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Biological and Medical Applications of Materials and Interfaces

Ternary-Responsive Drug Delivery with Activatable Dual Mode Contrast-Enhanced in Vivo Imaging Shuangshuang Ren, Jie Yang, Lan Ma, Xincong Li, Wenlei Wu, Chao Liu, Jian He, and Leiying Miao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10564 • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 5, 2018

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ACS Applied Materials & Interfaces

Ternary-Responsive Drug Delivery with Activatable Dual Mode Contrast-Enhanced in Vivo Imaging Shuangshuang Ren,‡,# Jie Yang,‡,# Lan Ma,† Xincong Li,† Wenlei Wu,‡ Chao Liu,§,* Jian He,⊥,* Leiying Miao†,* †

Department of Cariology and Endodontics, ‡Department of Periodontology, and

§

Department of Orthodontics, Nanjing Stomatological Hospital, Medical School of

Nanjing University, Nanjing 210093, China. ⊥

Department of Radiology, Nanjing Drum Tower Hospital, The Affiliated Hospital of

Nanjing University Medical School. Nanjing 210008, China

#

Those two authors contributed equally to this work.

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ACS Applied Materials & Interfaces 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

ABSTRACT: Designing a smart nanotheranostic system has recently attracted tremendous attention and is highly desirable for realizing targeted cancer therapy and early diagnosis. Herein we report the fabrication of smart nanotheranostic system using multi-responsive gatekeeping protocol of mesoporous silica nanoparticles (MSN). Acid, oxidative stress and redox sensitive manganese oxide (MnOx) coated superparamagnetic iron oxide nanoparticle (SPION) were employed as nanolids to regulate the camptothecin drug release from the channels of mesoporous silica and achieve responsive dual-mode MRI contrast. The nonvehicle showed high magnetization and T2 contrast in magnetic resonance imaging (MRI) due to the significant density of SPION onto the surface of MSN, and at the same time the MnOx shell degradation release Mn2+ which enhanced the T1 MRI visualization. The efficacy of responsive drug delivery system was investigated on pancreatic cancer cells and tumor-bearing mice, and results reinforced that MnOx-SPION@MSN@CPT nonvehicle is efficacious against cancer cells. We envision that our unique and multiresponsive nanoplatform may find applications in effective delivering of imaging and therapeutic agents to wide range of diseases besides cancer. Keywords: MRI, drug delivery, multi-responsive, mesoporous silica, manganese oxide


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1. INTRODUCTION Despite phenomenal advancement in early diagnosis and therapy, cancer treatment still have many challenges, among them is to design drug vehicles that can avoid systemic toxicity by targeted and controlled cargo release in response to the desired local environment.1,2 Development of burgeoning number of nanobased drug carrier can in this regard provide essential breakthroughs against cancer fight. Tumor sites microenvironments is distinctively different which can judiciously be exploited to realize triggered and controlled therapeutics release in response to the changes in inflamed and diseased tissues.3 Among the different physiological features, redox status and tumor acidity are quite different from normal ones, for instance, the concentration of glutathione, main intracellular reducing agent, is almost four times higher than that in normal tissues.4,5 Similarly, oxidative stress and low acidity of tumor site with pH about 7.2–6.5 are distinct characteristics, moreover after cellular uptake of nanoparticles pH value of early endosomes and lysosomes lowers to 6.2–5.0, thus providing opportunities to design smart drug delivery systems by exploiting oxidative stressed, redox and acidic environment.6 Over the years, diverse range of organic (polymers, liposomes etc.,) and inorganic smart drug delivery systems has been explored. Mesoporous silica nanoparticles (MSN) are among the most widely used smart nanovehicle for chemotherapeutic agents.7-9 Several mesoporous silica based smart stimuli responsive systems have been designed so far by particularly exploiting the redox homeostasis dysregulation and low pH of tumor.10-15 To realize redox responsiveness in



mesoporous silica systems, various inorganic nanoparticles (e.g., Fe3O4,16 CdS,17) and biomacromolecules (e.g., cyclodextrin,18-21 collagen,22 DNA23) were tethered through cleavable disulfide bond with mesoporous silica serving as the end-cappers of MSN-based stimuli-responsive drug delivery systems. However, a crucial issue in disulfide based systems is the complicated multi-step synthesis and high costs. In order to address these limitations, different nanolids dissolution in response to biostimuli based systems have also been reported. For instance, Zhu et al. applied Mn3O4 nanoparticles as mesoporous gate-cappers, and demonstrated that Mn3O4 nanolids could be dissolved into Mn2+ in tumor cells by reacting with endogenous metabolites (acidic and reducing environment).24 Recently, it has also been demonstrated in several studies that manganese oxide nanoparticles generate O2 bubbles when exposed to H2O2 oxidant and release Mn2+ ions,25 both oxygen bubbles and paramagnetic ions can enhance T1 magnetic resonance imaging (MRI) contrast. In addition to drug delivery, scientific community has also been attempting to integrate smart nanovahicles with imaging functionality to achieve multimodal imaging-guided therapy and tracking the outcomes. Among imaging techniques, MRI is the most powerful and robust imaging tool, and to date various nanobased MRI contrast agents have been explored to improve the contrast from the background.3,26 There are two types of MRI contrast agents, namely, T1-positive and T2-negative, both have their own pros and cons; therefore, combining the advantages of both positive and negative contrasts agents by designing a dual-mode T1–T2 contrast agents are highly desirable. One of the strategies is to combine paramagnetic


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T1-positive agent (e.g., Gd3+ and Mn2+ species) with magnetic T2-negative (e.g., superparamagnetic iron oxide) into one entity.27-31 To design a multi-responsive drug nonvehicle for effective cancer therapy and integration with a robust dual mode contrast agents for overcoming the disadvantages of single modality contrast agents, herein we developed an intelligent drug delivery system with activatable dual mode MRI contrast. In this system, superparamagnetic iron oxide nanoparticles (SPION) with ultra-thin layer of MnOx served as a gate-keeper for MSN, and upon exposure to low pH, reducing agent or oxidative stress environment, MnOx layer is rapidly disintegrated and thus SPION removes from MSN surface to achieve concurrent controlled drug release and enhance MR imaging.

2. EXPERIMENTAL SECTION Iron (III) acetylacetonate, Triethylene glycol (TEG),

2.1. Materials.

3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium

bromide

(MTT),

and

Manganese chloride (MnCl2•H2O) are purchased from Aladdin Reagent Company. Also,

rhodamine

6G,

Glutathione

(GSH),

1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide

Camptothecin hydrochloride

(CPT),

(EDC·HCl)

and are

bought from Sigma-Aldrich. 2.2. Instrumentation. The morphologies and detailed structure of the samples is recorded using JEOL JSM-6700F field-emission scanning electron microscope (SEM) and FEI Tecnai F20 transmission electron microscope (TEM).


A Rigaku X-ray


diffractometer (λ = 1.5418 Å) was used to analyze the XRD patterns. A Perkin-Elmer ICP-OES Optima 3300DV provided elemental composition while a Nicolet Impact 410 FTIR spectrometer gave Fourier transform infrared (FTIR) spectra (400-4000 cm−1). The nitrogen adsorption and desorption isotherms were measured at liquid N2 temperature by using a Quantachrom Autosorb-iQ2. Surface area was calculated according to the conventional Brunauer–Emmett–Teller (BET) method, and the adsorption branches of the isotherms were used for the calculation of the pore parameters using the BJH method. An ESCALAB 250 spectrometer displayed the X-ray photoelectron spectroscopy (XPS) data while a superconducting quantum interference device exhibited magnetic properties. 2.3. Synthesis of Water Dispersible SPION.

423.8 mg (1.2 mmol) of Iron(III)

acetylacetonate was introduced into 20 mL of TEG into a three-neck flask (100 mL), and stirred for 1 h at 140 °C until a form a transparent solution. The temperature was then quickly elevated to 200 °C and kept for 30 min, brownish slurry of SPION was produced. Acetone was used to separate SPION, and then washed with water and ethanol. In order to obtain water dispersible SPION, as-prepared SPION were dispersed into a saturated citric acid solution to modify SPION with –COOH. The mixture was sonicated for 30 min and isolated by thrice repeated magnetic separation to achieve a complete carboxylic acid functionalized SPION (SPION-COOH). The SPION-COOH was then redispersed into 5 mL of water and the solution was tuned into neutral (pH around 7) by adding 2M NaOH solution. 2.4. Synthesis of MnOx-SPION. A thin layer of MnOx-SPION was produced by


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adsorption-oxidation method. First, a saturated MnCl2 solution was dropwise added into 20 mg SPION-COOH (20 mL) solution until the brown transparent solution precipitated, indicating a complete adsorption of Mn2+ on the surface of SPION. After being separated by centrifugation, the precipitate of Mn2+ adsorbed SPION were redispersed into 20 mL water and then added 40 uL 2M NaOH and stirred the solution for 2h. After a complete oxidation of manganese hydroxide, a thin layer of MnOx was coated on SPION surface. Finally, 0.2 mg of citric solution was introduced into resulting MnOx-SPION to modify the surface with carboxylic acid for further use. 2.5. Loading, Capping and Release Experiments. Amine functionalized MSN (MSN-NH2) were prepared as described previously.32 In order to load cargo, MSN-NH2 powder (10 mg) was added into 1 mL DMSO with known amount of camptothecin or rhodamine 6G and the mixture was stirred overnight at room temperature. Camptothecin or rhodamine 6G loaded MSN was then centrifuged and wash two times with water to remove free CPT or rhodamine 6G. For capping experiment, CPT or rhodamine 6G loaded MSN solution was mixed with 25 mg EDC and 200 mg MnOx-SPION. After 10 min stirring, the precipitate was centrifuged and then dialyzed against distilled water for 2 days. To investigate the responsive properties of MnOx-SPION@MSN formulation in different stimulant circumstance, sample (10 mg) was dispersed in 10 mL of solutions with various concentrations of GSH, H+/H2O2 and at different pH solutions. The amount of unleased CPT/rhodamine 6G was collected at fixed time intervals and analyzed by UV/Vis spectroscopy at 365 nm.



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2.6. In Vitro and in Vivo MR Imaging. The MR imaging and relaxation times of the MnOx-SPION@MSN were tested by a MRI scanner (18.37MHz, 0.5T, MicroMR20-025V), for in vivo test a clinical MRI scanner system (23.315MHz, 0.55T, MesoMR-60) was used. With different concentrations of [Fe] aqueous samples, dispersed in 0.5% agarose gel, were placed in tubes. Representative images were then obtained by employing MicroMR20-025V. For T2 test: P90(us)=5, P180(us)=11, SW(KHz)=100, D3(us)=20, TR(ms)=18000, RG1=20, RG2=3, NS=2，Echo Time(us)=1000, Echo Count=18000. Relaxivity values of r2 were calculated by a series of T2 values, when plotted as 1/T2 according to different [Fe]. For T1 test: P90(us)=5, P180(us)=11, SW(KHz)=100, D3(us)=20, TR(ms)=18000, RG1=20, RG2=3, NS=2，NTI =36. Relaxivity values of r1 were obtained from T1 values and then plotted as 1/T1 according to different [Fe] concentrations. As to the in vivo MRI scans, a special mouse coil (23.315MHz) was utilized. The parameters used for T2 imaging were: FOVRead=100mm, FOV Phase=100mm, TR=1600ms, TE=55ms, Slices=5, NS=4, Slice Gap=0.6mm, Slice Width=2.5mm, K 192*256. The parameters used for T1 imaging were: NS=8, Slice Gap=0.6mm, Slice Width=2.5mm,

Slices=5,

TE=18.2ms,

TR=300ms,

FOVPhase=100mm,

FOVRead=100mm, K 192*256. 2.7. Cell Culture and in Vitro Cell Viability Assay. Pancreatic cancer cells (Panc-1) cultured in Medium (DMEM, Gibco) with a density of 8*103 cells per well were used to investigate the cell viability via MTT method. After 24 h incubation, the


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cells were co-cultured with various nanoparticle compositions for different incubation time. Another 4 h incubation was carried out after 10 µL per well of MTT solution was added. The color of precipitated formazan violet crystals reflected the cell growth condition. Neuro-2a cells (mouse neuroblastoma cell line) were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% heal inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin in a humidified 5% CO2 atm at 37 °C. Neurocytotoxicity was evaluated by MTT assay in Neuro-2a cells. Around 5*103 cells were seeded per well in a 96-well plate (total medium volume of 100 µL). After 24 h incubation, the cells were co-cultured with MnOx-SPION@MSN compositions of various concentrations by [Fe]. The MTT assays were performed after different incubation time. Cell without nanoparticles treatment was used as control. 2.8. In Vivo Antitumor Effect. 24 Pancreatic-tumor-bearing mice were split into three groups of eight for the in vivo antitumor effect investigation (saline group, CPT group, MnOx-SPION@MSN@CPT group). The tumor sizes and body weights were measured every 2 days and the relative tumor volume (RTV) was defined as RTV = V/V0, in which V0 represents the initial volume before treatment. On day 12 after treatment, tumors and major organs were isolated from the sacrificed mice for Hematoxylin and eosin (H&E) and Ki67 staining tests to further assess the antitumor effect. 2.9. Pathology Analysis.

The isolated organs including heart, lung, spleen, liver



and kidney were washed by PBS, fixed in 4% paraformaldehyde, embedded in paraffin. The sectioned tissues were then stained with hematoxylin and eosin (H&E) and their histopathologic changes were observed using an optical microscope (Olympus, Japan). 2.10. Hematology and Biochemical Assay. Hematology and biochemical assays were conducted by collecting mouse blood on day 7, day 15 and day 30 after the MnOx-SPION@MSN treatment or normal mice treated with nothing. Peripheral blood lymphocytes (LYM), blood urea nitrogen (BUN), monocytes (MON), platelets (PLT), behavioral approach system (BAS), alanine aminotransferase (ALT), aminopherase (AST), Red blood cells (RBC), serum globulin (GLB), eosinophils (EOS), alkaline phosphatase (ALP), and creatinine (Cr) were all monitored to assess the biocompatibility, in vivo toxicity, and the immune response. 2.11. Half-life detection. To determine the half-life, 16 Pancreatic-tumor-bearing mice were split into two groups with eight per group followed by i.v. injection of CPT and MnOx-SPION@MSN@CPT at a dose of 2.5 mg CPT/kg body weight. After that, Mice were sacrificed, and blood was harvested at different time intervals. CPT was extracted by methanol from the blood and the content was analyzed through the fluorescent data by employing HPLC (Shimadzu) equipment which had a RF-10A fluorescence detector. The fluorescence intensity was normalized and presented as percentage of dose/g. 2.12. Statistics. Data were analyzed with One-way ANOVA (SPSS software, Version 13.0). It was considered statistically significant when P < 0.05 in


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corresponding figures

3. Results and discussion 3.1. Characterization of NPs. The protocol of nanotheranostic system is illustrated in Figure 1. MSN based drug nonvehicle was first synthesized using previously reported method, and then functionalized with amine moiety through both co-condensation and grafting methods to improve the water stability and provide anchoring sites for nanolids. At the same time, we developed and optimized a synthetic strategy to obtain nonaggregated, monodisperse superparamagnetic iron oxide γ-Fe2O3 nanoparticle (SPION) with uniform particles size of 4 nm (Figure 2A). The surface of SPION was functionalized with citric acid. The presence of abundant carboxylate groups on surface of SPION later ensured the adsorption of manganese ions, which in turn facilitated the generation of ultra-thin manganese oxide layer onto SPION (MnOx-SPION). No extra aggregated MnOx impurities were found in low resolution TEM image (Figure S1). Notably, the morphology and size of SPION also remained unchanged following the coating of manganese oxide due to the ultra-thin nature of MnOx layer, shown in Figure 2B. The presence of MnOx layer was established by ICP and XPS analysis. While the surface chemistry of SPION, MnOx-SPION was investigated by infra-red (IR) and X-ray photoemission spectroscopic (XPS) techniques. Figure 2C presents the IR spectra of as-obtained SPIONs and citric acid functionalized SPION. The shifted vibration broad carboxyl band (C=O) of 1618 cm-1 in citric acid modified SPION validates the chemisorption of citric acid onto SPION surface.33 In XPS analysis (Figure 3), compared to as-prepared SPION, the oxidized Mn species are clearly discernible in the survey spectra of MnOx-SPION. Both inductively coupled plasma (ICP) and XPS analysis revealed the atomic ratio between Fe-Mn was 10:1. The modified MnOx-SPION was then used as nanocap to block the drug loaded MSN. The obtained nanocomposite is proposed to have targeted release properties due to the existence of redox, pH and H+/H2O2 sensitive MnOx layer. It should be remembered that SPION themselves are ACS Paragon Plus Environment


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quite stable against acidic or redox environment, actually the existence of MnOx shell imparts the pH or redox responsive functionality. To explore how this capped MSN system operate, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were first used to investigate the successes of the capping system. As-synthesized MSN revealed smooth surfaced nanoparticles (100 nm), as shown in SEM micrograph, whereas TEM images indicated 2 nm mesoporous channels for drug loading (Figure 4). After MnOx-SPION capping, it can be clearly observed in Figure 5A-C that numerous nanodots thoroughly covered the mesoporous channels and as a result scabrous surface can be clearly seen compared to original MSN. The MnOx-SPION is sufficiently significant to block the drugs from leaching out. Elemental mapping images (Figure 5D) show the distribution of Si, Fe and Mn compenents, revealing the coexistence of Si, Fe and Mn, in a one single MnOx-SPION capped MSN nanoparticle. During X-ray diffraction (XRD) analysis, the intensity of MSN type peak in small angle range is markedly reduced in drug loaded nanocarrier owing to the contrast and disordered product (Figure 6A). Figure 6B shows the XRD patterns of

SPION, MnOx-SPION

and MnOx-SPION@MSN, four characteristic peaks (2θ≈30.2°, 30.57°, 57.4° and 63.01°), marked by their indices [(2 0 6), (1 1 9), (1 1 15) and (4 0 12)] are observed and further validate the conjugation of SPION onto the surface. All diffraction peaks including the positions and relative intensities matched well with those from references for magnetite. The broadness of these peaks indicates the ultrasmall nature of SPION which is highly desired as nanolids for MSN. Moreover, the absence of MnOx related peak in MnOx-SPION patterns inferred the generation of ultrathin layer


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of MnOx onto SPION. Nitrogen adsorption analysis evaluate materials in terms of surface area and pore volume, therefore, we have also carried out nitrogen adsorption−desorption study to determine the effect of drug loading and pore filling on surface area. N2 sorption isotherms of different MSNs formulations were plotted in Figure S2. The BET surface area is decreased from 812.2 cm2 g−1 to 701.5 cm2 g−1 after CPT loading step, whereas the pore size likewise reduced from 2.66 to 2.49 nm respectively, indicating the successful drug loading. The capping step expectedly further lowered the surface area to 212.4 cm2g−1, and pore size distribution widened due to the generation of disordered mesoporous texture after MnOx-SPION capping. 3.2. Response and Release Experiments under Tumor-Like Conditions. As reported previously, manganese oxide MnOx nanoparticles are prone to the reductive and mildly acidic environments. Thus it is envisaged that thin layer of MnOx can readily be disintegrated under tumor-like conditions, and thus unleashing the dye or drugs which are loaded into silica channels. Tumor microenvironment and intracellular environment features low pH, reductive environment with higher concentrations of GSH and H2O2 contents than that in normal tissues. In order to imitate the tumor microenvironment and assess drug release, PBS buffer solutions with different pH, GSH and H+/H2O2 concentration were utilized for detailed biodegradation assays. PBS buffer (pH 7.4) played a role in simulating normal body conditions for comparison. In this study, the triggered and controlled drug release of poorly water-soluble antitumor drug (CPT) was monitored in various imitative tumor environments (Figure 7). At the normal condition (absence of any reducing agents, pH 7.4, Figure 7 A), a flat baseline with less than 10% CPT leaching was observed from drug formulation. In all systems, fast release of CPT was noticed in the first 5 h which was actually caused by physical adsorbed surface bound CPT. With the introduction of GSH even at lower amounts, gradual uncapping of the MSN channels started and led to slow release over 96 h. In higher stimulant concentrations, the uncapping speed ACS Paragon Plus Environment


was accelerated by the fast dissolution, with the passage of time a higher release profile trend similar with uncapping one was observed (shown in Figure 8A), suggesting the success of responsive controlled release system. At acidic simulation solutions, the similar release performance was also seen (Figure 7B). Faster and greater amount of drug was unleashed in acidic and oxidative stressed environ, even a higher release speed than pH=5.5 buffer solution was noticed at acidic H2O2 (pH 6.0) buffer (Figure 7C), such phenomena can be explain by acid accelerated the dissolution of MnOx layer in H2O2. Manganese oxide is already reported susceptible to oxidative stress and can act as nanoenzyme (such as catalase, SOD, HRP etc.) against various oxidants.25,34 Drug release data indicated the dissolution of MnOx in various simulated solutions, and this dissolution process was monitored using ICP tests. As shown in Figure 8B, in contrast to neutral and none-reductive PBS environment, the release of manganese ions is obviously augmented under reductive and mildly acidic solutions. Similarly, Fe ions dissolution was also tested to by ICP analysis (Figure S3), and as expected no obvious Fe ions leaching was detected under simulated tumor environment conditions. Thus we safely assumed that the thin layer of MnOx is actually the key factor to control the capping and uncapping phenomenon. Dissolution of MnOx layer causes the removal of SPION from the MSN surface in different simulation solutions, as revealed in TEM images as dark dots (notated with red arrow) removal from MSN surface (Figure 7D-F). 3.3. In Vitro and in Vivo MR Imaging. We evaluated the contrast generating ability of MnOx-SPION capped nanocarrier with changing concentrations. Magnetic moment curves measured for SPION, MnOx-SPION and Mn-SPION@MSN did not show magnetic hysteresis with zero coercivity (Figure 9A). All sample of SPION, MnOx-SPION and MnOx-SPION@MSN displayed good magnetization saturation values with 46.7, 42.8 and 56.1 emu/g, respectively. Notably, the assembled SPION on MSN exhibit significant higher magnetization saturation values than individual


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nanoparticles.35,36 The longitudinal relaxivity of MnOx-SPION@MSN was examined at 0.5 T in 37 °C, and the relaxivity value (r2) was calculated according to transverse relaxation time (T2), which came out to be 102.2 mM−1s−1. It is assumed that after exposing MnOx-SPION@MSN to mildly acidic (pH=5.0), reducing and oxidizing agents (H2O2, GSH), which are usually in high concentration in tumor compared to those in normal tissue, a positive contrast could be well observed. The relaxivity value (r1) was found to be 13.57 mM-1s-1 (Figure 9B). According to previous reports, sub-10nm SPIONs themselves can also furnish T1 MRI signals but with a relative low relaxivity values (r1 less than 5 mM-1s-1).37, 38 Thus, we deduce that the higher r1 value in our system can only be elucidated by leached manganese ions from MnOx thin layer.

Also,

both

of r1 and r2 value

of nanotheranostic system

(MnOx-SPION@MSN) are comparable with that of commercially available contrast agent (T2 MRI contrast agent r2 value of ferumoxtran (comibidex) is 65 mM−1s−1, ferumoxide (feridex) is 120 mM−1s−1. T1 MRI contrast agent r1 value of Magnevist is 5.6 mM−1s−1, DOTA-Gd3+ of 3.8 mM−1s−1) suggest the clinical applicability as T1 and T2 contrast-enhancement agents than those currently available. Furthermore, animal MRI test was performed by in vivo injection of MnOx-SPION@MSN formulations into the vein of the mouse’s tail. Series of T1 and T2 images can be seen in Figure 9C. In comparison with pre-contrast images, with increasing time an obvious T2 weighted contrast signal enhancement in kidney was observed, it approached the peak within 30 min. For T1 enhancement experiment, the peak signal appeared later compared to T2 test, the delayed signals are actually in agreement with the slow dissolution of the



MnOx thin layer. After 5 hours, the enhanced signals from the organ of the mice back to its original level. These nanomaterials were well tolerated by the mice. No apparent side effects were observed during the injection, immediately or days after the experiment. Indeed, 100% survival of the animals was achieved after more than 1 month, the indicating the potential utility of our system. 3.4. In Vitro Cell Viability Assay. Considering the fact that a good biocompatibility of nanomaterials is the precondition for clinical applications, MTT assay was performed to assess the feasibility of MnOx-SPION@MSN@CPT against Panc-1 cancer cells. Firstly, the cytotoxicity of SPION, MnOx-SPION and MnOx-SPION@MSN were compared (Figure 10A-C). Still 83% cells survive despite the concentration of iron up to 200 µg mL−1, and incubation time from 24h to 72h, thus validating the fact that all formulations without anticancer drug feature low cell cytotoxicity. Subsequently, we assessed the cytotoxicity of free CPT, MSN@CPT and MnOx-SPION capped MSN@CPT in various CPT concentrations and incubation time, both CPT and MnOx-SPION@MSN@CPT depicted dose-dependent cytotoxicity and effective inhibition of the growth of cancer cells. Comparing with free CPT groups, all MSN@CPT and MnOx-SPION@MSN@CPT groups exhibited much higher cytotoxicity (Figure 10D-F). The enhancement in anticancer efficacy of free hydrophobic CPT can be ascribed to hydrophilic carrier of MSN. Compared to MnOx-SPION@MSN@CPT group, MSN@CPT showed apparently higher toxicity after 24 h incubation, but identical toxicity is observed in both groups in prolonged incubation (3-4 days), implying the controlled and highly desired site specific targeted


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drug release of water insoluble CPT. Although trace amount of manganese is essential in humans, however overexposure to free Mn ions may result in neurotoxicity.39 Thus, we examine the neurotoxic effect of MnOx-SPION@MSN formulation using Neuro-2a cells (mouse neuroblastoma cell line) (Figure S4). After 6, 12, 24 h incubation, MnOx-SPION@MSN had no significant cytotoxic effects up to a Fe concentration of 200 µg/mL, manganese concentration of 20 µg/mL, and SPION@MSN concentration of nearly 1000 µg/mL. As shown in Figure 10G-I, confocal fluorescence microscope was used to visualize the release performance of rhodamine 6G, as a model dye loaded inside the MnOx-SPION@MSN nanocarrier, inside the cancer cells. A negligible staining of cancer cells was noticed after four hours incubation (Figure 11), however, prolonged incubation time (24h) ensured the more release of rhodamine 6G most likely due to the controlled release from nanocarrier.. Collectively, cell micrographs verify the slow dissolution of the MnOx layer and the subsequently concurrent release of drug. 3.5.

In

vivo

antitumor

effect.

Having

demonstrated

that

MnOx-SPION@MSN@CPT NPs had a highly efficient in vitro anti-tumor effect, we next investigated the efficacy of the in vivo MnOx-SPION@MSN@CPT treatment. The injection of saline was regarded as the negative control group while the injection of the CPT was used as the positive control group. As exhibited in Figure 12A, an unbridled growth of tumor tissues was observed in the saline group, and the tumor volume on day 12 almost increased to approximately 7 fold compared with day 0. While, alone CPT treatment was also not effective enough to inhibits the growth because the hydrophobic nature of drug hinders the cellular uptake. On the contrary, we observed that our nanoformaltion (MnOx-SPION@MSN@CPT) was effective in controlling the growth of tumor volume. Most likely, the stimuli responsive



functionality and improved cellular internalization of MnOx-SPION@MSN@CPT led to marked decreases in the tumor volume, indicating efficacy of our system against tumor, compared to CPT alone. Besides, no obvious changes happened in the mice body weight through the whole course regardless of the different treatments (Figure 12B), giving a solid evidence that MnOx-SPION@MSN@CPT had no acute toxicity. H&E and Ki67 stainings were further employed to evaluate the therapeutic outcome after the different treatments. Tumor tissues from the mice treated with CPT only or MnOx-SPION@MSN@CPT, both groups displayed obvious apoptosis and necrosis. Nevertheless, the degree of apoptosis and necrosis was the largest in the MnOx-SPION@MSN@CPT treatment (Figure 12C and 12D). Therefore, we concluded that the MnOx-SPION@MSN@CPT as the hydrophobic drug nonvehicle could have a significant therapeutic effect on tumor therapy.

3.6. In Vivo Long-Term Safety Evaluation. The in vivo cytotoxicity to normal tissues was evaluated by H&E staining for major organs. As indicated in Figure 13, compared with the control group, no apparent pathological changes, including necrosis, fibrosis, and hydropic degeneration, were observed in any organs isolated from MnOx-SPION@MSN@CPT-treated mice. These results confirmed the good biocompatibility of MnOx-SPION@MSN. The half-life of free drug and MnOx-SPION@MSN@CPT was also evaluated as displayed in Figure S5, the half-life of CPT and MnOx-SPION@MSN@CPT was found to be ~35 min and ~134 min, respectively, obviously demonstrating that MnOx-SPION@MSN encapsulation could enhance the half-life of CPT in blood, which in turn greatly benefits the tumor accumulation of CPT via the EPR effect. Hematology and biochemical assays were conducted by collecting mouse blood


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on day 7, day 15 and day 30 after the chemotherapy treatment rendered by MnOx-SPION@MSN@CPT or untreated normal mice. Negligible changes were found in the serum levels of BUN, TBIL, PLT,

ALT, TP, and CRE which were

associated with liver, kidney, and spleen function (Figure 14A and 14B) after the treatment compared with control group, demonstrating that the chemotherapy treatment rendered by MnOx-SPION@MSN@CPT did no harm to metabolism. The aerobic and hematopoietic capacity was analyzed by recording the counts of HB, WBC, and RBC (Figure 14C). Similarly, no detective changes over the period of the chemotherapy treatment rendered by MnOx-SPION@MSN@CPT were witnessed compared with control group. Figure 14D showed the counts of NEU, MON, and LYM which were used to evaluate the potential immune responses. We could find that the values on 7, 15, and 30 days after the chemotherapy treatment rendered by MnOx-SPION@MSN@CPT were similar to those in normal mice, implying negligible immune response had been aroused. Taken together, we can conclude that MnOx-SPION@MSN@CPT as a drug delivery nanocarrier hold a promising potential for clinical applications.

4. CONCLUSIONS We have developed a muti-responsive nanotheranostic system, wherein manganese oxide covered magnetite acted as T1 and T2 MRI contrast and nanolid to seal the drug loaded mesoporous silica for achieving multi-responsive drug delivery and MRI contrast enhancement at the same time. The thin layer of manganese oxide



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which is attached onto magnetite surface played a key role in this smart nonvehicle, on one hand, it worked as a vulnerable bridge between MSN and SPION to achieve uncapping in response to multiple endogenous triggers such as redox, acidic and oxidative stress. On another hand, tumor-responsive biodegradation of manganese oxide generated sufficient Mn ions which ensured greater T1 MRI contrast. The results of TEM, EDS, XPS, magnetization curves, and MRI verified the compositions, structures,

and

physicochemical

properties

of

MnOx-SPION@MSN@CPT.

Hematological assay and MTT demonstrated the good biocompatibility of theranostic nonvehicle

(MnOx-SPION@MSN).

In-Vivo

investigations

of

pancreatic

tumor-bearing mice with MnOx-SPION@MSN@CPT led to partial or even complete regression of tumors due to tumor microenvironment associated triggered release of poorly water-soluble CPT molecules. We think that our devloped nanoarchitecture which is capable of simultaneous responsive controlled drug release and enhanced magnetic resonance imaging for diagnosis and tracking the feedback of therapy can be a potential candidate for personalized medicine.

ASSOCIATED CONTENT Electronic Supplementary Information (ESI) available: Large scale TEM images of MnOx-SPION. Nitrogen adsorption−desorption isotherms of MSN, CPT loaded MSN (MSN@CPT) and MnOx-SPION capped MSN@CPT (MnOx-SPION@ MSN@CPT). The curve of pore size distribution. Inductively coupled plasma (ICP) analysis of [Fe] leaching after subjecting MnOx-SPION@MSN formulation in different concentration


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of stimulant. Neuro-2a cells (mouse neuroblastoma cell line) viability of MnOx-SPION@MSN nano-formulations. The half-life period of CPT with or without the encapsulation in MnOx-SPION@MSN.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (C. Liu); *E-mail: [email protected] (J. He); *E-mail: [email protected] (L. Y. Miao). ORCID Leiying Miao: 0000-0001-6915-8218 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by grants from the National Natural Science Foundation (81570952, 51772144), the Natural Science Foundation of Jiangsu Province (No. BK 20170143, BK20161114), the Major Project of Nanjing medical science and technology development project (No. 16008), the Medical special project of Nanjing science and Technology Commission (201605044).



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Figure 1. Schematic illustration for the synthesis of MnOx-SPION capped MSN and controlled drug release in response to tumor microenvironment.



Figure 2. TEM images of SPION, before (A) and after (B) MnOx layer coating. (C) FTIR spectra of SPION and citric acid functionalized SPION (SPION-COOH).


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Figure 3. (A)-(D) XPS spectrum of MnOx-SPION nanocomposite. (A) Full survey spectrum, (B) O 1s peak, (C) Fe 2p peak, (D) Mn 2p peak.



Figure 4. (A) SEM and (B) TEM micrographs of bared MSN.


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Figure 5. Morphological analysis of capping system. (A) SEM , (B) TEM and (C) HRTEM micrographs of MnOx-SPION capped MSN. (D) Typical EDS mapping with STEM-HAADF image and corresponding elemental maps of Si-K, Fe-K, and Mn-K.



Figure 6. (A) Low-angle X-ray diffraction (XRD) patterns of MnOx-SPION@MSN and bared MSN. (B) Wide-angle SPION, MnOx-SPION and MnOx-SPION@MSN and pattern. The black bar shown in (B) is standard pattern of bulk magnetite γ-Fe2O3 (PDF#25-1402).


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Figure 7. (A-C) Illustration of multi-responsive drug release from MnOx-SPION@MSN nanocarrier. Release profiles of nanocarrier when incubated in different concentration of GSH, acid and H+/H2O2. PBS media of pH=7.4. (D-F) The corresponding TEM micrographs of MnOx-SPION capped with MSN, after incubation in 0.5 mM GSH (D), pH=5.5 (E), and 0.2 mM H+/H2O2 (F) solutions.



Figure 8. Release profiles of uncapped CPT loaded MSN. (B) Inductively coupled plasma (ICP) analysis after subjecting MnOx-SPION@MSN formulation in different concentration of stimulant.


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Figure 9. Magnetism analysis of MnOx-SPION@MSN. (A) The room temperature hysteresis loop of SPION, MnOx-SPION and MnOx-SPION@MSN nonvehicle. (B) T2 and T1 weighted MRI images of various iron concentrations. (C) In vivo T2 and T1 MRI images of a mouse after the injection of a MnOx-SPION@MSN solution through tail vein.



Figure 10. Cell Viability of formulations. Panc-1 cells treated with SPION (A), MnOx-SPION (B), without drug loaded MnOx-SPION@MSN (C), free CPT (gray), MSN@CPT (blue) and drug loaded MnOx-SPION@MSN (cyan) (D-F) incubation time in various days. Three separate experiments were conducted, *p < 0.05. (G-I) Confocal fluorescence microscope images of dye (rhodamine 6G) loaded MnOx-SPION@MSN incubated in Panc-1 cancer cells for 24h.


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Figure 11. Confocal fluorescence microscopy images of dye (rhodamine 6G) loaded MnOx-SPION@MSN when incubated with Panc-1 cancer cells for 4h.



Figure 12. (A) Tumor volume curves and (B) Body weight curves of Panc-1 tumor-bearing mice after undergoing different treatments as exhibited. * p < 0.05, ** p < 0.01. (C) H&E

staining images of pancreatic tumor sections isolated from mice receiving different treatments. Scale bars: 50 µm. (D) Representative IHC for Ki67 staining images of pancreatic tumor sections isolated from mice receiving different treatments. Scale bars: 50 µm.


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Figure 13. H&E staining images for different organs collected from the mice after the

chemotherapy treatment rendered by MnOx-SPION@MSN@CPT or untreated normal mice.



Figure 14. Hematology study and serum biochemical assay conducted on the mice 7, 15 and 30 days after the chemotherapy treatment rendered by MnOx-SPION@MSN@CPT or

untreated normal mice. (A) Counts of total protein (TP), total bilirubin (TBIL), and alanine aminotransferase (ALT). (B) Counts of platelets (PLT), blood urea nitrogen (BUN), and Creatinine (CRE). (C) white blood cells (WBC), hemoglobin (HB), and Counts of Red blood cells (RBC). (D) Counts of neutrophils (NEU), lymphocyte (LYM), and Monocyte (MON).


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