Aqueous Self-Assembly of Block Copolymers to Form Manganese Oxide-Based Polymeric Vesicles for Tumor Microenvironment-Activated Drug Delivery

The formation of manganese oxide induces self-assembly of block copolymers to form polymeric vesicles. The polymeric vesicles possessed strong stability and high drug loading capacity. The drug-loaded polymeric vesicles have been demonstrated, especially in in vivo studies, to exhibit a higher efficacy of tumor suppression without known cardiotoxicity. The formation of manganese oxide induces self-assembly of block copolymers to form polymeric vesicles. The polymeric vesicles possessed strong stability and high drug loading capacity. The drug-loaded polymeric vesicles have been demonstrated, especially in in vivo studies, to exhibit a higher efficacy of tumor suppression without known cardiotoxicity. Molecular self-assembly is crucially fundamental to nature. However, the aqueous self-assembly of polymers is still a challenge. To achieve self-assembly of block copolymers [(polyacrylic acid–block–polyethylene glycol–block–polyacrylic acid (PAA68–b–PEG86–b–PAA68)] in an aqueous phase, manganese oxide (MnO2) is first generated to drive phase separation of the PAA block to form the PAA68–b–PEG86–b–PAA68/MnO2 polymeric assembly that exhibits a stable structure in a physiological medium. The polymeric assembly exhibits vesicular morphology with a diameter of approximately 30 nm and high doxorubicin (DOX) loading capacity of approximately 94%. The transformation from MnO2 to Mn2+ caused by endogenous glutathione (GSH) facilitates the disassembly of PAA68–b–PEG86–b–PAA68/MnO2 to enable its drug delivery at the tumor sites. The toxicity of DOX-loaded PAA68–b–PEG86–b–PAA68/MnO2 to tumor cells has been verified in vitro and in vivo. Notably, drug-loaded polymeric vesicles have been demonstrated, especially in in vivo studies, to overcome the cardiotoxicity of DOX. We expect this work to encourage the potential application of polymer self-assembly.


Introduction
In nature, self-assembly is one of the most crucial approaches that enables the building of micro-and nanostructures [1]. In biology, from structures of plant to those of multicellular organisms, it plays an important role in generating sophisticated superstructures through different interactions between a large number of building blocks [2][3][4]. Inspired by nature, polymer self-assembly has attracted extensive attention since the 1960s and serves as a "bottom-up" strategy for building complexes naturally [5,6]. The self-assembly of block copolymers in solution developed by Armes, Kataoka, Discher, and Eisenberg has been utilized to design a series of polymeric architectures [7][8][9][10].
In principle, block copolymers can be self-assembled into a wide variety of polymer architectures, but in practice this is usually achieved in co-solvents, often with the aid of both water and an organic solvent [8][9][10]. As far as we know, selfassembly in nature occurs in aqueous phases, not organic phases, and aqueous self-assembly of block copolymer is an environmentally friendly method with the absence of organic solvents or surfactants. It is important to note that the use of organic solvents or surfactants in traditional self-assembly methods has restricted their application because of the side effects to humans. No matter the application, polymeric architectures formed by block copolymer self-assembly in drug delivery have already become an intriguing focus for a number of research areas, especially in cancer treatments.
The advancement of high-quality therapeutic methods based on polymeric architectures has attracted considerable attention for the treatment of cancer over the past 10 years [11,12]. Moreover, we have gradually increased our understanding of tumor microenvironments and cells that contain various cooperating components (such as lysosomes and mitochondria) [13][14][15]. This understanding has facilitated the advancement of numerous nanoparticle-based drug delivery systems (nano-DDSs), both inorganic and organic, to fight against cancers [11,16,17]. Organic nanoparticlebased DDSs, especially the polymer micelle-based DDSs, are considered a major strategy to alleviate the side effects of antitumor drugs and improve their therapeutic efficacy [18,19]. A wide range of self-assembly methods have been extensively utilized to fabricate polymeric nanoparticles such as polymerization-or precipitation-induced self-assembly [20,21]. However, the inherently inferior stability of polymer micelle-based DDSs at low concentrations could be harmful to humans or impede high-quality therapy [22]. Both covalent cross-linking bonds (including disulfide, borate ester, Schiff base, and ketal bonds) and non-covalent interactions (including electrostatic, hydrophobic, and coordination interaction) have been widely used to develop nano-DDSs for overcoming this limitation [23][24][25][26][27][28][29]. However, only a few of these stable inorganic cross-linked block copolymer architectures with a redox potential-responsive property can be utilized to deliver anticancer agents by responding to tumor microenvironments. Therefore, researchers are enthusiastically developing stable block copolymer micelles that can fulfill their target delivery [30,31].
Although numerous advancements have been achieved, there are still some major barriers that need to be overcome, such as the need for enhanced stability to prevent premature leakage of anticancer agents during the delivery process, selective delivery at tumor sites, and biodegradability of polymeric micelle-based DDSs after completion of drug delivery. However, no easy-to-use fabrication system has been developed. Transformation of MnO 2 to Mn 2+ ions has attracted extensive attention because of the stability of MnO 2 in biological fluids and its disintegration by endogenous glutathione (GSH) inside lysosomes and endosomes at tumor sites [32,33].
Moreover, Mn 2+ ions released from the MnO 2 component within MnO 2 -based systems can be easily metabolized by the kidneys [34]. Therefore, the MnO 2 component was introduced in the fabrication of biomaterials because of its remarkable biocompatibility [35]. Few reported studies have described the in situ self-assembly of block copolymers using MnO 2 formation in an aqueous phase. Based on the current knowledge of self-assembly of block copolymers, we hypothesized that block copolymer self-assembly is achievable by MnO 2 formation in an aqueous phase without additional agents such as organic solvents or surfactants to form a stable polymeric architecture, which could extend the capability of self-assemblies beyond traditional methods.
Herein, we describe the use of the self-assembly approach in an aqueous phase in the fabrication of polymeric vesicles with high stability for selective delivery of anticancer agents. The formation of MnO 2 under mild conditions is used to induce the in situ self-assembly of block copolymers (PAA 68 -b-PEG 86 -b-PAA 68 ) to form PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 polymeric vesicles 1 3 (Scheme 1), which exhibit the advantage of stability, which prevents the diffusion of encapsulated anticancer agents during the delivery process.
Thus, for the PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 vesicles, MnO 2 not only serves as a nucleating agent, but it also acts as an interlocking agent in adjusting the self-assembly process of block copolymer with the goal of optimizing their morphology and structural stability. In this design, the introduction of MnO 2 enhances the structural stability of these PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 vesicles. Compared to the silica/organosilica cross-linked block copolymer architectures [22], the PAA 68 -b-PEG 86 -b-PAA 68 / MnO 2 vesicles can respond to GSH and weak acidic conditions to unload their cargos.
Moreover, these polymeric vesicles were further degraded to hydrophilic linear polymers and Mn 2+ ions after effectively delivery (Scheme 1). On the other hand, the as-released Mn 2+ ions from the decomposition of the PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 vesicles as a T 2 contrast agent could endow the DDSs with excellent characteristics for magnetic resonance imaging (MRI) [36]. Therefore, through the self-assembly of copolymers by the formation of MnO 2 in an aqueous phase, polymeric micelles with controlled morphology and stability were fabricated for the selective delivery of anticancer agents, which may be extended to the development of polymeric architectures. Leibovitz's L15 medium, 4′,6-diamidino-2-phenylindole (DAPI), paraformaldehyde, and cell counting kit-8 (CCK-8) were obtained from Boster Biotechnology (Wuhan, China). Dulbecco's modified Eagle's medium (DMEM) was obtained from Thermo Scientific. The human breast cancer MCF-7 (Catalog No.: SCSP-531) and human embryonic kidney HEK-293 (Catalog No.: GNHu43) cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The two cell lines were used within 20 passages in this study. Additionally, deionized water was adopted for all experiments.

Synthesis of PAA 68 -b-PEG 86 -b-PAA 68
The synthetic steps of these copolymers are shown in the Supporting Information according to our previously reported studies [18,19].

Aqueous Self-assembly of PAA 68 -b-PEG 86 -b-PAA 68
The formation of MnO 2 was used to induce self-assembly of block copolymers (PAA 68 -b-PEG 86 -b-PAA 68 ) to form the MnO 2 -polymer hybrid vesicles (PAA 68 -b-PEG 86 -b-PAA 68 / MnO 2 ). Briefly, 50 mg PAA 68 -b-PEG 86 -b-PAA 68 was dispersed in 50 mL water under magnetic stirring with slight ultrasound sonication for 10 min using the Ultrasonic Cleaner (KQ-300DE). Then, 10 mL MnCl 2 solution (360 mg) was slowly added with vigorous magnetic stirring. The mixture was transferred into dialysis tubes (molecular weight cutoff [MWCO], 1000) and then immersed in abundant deionized water to remove excess Mn 2+ ions. After dialysis against deionized water, the pH value of the mixture was adjusted to pH 11 using a 1.0 M NaOH aqueous solution. The PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 vesicles were obtained under vigorous stirring for 24 h and stored at 4 °C. To optimize the effect of the Mn 2+ concentrations on the morphology of the MnO 2 -polymer hybrids, 60 and 180 mg MnCl 2 ·4H 2 O were successively added to induce the self-assembly of PAA 68 -b-PEG 86 -b-PAA 68 according to the above procedures.
Additionally, the above measurements were repeated with three samples from different batches to obtain the final

Controlled Release
For controlled release evaluation of DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 , 50 mg was equally dispersed in samples of 10.0 mL PBS (pH 7.4, pH 7.4 in the presence of 10 μM GSH, pH 6.5, pH 5.0, and pH 5.0 in the presence of 10 mM GSH). After filtration using 0.2-μm ultrafiltration membranes, the PBS dispersions of DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 were transferred into dialysis tubes (MWCO, 1000) and immersed in 140.0 mL of PBS at the corresponding pH at 37 °C. A 5.0 mL aliquot of the solution was collected to detect the drug concentration using a UV spectrophotometer at 482 nm at specific time intervals (0, 1, 3, 6, 12, 24, 48, and 60 h), and 5.0 mL fresh PBS of the corresponding pH was supplemented after each sampling.
Each controlled release analysis was repeated three times, and the final cumulative release ratio of DOX was the average of three measurements. DOX release from both DOX 1 -loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 and DOX 2 -loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 was analyzed at pH 7.4 in the presence of 10 μM GSH at pH 5.0 and 10 mM GSH according to the above procedures.

Confocal Laser Scanning Microscopy Analysis
The confocal laser scanning microscopy (CLSM) technique was used to investigate the cellular uptake of DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 vesicles and free DOX by MCF-7 cells at excitation wavelengths of 482 nm for DOX and 406 nm for Hoechst, as reported previously [36].

Animal Experiments
Female nude BALB/c mice aged 5-6 weeks were obtained from Hunan Slack Scene of Laboratory Animal Co., Ltd. (Hunan, China) and Shanghai Laboratory Animal Center (Shanghai, China). Animals were treated according to protocols established by the ethics committee of Zhengzhou University, and the in vivo experiments were approved and conducted in accordance with the guidelines of the ethics committee of Zhengzhou University. MCF-7 cells (1 × 10 6 ) were subcutaneously injected into the right flank of the nude mice (n = 5 per group).
When the tumor volumes reached 100 mm 3 , the mice were randomized into saline, PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 , free DOX, and DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 / MnO 2 groups. The groups were treated with corresponding samples intravenously (5 mg kg −1 day −1 ) once every 3 days. Tumor size was then measured every 3 days using Vernier calipers. Tumor volume was calculated using the following formula: volume = (length × width 2 )/2. Tumors were weighed after 31 days, and, subsequently, the hearts and livers were sectioned for histological evaluation using hematoxylin and eosin (H&E) staining.

Characterization
Proton ( 1 H) nuclear magnetic resonance (NMR) spectra were recorded using a Bruker Avance (II) 400 MHz spectrometer at room temperature. Automatic tuning module (ATM) and double probe were utilized for the NMR spectrometer. 1 H NMR spectra were measured in deuterated chloroform (CDCl 3 ) or deuterium oxide (D 2 O, heavy water) using tetramethylsilane as the internal standard, and the concentration of the samples was 2 mg mL −1 . Fourier transform infrared (FT-IR) spectra were recorded using a Bruker IFS 66 v/s IR spectrometer at 4000-400 cm −1 with a resolution of 4 cm −1 . The number average molecular weight (M n ) and polydispersity (PDI) of the copolymers were measured using gel permeation chromatography (GPC) in THF at 35 °C. The vesicular morphology was analyzed using a JEM-1200 EX/S transmission electron microscope (TEM).
X-ray photoelectron spectroscopy (XPS) of PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 was performed using an Elementar Vario EL instrument (Elementar Analysensysteme GmbH, Munich, Germany). An Agilent 7700× inductively coupled plasma mass spectrometer (ICP-MS) was used to detect the content of MnO 2 in the PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 vesicles. For ICP-MS analysis, the samples were treated with 10% hydrochloric acid for 8 h and then heated to remove the hydrochloric acid. The dynamic light scattering (DLS) measurements were performed using a light scattering system BI-200SM device. The release performance of DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 was assessed using a PerkinElmer Lambda 35 UV-Vis spectrometer at room temperature.

Preparation of PAA 68 -b-PEG 86 -b-PAA 68
To achieve aqueous self-assembly, a water-soluble block copolymer PAA 68 -b-PEG 86 -b-PAA 68 was prepared using rational design. Furthermore, we selected biocompatible PEG as a unique block to surmount the biological barriers. In this work, the PAA 68 -b-PEG 86 -b-PAA 68 triblock copolymer was synthesized using a combination of acylation reaction, atomic transfer radical polymerization (ATRP), and hydrolysis reaction (Fig. S1). In the 1 H NMR spectrum of HO-PEG 86 -OH, the characteristic peak at 3.68 ppm was assigned to the inner methylene protons (Fig. 1a). After the acylation reaction, the presence of the -CH 3 proton peak at 1.94 ppm in the 1 H NMR spectrum of Br-PEG 86 -Br (Fig. 1a) clearly confirmed the successful acylation reaction.
In addition to the characteristic proton peaks of the inner methylene and methyl protons, the characteristic proton peaks at 1.44 ppm clearly revealed the presence of t-butyl groups in PtBA-b-PEG 86 -b-PtBA (Fig. 1a). The peak area ratio of a to c was used to evaluate the polymerization degree to tBA, and the well-defined PtBA 68 -b-PEG 86 -b-PtBA 68 block copolymer was successfully obtained. According to the gel permeation chromatography (GPC) results (Fig. 1b) We made an important discovery that well-defined assemblies of PAA 68 -b-PEG 86 -b-PAA 68 were not obtained with lower amounts of MnCl 2 ·4H 2 O (1 and 3 mg mL −1 ), but amounts higher than 6 mg mL −1 produced well-defined vesicles. TEM images (Fig. 2a) showed that PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 exhibited a vesicular morphology with a diameter of approximately 30 nm, which was consistent with the results of the determinations using the high-angle angular dark-field scanning transmission electron microscopy (HADDF-STEM, Fig. 2b) and atomic force microscopy (AFM, Fig. 2d, e). Moreover, the HADDF-STEM image of PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 also revealed the vesicular morphology.
Notably, the AFM result showed that PAA 68 -b-PEG 86b-PAA 68 /MnO 2 displayed a closed form, similar to vesicles with a hollow structure. Especially in the DLS (Fig. 3a), importantly, the hydrodynamic diameter of the PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 architecture was much bigger than the size observed with TEM. This finding was attributed to the extreme stretch of hydrophilic PEG segments in the PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 architecture in the aqueous phase, whereas these vesicles collapsed during the TEM analysis.
As shown in Fig. 2f, the oxygen and Mn were uniformly distributed in the polymeric vesicles, indicating that Mn 2+ ions were transformed into MnO 2 . Moreover, the result agreed with the energy-dispersive spectrometer (EDS) analysis (Fig. 2g) and XPS results (Fig. 2h), further indicating the existence of MnO 2 . To confirm the chemical valence of elemental Mn, the high-resolution (HR) XPS spectra of PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 were analyzed and are presented in Fig. 2k. As shown in Fig. 2i-k, the C 1 s spectra of these samples were deconvoluted into three peaks that were assigned to a C-H/C-C band at 284.8 eV, C-O-C band at 286.7 eV, and C=O band at 288.2 eV, indicating the existence of a copolymer. The deconvolutions of the O 1s spectra of PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 indicated the obvious formation of MnO 2 in these polymeric architectures because of the special peaks at 530.6 eV. In the Mn 2p spectrum (Fig. 2k), two specific peaks at binding energies of 653.1 and 641.1 eV were observed, indicating the existence of Mn 4+ in MnO 2 [39]. Additionally, the HRTEM image of the PAA 68 -b-PEG 86 -b-PAA 68 / MnO 2 vesicle (Fig. 2c) exhibited characteristic lattice fringes with interplanar distance of 1.57 and 3.14 Å, also demonstrating the presence of MnO 2 [40]. Moreover, the ICP-MS used to detect the content of MnO 2 in the PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 vesicles revealed that it was approximately 4 wt%.
Based on these results, polymeric vesicles with a controlled morphology and stability were successfully fabricated using the self-assembly approach in an aqueous phase. Compared to polymerization-induced self-assembly [41], MnO 2 formation gradually made the soluble PAA block insoluble, which drove in situ self-assembly to form the MnO 2 -polymer polymeric vesicles. During the self-assembly, formation of MnO 2 was used to drive the phase separation of one of the blocks and further induce in situ self-assembly of block copolymers (PAA 68 -b-PEG 86 -b-PAA 68 ) to form the MnO 2 -polymer hybrids (PAA 68 -b-PEG 86 -b-PAA 68 / MnO 2 ). Consequently, we determined that the self-assembly process in water should be performed via three steps: (1) complete dissolution of the copolymer segments in the aqueous phase at the initial stage, (2) reduction in the solvability of the PAA/MnO 2 segments to drive the phase separation in the MnO 2 formation process, and (3)   These degradable products, which have received US Food and Drug Administration (FDA) approval, included PEG and PAA [43,44]. Other products include the as-released Mn 2+ ions that can be metabolized easier by the kidneys [35,36]. The transformation of MnO 2 to Mn 2+ ions could contribute to the disintegration of PAA 68 -b-PEG 86 -b-PAA 68 / MnO 2 (Fig. 3i), which would facilitate the wider application of polymer-inorganic hybrids as DDS, especially in cancer treatments where the delivery of anticancer agents is the focus.

Dual-Responsive Drug Release from DOX-Loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 In Vitro
Taking advantage of the electrostatic interaction in an aqueous phase [45], the chemotherapeutic agent (DOX), a broad spectrum anticancer drug with known cardiotoxicity [46], was encapsulated in PAA 68 -b-PEG 86 -b-PAA 68 / MnO 2 . The PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 polymeric vesicles showed a high drug encapsulation efficiency and drug loading capacity (both up to ~ 94%) derived from their abundant carboxyl groups. Moreover, the interpenetrating network between the copolymer and MnO 2 in PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 polymeric vesicles also probably contributed to the efficient loading of DOX. After DOX loading, the vesicular morphology of DOXloaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 vesicles was observed (Fig. 3g) and its D h (Fig. 3e) increased more than that of the PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 vesicles (Fig. 3a), suggesting the introduction of DOX molecules. The structural stability of DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 / MnO 2 was evaluated as shown in Fig. 3f, implying that DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 exhibited a favorable stability in PBS during 110 h. In addition, the Tyndall effect, which depends on the support of the corresponding dispersion system at different points in time, was observed as shown in Fig. 3f, demonstrating the excellent stability of DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 / MnO 2 .
After treatment with an acidic and reductive dual-sensitive trigger (pH 5.0 and 10 mM GSH), the dissociation of DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 was observed as shown in Fig. 3h, j, indicating the disintegration of DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 . The DOX release curve showed that only 6% drug cumulative release occurred under normal conditions, whereas the drug cumulative release proportion reached 22% and 50% with decreasing pH from 6.5 to 5.0 within 60 h (Fig. 4a). In addition, the introduction of 10 μM GSH only slightly increased DOX release compared to that at pH 7.4 as presented in Fig. 4a.
After treatment with GSH as high as 10 mM (a biologically relevant level [2-10 mM]) at pH 5.0 [18,36], the DOX release was obviously facilitated, and a passive release plateau was observed over 24 h. Importantly, the final cumulative release ratio of DOX culminated at 75% at pH 5.0 in the presence of 10 mM GSH as presented in Fig. 4a. When the feed concentrations of DOX were decreased from 1.00 to 0.25 or 0.50 mg mL −1 , a similar drug encapsulation efficiency of 95% or 93% was obtained. After DOX loading, the DOX 1 Fig. 4b. At each incubation time, the DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 exhibited a slow cellular release in contrast to that of free DOX as shown in Fig. 4c, indicating that the DOX-loaded DDS was first degraded before and then the drugs were unloaded.
The DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 unloaded almost all its cargos when the incubation time was increased to 12 h, which almost agreed with the accumulative release of DOX from DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 at pH 5.0 in the presence of 10 mM GSH as presented in Fig. 4a. The CLSM technique was used to detect the intracellular distribution of the as-released DOX molecules from DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 as visualized in MCF-7 cells. After incubation for 9 h, the obvious red fluorescence of DOX molecules was observed at the cell nucleus as shown in Fig. 4d, suggesting that the DOX molecule was more efficiently delivered to the cell nucleus by the PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 than the free DOX was (Fig. 4e). These results demonstrate that the polymeric vesicles responded to tumor acidity and reduction-triggered activation, achieving on-demand release of DOX.  similar tumor suppression to that in the DOX group. During treatment, the body weight of mice in the DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 group exhibited a similar trend with that of the PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 group (Fig. 5c). In contrast, after 31 days, the body weight of mice in the free DOX group was obviously reduced by approximately 24%, revealing the severe toxicity of free DOX (Fig. 5c). As shown in Fig. 5d, the tumor size of the DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 and free DOX group slightly increase from ~ 100 to ~ 220 and 290 mm 3 , respectively.

Cytotoxicity of DOX-Loaded
In contrast, the tumor size of the PAA 68 -b-PEG 86b-PAA 68 group obviously increases from approximately 100 to approximately 910 mm 3 . According to the histological analyses of the major organs, DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 did not induce obvious pathological changes in these tissues in contrast to the obvious pathological changes induced by the free DOX group as shown in Fig. 5e. Particularly, the liver and heart of the DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 group over came the cardiotoxicity of DOX. All the observations demonstrated that DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 / MnO 2 exhibited higher tumor suppression without known cardiotoxicity, most probably because of the unique structure of the MnO 2 -polymer hybrid vesicles.

Conclusions
In conclusion, a simple and efficient aqueous self-assembly strategy was investigated for the construction of polymeric vesicles. In this self-assembly process, the formation of MnO 2 not only induced in situ self-assembly of block copolymers, but also served as an interlocking agent to endow the polymeric vesicles with a fairly stable structure for the delivery process. Accordingly, the process of self-assembly is performed in three steps: (1) complete dissolution of the copolymer segments in an aqueous phase at the initial stage, (2) reduction in the solvability of the PAA block to drive the phase separation in the nucleation process, and (3) nucleation of PAA/MnO 2 segments to induce self-assembly of the copolymer chains at the final stage.
Compared to other polymeric architectures formed using traditional self-assembly strategies, the polymeric vesicles respond to GSH and weak acidic conditions at tumor sites to unloading the cargo and are further degraded to hydrophilic linear polymers and Mn 2+ ions after achieving drug delivery. DOX-loaded PAA 68 -b-PEG 86 -b-PAA 68 /MnO 2 vesicles have been demonstrated, especially in in vivo studies, to overcome the cardiotoxicity of DOX. Moreover, other metallic oxides (including zinc oxide and ferroferric oxide) may also be utilized to induce in situ self-assembly of block copolymers using this strategy. Therefore, the as-proposed self-assembly strategy could facilitate the widening of the fabrication of a variety of polymeric architectures, and we expect that some versatile polymeric systems will be crafted using this environmentally friendly approach.