Acta Biomaterialia xxx (2015) xxx–xxx
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pH-responsive metallo-supramolecular nanogel for synergistic chemo-photodynamic therapy Xuemei Yao a, Li Chen a,⇑, Xiaofei Chen a, Zhigang Xie b, Jianxun Ding b, Chaoliang He b, Jingping Zhang a, Xuesi Chen b a b
Department of Chemistry, Northeast Normal University, Changchun 130024, PR China Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China
a r t i c l e
i n f o
Article history: Received 26 February 2015 Received in revised form 14 July 2015 Accepted 14 July 2015 Available online xxxx Keywords: Chemo-photodynamic therapy Metallo-supramolecular nanogel pH-responsiveness Synergistic treatment
a b s t r a c t Benefited from the high orientation of coordinated interaction, metallo-supramolecular materials have attracted enormous interest in many fields. Herein, a novel metallo-supramolecular nanogel (SNG)-based drug delivery system for synergistic chemo-photodynamic therapy is explored to enhance anticancer efficacy. It is fabricated by the metallo-supramolecular-coordinated interaction between tetraphenylporphyrin zinc (Zn-Por) and histidine. It can respond to tumor acid microenvironment to release the co-delivered anticancer drug and photosensitizer to kill the lesion cells. Zn-Por moieties in SNG keep the photosensitivity in the range of visible wavelength and possess the ability of generating active oxygen species for photodynamic therapy. The drug-loaded SNG provides a di-functional platform for chemotherapy and photodynamic therapy. Compared with the single chemotherapy of free doxorubicine (DOX) or photodynamic therapy of Zn-Por in SNG, DOX-loaded SNG with irradiation shows higher in vitro cytotoxicity and in vivo anticancer therapeutic activity, endowing the SNG with great potential in cancer treatments. The statement of significance A combination of multiple non-cross-resistant anticancer agents has been widely applied clinically. Applying multiple drugs with different molecular targets can raise the genetic barriers and delay the cancer adaption process. Multiple drugs targeting different cellular pathways can function synergistically, giving higher therapeutic efficacy and target selectivity. Overall, developing a combination therapeutic approach might even be the key to enhance anticancer efficacy and overcome chemo-resistance. Herein, a novel metallo-supramolecular nanogel (SNG) is fabricated by the metallo-supramolecular-coor dinated interaction between tetraphenylporphyrin zinc (Zn-Por) and histidine. The DOX-loaded SNG provides a di-functional platform for chemotherapy and photodynamic therapy because it can respond to tumor acid microenvironment to release the co-delivered anticancer drug and photosensitizer to kill the lesion cells. Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction In the biological system, supramolecular chemistry plays an important role, such as hemoglobin carrying oxygen efficiently from lungs to the tissues of the body by the metallo-supramolecular interaction between iron-porphyrin and L-histidine. It is only in recent years that supramolecular chemistry has progressively drawn increasing attention and has been successively employed to construct the functional structure-defined materials for application in
⇑ Corresponding author. E-mail address: [email protected] (L. Chen).
many fields. Compared with covalent interactions, supramolecular interactions [1], as a kind of non-covalent interaction, such as p–p conjugation [2], host–guest recognition [3,4], metallosupramolecular interaction [5], halogen bonding [6], and hydrogen bonding [7], exhibit their unique convenience and flexibility. On account of the dynamic and reversible nature of non-covalent interactions, the materials based on supramolecular interactions have the ability to adapt to their environment and possess some intriguing properties, such as degradability, stimuli-response, and self-healing, making them special candidates of intelligent materials [8,9]. Considering various mentioned physical interactions, metal– ligand coordination is a preeminent approach for constructing many types of supramolecular materials due to the high degree
http://dx.doi.org/10.1016/j.actbio.2015.07.024 1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: X. Yao et al., pH-responsive metallo-supramolecular nanogel for synergistic chemo-photodynamic therapy, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.07.024
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Scheme 1. Schematic illustration of DOX loading and in vivo microenvironment-triggered release from DOX-loaded SNG and photodynamic therapy.
of orientation of the coordination bond [10]. In addition, a wide range of easily functionalized ligands can be chosen, and the interaction strength can be well tuned by choosing appropriate metal ions and/or ligands. Moreover, the introduction of metal complexes in the polymer structure endows electrochemical [11], photo-physical [12], catalytic [13], and magnetic [14] properties, potentially, allowing these copolymers as precursors to generate the inorganic or hybrid structures. The presence of metal centers in polymers also offers many unique opportunities to construct external stimuli-responsive materials [15]. However, as we know, the stimuli-responsive materials based on metallosupramolecular interaction as ‘‘smart’’ drug delivery systems are seldom reported. Metalloporphyrins, as a group of organic compounds with a conjugated macrocyclic structure, have attracted increasing attention because of their potential applications in catalytic, photo-physical, and biological fields. In particular, some metalloporphyrins, such as zinc-porphyrins, have been used as a kind of photosensitizer in clinical photodynamic therapy because of the effective generation of cytotoxic reactive oxygen species, such as singlet oxygen [16]. Several examples of dual effect combining the photodynamic action with a known therapeutic agent are reported [17–20]. Zhang and co-workers synthesized two kinds of zinc(II) phthalocyanine–erlotinib conjugates. In vitro photodynamic activities and selective affinity of these conjugates toward HepG2 cancer cells and A431 tumor tissues were evaluated [21]. Recently, our group had fabricated a kind of dual pH-responsive mesoporous silica nanoparticle (MSN)-based drug delivery system for synergistic chemo-photodynamic therapy, which could respond to the cancer extracellular and intercellular pH stimuli. This dual pH-sensitive MSN-based drug delivery system showed
higher in vitro cytotoxicity than the single chemotherapy of free DOX or photodynamic therapy of Zn-Por [22]. Doxorubicin (DOX) has been widely used in chemotherapy to treat several types of cancers. However, severe heart toxicity limited its clinical efficiency and applications [23]. Moreover, it will be cleared fast from the blood system because of nonspecific protein adsorption and result in an immune response, or even severe systemic toxicity. To overcome this drawback, many nanoparticles with biocompatibility and biodegradability have been constructed for drug delivery systems [24,25]. Nanoparticles can deliver drugs to the cancer tissue under the help of the enhanced permeability and retention (EPR) effect, which is the property by which certain sizes of molecules (typically liposomes, nanoparticles, and macromolecular drugs) tend to accumulate in tumor tissue much more than they do in normal tissues [26]. Administering a single drug is unlikely to succeed in the treatment of cancer. A combination of multiple non-cross-resistant anticancer agents has been widely applied clinically [27]. Applying multiple drugs with different molecular targets can raise the genetic barriers and delay the cancer adaption process [28]. Multiple drugs targeting different cellular pathways can function synergistically, giving higher therapeutic efficacy and target selectivity. Overall, developing a combination therapeutic approach might even be the key to enhance anticancer efficacy and overcome chemoresistance. In our previous work [29–31], we have made a detailed study on the pH-responsive metallo-supramolecular coordinated interaction between histidine and porphyrin. By this interaction, pH-responsive supramolecular nanogels can be fabricated to delivery the anticancer drug for single chemo-treatment. Based on the above mentioned work, herein, a much deeper research has been done to realize the combination of chemo-treatment and
Please cite this article in press as: X. Yao et al., pH-responsive metallo-supramolecular nanogel for synergistic chemo-photodynamic therapy, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.07.024
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photodynamic treatment. In this article, tetraphenylporphyrin zinc (Zn-Por), a kind of photosensitizer, is used to coordinate with histidine to fabricate pH-responsive metallo-supramolecular nanogel (SNG), which can respond to tumor acid microenvironment to release not only the anticancer drug but also the photosensitizer to kill the lesion cells. Zn-Por moieties in SNG keep the photosensitivity in the range of visible wavelength and possess the ability of generating active oxygen species for photodynamic therapy. Several articles about tumor-site acidic activation of photosensitizers have been reported [32,33]. Considering the safety of materials, dextran and histidine were chosen, which are biocompatible and biodegradable [34,35]. Boc-histidine also acts as a hydrophobic group and can coordinate with Zn-Por to form a pH-responsive nanogel. The drug-loaded SNG provides a di-functional platform for chemotherapy and photodynamic therapy, endowing the SNG with great potential for cancer treatments. 2. Materials and methods 2.1. Materials Dextran (Dex, Mn = 40 kDa, Sigma) and Boc-His-OH (Sigma) were used directly. Doxorubicin hydrochloride (DOXHCl) was bought from Zhejiang Hisun Pharmaceutical Co., Ltd. 3-(4,5-Dime thyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was obtained from Sigma–Aldrich. Benzaldehyde, propionic acid and pyrrole were bought from Aladdin and pyrrole was used after distilling. 2.2. Characterization 1
H NMR spectra were collected on a Bruker AV 400 NMR spectrometer. Fourier transform infrared (FTIR) measurements were performed on a Bio-Rad Win-IR spectrometer. The size and distribution of particles were tested by a WyattQELS dynamic laser scattering (DLS) instrument. Confocal laser scanning microscopy (CLSM) was performed by Olympus FluoView 1000. MTT assay was conducted by a Bio-Rad 680 microplate reader. UV–vis
3
spectra were recorded on a Shimadzu UV-2401PC UV–vis spectrophotometer. 2.3. Synthesis of tetraphenylporphrin zinc (Zn-Por) Tetraphenylporphrin zinc (Zn-Por) was synthesized according to the previous literature [36] as shown in Scheme S1. TPP (1 g, 1.6 mmol) prepared using the Adler method as in previous work [29] and Zn(Ac)2 2H2O (1.4 g, 6.4 mmol) were dissolved in chloroform (CHCl3) (40 mL). Then the above mixture was refluxed and the reaction process was monitored by thin-layer chromatography (TLC). The resulting mixture was cooled. The solvent was removed and the resulting solid was washed with water and then vacuum-dried. 2.4. Synthesis of histidine modified dextran (DH) As shown in Scheme 2, dextran (Dex, 1.0 g, 0.025 mmol), histidine (0.47 g, 1.9 mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC) (0.75 g, 3.8 mmol) and 4-dimethylaminopyridine (46 mg, 0.38 mmol) were dissolved in DMSO (20 mL) at room temperature. After reacted 2 d, the solvent and unreacted substances were removed by dialysis against deionized water for 72 h. The product was obtained through lyophilization. 2.5. Synthesis of metallo-supramolecular nanogel (SNG) DH (0.004 mM, 0.5 g) and Zn-Por (0.5 mM, 0.35 g) were dissolved in 10 mL of DMSO. The mixture was stirred at 25 °C for 48 h. Then, the solvent and unreacted substances were removed by dialysis against deionized water for 72 h. The products were obtained by lyophilization. See Scheme 2. 2.6. In vitro drug loading and release Doxorubicin (DOX) was used as a model drug for in vitro drug loading and release. DOX loaded SNGs were prepared by a simple
Scheme 2. Synthetic route for DH and SNG.
Please cite this article in press as: X. Yao et al., pH-responsive metallo-supramolecular nanogel for synergistic chemo-photodynamic therapy, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.07.024
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dialysis technique. Typically, SNG (20.0 mg) and drug (4.0 mg) were mixed in 2.0 mL of DMSO. The mixture was stirred at room temperature for 24 h and then added dropwise into 20.0 mL of PBS at pH 7.4. The DMSO was removed by dialysis against water at pH 7.4 for 24 h. The dialysis medium was refreshed four times and the whole procedure was performed in the dark. Finally, the solution was filtered and lyophilized. To determine the drug loading content (DLC) and drug loading efficiency (DLE), the drug-loaded SNGs were dissolved in DMSO and analyzed by fluorescence measurement (Perkin-Elmer LS50B luminescence spectrometer) using a standard curve method (kex = 480 nm). DLC and DLE of drug-loaded SNGs were calculated according to Eqs. (1) and (2), respectively:
DLC ðwt%Þ ¼ amount of drug in SNG=amount of drug loaded SNG 100
ð1Þ
DLE ðwt%Þ ¼ amount of drug in SNG=total amount of feeding drug 100
ð2Þ
In vitro drug release profiles of drug-loaded SNGs were investigated in PBS (pH 5.3, 6.8 or 7.4). The pre-weighed freeze-dried DOX loaded SNGs were suspended in 4 mL of release medium and transferred into a dialysis bag (MWCO 3500 Da). The release experiment was initiated by placing the end-sealed dialysis bag into 50 mL of release medium at 37 °C with continuous shaking at 70 rpm. At predetermined intervals, 2 mL of external release medium was taken out and an equal volume of fresh release medium was replenished. The amount of released DOX was determined by using fluorescence measurement (kex = 480 nm). The release experiments were conducted in triplicate. 2.7. Confocal laser scanning microscopy (CLSM) For CLSM study, HepG2 cells were seeded in 6-well plates at a density of 105 cells per well in 2.0 mL of complete Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, supplemented with 50 IU mL1 penicillin and 50 IU mL1 streptomycin. After incubation for 24 h, the culture media were withdrawn and culture media containing SNG or Zn-Por were supplemented. The cells were incubated for another 2 or 6 h and washed with PBS three times. The cells were then fixed in 4% paraformaldehyde for 20 min and washed with PBS thrice. For staining the nuclei, the cells were incubated with 40 ,6-diamidi no-2-phenylindole (DAPI, blue) for 1.5 min. The images of cells were observed using a laser scanning confocal microscope (Olympus FluoView 1000). 2.8. Flow cytometric analyses HepG2 cells were incubated into 6-well plates based on 2 105 cells/well and cultured in 2.0 mL of DMEM for 24 h. The culture media were then replaced by culture media with Zn-Por or SNG at a final Zn-Por concentration of 0.72 mg mL1. The cells were incubated for an additional 2 or 6 h, then washed with PBS three times and trypsinized. 1.0 mL of DMEM was added, and the solutions were centrifuged for 5 min at 1000 rpm, then 1.0 mL of PBS was used to wash twice and the cells were resuspended in 0.2 mL of PBS. The analysis was performed by flow cytometer (Beckman, California, U.S.A.) for 1 104 cells.
DMEM and incubated at 37 °C in 5% CO2 atmosphere for 24 h. The culture medium was then removed and SNG solutions in complete DMEM at different concentrations (0–50 mg L1) were added. The cells were subjected to MTT assay after being incubated for an additional 24 h under light irradiation (2 mW, 400–700 nm). The absorbance of the formazan was measured on a Bio-Rad 680 microplate reader at 490 nm. Cell viability (%) was calculated based on Eq. (3):
Cell viability ð%Þ ¼ Asample =Acontrol 100
ð3Þ
where, Asample and Acontrol represent the absorbances of the sample and control wells, respectively. The cytotoxicities of DOX-loaded SNG against HepG2 and HeLa cells were also evaluated in vitro by an MTT assay. Similarly, cells were seeded into 96-well plates at 7 103 cells per well in 200.0 lL of complete DMEM and further incubated for 24 h. After washing cells with PBS, 180.0 lL of complete DMEM and 20.0 lL of DOX-loaded SNG solutions in PBS were added to form culture media with different DOX concentrations (0–10.0 mg L1 DOX). The cells were subjected to MTT assay after being incubated for 24 h. The absorbance of the solution was measured on a Bio-Rad 680 microplate reader at 490 nm. Cell viability (%) was also calculated based on Eq. (3). 2.10. Pharmacokinetics Wistar rats were randomly divided into two groups, containing three mice (average weight 250 g). DOX, DOX-loaded SNG were administered via the tail vein (Dosage, 6 mg DOX equivalent/kg body weight). At defined time periods (5, 10 min, 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 h), blood samples were collected from the orbital cavity, heparinized, and centrifuged (11,000 r/min, 5 min) to obtain the plasma. The concentrations of DOX in samples were detected by HPLC methods based on previous literature [37] with minor modifications. Briefly, 200 lL of plasma sample was deproteinized with 800 mL of methanol and 10 lL of daunorubicin hydrochloride (1 lg mL1, internal standard), then vortexed for 10 min, and centrifuged at 11,000 rpm for 5 min. Then supernatant was collected. The precipitation was added 800 mL of methanol then vortexed for 10 min, and centrifuged at 11,000 rpm for 5 min. Then supernatant was collected and dried under a stream of nitrogen at 25 °C. The dried sample was then dissolved in the mobile phase for HPLC analysis. 2.11. Anti-tumor efficacy The in vivo anti-tumor efficacy of the drug-loaded SNG was evaluated utilizing the xenograft B16F10 melanoma xeno-implanted on BALB/C mice. Treatments were started 5 days after cell implantation, when tumor in the mice reached a volume of 35–70 mm3, and this day was designated as day 0. The mice were weighed and randomly divided into 6 groups (8 mice per group): saline, SNG, SNG with irradiation, free DOX, DOX-loaded SNG and DOX-loaded SNG with irradiation (dosage: 15 mg Zn-Por/kg body weight, 3.0 mg DOX/kg body weight). The injection was carried out on day 1, 5, 9, 13 via tail vein, then irradiating (40 mW/cm2, 400–700 nm) for 5 min after 24, 48 and 72 h. The treatment efficacy and safety were assessed by measuring the tumor volume and body weight, respectively. Tumor volume was calculated by the following formula: 2
2.9. Cell viability assays The relative cytotoxicities of SNG against HepG2 and HeLa cells were evaluated in vitro by a standard MTT assay. The cells were seeded in 96-well plates at 1 104 cells per well in 200.0 lL of complete
Tumor volume ðVt Þ ¼ a b =2 Tumor growth rate ðTGR; %Þ ¼ ðVt =V0 Þ 100% where, a and b are the major and minor axes of the tumors measured by caliper; V0 represented the initial volume.
Please cite this article in press as: X. Yao et al., pH-responsive metallo-supramolecular nanogel for synergistic chemo-photodynamic therapy, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.07.024
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2.12. Detection of singlet oxygen Generation of singlet oxygen can be usually detected by the characteristic photoluminescence peak of singlet oxygen at 1270 nm or by chemical method using 1,3-diphenylisobenzofuran (DPBF) or 9, 10-anthracenediyl-bis(methylene) dimalonic acid (ABDA) as a singlet oxygen sensor. In our experiment, ABDA was chosen to monitor the release of singlet oxygen into solution by recording the decrease in absorption of ABDA at 376 nm via UV–vis spectroscopy. 50 lL of ABDA solution (1 g/mL) was mixed well with SNG and DOX-loaded SNG (0.5 g/mL) and placed in a cuvette, respectively. The ABDA alone in PBS was used as the control. The absorption intensity of ABDA at 376 nm was monitored under different blue light illumination periods. 2.13. Statistical analysis All experiments were conducted at least three times and expressed as mean ± standard deviation (SD).
Fig. 2. In vitro DOX release profiles for DOX-loaded SNG pH 5.3 (a), DOX-loaded SNG pH 6.8 b), DOX-loaded SNG pH 7.4 (c), DOX-loaded DH pH 5.3 (d), DOX-loaded DH pH 6.8 (e) and DOX-loaded DH pH 7.4 (f) in PBS at 37 °C, respectively (n = 3, mean ± SD).
3. Results and discussion 3.1. Preparation and properties of SNG Along this line, at the first step, we synthesized Zn-Por (Scheme S1, Figs. S1–S3) and dextran-g-histidine copolymer (DH). Then SNG was fabricated by the metallo-supramolecular coordination between Zn-Por and histidine in DH (Scheme 2). By adjusting the feed ratio, SNG was synthesized as listed in Table S1. The 1H NMR spectrum shown in Fig. S4 verified the successful preparation of SNG. Compared with that of DH (Fig. S4A), peak 12 (–C(N@)@CH–NH–) signed to histidine showed a shift from 6.95 to 6.9 ppm in the spectrum of SNG (Fig. S4B). A similar peak shift from 7.7 to 7.6 ppm also could be observed for peak
11 (–N@CH–NH–). These peaks shifts were attributed to the coordinated interaction between Zn-Por and histidine and thus evidenced the successful synthesis of SNG. And the signals at 8.9 (a), 8.2 (b) and 7.9 ppm (c) were signed to Zn-Por in SNG. As shown in Fig. S5B, the peak at 1742 cm1 attributed to C@O inferred that histidine was grafted onto dextran successfully. The characteristic peaks at 1588 and 1480 cm1 in Fig. S5C were signed to phenyl group in Zn-Por and the peak at 1003 cm1 was attributed to the conjugated macrocycle. All of them further confirmed the structures of SNG. It is known that two histidine moieties can bind to one Zn-Por via metallo-coordinated interactions at the normal biological pH of 7.4, while under acidic conditions (e.g., pH < 6), histidine will
Fig. 1. CMC values of SNG at pH 7.4 and 5.3 (A); hydrodynamic radii (Rh) of SNG in PBS at pH 7.4 and 5.3 (B); TEM micrographs of SNG at pH 7.4, 0 h (C), pH 7.4, 72 h (D), and pH 5.3, 0 h (E).
Please cite this article in press as: X. Yao et al., pH-responsive metallo-supramolecular nanogel for synergistic chemo-photodynamic therapy, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.07.024
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distribution of SNG become smaller. To test the formation and pH-response of SNG, Nile red was used as a probe to measure the CMC values. As shown in Fig. 1A, the CMC value of SNG in PBS at pH 7.4 was 18.6 mg/L while it was 79.0 mg/L at pH 5.3. Dynamic light scattering (DLS) measurements also indicated the size and size distribution of SNG. As shown in Fig. 1B, SNG exhibited an average hydrodynamic radius (Rh) of 73 nm with quite narrow size distribution at pH 7.4, which can preferentially reach and accumulate in tumors by the EPR effect. At pH 5.3 two DLS peaks were detected with one centered at 5 nm and the other at 165 nm, indicating dissociation and re-association of SNG at pH 5.3. TEM micrographs of SNG at pH 7.4 (Fig. 1C) and 5.3 (Fig. 1E) also confirmed these results. These changes of CMC value and particle size of SNG with pH variation demonstrated that SNG could respond to different pH environments. Moreover, as shown in Fig. 1C and D, the size of SNG remained almost unchanged after standing for 72 h at pH 7.4, confirming again the stability of SNG in normal biophysical microenvironment. 3.2. In vitro DOX loading and triggered release Fig. 3. Flow cytometry profiles of HepG2 cells incubated with PBS, Zn-Por for 2 and 6 h, SNG for 2 and 6 h.
be protonated (shown in Fig. S6) and the histidine/Zn-Por coordinates would disassemble dramatically, resulting in the dissociation of Zn-Por from histidine stalk. In order to comment the effect of the presence of Zn-Por on the size of SNG, the size and the critical micelle concentration (CMC) values in pH 7.4 for SNG and DH were measured, which were 73 ± 13 nm and 18.6 lg/mL and 286 ± 184 nm and 106.4 lg/mL, respectively (Table S1). The results demonstrated the presence of Zn-Por made the size and
To verify the feasibility of SNG as a delivery platform for antitumor drugs, doxorubicin (DOX) was chosen as a model drug and encapsulated into SNG via a typical dialysis method (Scheme 1). In order to indicate the stability of DOX-loaded SNG, DOX-loaded DH was chosen as the comparable group. Compared with DOX-loaded DH, DOX-loaded SNG presented higher DLC and DLE, which were 5.32% and 36.7% for DOX-loaded SNG and 3.43% and 25.6% for DOX-loaded DH, respectively. These results manifested SNG was more stable than DH, which could be ascribed to the cross of SNG. The in vitro release experiments were performed at pH 5.3, 6.8 and 7.4, mimicking the pH values under tumor intracellular,
Fig. 4. Representative CLSM images of HepG2 cells incubated with Zn-Por for 2 h (A), SNG for 2 h (B), Zn-Por for 6 h (C), and SNG for 6 h (D). For each panel, the microimages from left to right show a differential interference contrast (DIC) image, cell nuclei stained by DAPI (blue), Zn-Por fluorescence in cells (red), and overlays of the three images. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: X. Yao et al., pH-responsive metallo-supramolecular nanogel for synergistic chemo-photodynamic therapy, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.07.024
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Fig. 5. The photodynamic cytotoxicity toward HepG2 (A) and HeLa cells (B) incubated with SNG under different light irradiation times; Cytotoxicity of Zn-Por under light irradiation for 30 min, SNG without light irradiation, SNG under 30 min of light irradiation, DOX-loaded SNG without light irradiation, DOX-loaded SNG under 30 min of light irradiation and DOX toward HepG2 (C) and HeLa cells (D) (n = 3, mean ± SD).
tissular and normal biophysical conditions, respectively. The cumulative release percentages of DOX loaded in SNG were plotted versus time in Fig. 2. The DOX release rate was in the order of pH 7.4 < pH 6.8 < pH 5.3. Up to about 80% of DOX was released from the DOX-loaded SNG in PBS at pH 5.3 in 48 h, while only about 25% of DOX was released at pH 7.4 in the same time interval. These different release behaviors should be attributed to the pH-triggered dissociation of Zn-Por/histidine coordinates, i.e., SNG could effectively hinder the loaded drug from releasing during blood circulation, while releasing it rapidly at lower pH in tumor microenvironment. This pH-sensitive property makes it possible for SNG to efficiently encapsulate hydrophobic drugs under neutral condition and release them in intracellular condition, suggesting tremendous potential of SNG in drug delivery. 3.3. Intracellular endocytosis Zn-Por is known as not only a clinical photosensitizer but also a fluorescent probe (red) for cell imaging. So flow cytometric analyses and confocal laser scanning microscopy (CLSM) were employed to monitor the cellular uptake of Zn-Por and SNG by HepG2 cells. As shown in Fig. 3, the fluorescence intensity form high to low was in the following order: SNG for 6 h > SNG for 2 h > Zn-Por for 6 h > Zn-Por for 2 h, respectively. The same phnomenona also can be observed in Fig. 4. After incubation with SNG for 2 or 6 h, the red fluorescence intensity was stronger than that of the cells treated with Zn-Por alone. The intensity difference was bigger at 6 h
than that at 2 h, implying both time and micellarization of Zn-Por had effect on the cell uptake. Furthermore, the red fluorescence of Zn-Por looked as coming from both the nucleus and cytoplasm areas in the SNG-treated cells at 2 h, and mainly from the nuclei at 6 h, while mainly from the cytoplasm area in the free Zn-Por-treated cells at both 2 and 6 h. This is because Zn-Por has limited solubility in cell culture medium and its entry into cells depends on its molecular diffusion through the cell membranes, whereas SNG particles diffuse through the cell membranes and enter the cells efficiently by an active mechanism [38]. The red fluorescence from the nucleus area means that the SNG as a whole or its dissociation products have good affinity to the nuclear membrane and can enter the nuclei with ease. This ability might be ascribed to the co-existence of the histidine residues and Zn-Por in the dissociation products. The former provides affinity to the nuclear membrane and enhances the nuclear uptake and the latter is used as a fluorescent probe to make the detection possible. 3.4. In vitro anti-tumor efficacy As a nanocarrier for drug delivery, biocompatibility is extremely important. The in vitro cytotoxicity of SNG against HepG2 and HeLa cells were examined by an MTT assay. Free SNG did not show appreciable cytotoxicity at different concentrations, up to 10.0 g L1 (Fig. 5), indicating the excellent biocompatibility of SNG itself as a drug carrier.
Please cite this article in press as: X. Yao et al., pH-responsive metallo-supramolecular nanogel for synergistic chemo-photodynamic therapy, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.07.024
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Fig. 6. In vivo pharmacokinetics profiles after intravenous injection of free DOX and DOX-loaded SNG in mice (n = 3, mean ± SD).
To investigate the combined efficiency of chemotherapy and photodynamic therapy, we explored the anticancer activity in vitro and in vivo. Firstly, singlet oxygen generated by SNG was monitored by a chemical method involving the photo-oxidation of 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) (Fig. S7) [39]. Then, MTT assays were employed to investigate the effect of light irradiation (2.0 mW/cm2, 400–700 nm) with different durations on the cytotoxicity toward HepG2 and HeLa cells in vitro. To rule out the potential effect induced by light irradiation, we irradiated HepG2 and HeLa cells directly without SNG or Zn-Por. As shown in Fig. S8, there was no harm to cells by a single light irradiation. Therefore, the effects of light irradiation on the growth of HepG2 cells incubated with Zn-Por and SNG were studied. With increasing light irradiation time, the cytotoxicity of SNG increased (Fig. 5A and B). However, the cytotoxicity of Zn-Por had less change (Fig. S9). We also observed the same phenomenon toward HeLa cells (Figs. 5B and S10). Obviously, this was corresponding to the cell uptake shown in Figs. 3 and 4. Subsequently, HepG2 and HeLa cells were treated with a gradient concentration of Zn-Por, SNG, DOX-loaded SNG and free DOX under or without light irradiation for 30 min. Cell viability data are shown in Fig. 5C and D. For simplicity, only the data in the case of 10.0 mg/L DOX and 0.72 mg/mL Zn-Por were samplified. Firstly,
the three formulations without DOX show the cytotoxicity order of free SNG < Zn-Por under irradiation
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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
pH-responsive metallo-supramolecular nanogel for synergistic chemo-photodynamic therapy Xuemei Yao a, Li Chen a,⇑, Xiaofei Chen a, Zhigang Xie b, Jianxun Ding b, Chaoliang He b, Jingping Zhang a, Xuesi Chen b a b
Department of Chemistry, Northeast Normal University, Changchun 130024, PR China Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China
a r t i c l e
i n f o
Article history: Received 26 February 2015 Received in revised form 14 July 2015 Accepted 14 July 2015 Available online xxxx Keywords: Chemo-photodynamic therapy Metallo-supramolecular nanogel pH-responsiveness Synergistic treatment
a b s t r a c t Benefited from the high orientation of coordinated interaction, metallo-supramolecular materials have attracted enormous interest in many fields. Herein, a novel metallo-supramolecular nanogel (SNG)-based drug delivery system for synergistic chemo-photodynamic therapy is explored to enhance anticancer efficacy. It is fabricated by the metallo-supramolecular-coordinated interaction between tetraphenylporphyrin zinc (Zn-Por) and histidine. It can respond to tumor acid microenvironment to release the co-delivered anticancer drug and photosensitizer to kill the lesion cells. Zn-Por moieties in SNG keep the photosensitivity in the range of visible wavelength and possess the ability of generating active oxygen species for photodynamic therapy. The drug-loaded SNG provides a di-functional platform for chemotherapy and photodynamic therapy. Compared with the single chemotherapy of free doxorubicine (DOX) or photodynamic therapy of Zn-Por in SNG, DOX-loaded SNG with irradiation shows higher in vitro cytotoxicity and in vivo anticancer therapeutic activity, endowing the SNG with great potential in cancer treatments. The statement of significance A combination of multiple non-cross-resistant anticancer agents has been widely applied clinically. Applying multiple drugs with different molecular targets can raise the genetic barriers and delay the cancer adaption process. Multiple drugs targeting different cellular pathways can function synergistically, giving higher therapeutic efficacy and target selectivity. Overall, developing a combination therapeutic approach might even be the key to enhance anticancer efficacy and overcome chemo-resistance. Herein, a novel metallo-supramolecular nanogel (SNG) is fabricated by the metallo-supramolecular-coor dinated interaction between tetraphenylporphyrin zinc (Zn-Por) and histidine. The DOX-loaded SNG provides a di-functional platform for chemotherapy and photodynamic therapy because it can respond to tumor acid microenvironment to release the co-delivered anticancer drug and photosensitizer to kill the lesion cells. Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction In the biological system, supramolecular chemistry plays an important role, such as hemoglobin carrying oxygen efficiently from lungs to the tissues of the body by the metallo-supramolecular interaction between iron-porphyrin and L-histidine. It is only in recent years that supramolecular chemistry has progressively drawn increasing attention and has been successively employed to construct the functional structure-defined materials for application in
⇑ Corresponding author. E-mail address: [email protected] (L. Chen).
many fields. Compared with covalent interactions, supramolecular interactions [1], as a kind of non-covalent interaction, such as p–p conjugation [2], host–guest recognition [3,4], metallosupramolecular interaction [5], halogen bonding [6], and hydrogen bonding [7], exhibit their unique convenience and flexibility. On account of the dynamic and reversible nature of non-covalent interactions, the materials based on supramolecular interactions have the ability to adapt to their environment and possess some intriguing properties, such as degradability, stimuli-response, and self-healing, making them special candidates of intelligent materials [8,9]. Considering various mentioned physical interactions, metal– ligand coordination is a preeminent approach for constructing many types of supramolecular materials due to the high degree
http://dx.doi.org/10.1016/j.actbio.2015.07.024 1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: X. Yao et al., pH-responsive metallo-supramolecular nanogel for synergistic chemo-photodynamic therapy, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.07.024
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Scheme 1. Schematic illustration of DOX loading and in vivo microenvironment-triggered release from DOX-loaded SNG and photodynamic therapy.
of orientation of the coordination bond [10]. In addition, a wide range of easily functionalized ligands can be chosen, and the interaction strength can be well tuned by choosing appropriate metal ions and/or ligands. Moreover, the introduction of metal complexes in the polymer structure endows electrochemical [11], photo-physical [12], catalytic [13], and magnetic [14] properties, potentially, allowing these copolymers as precursors to generate the inorganic or hybrid structures. The presence of metal centers in polymers also offers many unique opportunities to construct external stimuli-responsive materials [15]. However, as we know, the stimuli-responsive materials based on metallosupramolecular interaction as ‘‘smart’’ drug delivery systems are seldom reported. Metalloporphyrins, as a group of organic compounds with a conjugated macrocyclic structure, have attracted increasing attention because of their potential applications in catalytic, photo-physical, and biological fields. In particular, some metalloporphyrins, such as zinc-porphyrins, have been used as a kind of photosensitizer in clinical photodynamic therapy because of the effective generation of cytotoxic reactive oxygen species, such as singlet oxygen [16]. Several examples of dual effect combining the photodynamic action with a known therapeutic agent are reported [17–20]. Zhang and co-workers synthesized two kinds of zinc(II) phthalocyanine–erlotinib conjugates. In vitro photodynamic activities and selective affinity of these conjugates toward HepG2 cancer cells and A431 tumor tissues were evaluated [21]. Recently, our group had fabricated a kind of dual pH-responsive mesoporous silica nanoparticle (MSN)-based drug delivery system for synergistic chemo-photodynamic therapy, which could respond to the cancer extracellular and intercellular pH stimuli. This dual pH-sensitive MSN-based drug delivery system showed
higher in vitro cytotoxicity than the single chemotherapy of free DOX or photodynamic therapy of Zn-Por [22]. Doxorubicin (DOX) has been widely used in chemotherapy to treat several types of cancers. However, severe heart toxicity limited its clinical efficiency and applications [23]. Moreover, it will be cleared fast from the blood system because of nonspecific protein adsorption and result in an immune response, or even severe systemic toxicity. To overcome this drawback, many nanoparticles with biocompatibility and biodegradability have been constructed for drug delivery systems [24,25]. Nanoparticles can deliver drugs to the cancer tissue under the help of the enhanced permeability and retention (EPR) effect, which is the property by which certain sizes of molecules (typically liposomes, nanoparticles, and macromolecular drugs) tend to accumulate in tumor tissue much more than they do in normal tissues [26]. Administering a single drug is unlikely to succeed in the treatment of cancer. A combination of multiple non-cross-resistant anticancer agents has been widely applied clinically [27]. Applying multiple drugs with different molecular targets can raise the genetic barriers and delay the cancer adaption process [28]. Multiple drugs targeting different cellular pathways can function synergistically, giving higher therapeutic efficacy and target selectivity. Overall, developing a combination therapeutic approach might even be the key to enhance anticancer efficacy and overcome chemoresistance. In our previous work [29–31], we have made a detailed study on the pH-responsive metallo-supramolecular coordinated interaction between histidine and porphyrin. By this interaction, pH-responsive supramolecular nanogels can be fabricated to delivery the anticancer drug for single chemo-treatment. Based on the above mentioned work, herein, a much deeper research has been done to realize the combination of chemo-treatment and
Please cite this article in press as: X. Yao et al., pH-responsive metallo-supramolecular nanogel for synergistic chemo-photodynamic therapy, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.07.024
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photodynamic treatment. In this article, tetraphenylporphyrin zinc (Zn-Por), a kind of photosensitizer, is used to coordinate with histidine to fabricate pH-responsive metallo-supramolecular nanogel (SNG), which can respond to tumor acid microenvironment to release not only the anticancer drug but also the photosensitizer to kill the lesion cells. Zn-Por moieties in SNG keep the photosensitivity in the range of visible wavelength and possess the ability of generating active oxygen species for photodynamic therapy. Several articles about tumor-site acidic activation of photosensitizers have been reported [32,33]. Considering the safety of materials, dextran and histidine were chosen, which are biocompatible and biodegradable [34,35]. Boc-histidine also acts as a hydrophobic group and can coordinate with Zn-Por to form a pH-responsive nanogel. The drug-loaded SNG provides a di-functional platform for chemotherapy and photodynamic therapy, endowing the SNG with great potential for cancer treatments. 2. Materials and methods 2.1. Materials Dextran (Dex, Mn = 40 kDa, Sigma) and Boc-His-OH (Sigma) were used directly. Doxorubicin hydrochloride (DOXHCl) was bought from Zhejiang Hisun Pharmaceutical Co., Ltd. 3-(4,5-Dime thyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was obtained from Sigma–Aldrich. Benzaldehyde, propionic acid and pyrrole were bought from Aladdin and pyrrole was used after distilling. 2.2. Characterization 1
H NMR spectra were collected on a Bruker AV 400 NMR spectrometer. Fourier transform infrared (FTIR) measurements were performed on a Bio-Rad Win-IR spectrometer. The size and distribution of particles were tested by a WyattQELS dynamic laser scattering (DLS) instrument. Confocal laser scanning microscopy (CLSM) was performed by Olympus FluoView 1000. MTT assay was conducted by a Bio-Rad 680 microplate reader. UV–vis
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spectra were recorded on a Shimadzu UV-2401PC UV–vis spectrophotometer. 2.3. Synthesis of tetraphenylporphrin zinc (Zn-Por) Tetraphenylporphrin zinc (Zn-Por) was synthesized according to the previous literature [36] as shown in Scheme S1. TPP (1 g, 1.6 mmol) prepared using the Adler method as in previous work [29] and Zn(Ac)2 2H2O (1.4 g, 6.4 mmol) were dissolved in chloroform (CHCl3) (40 mL). Then the above mixture was refluxed and the reaction process was monitored by thin-layer chromatography (TLC). The resulting mixture was cooled. The solvent was removed and the resulting solid was washed with water and then vacuum-dried. 2.4. Synthesis of histidine modified dextran (DH) As shown in Scheme 2, dextran (Dex, 1.0 g, 0.025 mmol), histidine (0.47 g, 1.9 mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC) (0.75 g, 3.8 mmol) and 4-dimethylaminopyridine (46 mg, 0.38 mmol) were dissolved in DMSO (20 mL) at room temperature. After reacted 2 d, the solvent and unreacted substances were removed by dialysis against deionized water for 72 h. The product was obtained through lyophilization. 2.5. Synthesis of metallo-supramolecular nanogel (SNG) DH (0.004 mM, 0.5 g) and Zn-Por (0.5 mM, 0.35 g) were dissolved in 10 mL of DMSO. The mixture was stirred at 25 °C for 48 h. Then, the solvent and unreacted substances were removed by dialysis against deionized water for 72 h. The products were obtained by lyophilization. See Scheme 2. 2.6. In vitro drug loading and release Doxorubicin (DOX) was used as a model drug for in vitro drug loading and release. DOX loaded SNGs were prepared by a simple
Scheme 2. Synthetic route for DH and SNG.
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dialysis technique. Typically, SNG (20.0 mg) and drug (4.0 mg) were mixed in 2.0 mL of DMSO. The mixture was stirred at room temperature for 24 h and then added dropwise into 20.0 mL of PBS at pH 7.4. The DMSO was removed by dialysis against water at pH 7.4 for 24 h. The dialysis medium was refreshed four times and the whole procedure was performed in the dark. Finally, the solution was filtered and lyophilized. To determine the drug loading content (DLC) and drug loading efficiency (DLE), the drug-loaded SNGs were dissolved in DMSO and analyzed by fluorescence measurement (Perkin-Elmer LS50B luminescence spectrometer) using a standard curve method (kex = 480 nm). DLC and DLE of drug-loaded SNGs were calculated according to Eqs. (1) and (2), respectively:
DLC ðwt%Þ ¼ amount of drug in SNG=amount of drug loaded SNG 100
ð1Þ
DLE ðwt%Þ ¼ amount of drug in SNG=total amount of feeding drug 100
ð2Þ
In vitro drug release profiles of drug-loaded SNGs were investigated in PBS (pH 5.3, 6.8 or 7.4). The pre-weighed freeze-dried DOX loaded SNGs were suspended in 4 mL of release medium and transferred into a dialysis bag (MWCO 3500 Da). The release experiment was initiated by placing the end-sealed dialysis bag into 50 mL of release medium at 37 °C with continuous shaking at 70 rpm. At predetermined intervals, 2 mL of external release medium was taken out and an equal volume of fresh release medium was replenished. The amount of released DOX was determined by using fluorescence measurement (kex = 480 nm). The release experiments were conducted in triplicate. 2.7. Confocal laser scanning microscopy (CLSM) For CLSM study, HepG2 cells were seeded in 6-well plates at a density of 105 cells per well in 2.0 mL of complete Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, supplemented with 50 IU mL1 penicillin and 50 IU mL1 streptomycin. After incubation for 24 h, the culture media were withdrawn and culture media containing SNG or Zn-Por were supplemented. The cells were incubated for another 2 or 6 h and washed with PBS three times. The cells were then fixed in 4% paraformaldehyde for 20 min and washed with PBS thrice. For staining the nuclei, the cells were incubated with 40 ,6-diamidi no-2-phenylindole (DAPI, blue) for 1.5 min. The images of cells were observed using a laser scanning confocal microscope (Olympus FluoView 1000). 2.8. Flow cytometric analyses HepG2 cells were incubated into 6-well plates based on 2 105 cells/well and cultured in 2.0 mL of DMEM for 24 h. The culture media were then replaced by culture media with Zn-Por or SNG at a final Zn-Por concentration of 0.72 mg mL1. The cells were incubated for an additional 2 or 6 h, then washed with PBS three times and trypsinized. 1.0 mL of DMEM was added, and the solutions were centrifuged for 5 min at 1000 rpm, then 1.0 mL of PBS was used to wash twice and the cells were resuspended in 0.2 mL of PBS. The analysis was performed by flow cytometer (Beckman, California, U.S.A.) for 1 104 cells.
DMEM and incubated at 37 °C in 5% CO2 atmosphere for 24 h. The culture medium was then removed and SNG solutions in complete DMEM at different concentrations (0–50 mg L1) were added. The cells were subjected to MTT assay after being incubated for an additional 24 h under light irradiation (2 mW, 400–700 nm). The absorbance of the formazan was measured on a Bio-Rad 680 microplate reader at 490 nm. Cell viability (%) was calculated based on Eq. (3):
Cell viability ð%Þ ¼ Asample =Acontrol 100
ð3Þ
where, Asample and Acontrol represent the absorbances of the sample and control wells, respectively. The cytotoxicities of DOX-loaded SNG against HepG2 and HeLa cells were also evaluated in vitro by an MTT assay. Similarly, cells were seeded into 96-well plates at 7 103 cells per well in 200.0 lL of complete DMEM and further incubated for 24 h. After washing cells with PBS, 180.0 lL of complete DMEM and 20.0 lL of DOX-loaded SNG solutions in PBS were added to form culture media with different DOX concentrations (0–10.0 mg L1 DOX). The cells were subjected to MTT assay after being incubated for 24 h. The absorbance of the solution was measured on a Bio-Rad 680 microplate reader at 490 nm. Cell viability (%) was also calculated based on Eq. (3). 2.10. Pharmacokinetics Wistar rats were randomly divided into two groups, containing three mice (average weight 250 g). DOX, DOX-loaded SNG were administered via the tail vein (Dosage, 6 mg DOX equivalent/kg body weight). At defined time periods (5, 10 min, 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 h), blood samples were collected from the orbital cavity, heparinized, and centrifuged (11,000 r/min, 5 min) to obtain the plasma. The concentrations of DOX in samples were detected by HPLC methods based on previous literature [37] with minor modifications. Briefly, 200 lL of plasma sample was deproteinized with 800 mL of methanol and 10 lL of daunorubicin hydrochloride (1 lg mL1, internal standard), then vortexed for 10 min, and centrifuged at 11,000 rpm for 5 min. Then supernatant was collected. The precipitation was added 800 mL of methanol then vortexed for 10 min, and centrifuged at 11,000 rpm for 5 min. Then supernatant was collected and dried under a stream of nitrogen at 25 °C. The dried sample was then dissolved in the mobile phase for HPLC analysis. 2.11. Anti-tumor efficacy The in vivo anti-tumor efficacy of the drug-loaded SNG was evaluated utilizing the xenograft B16F10 melanoma xeno-implanted on BALB/C mice. Treatments were started 5 days after cell implantation, when tumor in the mice reached a volume of 35–70 mm3, and this day was designated as day 0. The mice were weighed and randomly divided into 6 groups (8 mice per group): saline, SNG, SNG with irradiation, free DOX, DOX-loaded SNG and DOX-loaded SNG with irradiation (dosage: 15 mg Zn-Por/kg body weight, 3.0 mg DOX/kg body weight). The injection was carried out on day 1, 5, 9, 13 via tail vein, then irradiating (40 mW/cm2, 400–700 nm) for 5 min after 24, 48 and 72 h. The treatment efficacy and safety were assessed by measuring the tumor volume and body weight, respectively. Tumor volume was calculated by the following formula: 2
2.9. Cell viability assays The relative cytotoxicities of SNG against HepG2 and HeLa cells were evaluated in vitro by a standard MTT assay. The cells were seeded in 96-well plates at 1 104 cells per well in 200.0 lL of complete
Tumor volume ðVt Þ ¼ a b =2 Tumor growth rate ðTGR; %Þ ¼ ðVt =V0 Þ 100% where, a and b are the major and minor axes of the tumors measured by caliper; V0 represented the initial volume.
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2.12. Detection of singlet oxygen Generation of singlet oxygen can be usually detected by the characteristic photoluminescence peak of singlet oxygen at 1270 nm or by chemical method using 1,3-diphenylisobenzofuran (DPBF) or 9, 10-anthracenediyl-bis(methylene) dimalonic acid (ABDA) as a singlet oxygen sensor. In our experiment, ABDA was chosen to monitor the release of singlet oxygen into solution by recording the decrease in absorption of ABDA at 376 nm via UV–vis spectroscopy. 50 lL of ABDA solution (1 g/mL) was mixed well with SNG and DOX-loaded SNG (0.5 g/mL) and placed in a cuvette, respectively. The ABDA alone in PBS was used as the control. The absorption intensity of ABDA at 376 nm was monitored under different blue light illumination periods. 2.13. Statistical analysis All experiments were conducted at least three times and expressed as mean ± standard deviation (SD).
Fig. 2. In vitro DOX release profiles for DOX-loaded SNG pH 5.3 (a), DOX-loaded SNG pH 6.8 b), DOX-loaded SNG pH 7.4 (c), DOX-loaded DH pH 5.3 (d), DOX-loaded DH pH 6.8 (e) and DOX-loaded DH pH 7.4 (f) in PBS at 37 °C, respectively (n = 3, mean ± SD).
3. Results and discussion 3.1. Preparation and properties of SNG Along this line, at the first step, we synthesized Zn-Por (Scheme S1, Figs. S1–S3) and dextran-g-histidine copolymer (DH). Then SNG was fabricated by the metallo-supramolecular coordination between Zn-Por and histidine in DH (Scheme 2). By adjusting the feed ratio, SNG was synthesized as listed in Table S1. The 1H NMR spectrum shown in Fig. S4 verified the successful preparation of SNG. Compared with that of DH (Fig. S4A), peak 12 (–C(N@)@CH–NH–) signed to histidine showed a shift from 6.95 to 6.9 ppm in the spectrum of SNG (Fig. S4B). A similar peak shift from 7.7 to 7.6 ppm also could be observed for peak
11 (–N@CH–NH–). These peaks shifts were attributed to the coordinated interaction between Zn-Por and histidine and thus evidenced the successful synthesis of SNG. And the signals at 8.9 (a), 8.2 (b) and 7.9 ppm (c) were signed to Zn-Por in SNG. As shown in Fig. S5B, the peak at 1742 cm1 attributed to C@O inferred that histidine was grafted onto dextran successfully. The characteristic peaks at 1588 and 1480 cm1 in Fig. S5C were signed to phenyl group in Zn-Por and the peak at 1003 cm1 was attributed to the conjugated macrocycle. All of them further confirmed the structures of SNG. It is known that two histidine moieties can bind to one Zn-Por via metallo-coordinated interactions at the normal biological pH of 7.4, while under acidic conditions (e.g., pH < 6), histidine will
Fig. 1. CMC values of SNG at pH 7.4 and 5.3 (A); hydrodynamic radii (Rh) of SNG in PBS at pH 7.4 and 5.3 (B); TEM micrographs of SNG at pH 7.4, 0 h (C), pH 7.4, 72 h (D), and pH 5.3, 0 h (E).
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distribution of SNG become smaller. To test the formation and pH-response of SNG, Nile red was used as a probe to measure the CMC values. As shown in Fig. 1A, the CMC value of SNG in PBS at pH 7.4 was 18.6 mg/L while it was 79.0 mg/L at pH 5.3. Dynamic light scattering (DLS) measurements also indicated the size and size distribution of SNG. As shown in Fig. 1B, SNG exhibited an average hydrodynamic radius (Rh) of 73 nm with quite narrow size distribution at pH 7.4, which can preferentially reach and accumulate in tumors by the EPR effect. At pH 5.3 two DLS peaks were detected with one centered at 5 nm and the other at 165 nm, indicating dissociation and re-association of SNG at pH 5.3. TEM micrographs of SNG at pH 7.4 (Fig. 1C) and 5.3 (Fig. 1E) also confirmed these results. These changes of CMC value and particle size of SNG with pH variation demonstrated that SNG could respond to different pH environments. Moreover, as shown in Fig. 1C and D, the size of SNG remained almost unchanged after standing for 72 h at pH 7.4, confirming again the stability of SNG in normal biophysical microenvironment. 3.2. In vitro DOX loading and triggered release Fig. 3. Flow cytometry profiles of HepG2 cells incubated with PBS, Zn-Por for 2 and 6 h, SNG for 2 and 6 h.
be protonated (shown in Fig. S6) and the histidine/Zn-Por coordinates would disassemble dramatically, resulting in the dissociation of Zn-Por from histidine stalk. In order to comment the effect of the presence of Zn-Por on the size of SNG, the size and the critical micelle concentration (CMC) values in pH 7.4 for SNG and DH were measured, which were 73 ± 13 nm and 18.6 lg/mL and 286 ± 184 nm and 106.4 lg/mL, respectively (Table S1). The results demonstrated the presence of Zn-Por made the size and
To verify the feasibility of SNG as a delivery platform for antitumor drugs, doxorubicin (DOX) was chosen as a model drug and encapsulated into SNG via a typical dialysis method (Scheme 1). In order to indicate the stability of DOX-loaded SNG, DOX-loaded DH was chosen as the comparable group. Compared with DOX-loaded DH, DOX-loaded SNG presented higher DLC and DLE, which were 5.32% and 36.7% for DOX-loaded SNG and 3.43% and 25.6% for DOX-loaded DH, respectively. These results manifested SNG was more stable than DH, which could be ascribed to the cross of SNG. The in vitro release experiments were performed at pH 5.3, 6.8 and 7.4, mimicking the pH values under tumor intracellular,
Fig. 4. Representative CLSM images of HepG2 cells incubated with Zn-Por for 2 h (A), SNG for 2 h (B), Zn-Por for 6 h (C), and SNG for 6 h (D). For each panel, the microimages from left to right show a differential interference contrast (DIC) image, cell nuclei stained by DAPI (blue), Zn-Por fluorescence in cells (red), and overlays of the three images. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. The photodynamic cytotoxicity toward HepG2 (A) and HeLa cells (B) incubated with SNG under different light irradiation times; Cytotoxicity of Zn-Por under light irradiation for 30 min, SNG without light irradiation, SNG under 30 min of light irradiation, DOX-loaded SNG without light irradiation, DOX-loaded SNG under 30 min of light irradiation and DOX toward HepG2 (C) and HeLa cells (D) (n = 3, mean ± SD).
tissular and normal biophysical conditions, respectively. The cumulative release percentages of DOX loaded in SNG were plotted versus time in Fig. 2. The DOX release rate was in the order of pH 7.4 < pH 6.8 < pH 5.3. Up to about 80% of DOX was released from the DOX-loaded SNG in PBS at pH 5.3 in 48 h, while only about 25% of DOX was released at pH 7.4 in the same time interval. These different release behaviors should be attributed to the pH-triggered dissociation of Zn-Por/histidine coordinates, i.e., SNG could effectively hinder the loaded drug from releasing during blood circulation, while releasing it rapidly at lower pH in tumor microenvironment. This pH-sensitive property makes it possible for SNG to efficiently encapsulate hydrophobic drugs under neutral condition and release them in intracellular condition, suggesting tremendous potential of SNG in drug delivery. 3.3. Intracellular endocytosis Zn-Por is known as not only a clinical photosensitizer but also a fluorescent probe (red) for cell imaging. So flow cytometric analyses and confocal laser scanning microscopy (CLSM) were employed to monitor the cellular uptake of Zn-Por and SNG by HepG2 cells. As shown in Fig. 3, the fluorescence intensity form high to low was in the following order: SNG for 6 h > SNG for 2 h > Zn-Por for 6 h > Zn-Por for 2 h, respectively. The same phnomenona also can be observed in Fig. 4. After incubation with SNG for 2 or 6 h, the red fluorescence intensity was stronger than that of the cells treated with Zn-Por alone. The intensity difference was bigger at 6 h
than that at 2 h, implying both time and micellarization of Zn-Por had effect on the cell uptake. Furthermore, the red fluorescence of Zn-Por looked as coming from both the nucleus and cytoplasm areas in the SNG-treated cells at 2 h, and mainly from the nuclei at 6 h, while mainly from the cytoplasm area in the free Zn-Por-treated cells at both 2 and 6 h. This is because Zn-Por has limited solubility in cell culture medium and its entry into cells depends on its molecular diffusion through the cell membranes, whereas SNG particles diffuse through the cell membranes and enter the cells efficiently by an active mechanism [38]. The red fluorescence from the nucleus area means that the SNG as a whole or its dissociation products have good affinity to the nuclear membrane and can enter the nuclei with ease. This ability might be ascribed to the co-existence of the histidine residues and Zn-Por in the dissociation products. The former provides affinity to the nuclear membrane and enhances the nuclear uptake and the latter is used as a fluorescent probe to make the detection possible. 3.4. In vitro anti-tumor efficacy As a nanocarrier for drug delivery, biocompatibility is extremely important. The in vitro cytotoxicity of SNG against HepG2 and HeLa cells were examined by an MTT assay. Free SNG did not show appreciable cytotoxicity at different concentrations, up to 10.0 g L1 (Fig. 5), indicating the excellent biocompatibility of SNG itself as a drug carrier.
Please cite this article in press as: X. Yao et al., pH-responsive metallo-supramolecular nanogel for synergistic chemo-photodynamic therapy, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.07.024
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Fig. 6. In vivo pharmacokinetics profiles after intravenous injection of free DOX and DOX-loaded SNG in mice (n = 3, mean ± SD).
To investigate the combined efficiency of chemotherapy and photodynamic therapy, we explored the anticancer activity in vitro and in vivo. Firstly, singlet oxygen generated by SNG was monitored by a chemical method involving the photo-oxidation of 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) (Fig. S7) [39]. Then, MTT assays were employed to investigate the effect of light irradiation (2.0 mW/cm2, 400–700 nm) with different durations on the cytotoxicity toward HepG2 and HeLa cells in vitro. To rule out the potential effect induced by light irradiation, we irradiated HepG2 and HeLa cells directly without SNG or Zn-Por. As shown in Fig. S8, there was no harm to cells by a single light irradiation. Therefore, the effects of light irradiation on the growth of HepG2 cells incubated with Zn-Por and SNG were studied. With increasing light irradiation time, the cytotoxicity of SNG increased (Fig. 5A and B). However, the cytotoxicity of Zn-Por had less change (Fig. S9). We also observed the same phenomenon toward HeLa cells (Figs. 5B and S10). Obviously, this was corresponding to the cell uptake shown in Figs. 3 and 4. Subsequently, HepG2 and HeLa cells were treated with a gradient concentration of Zn-Por, SNG, DOX-loaded SNG and free DOX under or without light irradiation for 30 min. Cell viability data are shown in Fig. 5C and D. For simplicity, only the data in the case of 10.0 mg/L DOX and 0.72 mg/mL Zn-Por were samplified. Firstly,
the three formulations without DOX show the cytotoxicity order of free SNG < Zn-Por under irradiation
