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photoluminescence imaging-guided photodynamic therapy by multiple pathways
Biomaterials 199 (2019) 52–62
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Enhancing magnetic resonance/photoluminescence imaging-guided photodynamic therapy by multiple pathways
T
Pei Liua, Jinghua Rend, Yuxuan Xionga, Zhe Yanga, Wei Zhua, Qianyuan Hea, Zushun Xua,∗, Wenshan Heb,∗∗, Jing Wangc,∗∗∗ a Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry of Education Key Laboratory for the Green Preparation and Application of Functional Materials, Hubei University, Wuhan, Hubei, 430062, China b Department of Breast and Thyroid Surgery, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei, 430030, China c Department of Radiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China d Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Endogenously biosynthetic photosensitizer Mitochondria Dual-imaging nanoplatform
Mitochondria, which are a major source of adenosine triphosphate (ATP) and apoptosis regulators, are the key organelles that promote tumor cell proliferation, and their dysfunction affects tumor cell behavior. Additionally, mitochondria have been shown to play a central role in the biosynthesis of protoporphyrin IX (PpIX), which is a widely used photosensitizer that has been used for tumor detection, monitoring and photodynamic therapy. Nevertheless, photosensitizers administrated exogenously are often restricted by limited bioavailability.δAminolevulinic acid (δ-ALA) is a naturally occurring delta amino acid that can be converted in situ to PpIX via the heme biosynthetic pathway in mitochondria. Because δ-ALA is the precursor for PpIX, δ-ALA-based photodynamic therapy (PDT) shows promise in treating cancer. However, the accumulation of δ-ALA within endosomal system limits the production of PpIX and eventually impedes its effectiveness. Theranostic nanoparticles (NPs) capable of endosomal escape are expected to optimize the endogenous biosynthetic yield. In this study, δ-ALA was improved with triphenylphosphoniumcation (TPP+), a high net position cation that functions in endosomal escape and as a mitochondria-targeting ligand, and was further modified with bovine serum albumin stabilized manganese dioxide (MnO2). The tumor microenvironment (TME) responsive MnO2 in this system can elevate oxygen content to relieve hypoxia. Both enhanced photosensitizer yield and elevated oxygen contributing to the final therapeutic effect. Moreover, the enhancement of magnetic resonance imaging (MRI) (r1 = 5.410 s−1mM−1) stemming from the degradation of MnO2 by the TME could serve as a guide prior to treatment for accurate location, while in situ hysteretic photoluminescence imaging derived from PpIX can be utilize as a supervisor for prognosis evaluation. This systematic design could broaden the biomedical application and highlight the considerable therapeutic promise of PDT.
1. Introduction Rapid-cell proliferation is a distinctive characteristic of solid tumor that require a compatible level of ATP, which is mainly produced by the mitochondria [1]. This demand for ATP results in uncommon vitality in mitochondrial physiology [2,3]. Additionally, mitochondria are crucial regulators of the intrinsic pathway of apoptosis [4]. Both of these characteristics suggest that dysfunctions of mitochondria can be lethal to tumor cells. From another perspective, tumor cells are heterogeneity; they differ from patients, even within the same lesion of an individual
patient [5]. In fact, initial tumorigenesis and eventual metastasis is not determined exclusively by genetic alteration. However, the initial tumorigenesis and eventual metastasis involve the reciprocal influence between mutations and the state of the stromal microenvironment [6]. A high degree of angiogenesis in solid tumor accounts for nutrient deficiency [7], hypoxia [8] and acidification [9]. Tumor cells within a unique TME differ from normal tissue, and this unique environment is eventually responsible for failure of some treatments due to therapeutic resistance [10]. Therefore, it is necessary to develop nanoplatforms that target the tumor milieu to enhance therapeutic outcome.
∗
Corresponding author. Corresponding author. ∗∗∗ Corresponding author. E-mail addresses: [email protected] (Z. Xu), [email protected] (W. He), jjwinfl[email protected] (J. Wang). ∗∗
https://doi.org/10.1016/j.biomaterials.2019.01.044 Received 11 September 2018; Received in revised form 21 January 2019; Accepted 30 January 2019 Available online 02 February 2019 0142-9612/ © 2019 Elsevier Ltd. All rights reserved.
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δ-Aminolevulinic acid (δ-ALA), a biological endogenous molecular that is widely used as a non-photoactivatable clinical molecule approved by U.S Food and Drug Administration [11], has been used mostly as a biological precursor of the protoporphyrin IX (PpIX) via the cellular heme biosynthesis pathway for photodynamic therapy [12]. Compared to other synthetic photosensitizers [13,14], δ-ALA is distinct because of its easy operation, biosecurity and non-photobleaching characteristic due to its endogeneity. Because of the negative feedback mechanism that causes free heme to repress endogenous production of δ-ALA [15], it is more favorable for exogenous administration of δ-ALA to synthesize PpIX for intracellular accumulation as result of bypassing it. Moreover, this accumulation is more pronounced in malignant cells than in their normal counterparts in vitro and in vivo [16]. Indeed, δALA itself has some limitations on photodynamic therapy, such as poor penetrability of the lipidic barrier due to high polar characteristics and zwitterionic nature [15]. Many improvements have been made to δALA. Ma et al. [17] designed a multifunctional hollow mesoporous silica coating on δ-ALA for treatment against superficial cancer cells. Shi et al. [18] fabricated δ-ALA loaded PLGA nanoparticles that targeted human squamous skin cells. Nevertheless, under normal physiological condition, δ-ALA was transported to coproporphyrinogen III under various enzymolytic mechanism in the cytosol, and the photosensitizer agent PpIX was transported to mitochondria in the presence of oxidase [16]. After nanoparticles passively accumulate in tumor sections via enhanced permeability and retention (EPR) effects, they are usually taken up by endocytosis and enclosed within an early endosome. These vesicles that are loaded with nanoparticles mature to become late endosomes that gradually fuse with lysosomes [19]. This endosomal pathway in cells limits the adequate accumulation of precursors to mitochondria. Because of the inadequate endogenous production of PpIX, drug resistance and weakened anticancer therapy occur sequentially. Therefore, it's essential to increase the bioavailability of δ-ALA to enhance photodynamic therapy. Finally, visualization of a therapeutic system is of great significance for scrutiny and guidance to improve therapeutic outcome [20]. Considering both time-consuming biosynthesis and the fluorescent property of PpIX, we speculate the fluorescence imaging can be used to supervise prognosis evaluation. To accurately locate NPs at tumor regions prior to treatment, another auxiliary imaging technique should be used for more accurate guidance. Among all the diagnose methods, MR imaging stands out as a clinical noninvasive and excellent 3D spatial resolution diagnose technology for soft tissue detection [21]. Manganese is a considerable candidate as a potential T1 contrast agents for MRI. Additionally, some studies [22,23] have recently reported that −MneO− bond is sensitive to conditions in the TME, such as reducing or acidic conditions, and can be utilized to deliver therapeutic agent effectively. In addition, the catalytic reaction between MnO2 and endogenously abundant H2O2 in tumor area to produce oxygen could relieve hypoxia condition, contributing to the promotion of therapeutic effect. Therefore, a smart design for the δ-ALA-based system was demonstrated as dual imaging-guided enhanced photodynamic therapy. First, dual-model imaging using in this system can provide complete monitoring prior to treatment and prognosis evaluation. Second, based on our design, the multi-responsive MnO2 in the TME guarantees effective accumulation in the tumor region. Enhanced precursor accumulation in tumor cells could further elevate the endogenous biosynthetic yield of photosensitizer, thereby promoting the bioavailability. Finally, with increased oxygen and sufficient photosensitizer, the therapeutic efficacy can be enhanced at a significant level.
(GSH), (3-Carboxypropyl) triphenylphosphonium bromide (CTPP), Nhydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), Fluorescamine, were purchased from Aladdin Reagents Co. Ltd. Cell Counting Kit-8 (CCK-8) was obtained from MedChemExpress. CM-H2DCFDA, MitoTracker™ Green FM, LysoTracker™ Green were obtained from Thermo Fisher Scientific. JC10 Mitochondrion Membrane Potential Assay Kit was brought from AAT Bioquest. Hoechst 33258, Enhanced ATP Assay Kit, Annexin VFITC Apoptosis Detection Kit were obtained from Beyotime Ltd. Minimum essential medium (MEM) was brought from Invitrogen Corp. 2.2. Instrument Material and Regions. Transmission electron microscopy (TEM) images were obtained by Tecnai G20, FEI Corp. USA. Nanosize was measured by ZetaSizer Nano series Nano-ZS (Malvern Instruments Ltd, Malvern, UK). UV–vis spectra (UV–vis spectrophotometer, 1800 PC, Mapada, China) were used to identify the characteristic Peaks. The functional groups were determined by FTIR (Nicolet 570). Fluorescence spectra were tested by fluorospectro photometer (F-2500, HITASCHI). The X-ray photoelectron spectroscopy (XPS) measurement was conducted on Thermo Fisher Scientific 250Xi. The cell viability was measured using a micro-plate reader (1420 multilabel counter, Perkin Elmer, MA, USA). Confocal laser scanning microscope (CLSM) images were obtained from ZEISS LSM 710 (Germany). Clinical Siemens Magnetom Trio 3.0 T MR scanner was used to evaluate the MRI property. Small animal fluorescence imaging was carried on Bruker In-Vivo optical imaging system (Billerica, USA, Lago X). 2.3. Preparation of the TarA@bM, NTarA@bM, TarA@bN and BM First, 100 mM of δ-ALA solution was prepared as follows: 50.28 mg δ-ALA (HCl) was dissolved in 3 mL PBS, the pH of solution was adjusted at 7.0 by 5 M NaOH. Subsequently, 0.22 μm filter was used to degerm for store in a certain period. 10 mg CTPP was dissolved in 200 μL DMSO, 4.5 mg EDC and 3 mg NHS was added in sequence and mixed for 2 h at room temperature. Then, 200 μL 100 mM δ-ALA was added for another 1 h to obtained the TarA. 10 mL 100 mg BSA was added in the above-mentioned solution for 6 h. Finally, 2 mL of 0.03 M MnCl2 4H2O was added dropwise, 1 M NaOH was used to adjust the pH at 10 for 4 h to obtain the final TarA@bM. Correspondingly, NTarA@bM and TarA@ bN were synthesized as control nanoparticles. Briefly, NTarA@bM was synthesized as the same procedure as TarA@bM's but without chemical combination with CTPP. TarA@bN was obtained with the same procedure as TarA@bM's before add MnCl2 4H2O. After that, the obtained solution was reacted with glutaraldehyde (2.5%) to acquire cross-linked nanoparticles. All the ultimate productions were dialyzing in a dialysis bag (MWCO 100 kDa). Additionally, TarA was dialyzing against ultrapure water in a dialysis bag (MWCO 100) and frozen, lyophilized. Mass spectrum (Fig. S6): The m/z peak was 462. 1H NMR (600 MHz, DMSO, Fig. S7):7.95–7.75 (m, 15H), 4.00–3.95 (s, 2H), 3.65–3.55 (dt, 2H), 3.50–3.40 (dt, 2H), 2.79–2.73 (dt, 2H), 1.79–1.70 (dt, 2H). The BSA stabilized MnO2 (BM) was synthesized as follows: 2 mL of 0.03 M MnCl2 4H2O was added into 10 mL of 100 mg BSA solution dropwise, 1 M NaOH was used to adjust the pH at 10 for 4 h, and finally were dialyzing in a dialysis bag (MWCO 100 kDa) to obtain the final BM. 2.4. Detection of stimuli-responsive properties The release of δ-ALA in the tumor microenvironment (TME) was estimated by a fluorescamine -based fluorescence assay [15]. Briefly, 20 mL simulated fluid (pH = 7.4 or 6.5, glutathione (GSH) = 10 mM) was added to a 50 mL centrifuge tube. Two milliliters of TarA@bM or BM solution was added to a dialysis tube (molecular weight cut-off (MWCO) = 8000 Da) and disposed of in the centrifuge tube mentioned above. To stimulate the enzyme hydrolysis condition, animal hydrolase
2. Experimental section 2.1. Materials δ-Aminolevulinic acid Hydrochloride, Bovine Serum Albumin (BSA), Manganese Chloride Tetrahydrate, (MnCl2 4H2O), Glutathione 53
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2.8. JC-10 mitochondria membrane potential assay
(Shanghai, Jianglai. Ltd, 0.05 mg/mL) was added in TarA@bM or BM solution and then disposed of in the centrifuge tube containing simulated fluid (pH = 6.5). 200 μL of solution was removed from the centrifuge tubes at predetermined timepoints. Fluorescamine reagent (0.1% w/v) was prepared in acetonitrile prior to used. Then, the removed solution was mixed with both 200 μL fluorescamine reagent and 800 μL of 0.1 M borate buffer (pH = 8) for 10 min at room temperature. The fluorescent derivative was analyzed by a fluorescent spectrophotometer (λex = 395 nm λem = 480 nm). The final released δ-ALA was calculated as follows: Ireleased [δ-ALA] = I TarA@bM - IBM. “I” is the shorten form of fluorescence intensity. A standard curve was obtained by analyzing a known concentration of the δ-ALA solution. Similarly, quantification of [Mn2+] in the TME was nearly the same as the method for quantifying δ-ALA but with a small modification. When a certain solution was withdrawn from the centrifuge tube, ICP-AES was used to quantify [Mn2+]. The detection of dissolved oxygen was conducted according to the following method. Four groups were prepared as follows, phosphate-buffered saline (PBS), TarA@bM ([Mn2+] = 0.025 mM), 100 mM H2O2 with TarA@bM ([Mn2+] = 0.025 mM, pH = 6.5) and 100 mM H2O2 with TarA@bM ([Mn2+] = 0.05 mM, pH = 6.5). The nanoparticle solution was added when it was ready for testing. The generation of dissolved oxygen was quantified by a portable dissolved oxygen meter (Ruosull, RDB100) every 10 s.
4T1 cells seeded in 6-well plates (3 × 105 cells/well) were coincubated with TarA@bM and NTarA@bM (δ-ALA = 1.6 mM equivalent) for 4 h. Then, each well was irradiated for 5 min (660 nm, 21.5 mW cm−2). Finally, the cells were labeled by JC-10 and analyzed by flow cytometry. 2.9. Apoptosis assay To investigate the proapoptotic ability of different samples, TarA@ bM, NTarA@bM, and TarA@bN (δ-ALA = 1.6 mM equivalent) were added into 6-well plates seeded with 4T1 cell (3 × 105 cells) for 4 h of incubation. Then, each well was irradiated for 5 min (660 nm, 21.5 mW cm−2). After 4 h of incubation, the cells were stained with annexin-V/propidium iodide (PI) and analyzed by flow cytometry. 2.10. In vitro ATP detection The influence of each sample on ATP production in cells was detected by an enhanced ATP assay kit. Briefly, 4T1 cells were incubated in 12-well plates (1 × 105 cells/well). Twelve hours later, each sample (δ-ALA = 1.6 mM equivalent) was coincubated for 4 h. The rest of the procedure was conducted according the manufacturer's protocol. 2.11. Cell viability assay
2.5. Mitochondrial-targeting assay
4T1 cells were incubated in 96-well plates (8000 cells/well). After 24 h, TarA@bM, NTarA@bM and TarA@bN in gradient concentrations were added into each well. Four hours later, the cells were irradiated by visible light (bandpass: 400–700 nm, 3.3 mW cm−2) for 0 and 30 min. After for an additional 24 h, each well was treated with 20 μL cell counting kit (CCK-8) solution for 1 h. The absorbance was measured at a wavelength of 450 nm by a microplate reader. The cell viability was calculated as follows: (OD450samples – OD450blank)/(OD450control – OD450blank) × 100%.
To verify the mitochondrial-targeting ability, 4T1 cells (7 × 104 cells/well) were seeded on glass-bottom culture dishes (NEST). Different samples were incubated with cells for 4 h. After washing with PBS twice, 100 nM MitoTracker™ Green FM was used to locate the mitochondria. Additionally, the lysosome escape ability was detected by LysoTracker™ Green (50 nM). Both probes were incubated for 15 min. The nuclei were stained with Hoechst 33258 for an additional 5 min. Fluorescence images were obtained via confocal laser scanning microscopy (CLSM) as soon as possible.
2.12. Western blot assay 2.6. Quantification of the endogenously biosynthesized PpIX The endogenous biosynthesis of PpIX in free δ-ALA, TarA@bM and NTarA@bM groups were quantified by flow cytometry. Briefly, 4T1 cells (1 × 105) were seeded in 12-well plates for 24 h for cell adhesion. Free δ-ALA, TarA@bM and NTarA@bM (δ-ALA = 1.6 mM equivalent) were co-incubated with the cells and at different timepoints, the cells were trypsinized and aspirated. The fluorescence intensity of biosynthetic PpIX (λex = 405 nm, λem = 635 nm) was quantified by flow cytometry.
The expression of proteins of interest was detected by Western blot. Briefly, cells coincubated with different samples (δ-ALA = 1.6 mM equivalent) were lysed by radio immunoprecipitation (RIPA) assay buffer and the protein content was quantified by a BCA protein assay kit. After electrophoresis, the proteins were transferred to PVDF membranes (Millipore) and blocked with 5% skim milk. One hour later, the membranes were incubated with Bcl-2 and Bax, (1:1000, dilution, Abcam, rabbit) at 4 °C overnight followed by incubation with an HRPconjugated goat anti-rabbit (1:10000 dilution, ASPEN) for 1 h. Finally, the proteins of interest were detected by enhanced chemiluminescence.
2.7. In vitro reactive oxygen species (ROS) detection
2.13. Establishment of animal model
The generation of ROS derived from δ-ALA biosynthesized PpIX under irradiation was detected by CM-H2DCFDA on an inverted fluorescence microscope. First, 1 × 105 4T1 cells/well were seeded in 12well plates. TarA@bM and TarA@bN (δ-ALA = 1.6 mM equivalent) were coincubated for 4 h. The negative controls consisted of cells cultured without addition of ant samples. Then each well was irradiated for 5 min (660 nm, 21.5 mW). After washing three times with PBS, 1 μM CM-H2DCFDA (λex = 492–495 nm, λem = 517–527 nm) solution in minimum essential medium (MEM) was added for another 15 min at 37 °C. The nuclei were stained with Hoechst 33258 (λex = 350 nm, λem = 440–460 nm) for 5 min. Fluorescence images were obtained by fluorescence microscopy. After trypsinization and aspiration, flow cytometry analysis was used to quantify the fluorescence intensity of ROS using the FITC channel.
Female Balb/c mice (aged 4–5 weeks) were obtained from Beijing Huafukang Biological Technology Co. Ltd. A total of 2 × 106 4T1 cells in 150 μL PBS were hypodermically injected into the right back leg. All the in vivo experiments were initiated when the tumor size reached ∼100 mm3 (volume = length × width2/2). All animals were treated under the regulation of the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology. 2.14. In vitro and in vivo MRI measurements For MR imaging in vitro, TarA@bM solutions with concentrations ranging from 0.03 to 0.5 mM Mn2+ were scanned using a Clinical Siemens Magnetom Trio 3.0 T MR scanner. The relaxivity value was 54
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calculated by linear fitting of 1/T1 relaxation time. For the in vivo experiments, mice were scanned at different time points before and after intravenous injection of TarA@bM ([Mn2+]: 5 mg kg−1).
new absorption band was observed for TarA@bM at approximately 300–400 nm that could be contributed to the surface plasmon band of MnO2. Additionally, the survey spectrum determined by X-ray photoelectron spectroscopy (XPS) (Fig. S4) showed the primary elements C, N, O, P, S and Mn of TarA@bM. To further verify the existence of MnO2, the XPS peaks of Mn were analyzed by Peak 4.1 software. Two characteristic peaks at 654.1 and 642.0 eV in the XPS results (Fig. 1d) showed the Mn [Ⅳ] 2p1/2 and 2p3/2 spin-orbit peaks respectively, further collectively verifying the successful mineralization of MnO2. The size of the obtained nanoparticle was detected by transmission electron microscopy (TEM) to ensure valid accumulation by the EPR effect at tumor regions. As shown in Fig. 1a, the final TarA@bM had a spheroidal morphology with an average diameter of 60 nm. For the controls, NTarA@bM was synthesized without the conjugation with (3Carboxypropyl) triphenylphosphonium bromide (CTPP), and TarA@bN was prepared by glutaraldehyde-induced cross-linking of BSA. All the experimental nanoparticles were examined by dynamic light scattering (DLS) were shown to have a similar hydrodynamic size (≈90 nm) (Fig. S1). The slightly larger size might be due to the hydrophilic property of the obtained nanoparticles. The size stability of TarA@bM was measured at 30 days (Fig. S3) and showed little change indicating the size stability of this nanoplatform under physiological conditions. When the nanoparticles were localized to the heterogeneous tumor regions, the intolerance of MnO2 to the unique TME broke down, which was detected carefully. As shown in Fig. 2a, compared with the normal environment (pH = 7.4), the simulated tumor environment (either pH = 5.5 or GSH = 10 mM) showed elevated release of [Mn2+]. These changes could attribute to the sensitive properties of MnO2 against these changes. A fluorescanmine-based fluorescence assay was used to quantity the released δ-ALA. The fluorescanmine is non-fluorescence. However, it can produce cyan fluorescent derivative when react with some amines, include the released δ-ALA, therefore to quantify the released δ-ALA. Withal, the structure of BSA stabilized MnO2 was destroyed under such stimulus, the tryptophan and tyrosine, which consisting of BSA，would be interference factor to cause unfavorable fluorescent intensity. Therefore, the BM solution group was used to eliminate this interference. As shown in Fig. 2b, the high release rate in hydrolase may be attributed to the similar hydrolysis manner on amide bond and peptide bond, which guarantee the effective release of δ-ALA in lysosome. Other simulated environment in vitro exhibited a low release rate. This may be attribute to that amide bonds are stable in the systemic circulation and also ensures safety before uptake by tumor cells and ensures adequate accumulation in cells. Except for the acidification of the TME, the H2O2 level was also significantly increased inside tumors due to insufficient blood supply [26]. Therefore, the catalytic effect was analyzed (Fig. 2c). The results showed that the presence of both TarA@bM and H2O2 under acidic conditions increased the generation of O2 compared to either H2O2 (pH 6.5) or TarA@bM. It was favorable in relieving hypoxia and could further promote the effect of photodynamic therapy.
2.15. In vivo photoluminescence imaging measurements 4T1 bearing mice were intravenously injected with TarA@bM ([δALA]: 1.6 mM, 300 μL) and photoluminescence imaging was conducted at different timepoints. Mice that were scanned before administration of TarA@bM were used as controls. (λex = 640 nm, λem = 730 nm). 2.16. In vivo antitumor therapy, metabonomic and histological assay For in vivo photodynamic therapy, thirty mice were randomly divided into six groups as follows: control, laser only, δ-ALA (in PBS without laser), δ-ALA (in PBS with laser), TarA@bM (without laser) and TarA@bM (with laser). Each group was intravenously injected with the corresponding sample ([δ-ALA] = 1.6 mM equivalent, 300 μL). The control group was administrated the same volume of PBS. Twenty-four hours later, the groups treated with the laser were illuminated (660 nm, 21.5 mW cm−2) for 10 min. The tumor volume and body weight of each mouse were recorded in 14 days. The relative tumor volumes were calculated as V/V0, where V0 is the initial tumor volume and V is the tumor volume every day after illumination. On the fifteenth day after illumination, whole blood (500 μL) was collected for routine analysis by a veterinary automatic blood cell analyzer (Mindray BC-2800 Vet). Subsequently, all mice were sacrificed, and representative hearts, livers, spleens, lungs, kidneys and tumors were collected for hematoxylin and eosin (H&E) staining and a Tdt-mediated dUTP nick-end labeling (TUNEL) assay according to protocols [24]. To detect the distribution of TarA@bM in the main organs, mice (n = 3) were sacrificed at 24 and 48 h after intravenously injecting 300 μL of sample, and the heart, liver, spleen, lung, kidney and tumor were collected and dissolved in aqua regia. The supernatant was analyzed to quantify [Mn2+] by ICP-AES. For the calculation of blood halflife time, blood samples (10 μL) were collected from TarA@bM treated Balb/c mice at predetermined timepoints, and [Mn2+] was quantified by ICP-AES. 2.17. Statistical analysis Experimental data are presented as the means ± standard deviation (n = 3). Differences among the groups were analyzed by one-way ANOVA followed by Tukey's posttest using Statistical Package for the Social Sciences (SPSS) software. Asterisks indicate that differences between the control group and other treatment groups are statistically significant as follows: *p＜0.05, **p＜0.01, ***p＜0.001. 3. Results and discussion 3.1. Synthesis and characterization of TarA@bM
3.2. Localization of TarA@bM on the subcellular level A simple and biocompatible theranostic agent was developed by integrating mitochondrial-targeting δ-ALA with an intelligent housing for synthetic cancer therapy. As shown in Scheme 1, the TPP group was introduced to the therapeutic precursor δ-ALA by an activated carboxy -amine coupling reaction to obtain an amphipathic mitochondria-targeting component. Subsequently, bovine serum albumin (BSA), as both a proactive cap and a site for biomineralization became entangled with the molecule obtained by self-assembly via intermolecular forces. Finally, Mn2+ was anchored on BSA to mineralize MnO2 under alkaline conditions. The Fourier transform infrared (FITR) spectra (Fig. 1b) of TarA@bM showed the typical stretching vibration of CeN at 1242 cm−1 which can be attributed to amide bonds suggesting successful conjunction. Next, the modified MnO2 was verified as reported in the literature [25]. In the UV–vis–NIR absorption spectra (Fig. 1c), a
The intracellular trafficking of nanoparticles occurs through several competing pathways. Generally, the use of nanoparticles for mitochondrial-targeting is often impeded by the endosomal pathway [2]. For instance, in this system, TarA@bM was taken up by endocytosis and enclosed within the early endosome. These vesicles loaded with nanoparticles matured as late endosomes where the majority of the substance in cells was degraded. Therefore, the localization of nanoparticles after endocytosis was monitored. As mentioned before, the high net positive charge of cationic TPP+ could break through lysosomes besieging by proton-sponge effect [27]. In physiological conditions, the mitochondria have a high mitochondrial transmembrane potential (Δψm), which is generated by the respiratory chain and exploited for ATP generation [4], and attracts the positively charged 55
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Scheme 1. Schematic illustration of the theranostic process of TarA@bM
TPP+ to orient to mitochondria. As shown in Fig. 3, both TarA@bM and NTarA@bM were coincubated with cells for 4 h, respectively. Blue fluorescence indicated the stained nucleus and the lysosome (above) and mitochondria (below) were stained with green fluorescence. Furthermore, red fluorescence indicated the biosynthesized PpIX was used to verify the successful internalization of δ-ALA. As shown in the merged image, the green fluorescence of mitochondria was nearly overlapped with that of TarA@bM, while this overlap was partially
observed in NTarA@bM treated cells, indicating the localization of TarA@bM to mitochondria.
3.3. In vitro assessment of photodynamic therapy Photodynamic therapy (PDT) composed of three components: photosensitizer, illuminant and oxygen. In this system, two complementary aspects were designed cooperatively to enhance the therapeutic
Fig. 1. a) TEM image of TarA@bM. b) FTIR spectra of TarA@bM and BSA. c) UV–vis absorbance spectrum of TarA@bN and TarA@bM. d) The XPS spectrum analysis of Mn[Ⅳ] 2p peak from TarA@bM 56
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Fig. 2. a) The degradation behavior of TarA@bM under normal and simulated conditions. b) The release behavior of δ-ALA under normal and simulated conditions. c) Oxygen generation in different condition. Fig. 3. Subcellular localization of δ-ALA biosynthesized PpIX induced red fluorescent-targeted TarA@ bM and nontargeted NTarA@bM. 4T1 cells were stained with the mitochondrial marker MitoTracker Green and lysosome probe LysoTracker Green, respectively. The rightmost image (bar: 50 μm) was the enlarged view of its left one (bar: 20 μm). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
besieging by the proton-sponge effect and then the lysosome breaks up. Therefore, the residual nanoparticles and proteolytic enzymes were removed. The linker between δ-ALA and TPP+ could be cleaved by proteolytic enzymes to free δ-ALA, which was in agreement with the in vitro analysis of the released δ-ALA. This result indicated that more endogenous PpIX was synthesized with a mitochondrial-targeting ligand. Next, we investigated the intracellular ROS generation properties. An ROS probe CM-H2DCFDA which can be oxidized by ROS to induce bright green fluorescence, was used. As shown in Fig. 4b, all groups were treated with irradiation to preclude the influence on illumination. Seldom green fluorescence in the control group indicated that light had little influence on cells to produce ROS and the self-produced ROS in cells was negligible. It was obvious that the TarA@bM group (p＜0.05) exhibited stronger fluorescence intensity in both fluorescent image and flow cytometry than the TarA@bN group (p＜0.05), which verified that the existence of MnO2 induced increase in oxygen could finally enhance ROS generation. It is widely believed that mitochondrial depolarization reflects the early stages of apoptosis. The JC-10 assay was used to verify whether mitochondria function well. When mitochondria were in good
outcome. A signal motivates a proapoptotic process with the overload of ROS accumulated intracellularly [28], and contributed to the catalysis of MnO2 with endogenous H2O2. In addition, the elevated endogenously biosynthesized PpIX could elevate the yield of the photosensitizer. Both of these characteristics contribute to photodynamic therapy under illumination. First, we evaluated the photosensitizer yield among the free δ-ALA, NTarA@bM and TarA@bM groups. It was obvious that the fluorescence intensity in all groups showed a timedependent manner (Fig. 4a). At the same time point, the fluorescence intensity induced by free δ-ALA was weaker than those of both NTar@ bM and TarA@bM. This result can be explained by the fact that compared to free small molecules, nanoparticles more favorably accumulated in the tumor area by the EPR effect. Furthermore, the TarA@bM group demonstrated stronger fluorescence intensity than the NTarA@ bM group. This result could be explained as follows: before PpIX was synthesized in mitochondria, the precursor was detained in a lysosome. Lysosomes are indigestive components of cells that contain many proteolytic enzymes. The similar hydrolysis manner of amide bond and peptide bond can take into effect in this condition to free δ-ALA. According to our design, the CTPP group can break through lysosome 57
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Fig. 4. a) The fluorescence intensity of endogenous biosynthetic of PpIX at different time point. The orange one is TarA@bM，the blue curve represents NTarA@bM and the red one is free δ-ALA. b) The fluorescence images of ROS generation of each sample. The fluorescence intensity was quantified by flow cytometry in the rightmost images. c) JC-10 assay on each group. d, e) The statistical analysis data of mean intensity ROS fluorescence and rate of depolarization/polarization, respectively. f) The ATP detection on different samples. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
condition, JC-10 was observed in the form of green fluorescent aggregates. The JC- aggregate transformed to a JC-monomer if the mitochondria were dysfunctional and induced red fluorescence along with
the reduction of Δψm or, in other words, the mitochondria membrane became depolarized. Therefore, the decreased Δψm not only indicates mitochondrial dysfunction but also is an important indicator of
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flow cytometry. The results demonstrated that TarA@bM with illumination caused the highest apoptosis rate, which was in agreement with the result from the CCK-8 assay of cell viability (Fig. 5b). As previously reported, high concentrations of ROS cause severe oxidative stress in mitochondria, and the collapse of Δψm could lead to mitochondrial permeability transition (PT); the opening of the mitochondrial permeability transition pore (PTP) results in endosmosis of small molecules, thereby resulting in cytotoxicity [4]. This disturbance might ultimately make mitochondria dysfunctional as well as reduce the ATP level in cells. To investigate the potential molecular mechanism of this nanoplatform in apoptosis, the relative protein expression in the Bcl-2 family was analyzed by Western blot. The ratio of Bax/Bcl-2 could be a useful indicator of the cell threshold for apoptosis-induced PDT [29,30]. As shown in Fig. 5d, the overexpression of the proapoptotic protein Bax was significantly detected. In parallel, the expression level of the antiapoptotic protein Bcl-2 was decreased. Based on these various impacts, it could be concluded that this systematic nanoplatform had a synergistic effect to promote PDT.
apoptosis. Then, we evaluated the impact on mitochondria. Flow cytometry was used to measure the fluorescence intensity ratio of red/ green fluorescence, further accounting for the rate of depolarization/ polarization, respectively. As shown in Fig. 4c, the upper area indicated the ratio of the JC-aggregate, while the area below indicates the JCmonomer. In this light-triggered system, it was obvious that under dark conditions, the ratio of fluorescence intensity changed negligibly suggesting that simple nanoparticles in the dark have little influence on Δψm. When the nanoparticles were administrated and illuminated, the Δψm in both the NTar@bM (p＜0.01) and TarA@bM (p＜0.01) groups decreased to certain levels, but the latter group decreased more. This study elucidated that δ-ALA based photodynamic therapy damaged mitochondria, but the targeted component promoted the attack efficiency as a result of the increased accumulation of effective constituents, which was in accord with the localization and biosynthesis assay. ATP is crucial for providing intracellular energy, therefore, the detection of ATP levels is an important indicator assessing cell viability. As shown in Fig. 4f, the ATP levels in the treatment groups were slightly different from those in the control group when treated in the dark. The ATP level decreased most in the TarA@bM group (p＜0.01). Similar results were observed in the apoptosis and cell viability assays. As shown in Fig. 5a, the apoptosis/necrosis rate was detected by
3.4. In vitro and in vivo MR/FL imaging To investigate enhanced MRI as a result of the decomposition of MnO2 in acidic tumor conditions, different buffers (pH = 6.5 and 7.4)
Fig. 5. a) Apoptosis assay of different treated 4T1 cells under different conditions. b,c) Cell viability assay on different treatments by CCK-8. b was in the dark condition while c was under illumination. d) Western blot assay of different treated 4T1 cells. 59
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Fig. 6. a) In vitro MRI measurement of TarA@bM in concentration gradient under different condition. b,d) In vivo MRI measurement at different time point. d was the images of different time point and b was calculated relative MR signal intensity. c.e) In vivo photoluminescence imaging measurement of TarA@bM. e was the images at different time point and c was calculated relative mean intensity.
3.5. In vivo biodistribution measurement
were used to assess the relaxivity value (r1) of TarA@bM. As shown in Fig. 6a, TarA@bM in both neutral and acidic conditions showed concentration-dependent manners. Notably, when treated in acidic conditions (pH = 6.5), the imaging was brighter than its neutral counterpart. The calculated relaxivity value (r1) at pH = 6.5 was 5.410 s−1mM−1, which was three times greater than that in a normal environment. There results might be attributed to the increased release of Mn2+ compared with manganese dioxide, which functioned as a T1 contrast agent [31] in an acidic environment and therefore enhanced the MR imaging properties. Next, we carried out an in vivo experiment on 4T1 cell-bearing-mice to verify its final image-guided effect on small mammals. From the MR images at different timepoints (Fig. 6d), after 12 h injection, the tumor region showed the brightest imaging, indicating effective accumulation, while the MR signal intensity showed a slightly decreased in brightness after 24 h, which may be attributed to the clearance of small molecular Mn2+ by the reticuloendothelial system (RES). Similarly, photoluminescence (PL) imaging was demonstrated in the same manner to assess the imaging capability. Interestingly, a hysteresis effect was observed by FL imaging (Fig. 6e), and at 24 h injection, the fluorescence intensity was stronger than that at 12 h postinjection. We speculated that this could be attributed to the timeconsuming biosynthetic pathway of PpIX, which induces the final fluorescence. Furthermore, the lasting fluorescence signal at 36 h may be attributed to effective retention in the tumor area that could be used to monitor the posttreatment process and provide prognosis assessment.
To further understand the biodistribution of TarA@bM, we monitored the blood circulation and tissue distribution by measuring the [Mn2+] in the blood samples and major organs of TarA@bM treated mice by ICP-AES. Fig. 7a shows the distribution of the Mn2+ in main tissues. The amount of Mn was measured at 12 and 24 h by dissolving the main tissue in aqua regia. The high concentration of TarA@bM resulted in an effective accumulation of TarA@bM in the tumor section for 12 h. Notably, the results also demonstrated that the liver and spleen showed rather high levels of nanoparticle accumulation, which could be contributed to the function of the reticuloendothelial system. After 48 h, the concentration in each tissue decreased correspondingly, which might be attributed to the biodegradation of TarA@bM, but still remained at a considerable concentration in the tumor region (9.7%). The half-life time was calculated by fitting with a two-compartment model. The results (Fig. 7b) of the calculation showed that the first (t1/2(α)) and second (t1/2(β)) phases were 1.36 ± 0.21 h and 8.5 ± 1.12 h, respectively. The relatively long blood half-life could be contributed to the existence of a protein-based carrier that could promote circulation time, as previously reported [32].
3.6. In vivo photodynamic therapy 4T1 tumor-bearing Balb/c mice were divided randomly into six groups as follows: PBS, laser only, δ-ALA without laser, δ-ALA with 60
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Fig. 7. a) The biodistribution of TarA@bM in the main organs in 12 h and 24 h. b) The blood circulation curve of TarA@bM in 36 h. c) The photodynamic effect on tumors of different treatments. d) The relative tumor volume on different treatments. e) The relative body weight on different treatments. f) Heat map of the blood routine examination on different treatments. g) Histological H&E (bar is 100 μm) staining and TUNEL (bar is 50 μm) staining of the tumor tissues on different treatments.
stained major organs, including heart, liver, spleen, lung, and kidney, in all the groups showed no conspicuous necrosis or physiological morphology changes (Fig. S5). Moreover, the routine blood examination (Fig. 7f) showed that all the groups had little influence on normal physiological activity. All the results demonstrated that this nanoplatform exerted no obvious toxicity to animals.
laser, TarA@bM without laser and TarA@bM with laser. According to the results of fluorescence imaging, we administrated illumination 24 h after intravenous tail injection. The tumor area was exposed to light illumination for 10 min. All the body weight and tumor areas were recorded at 14 days after illumination. The laser only group had no effect on either the tumor or body weight compared with the control group. The δ-ALA and TarA@bM groups without laser illumination exhibited slight inhibition of tumor proliferation, which could be attributed to the influence of sunlight. The relative tumor volume on both TarA@bM and δ-ALA with laser illumination showed a considerable decrease compared with the same injection groups without laser illumination (Fig. 7c and d). To further understand the therapeutic effects on tumor tissues, relevant organs and tumors were harvested for histological analysis. As shown in Fig. 7g, obvious cytoplasmic leakage and nuclear shrinkage of apoptotic cells were observed in the H&E stained tumor slices from the TarA@bM treated PDT group. Furthermore, TUNEL staining was used to locate the apoptotic cells. The strong green fluorescence was attributed to the nucleus of apoptotic cells in the TarA@bM PDT group. These results showed that compared to the traditional therapeutic molecule, TarA@bM showed an enhanced therapeutic effect, which could be considered for clinic application. Finally, the safety of this nanoplatform was assessed. From the macroscopic perspective, the body weight (Fig. 7e) in all the groups slightly increased, indicating that all the treatments had no obvious side-effects on the mice. Next, the histological examination of the H&E
4. Conclusion In summary, we designed a complete theranostics system through a simply modified photosensitive precursor, δ-ALA. Two complementary components were fabricated for enhanced photodynamic therapy. From one of the components, mitochondrial ligands were introduced to the precursor that were converted into photosensitizer PpIX for more specific delivery to enhance the yield of PpIX and make mitochondria dysfunctional. For the other component the modified MnO2 could interact with endogenous H2O2 to elevate oxygen, resulting in severe oxidative stress. Both components contributed to apoptosis, leading to the final lethal signal. Moreover, with the dual imaging system, the entire process of treatment was monitored in a pivotal period for more accurate treatment. The in vivo experiments demonstrated superior imaging results and tumor inhibition, suggesting that this nanoplatform can function as favorable therapeutic agent and broaden the clinical application of δ-ALA. 61
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Notes
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The authors declare no competing financial interests. Acknowledgment We would like to appreciate Mr. Hongyi Zheng for his guidance in flow cytometry analysis. This work was supported by the National Natural Science Foundation of China (Grant NO. 51573039, 81701653, 81874084). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biomaterials.2019.01.044. References [1] V.R. Fantin, J. St-Pierre, P. Leder, Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance, Cancer Cell 9 (2006) 425–434. [2] S. Marrache, S. Dhar, Engineering of blended nanoparticle platform for delivery of mitochondria-acting therapeutics, Proceedings of the National Academy of Sciences of the United States of America, vol. 109, 2012, pp. 16288–16293. [3] S.P. Mathupala, Y.H. Ko, P.L. Pedersen, Hexokinase II: cancer's double-edged sword acting as both facilitator and gatekeeper of malignancy when bound to mitochondria, Oncogene 25 (2006) 4777–4786. [4] S. Fulda, L. Galluzzi, G. Kroemer, Targeting mitochondria for cancer therapy, Nat. Rev. Drug Discov. 9 (2010) 447–464. [5] S. Jones, X. Zhang, D.W. Parsons, J.C. Lin, R.J. Leary, P. Angenendt, et al., Core signaling pathways in human pancreatic cancers revealed by global genomic analyses, Science 321 (2008) 1801–1806. [6] M.R. Junttila, F.J. de Sauvage, Influence of tumour micro-environment heterogeneity on therapeutic response, Nature 501 (2013) 346–354. [7] F. Danhier, O. Feron, V. Preat, To exploit the tumor microenvironment: passive and active tumor targeting of nanocarriers for anti-cancer drug delivery, J. Contr. Release 148 (2010) 135–146 official journal of the Controlled Release Society. [8] M. Hockel, P. Vaupel, Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects, J. Natl. Cancer Inst. 93 (2001) 266–276. [9] M.F. McCarty, J. Whitaker, Manipulating tumor acidification as a cancer treatment strategy, Altern. Med. Rev. 15 (2010) 264–272 a journal of clinical therapeutic. [10] O. Tredan, C.M. Galmarini, K. Patel, I.F. Tannock, Drug resistance and the solid tumor microenvironment, J. Natl. Cancer Inst. 99 (2007) 1441–1454. [11] D.W. Zheng, J.X. Fan, X.H. Liu, X. Dong, P. Pan, L. Xu, et al., A simply modified lymphocyte for systematic cancer therapy, Adv. Mater. (2018) e1801622. [12] W.J. Cottrell, A.D. Paquette, K.R. Keymel, T.H. Foster, A.R. Oseroff, Irradiancedependent photobleaching and pain in delta-aminolevulinic acid-photodynamic therapy of superficial basal cell carcinomas, Clin. Canc. Res. 14 (2008) 4475–4483 an official journal of the American Association for Cancer Research. [13] S.G. Awuah, Y. You, Boron dipyrromethene (BODIPY)-based photosensitizers for photodynamic therapy, RSC Adv. 2 (2012) 11169.
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Enhancing magnetic resonance/photoluminescence imaging-guided photodynamic therapy by multiple pathways
T
Pei Liua, Jinghua Rend, Yuxuan Xionga, Zhe Yanga, Wei Zhua, Qianyuan Hea, Zushun Xua,∗, Wenshan Heb,∗∗, Jing Wangc,∗∗∗ a Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry of Education Key Laboratory for the Green Preparation and Application of Functional Materials, Hubei University, Wuhan, Hubei, 430062, China b Department of Breast and Thyroid Surgery, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei, 430030, China c Department of Radiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China d Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Endogenously biosynthetic photosensitizer Mitochondria Dual-imaging nanoplatform
Mitochondria, which are a major source of adenosine triphosphate (ATP) and apoptosis regulators, are the key organelles that promote tumor cell proliferation, and their dysfunction affects tumor cell behavior. Additionally, mitochondria have been shown to play a central role in the biosynthesis of protoporphyrin IX (PpIX), which is a widely used photosensitizer that has been used for tumor detection, monitoring and photodynamic therapy. Nevertheless, photosensitizers administrated exogenously are often restricted by limited bioavailability.δAminolevulinic acid (δ-ALA) is a naturally occurring delta amino acid that can be converted in situ to PpIX via the heme biosynthetic pathway in mitochondria. Because δ-ALA is the precursor for PpIX, δ-ALA-based photodynamic therapy (PDT) shows promise in treating cancer. However, the accumulation of δ-ALA within endosomal system limits the production of PpIX and eventually impedes its effectiveness. Theranostic nanoparticles (NPs) capable of endosomal escape are expected to optimize the endogenous biosynthetic yield. In this study, δ-ALA was improved with triphenylphosphoniumcation (TPP+), a high net position cation that functions in endosomal escape and as a mitochondria-targeting ligand, and was further modified with bovine serum albumin stabilized manganese dioxide (MnO2). The tumor microenvironment (TME) responsive MnO2 in this system can elevate oxygen content to relieve hypoxia. Both enhanced photosensitizer yield and elevated oxygen contributing to the final therapeutic effect. Moreover, the enhancement of magnetic resonance imaging (MRI) (r1 = 5.410 s−1mM−1) stemming from the degradation of MnO2 by the TME could serve as a guide prior to treatment for accurate location, while in situ hysteretic photoluminescence imaging derived from PpIX can be utilize as a supervisor for prognosis evaluation. This systematic design could broaden the biomedical application and highlight the considerable therapeutic promise of PDT.
1. Introduction Rapid-cell proliferation is a distinctive characteristic of solid tumor that require a compatible level of ATP, which is mainly produced by the mitochondria [1]. This demand for ATP results in uncommon vitality in mitochondrial physiology [2,3]. Additionally, mitochondria are crucial regulators of the intrinsic pathway of apoptosis [4]. Both of these characteristics suggest that dysfunctions of mitochondria can be lethal to tumor cells. From another perspective, tumor cells are heterogeneity; they differ from patients, even within the same lesion of an individual
patient [5]. In fact, initial tumorigenesis and eventual metastasis is not determined exclusively by genetic alteration. However, the initial tumorigenesis and eventual metastasis involve the reciprocal influence between mutations and the state of the stromal microenvironment [6]. A high degree of angiogenesis in solid tumor accounts for nutrient deficiency [7], hypoxia [8] and acidification [9]. Tumor cells within a unique TME differ from normal tissue, and this unique environment is eventually responsible for failure of some treatments due to therapeutic resistance [10]. Therefore, it is necessary to develop nanoplatforms that target the tumor milieu to enhance therapeutic outcome.
∗
Corresponding author. Corresponding author. ∗∗∗ Corresponding author. E-mail addresses: [email protected] (Z. Xu), [email protected] (W. He), jjwinfl[email protected] (J. Wang). ∗∗
https://doi.org/10.1016/j.biomaterials.2019.01.044 Received 11 September 2018; Received in revised form 21 January 2019; Accepted 30 January 2019 Available online 02 February 2019 0142-9612/ © 2019 Elsevier Ltd. All rights reserved.
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δ-Aminolevulinic acid (δ-ALA), a biological endogenous molecular that is widely used as a non-photoactivatable clinical molecule approved by U.S Food and Drug Administration [11], has been used mostly as a biological precursor of the protoporphyrin IX (PpIX) via the cellular heme biosynthesis pathway for photodynamic therapy [12]. Compared to other synthetic photosensitizers [13,14], δ-ALA is distinct because of its easy operation, biosecurity and non-photobleaching characteristic due to its endogeneity. Because of the negative feedback mechanism that causes free heme to repress endogenous production of δ-ALA [15], it is more favorable for exogenous administration of δ-ALA to synthesize PpIX for intracellular accumulation as result of bypassing it. Moreover, this accumulation is more pronounced in malignant cells than in their normal counterparts in vitro and in vivo [16]. Indeed, δALA itself has some limitations on photodynamic therapy, such as poor penetrability of the lipidic barrier due to high polar characteristics and zwitterionic nature [15]. Many improvements have been made to δALA. Ma et al. [17] designed a multifunctional hollow mesoporous silica coating on δ-ALA for treatment against superficial cancer cells. Shi et al. [18] fabricated δ-ALA loaded PLGA nanoparticles that targeted human squamous skin cells. Nevertheless, under normal physiological condition, δ-ALA was transported to coproporphyrinogen III under various enzymolytic mechanism in the cytosol, and the photosensitizer agent PpIX was transported to mitochondria in the presence of oxidase [16]. After nanoparticles passively accumulate in tumor sections via enhanced permeability and retention (EPR) effects, they are usually taken up by endocytosis and enclosed within an early endosome. These vesicles that are loaded with nanoparticles mature to become late endosomes that gradually fuse with lysosomes [19]. This endosomal pathway in cells limits the adequate accumulation of precursors to mitochondria. Because of the inadequate endogenous production of PpIX, drug resistance and weakened anticancer therapy occur sequentially. Therefore, it's essential to increase the bioavailability of δ-ALA to enhance photodynamic therapy. Finally, visualization of a therapeutic system is of great significance for scrutiny and guidance to improve therapeutic outcome [20]. Considering both time-consuming biosynthesis and the fluorescent property of PpIX, we speculate the fluorescence imaging can be used to supervise prognosis evaluation. To accurately locate NPs at tumor regions prior to treatment, another auxiliary imaging technique should be used for more accurate guidance. Among all the diagnose methods, MR imaging stands out as a clinical noninvasive and excellent 3D spatial resolution diagnose technology for soft tissue detection [21]. Manganese is a considerable candidate as a potential T1 contrast agents for MRI. Additionally, some studies [22,23] have recently reported that −MneO− bond is sensitive to conditions in the TME, such as reducing or acidic conditions, and can be utilized to deliver therapeutic agent effectively. In addition, the catalytic reaction between MnO2 and endogenously abundant H2O2 in tumor area to produce oxygen could relieve hypoxia condition, contributing to the promotion of therapeutic effect. Therefore, a smart design for the δ-ALA-based system was demonstrated as dual imaging-guided enhanced photodynamic therapy. First, dual-model imaging using in this system can provide complete monitoring prior to treatment and prognosis evaluation. Second, based on our design, the multi-responsive MnO2 in the TME guarantees effective accumulation in the tumor region. Enhanced precursor accumulation in tumor cells could further elevate the endogenous biosynthetic yield of photosensitizer, thereby promoting the bioavailability. Finally, with increased oxygen and sufficient photosensitizer, the therapeutic efficacy can be enhanced at a significant level.
(GSH), (3-Carboxypropyl) triphenylphosphonium bromide (CTPP), Nhydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), Fluorescamine, were purchased from Aladdin Reagents Co. Ltd. Cell Counting Kit-8 (CCK-8) was obtained from MedChemExpress. CM-H2DCFDA, MitoTracker™ Green FM, LysoTracker™ Green were obtained from Thermo Fisher Scientific. JC10 Mitochondrion Membrane Potential Assay Kit was brought from AAT Bioquest. Hoechst 33258, Enhanced ATP Assay Kit, Annexin VFITC Apoptosis Detection Kit were obtained from Beyotime Ltd. Minimum essential medium (MEM) was brought from Invitrogen Corp. 2.2. Instrument Material and Regions. Transmission electron microscopy (TEM) images were obtained by Tecnai G20, FEI Corp. USA. Nanosize was measured by ZetaSizer Nano series Nano-ZS (Malvern Instruments Ltd, Malvern, UK). UV–vis spectra (UV–vis spectrophotometer, 1800 PC, Mapada, China) were used to identify the characteristic Peaks. The functional groups were determined by FTIR (Nicolet 570). Fluorescence spectra were tested by fluorospectro photometer (F-2500, HITASCHI). The X-ray photoelectron spectroscopy (XPS) measurement was conducted on Thermo Fisher Scientific 250Xi. The cell viability was measured using a micro-plate reader (1420 multilabel counter, Perkin Elmer, MA, USA). Confocal laser scanning microscope (CLSM) images were obtained from ZEISS LSM 710 (Germany). Clinical Siemens Magnetom Trio 3.0 T MR scanner was used to evaluate the MRI property. Small animal fluorescence imaging was carried on Bruker In-Vivo optical imaging system (Billerica, USA, Lago X). 2.3. Preparation of the TarA@bM, NTarA@bM, TarA@bN and BM First, 100 mM of δ-ALA solution was prepared as follows: 50.28 mg δ-ALA (HCl) was dissolved in 3 mL PBS, the pH of solution was adjusted at 7.0 by 5 M NaOH. Subsequently, 0.22 μm filter was used to degerm for store in a certain period. 10 mg CTPP was dissolved in 200 μL DMSO, 4.5 mg EDC and 3 mg NHS was added in sequence and mixed for 2 h at room temperature. Then, 200 μL 100 mM δ-ALA was added for another 1 h to obtained the TarA. 10 mL 100 mg BSA was added in the above-mentioned solution for 6 h. Finally, 2 mL of 0.03 M MnCl2 4H2O was added dropwise, 1 M NaOH was used to adjust the pH at 10 for 4 h to obtain the final TarA@bM. Correspondingly, NTarA@bM and TarA@ bN were synthesized as control nanoparticles. Briefly, NTarA@bM was synthesized as the same procedure as TarA@bM's but without chemical combination with CTPP. TarA@bN was obtained with the same procedure as TarA@bM's before add MnCl2 4H2O. After that, the obtained solution was reacted with glutaraldehyde (2.5%) to acquire cross-linked nanoparticles. All the ultimate productions were dialyzing in a dialysis bag (MWCO 100 kDa). Additionally, TarA was dialyzing against ultrapure water in a dialysis bag (MWCO 100) and frozen, lyophilized. Mass spectrum (Fig. S6): The m/z peak was 462. 1H NMR (600 MHz, DMSO, Fig. S7):7.95–7.75 (m, 15H), 4.00–3.95 (s, 2H), 3.65–3.55 (dt, 2H), 3.50–3.40 (dt, 2H), 2.79–2.73 (dt, 2H), 1.79–1.70 (dt, 2H). The BSA stabilized MnO2 (BM) was synthesized as follows: 2 mL of 0.03 M MnCl2 4H2O was added into 10 mL of 100 mg BSA solution dropwise, 1 M NaOH was used to adjust the pH at 10 for 4 h, and finally were dialyzing in a dialysis bag (MWCO 100 kDa) to obtain the final BM. 2.4. Detection of stimuli-responsive properties The release of δ-ALA in the tumor microenvironment (TME) was estimated by a fluorescamine -based fluorescence assay [15]. Briefly, 20 mL simulated fluid (pH = 7.4 or 6.5, glutathione (GSH) = 10 mM) was added to a 50 mL centrifuge tube. Two milliliters of TarA@bM or BM solution was added to a dialysis tube (molecular weight cut-off (MWCO) = 8000 Da) and disposed of in the centrifuge tube mentioned above. To stimulate the enzyme hydrolysis condition, animal hydrolase
2. Experimental section 2.1. Materials δ-Aminolevulinic acid Hydrochloride, Bovine Serum Albumin (BSA), Manganese Chloride Tetrahydrate, (MnCl2 4H2O), Glutathione 53
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2.8. JC-10 mitochondria membrane potential assay
(Shanghai, Jianglai. Ltd, 0.05 mg/mL) was added in TarA@bM or BM solution and then disposed of in the centrifuge tube containing simulated fluid (pH = 6.5). 200 μL of solution was removed from the centrifuge tubes at predetermined timepoints. Fluorescamine reagent (0.1% w/v) was prepared in acetonitrile prior to used. Then, the removed solution was mixed with both 200 μL fluorescamine reagent and 800 μL of 0.1 M borate buffer (pH = 8) for 10 min at room temperature. The fluorescent derivative was analyzed by a fluorescent spectrophotometer (λex = 395 nm λem = 480 nm). The final released δ-ALA was calculated as follows: Ireleased [δ-ALA] = I TarA@bM - IBM. “I” is the shorten form of fluorescence intensity. A standard curve was obtained by analyzing a known concentration of the δ-ALA solution. Similarly, quantification of [Mn2+] in the TME was nearly the same as the method for quantifying δ-ALA but with a small modification. When a certain solution was withdrawn from the centrifuge tube, ICP-AES was used to quantify [Mn2+]. The detection of dissolved oxygen was conducted according to the following method. Four groups were prepared as follows, phosphate-buffered saline (PBS), TarA@bM ([Mn2+] = 0.025 mM), 100 mM H2O2 with TarA@bM ([Mn2+] = 0.025 mM, pH = 6.5) and 100 mM H2O2 with TarA@bM ([Mn2+] = 0.05 mM, pH = 6.5). The nanoparticle solution was added when it was ready for testing. The generation of dissolved oxygen was quantified by a portable dissolved oxygen meter (Ruosull, RDB100) every 10 s.
4T1 cells seeded in 6-well plates (3 × 105 cells/well) were coincubated with TarA@bM and NTarA@bM (δ-ALA = 1.6 mM equivalent) for 4 h. Then, each well was irradiated for 5 min (660 nm, 21.5 mW cm−2). Finally, the cells were labeled by JC-10 and analyzed by flow cytometry. 2.9. Apoptosis assay To investigate the proapoptotic ability of different samples, TarA@ bM, NTarA@bM, and TarA@bN (δ-ALA = 1.6 mM equivalent) were added into 6-well plates seeded with 4T1 cell (3 × 105 cells) for 4 h of incubation. Then, each well was irradiated for 5 min (660 nm, 21.5 mW cm−2). After 4 h of incubation, the cells were stained with annexin-V/propidium iodide (PI) and analyzed by flow cytometry. 2.10. In vitro ATP detection The influence of each sample on ATP production in cells was detected by an enhanced ATP assay kit. Briefly, 4T1 cells were incubated in 12-well plates (1 × 105 cells/well). Twelve hours later, each sample (δ-ALA = 1.6 mM equivalent) was coincubated for 4 h. The rest of the procedure was conducted according the manufacturer's protocol. 2.11. Cell viability assay
2.5. Mitochondrial-targeting assay
4T1 cells were incubated in 96-well plates (8000 cells/well). After 24 h, TarA@bM, NTarA@bM and TarA@bN in gradient concentrations were added into each well. Four hours later, the cells were irradiated by visible light (bandpass: 400–700 nm, 3.3 mW cm−2) for 0 and 30 min. After for an additional 24 h, each well was treated with 20 μL cell counting kit (CCK-8) solution for 1 h. The absorbance was measured at a wavelength of 450 nm by a microplate reader. The cell viability was calculated as follows: (OD450samples – OD450blank)/(OD450control – OD450blank) × 100%.
To verify the mitochondrial-targeting ability, 4T1 cells (7 × 104 cells/well) were seeded on glass-bottom culture dishes (NEST). Different samples were incubated with cells for 4 h. After washing with PBS twice, 100 nM MitoTracker™ Green FM was used to locate the mitochondria. Additionally, the lysosome escape ability was detected by LysoTracker™ Green (50 nM). Both probes were incubated for 15 min. The nuclei were stained with Hoechst 33258 for an additional 5 min. Fluorescence images were obtained via confocal laser scanning microscopy (CLSM) as soon as possible.
2.12. Western blot assay 2.6. Quantification of the endogenously biosynthesized PpIX The endogenous biosynthesis of PpIX in free δ-ALA, TarA@bM and NTarA@bM groups were quantified by flow cytometry. Briefly, 4T1 cells (1 × 105) were seeded in 12-well plates for 24 h for cell adhesion. Free δ-ALA, TarA@bM and NTarA@bM (δ-ALA = 1.6 mM equivalent) were co-incubated with the cells and at different timepoints, the cells were trypsinized and aspirated. The fluorescence intensity of biosynthetic PpIX (λex = 405 nm, λem = 635 nm) was quantified by flow cytometry.
The expression of proteins of interest was detected by Western blot. Briefly, cells coincubated with different samples (δ-ALA = 1.6 mM equivalent) were lysed by radio immunoprecipitation (RIPA) assay buffer and the protein content was quantified by a BCA protein assay kit. After electrophoresis, the proteins were transferred to PVDF membranes (Millipore) and blocked with 5% skim milk. One hour later, the membranes were incubated with Bcl-2 and Bax, (1:1000, dilution, Abcam, rabbit) at 4 °C overnight followed by incubation with an HRPconjugated goat anti-rabbit (1:10000 dilution, ASPEN) for 1 h. Finally, the proteins of interest were detected by enhanced chemiluminescence.
2.7. In vitro reactive oxygen species (ROS) detection
2.13. Establishment of animal model
The generation of ROS derived from δ-ALA biosynthesized PpIX under irradiation was detected by CM-H2DCFDA on an inverted fluorescence microscope. First, 1 × 105 4T1 cells/well were seeded in 12well plates. TarA@bM and TarA@bN (δ-ALA = 1.6 mM equivalent) were coincubated for 4 h. The negative controls consisted of cells cultured without addition of ant samples. Then each well was irradiated for 5 min (660 nm, 21.5 mW). After washing three times with PBS, 1 μM CM-H2DCFDA (λex = 492–495 nm, λem = 517–527 nm) solution in minimum essential medium (MEM) was added for another 15 min at 37 °C. The nuclei were stained with Hoechst 33258 (λex = 350 nm, λem = 440–460 nm) for 5 min. Fluorescence images were obtained by fluorescence microscopy. After trypsinization and aspiration, flow cytometry analysis was used to quantify the fluorescence intensity of ROS using the FITC channel.
Female Balb/c mice (aged 4–5 weeks) were obtained from Beijing Huafukang Biological Technology Co. Ltd. A total of 2 × 106 4T1 cells in 150 μL PBS were hypodermically injected into the right back leg. All the in vivo experiments were initiated when the tumor size reached ∼100 mm3 (volume = length × width2/2). All animals were treated under the regulation of the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology. 2.14. In vitro and in vivo MRI measurements For MR imaging in vitro, TarA@bM solutions with concentrations ranging from 0.03 to 0.5 mM Mn2+ were scanned using a Clinical Siemens Magnetom Trio 3.0 T MR scanner. The relaxivity value was 54
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calculated by linear fitting of 1/T1 relaxation time. For the in vivo experiments, mice were scanned at different time points before and after intravenous injection of TarA@bM ([Mn2+]: 5 mg kg−1).
new absorption band was observed for TarA@bM at approximately 300–400 nm that could be contributed to the surface plasmon band of MnO2. Additionally, the survey spectrum determined by X-ray photoelectron spectroscopy (XPS) (Fig. S4) showed the primary elements C, N, O, P, S and Mn of TarA@bM. To further verify the existence of MnO2, the XPS peaks of Mn were analyzed by Peak 4.1 software. Two characteristic peaks at 654.1 and 642.0 eV in the XPS results (Fig. 1d) showed the Mn [Ⅳ] 2p1/2 and 2p3/2 spin-orbit peaks respectively, further collectively verifying the successful mineralization of MnO2. The size of the obtained nanoparticle was detected by transmission electron microscopy (TEM) to ensure valid accumulation by the EPR effect at tumor regions. As shown in Fig. 1a, the final TarA@bM had a spheroidal morphology with an average diameter of 60 nm. For the controls, NTarA@bM was synthesized without the conjugation with (3Carboxypropyl) triphenylphosphonium bromide (CTPP), and TarA@bN was prepared by glutaraldehyde-induced cross-linking of BSA. All the experimental nanoparticles were examined by dynamic light scattering (DLS) were shown to have a similar hydrodynamic size (≈90 nm) (Fig. S1). The slightly larger size might be due to the hydrophilic property of the obtained nanoparticles. The size stability of TarA@bM was measured at 30 days (Fig. S3) and showed little change indicating the size stability of this nanoplatform under physiological conditions. When the nanoparticles were localized to the heterogeneous tumor regions, the intolerance of MnO2 to the unique TME broke down, which was detected carefully. As shown in Fig. 2a, compared with the normal environment (pH = 7.4), the simulated tumor environment (either pH = 5.5 or GSH = 10 mM) showed elevated release of [Mn2+]. These changes could attribute to the sensitive properties of MnO2 against these changes. A fluorescanmine-based fluorescence assay was used to quantity the released δ-ALA. The fluorescanmine is non-fluorescence. However, it can produce cyan fluorescent derivative when react with some amines, include the released δ-ALA, therefore to quantify the released δ-ALA. Withal, the structure of BSA stabilized MnO2 was destroyed under such stimulus, the tryptophan and tyrosine, which consisting of BSA，would be interference factor to cause unfavorable fluorescent intensity. Therefore, the BM solution group was used to eliminate this interference. As shown in Fig. 2b, the high release rate in hydrolase may be attributed to the similar hydrolysis manner on amide bond and peptide bond, which guarantee the effective release of δ-ALA in lysosome. Other simulated environment in vitro exhibited a low release rate. This may be attribute to that amide bonds are stable in the systemic circulation and also ensures safety before uptake by tumor cells and ensures adequate accumulation in cells. Except for the acidification of the TME, the H2O2 level was also significantly increased inside tumors due to insufficient blood supply [26]. Therefore, the catalytic effect was analyzed (Fig. 2c). The results showed that the presence of both TarA@bM and H2O2 under acidic conditions increased the generation of O2 compared to either H2O2 (pH 6.5) or TarA@bM. It was favorable in relieving hypoxia and could further promote the effect of photodynamic therapy.
2.15. In vivo photoluminescence imaging measurements 4T1 bearing mice were intravenously injected with TarA@bM ([δALA]: 1.6 mM, 300 μL) and photoluminescence imaging was conducted at different timepoints. Mice that were scanned before administration of TarA@bM were used as controls. (λex = 640 nm, λem = 730 nm). 2.16. In vivo antitumor therapy, metabonomic and histological assay For in vivo photodynamic therapy, thirty mice were randomly divided into six groups as follows: control, laser only, δ-ALA (in PBS without laser), δ-ALA (in PBS with laser), TarA@bM (without laser) and TarA@bM (with laser). Each group was intravenously injected with the corresponding sample ([δ-ALA] = 1.6 mM equivalent, 300 μL). The control group was administrated the same volume of PBS. Twenty-four hours later, the groups treated with the laser were illuminated (660 nm, 21.5 mW cm−2) for 10 min. The tumor volume and body weight of each mouse were recorded in 14 days. The relative tumor volumes were calculated as V/V0, where V0 is the initial tumor volume and V is the tumor volume every day after illumination. On the fifteenth day after illumination, whole blood (500 μL) was collected for routine analysis by a veterinary automatic blood cell analyzer (Mindray BC-2800 Vet). Subsequently, all mice were sacrificed, and representative hearts, livers, spleens, lungs, kidneys and tumors were collected for hematoxylin and eosin (H&E) staining and a Tdt-mediated dUTP nick-end labeling (TUNEL) assay according to protocols [24]. To detect the distribution of TarA@bM in the main organs, mice (n = 3) were sacrificed at 24 and 48 h after intravenously injecting 300 μL of sample, and the heart, liver, spleen, lung, kidney and tumor were collected and dissolved in aqua regia. The supernatant was analyzed to quantify [Mn2+] by ICP-AES. For the calculation of blood halflife time, blood samples (10 μL) were collected from TarA@bM treated Balb/c mice at predetermined timepoints, and [Mn2+] was quantified by ICP-AES. 2.17. Statistical analysis Experimental data are presented as the means ± standard deviation (n = 3). Differences among the groups were analyzed by one-way ANOVA followed by Tukey's posttest using Statistical Package for the Social Sciences (SPSS) software. Asterisks indicate that differences between the control group and other treatment groups are statistically significant as follows: *p＜0.05, **p＜0.01, ***p＜0.001. 3. Results and discussion 3.1. Synthesis and characterization of TarA@bM
3.2. Localization of TarA@bM on the subcellular level A simple and biocompatible theranostic agent was developed by integrating mitochondrial-targeting δ-ALA with an intelligent housing for synthetic cancer therapy. As shown in Scheme 1, the TPP group was introduced to the therapeutic precursor δ-ALA by an activated carboxy -amine coupling reaction to obtain an amphipathic mitochondria-targeting component. Subsequently, bovine serum albumin (BSA), as both a proactive cap and a site for biomineralization became entangled with the molecule obtained by self-assembly via intermolecular forces. Finally, Mn2+ was anchored on BSA to mineralize MnO2 under alkaline conditions. The Fourier transform infrared (FITR) spectra (Fig. 1b) of TarA@bM showed the typical stretching vibration of CeN at 1242 cm−1 which can be attributed to amide bonds suggesting successful conjunction. Next, the modified MnO2 was verified as reported in the literature [25]. In the UV–vis–NIR absorption spectra (Fig. 1c), a
The intracellular trafficking of nanoparticles occurs through several competing pathways. Generally, the use of nanoparticles for mitochondrial-targeting is often impeded by the endosomal pathway [2]. For instance, in this system, TarA@bM was taken up by endocytosis and enclosed within the early endosome. These vesicles loaded with nanoparticles matured as late endosomes where the majority of the substance in cells was degraded. Therefore, the localization of nanoparticles after endocytosis was monitored. As mentioned before, the high net positive charge of cationic TPP+ could break through lysosomes besieging by proton-sponge effect [27]. In physiological conditions, the mitochondria have a high mitochondrial transmembrane potential (Δψm), which is generated by the respiratory chain and exploited for ATP generation [4], and attracts the positively charged 55
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Scheme 1. Schematic illustration of the theranostic process of TarA@bM
TPP+ to orient to mitochondria. As shown in Fig. 3, both TarA@bM and NTarA@bM were coincubated with cells for 4 h, respectively. Blue fluorescence indicated the stained nucleus and the lysosome (above) and mitochondria (below) were stained with green fluorescence. Furthermore, red fluorescence indicated the biosynthesized PpIX was used to verify the successful internalization of δ-ALA. As shown in the merged image, the green fluorescence of mitochondria was nearly overlapped with that of TarA@bM, while this overlap was partially
observed in NTarA@bM treated cells, indicating the localization of TarA@bM to mitochondria.
3.3. In vitro assessment of photodynamic therapy Photodynamic therapy (PDT) composed of three components: photosensitizer, illuminant and oxygen. In this system, two complementary aspects were designed cooperatively to enhance the therapeutic
Fig. 1. a) TEM image of TarA@bM. b) FTIR spectra of TarA@bM and BSA. c) UV–vis absorbance spectrum of TarA@bN and TarA@bM. d) The XPS spectrum analysis of Mn[Ⅳ] 2p peak from TarA@bM 56
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Fig. 2. a) The degradation behavior of TarA@bM under normal and simulated conditions. b) The release behavior of δ-ALA under normal and simulated conditions. c) Oxygen generation in different condition. Fig. 3. Subcellular localization of δ-ALA biosynthesized PpIX induced red fluorescent-targeted TarA@ bM and nontargeted NTarA@bM. 4T1 cells were stained with the mitochondrial marker MitoTracker Green and lysosome probe LysoTracker Green, respectively. The rightmost image (bar: 50 μm) was the enlarged view of its left one (bar: 20 μm). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
besieging by the proton-sponge effect and then the lysosome breaks up. Therefore, the residual nanoparticles and proteolytic enzymes were removed. The linker between δ-ALA and TPP+ could be cleaved by proteolytic enzymes to free δ-ALA, which was in agreement with the in vitro analysis of the released δ-ALA. This result indicated that more endogenous PpIX was synthesized with a mitochondrial-targeting ligand. Next, we investigated the intracellular ROS generation properties. An ROS probe CM-H2DCFDA which can be oxidized by ROS to induce bright green fluorescence, was used. As shown in Fig. 4b, all groups were treated with irradiation to preclude the influence on illumination. Seldom green fluorescence in the control group indicated that light had little influence on cells to produce ROS and the self-produced ROS in cells was negligible. It was obvious that the TarA@bM group (p＜0.05) exhibited stronger fluorescence intensity in both fluorescent image and flow cytometry than the TarA@bN group (p＜0.05), which verified that the existence of MnO2 induced increase in oxygen could finally enhance ROS generation. It is widely believed that mitochondrial depolarization reflects the early stages of apoptosis. The JC-10 assay was used to verify whether mitochondria function well. When mitochondria were in good
outcome. A signal motivates a proapoptotic process with the overload of ROS accumulated intracellularly [28], and contributed to the catalysis of MnO2 with endogenous H2O2. In addition, the elevated endogenously biosynthesized PpIX could elevate the yield of the photosensitizer. Both of these characteristics contribute to photodynamic therapy under illumination. First, we evaluated the photosensitizer yield among the free δ-ALA, NTarA@bM and TarA@bM groups. It was obvious that the fluorescence intensity in all groups showed a timedependent manner (Fig. 4a). At the same time point, the fluorescence intensity induced by free δ-ALA was weaker than those of both NTar@ bM and TarA@bM. This result can be explained by the fact that compared to free small molecules, nanoparticles more favorably accumulated in the tumor area by the EPR effect. Furthermore, the TarA@bM group demonstrated stronger fluorescence intensity than the NTarA@ bM group. This result could be explained as follows: before PpIX was synthesized in mitochondria, the precursor was detained in a lysosome. Lysosomes are indigestive components of cells that contain many proteolytic enzymes. The similar hydrolysis manner of amide bond and peptide bond can take into effect in this condition to free δ-ALA. According to our design, the CTPP group can break through lysosome 57
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Fig. 4. a) The fluorescence intensity of endogenous biosynthetic of PpIX at different time point. The orange one is TarA@bM，the blue curve represents NTarA@bM and the red one is free δ-ALA. b) The fluorescence images of ROS generation of each sample. The fluorescence intensity was quantified by flow cytometry in the rightmost images. c) JC-10 assay on each group. d, e) The statistical analysis data of mean intensity ROS fluorescence and rate of depolarization/polarization, respectively. f) The ATP detection on different samples. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
condition, JC-10 was observed in the form of green fluorescent aggregates. The JC- aggregate transformed to a JC-monomer if the mitochondria were dysfunctional and induced red fluorescence along with
the reduction of Δψm or, in other words, the mitochondria membrane became depolarized. Therefore, the decreased Δψm not only indicates mitochondrial dysfunction but also is an important indicator of
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flow cytometry. The results demonstrated that TarA@bM with illumination caused the highest apoptosis rate, which was in agreement with the result from the CCK-8 assay of cell viability (Fig. 5b). As previously reported, high concentrations of ROS cause severe oxidative stress in mitochondria, and the collapse of Δψm could lead to mitochondrial permeability transition (PT); the opening of the mitochondrial permeability transition pore (PTP) results in endosmosis of small molecules, thereby resulting in cytotoxicity [4]. This disturbance might ultimately make mitochondria dysfunctional as well as reduce the ATP level in cells. To investigate the potential molecular mechanism of this nanoplatform in apoptosis, the relative protein expression in the Bcl-2 family was analyzed by Western blot. The ratio of Bax/Bcl-2 could be a useful indicator of the cell threshold for apoptosis-induced PDT [29,30]. As shown in Fig. 5d, the overexpression of the proapoptotic protein Bax was significantly detected. In parallel, the expression level of the antiapoptotic protein Bcl-2 was decreased. Based on these various impacts, it could be concluded that this systematic nanoplatform had a synergistic effect to promote PDT.
apoptosis. Then, we evaluated the impact on mitochondria. Flow cytometry was used to measure the fluorescence intensity ratio of red/ green fluorescence, further accounting for the rate of depolarization/ polarization, respectively. As shown in Fig. 4c, the upper area indicated the ratio of the JC-aggregate, while the area below indicates the JCmonomer. In this light-triggered system, it was obvious that under dark conditions, the ratio of fluorescence intensity changed negligibly suggesting that simple nanoparticles in the dark have little influence on Δψm. When the nanoparticles were administrated and illuminated, the Δψm in both the NTar@bM (p＜0.01) and TarA@bM (p＜0.01) groups decreased to certain levels, but the latter group decreased more. This study elucidated that δ-ALA based photodynamic therapy damaged mitochondria, but the targeted component promoted the attack efficiency as a result of the increased accumulation of effective constituents, which was in accord with the localization and biosynthesis assay. ATP is crucial for providing intracellular energy, therefore, the detection of ATP levels is an important indicator assessing cell viability. As shown in Fig. 4f, the ATP levels in the treatment groups were slightly different from those in the control group when treated in the dark. The ATP level decreased most in the TarA@bM group (p＜0.01). Similar results were observed in the apoptosis and cell viability assays. As shown in Fig. 5a, the apoptosis/necrosis rate was detected by
3.4. In vitro and in vivo MR/FL imaging To investigate enhanced MRI as a result of the decomposition of MnO2 in acidic tumor conditions, different buffers (pH = 6.5 and 7.4)
Fig. 5. a) Apoptosis assay of different treated 4T1 cells under different conditions. b,c) Cell viability assay on different treatments by CCK-8. b was in the dark condition while c was under illumination. d) Western blot assay of different treated 4T1 cells. 59
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Fig. 6. a) In vitro MRI measurement of TarA@bM in concentration gradient under different condition. b,d) In vivo MRI measurement at different time point. d was the images of different time point and b was calculated relative MR signal intensity. c.e) In vivo photoluminescence imaging measurement of TarA@bM. e was the images at different time point and c was calculated relative mean intensity.
3.5. In vivo biodistribution measurement
were used to assess the relaxivity value (r1) of TarA@bM. As shown in Fig. 6a, TarA@bM in both neutral and acidic conditions showed concentration-dependent manners. Notably, when treated in acidic conditions (pH = 6.5), the imaging was brighter than its neutral counterpart. The calculated relaxivity value (r1) at pH = 6.5 was 5.410 s−1mM−1, which was three times greater than that in a normal environment. There results might be attributed to the increased release of Mn2+ compared with manganese dioxide, which functioned as a T1 contrast agent [31] in an acidic environment and therefore enhanced the MR imaging properties. Next, we carried out an in vivo experiment on 4T1 cell-bearing-mice to verify its final image-guided effect on small mammals. From the MR images at different timepoints (Fig. 6d), after 12 h injection, the tumor region showed the brightest imaging, indicating effective accumulation, while the MR signal intensity showed a slightly decreased in brightness after 24 h, which may be attributed to the clearance of small molecular Mn2+ by the reticuloendothelial system (RES). Similarly, photoluminescence (PL) imaging was demonstrated in the same manner to assess the imaging capability. Interestingly, a hysteresis effect was observed by FL imaging (Fig. 6e), and at 24 h injection, the fluorescence intensity was stronger than that at 12 h postinjection. We speculated that this could be attributed to the timeconsuming biosynthetic pathway of PpIX, which induces the final fluorescence. Furthermore, the lasting fluorescence signal at 36 h may be attributed to effective retention in the tumor area that could be used to monitor the posttreatment process and provide prognosis assessment.
To further understand the biodistribution of TarA@bM, we monitored the blood circulation and tissue distribution by measuring the [Mn2+] in the blood samples and major organs of TarA@bM treated mice by ICP-AES. Fig. 7a shows the distribution of the Mn2+ in main tissues. The amount of Mn was measured at 12 and 24 h by dissolving the main tissue in aqua regia. The high concentration of TarA@bM resulted in an effective accumulation of TarA@bM in the tumor section for 12 h. Notably, the results also demonstrated that the liver and spleen showed rather high levels of nanoparticle accumulation, which could be contributed to the function of the reticuloendothelial system. After 48 h, the concentration in each tissue decreased correspondingly, which might be attributed to the biodegradation of TarA@bM, but still remained at a considerable concentration in the tumor region (9.7%). The half-life time was calculated by fitting with a two-compartment model. The results (Fig. 7b) of the calculation showed that the first (t1/2(α)) and second (t1/2(β)) phases were 1.36 ± 0.21 h and 8.5 ± 1.12 h, respectively. The relatively long blood half-life could be contributed to the existence of a protein-based carrier that could promote circulation time, as previously reported [32].
3.6. In vivo photodynamic therapy 4T1 tumor-bearing Balb/c mice were divided randomly into six groups as follows: PBS, laser only, δ-ALA without laser, δ-ALA with 60
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Fig. 7. a) The biodistribution of TarA@bM in the main organs in 12 h and 24 h. b) The blood circulation curve of TarA@bM in 36 h. c) The photodynamic effect on tumors of different treatments. d) The relative tumor volume on different treatments. e) The relative body weight on different treatments. f) Heat map of the blood routine examination on different treatments. g) Histological H&E (bar is 100 μm) staining and TUNEL (bar is 50 μm) staining of the tumor tissues on different treatments.
stained major organs, including heart, liver, spleen, lung, and kidney, in all the groups showed no conspicuous necrosis or physiological morphology changes (Fig. S5). Moreover, the routine blood examination (Fig. 7f) showed that all the groups had little influence on normal physiological activity. All the results demonstrated that this nanoplatform exerted no obvious toxicity to animals.
laser, TarA@bM without laser and TarA@bM with laser. According to the results of fluorescence imaging, we administrated illumination 24 h after intravenous tail injection. The tumor area was exposed to light illumination for 10 min. All the body weight and tumor areas were recorded at 14 days after illumination. The laser only group had no effect on either the tumor or body weight compared with the control group. The δ-ALA and TarA@bM groups without laser illumination exhibited slight inhibition of tumor proliferation, which could be attributed to the influence of sunlight. The relative tumor volume on both TarA@bM and δ-ALA with laser illumination showed a considerable decrease compared with the same injection groups without laser illumination (Fig. 7c and d). To further understand the therapeutic effects on tumor tissues, relevant organs and tumors were harvested for histological analysis. As shown in Fig. 7g, obvious cytoplasmic leakage and nuclear shrinkage of apoptotic cells were observed in the H&E stained tumor slices from the TarA@bM treated PDT group. Furthermore, TUNEL staining was used to locate the apoptotic cells. The strong green fluorescence was attributed to the nucleus of apoptotic cells in the TarA@bM PDT group. These results showed that compared to the traditional therapeutic molecule, TarA@bM showed an enhanced therapeutic effect, which could be considered for clinic application. Finally, the safety of this nanoplatform was assessed. From the macroscopic perspective, the body weight (Fig. 7e) in all the groups slightly increased, indicating that all the treatments had no obvious side-effects on the mice. Next, the histological examination of the H&E
4. Conclusion In summary, we designed a complete theranostics system through a simply modified photosensitive precursor, δ-ALA. Two complementary components were fabricated for enhanced photodynamic therapy. From one of the components, mitochondrial ligands were introduced to the precursor that were converted into photosensitizer PpIX for more specific delivery to enhance the yield of PpIX and make mitochondria dysfunctional. For the other component the modified MnO2 could interact with endogenous H2O2 to elevate oxygen, resulting in severe oxidative stress. Both components contributed to apoptosis, leading to the final lethal signal. Moreover, with the dual imaging system, the entire process of treatment was monitored in a pivotal period for more accurate treatment. The in vivo experiments demonstrated superior imaging results and tumor inhibition, suggesting that this nanoplatform can function as favorable therapeutic agent and broaden the clinical application of δ-ALA. 61
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Notes
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