, the as-released DOX could insert the single and double-strand of DNAs to cause the death of cancer cells.43 Most importantly, the PMAABACy/DOX/MnO2-2/PEG TNs would deliver DOX to cell nucleus, demonstrating the growth of cancer cells being inhibited. In conclusion, the CLSM analysis indicated that the PMAABACy/DOX/MnO2-2/PEG TNs are efficiently internalized for HeLa cells, and the as-released DOX molecules from the PMAABACy/DOX/MnO2-2/PEG TNs are mainly accumulated in the cell nucleus to kill cancer cells.

0

Concentration of PMAABACy/MnO2-2/PEG (µg/mL)

50

100

150

200

250

300

350

c

100 Cell Viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 21

80 60

a b

40 20 0 0

2

4

6

8

10

12

14

16

DOX equivalent dose (µg/mL)

Figure

8.

Cell

viability

assay

in

HeLa

cells

treated

with

PMAABACy/MnO2-2/PEG

(c),

PMAABACy/DOX/MnO2-2/PEG TNs (a), and free DOX (b) at 37 °C for 24 h. 3.6. Cytotoxicity and HeLa Cell Growth Inhibition Assays. The favorable biocompatibility is of importance for

DDS. The PMAABACy/MnO2-2/PEG complexes, which have same components for comparison with PMAABACy/DOX/MnO2-2/PEG TNs excepting DOX, were conducted to assess the cytotoxicity of the PMAABACy/DOX/MnO2-2/PEG TNs using HeLa cells by MTT assays. The viability of the HeLa cells treated with the PMAABACy/MnO2-2/PEG was higher than approximately 93% during all the testing concentrations, further revealing that the hybrids exhibited favorable biocompatibility on the HeLa cells. In brief, along with the increasing of PMAABACy/MnO2-2/PEG concentration from 20, 40, 80,160 to 320 µg/mL, the viability of HeLa cells were 97.5% ± 4.2%, 95.8% ± 3.9%, 96.5% ± 4.0%, 94.7% ±4.2% and 92.6% ± 4.3%, respectively. As a desired chemotherapeutic agent, the fast release of chemotherapeutic drug under typical stimuli plays an important role in cancer treatment. To explore the inhibition growth of HeLa cell via MTT assays, the PMAABACy/DOX/MnO2-2/PEG TNs showed obviously cytotoxic effects toward HeLa cells after co-incubation for 24 h as shown in Figure 8. As the disintegration of the PMAABACy/DOX/MnO2-2/PEG TNs, the release of DOX components from the DDS were accelerated in an intracellular reductive and acidic environment of tumor tissues.4,42 Along with increasing of DOX concentration, as-released DOX resulted in a significant reduction in cell viabilities as shown in Figure 8. Furthermore, the PMAABACy/DOX/MnO2-2/PEG TNs kept similar killing capacity compared with free DOX as presented in Figure 8. On the basis of these results from the CLSM and MTT, the as-prepared PMAABACy/DOX/MnO2-2/PEG TNs therefore possess remarkable inhibition capacity to cancer cells.


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4.

Conclusions

In summary, for the first time, a kind of novel PMAABACy/DOX/MnO2-2/PEG TNs were successfully fabricated as a new generation of reasonable theranostic nanohybrids. Amorphous MnO2 in the theranostic nanohybrids served as a gatekeeper to inhibit the premature leakage of loaded DOX during blood circulation. In the presence of intracellular acid and GSH, Mn2+ ions reduced from amorphous MnO2 as a dual MRI contrast agent endowed the DDS with enhanced acid/GSH-activated MRI. Amorphization of MnO2 could pave a general roadway to improve the biodegradability and magnetism-related properties of DDS because of the disordered atomic arrangement. Owing to the introduction of disulfide linker, the as-loaded DOX was also site-specific released from the disintegration of PMAABACy nanohydrogels in response to reductive medium as mimicking the intracellular environment of tumor tissues. Therefore, the PMAABACy with reduction-sensitive property, chemopeutic drug, and the pH/GSH dual-sensitive amorphous MnO2 were integrated into the “all-in-one” multifunctional PMAABACy/DOX/MnO2-2/PEG TNs with biodegradability. To the best of our knowledge, this is the first study that the amorphization of MnO2 is employed as a smart gatekeeper to mediate the characters of PMAA-based DDS. This proof of concept might explore a novel strategy to provide a new tumor microenvironment-responsive DDS for real-time MRI-guided cancer therapy.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This project were granted financial support from the National Natural Science Foundation of China (No. 21704093), China Postdoctoral Science Foundation (No. 2018M632795), Plan for College Science and

Technology Innovation Team of Henan Province (No. 16IRTSTHN001), and Science & Technology Innovation Talent Plan of Henan Province (No. 174200510018).

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For Graphic Table of Contents Use Only:

NH2-PEG

Mn2+

DOX

OH-+O2

pH 7.4

Uptaking

Tumor Cell I MR 2+

M

n

Che mot hera py DO X

+

PMAA PMAABACy

GSH

H

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PMAABACy/DOX

PMAABACy/DOX/MnO2


PMAABACy/DOX/MnO2-2/PEG

Amorphous Manganese

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