Article pubs.acs.org/cm
Dual-Targeted Cascade-Responsive Prodrug Micelle System for Tumor Therapy in Vivo Liangliang Dai, Ruisi Cai, Menghuan Li, Zhong Luo, Yonglin Yu, Weizhen Chen, Xinkun Shen, Yuxia Pei, Xiaojing Zhao, and Kaiyong Cai* Key Laboratory of Biorheological Science and Technology, Ministry of Education; College of Bioengineering, Chongqing University, Chongqing 400044, People’s Republic of China S Supporting Information *
ABSTRACT: This study reports a cascade-responsive disassemble micellar drug delivery system with dual-targeting potential (cell and mitochondria targeting), which optimizes the distribution of antitumor drugs on systemic, local, and subcellular levels to enhance antitumor efficacy. A new cationic porphyrin derivative 5-(3-hydroxy-p-(4trimethylammonium)butoxyphenyl)-10,15,20-triphenylporphyrin chlorine (MTPP) is synthesized as a mitochondria-targeting photosensitizer. After accumulating at a tumor site, the micellar nanosystem is endocytosed by tumor cells facilitated by the folate receptor-mediated pathway. Then, the hydrophobic PDEA block would be protonated in intracellular acidic endo-/lysosomes and promote the escape of prodrug micelles from endo-/lysosome to cytoplasm, resulting in the first-stage destabilization of micelles. Subsequently, the CPT is released in response to high concentration of GSH in cytoplasm, which would greatly increase the hydrophilicity of the BOH block and initiate the complete disassembly of the polymer micelles owing to the damage of the hydrophilic−hydrophobic balance. Additionally, the released MTPP is selectively accumulated in mitochondria and activates mitochondria apoptotic pathway upon light irradiation as a result of ROS generation. Both in vitro and in vivo studies indicate that the polymeric micelle not only effectively improves the targeted delivery efficiency but also dramatically enhances the combinational antitumor efficacy while reducing the side effects associated with the laser irradiation and mitochondria-targeted tumor therapy.
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INTRODUCTION Most clinically used chemotherapeutic drugs have low bioavailability and suboptimal therapeutic effect due to the poor solubility and inevitable side effects.1,2 Besides, multidrug resistance (MDR) is another culprit that leads to unsatisfactory antitumor efficacy.3 To address these problems, polymeric prodrug micellar drug delivery systems (PPM-DDS) combined the advantages of micellar nanocarriers (e.g., good biocompatibility and low risk of immunogenicity) and prodrug strategy (e.g., structural stability and high drug loading content) were proposed for tumor therapy.4,5 Taking advantage of the relatively stable “core−shell” structures in the physiological environment and stimuli-responsive prodrug release, polymeric prodrug micelles can significantly improve the bioavailability of chemotherapeutic agents, prolong blood circulation, and reduce adverse effects.6,7 The solution to the critical challenges hindering clinical application for advanced PPM-DDSs depends on precisely controlling the cellular internalization, lysosomal sequestration, and prodrug release. Consequently, many targeting ligands were previously conjugated to the surface of polymeric prodrug micelles to enhance cell uptake efficiency; notable examples include folate (FA), RGD peptide, and transferrin.8−10 These targeted prodrug micelles therefore could be selectively uptaken by tumor cells via receptor-mediated endocytosis pathway since the specific receptors are all overexpressed in many tumors but © XXXX American Chemical Society
not in normal cells; after the endocytosis, most of these nanopreparations are spatially confined in endo-/lysosomes. In order to avoid drug degradation caused by low acidic environment and various enzymes in lysosomes, prodrug micelles need to rapidly escape from the lysosomes to the cytoplasm. The ultra-pH-responsive polymers with protonation property are developed to solve this problem.11 With the help of acidic endo-/lysosome environment,12 the rapid protonation of prodrug micelles would result in the lysosome swelling and destabilization and efficient endo-/lysosomal escape through the known “proton sponge” effect,13 which is one of the major strategies to overcome MDR and improve the bioavailability of chemotherapeutic drugs.14 Considering the redox state− gradient (e.g., GSH) between tumor extracellular and cytoplasm (2−20 μM vs 2−10 mM), the redox stimulus-responsive drug release mechanism is a promising strategy for the development of PPM-DDS tumor therapy.15,16 However, most of the reported PPM-DDSs for tumor therapy have relatively long term biodegradation, which limits their clinical application as well.17 Therefore, redox-responsive degradable PPM-DDS with tumor-specific targeting ability is a promising alternative strategy for targeted tumor therapy. Received: June 17, 2017 Revised: July 28, 2017
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DOI: 10.1021/acs.chemmater.7b02513 Chem. Mater. XXXX, XXX, XXX−XXX
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Scheme 1. (A) Synthesis Routes of Cascade-Responsive Disassemble Prodrug FA-PEG-PDBO-BPT; (B) Mechanism of CPT Release from Polymeric Prodrug in the Presence of GSH; (C) Illustration of Cascade-Responsive Disassemble Micelles with Dual-Targeting Capability for Tumor Therapy in Vivo
Mitochondria are not only the energy factories of cells but also the executioners of programmed cell death (apoptosis).18,19 The activation of cancer cell apoptosis could be triggered by stimulating mitochondrial membrane permeabilization, which leads to the release of pro-apoptotic proteins from the mitochondria to cytosol, initiating the subsequently mitochondria-mediated apoptotic pathways.20−22 Moreover, photosensitizers could effectively induce depolarization of mitochondria and initiate the subsequent apoptosis of tumor cells upon irradiation as a result of ROS generation. However, the primary challenge is to deliver the photosensitizer directly to mitochondria. Given the high mitochondrial membrane potential (∼180 mV),23 lipophilic cations, typically triphenylphosphine (TPP) and ammonium-containing cations, acting as superior mitochondria-targeting agents, could efficiently attach to mitochondria.24−26 Therefore, a sound strategy is to develop ammonium-functionalized porphyrin photosensitizers that combine the advantages of ammonium-functionalized cations (special mitochondria targeting) with porphyrin photosensitizer (high absorption coefficient in the near-IR wavelength region and efficient production of singlet oxygen), which could achieve highly selective mitochondria killing for tumor therapy. Based on the above concept, in this study, a caspaseresponsive disassemble micellar drug delivery system denoted as FA-PEG-PDBO-BPT is constructed and loaded with mitochondria-targeting photosensitizer to improve the antitumor efficacy (Scheme 1). Briefly, the polymeric prodrug was
synthesized by ring-opening copolymerization reaction with PEG as the outer hydrophilic layer; the pH-responsive segments of poly(2-(diethylamino)ethyl methacrylate) (PDEA) was integrated into the polymer backbone as a middle layer, which could be packed into a hydrophobic core due to its intrinsic hydrophobicity at the physiological condition (pH 7.4), while becoming positively charged hydrophilic residues and protrude out at low acid environment (pH < 6.5) by the protonation of the tertiary amine groups; camptothecin (CPT) was covalently conjugated to the polymer backbone via a reduction-sensitive disulfide bond as a hydrophobic core. Folate was anchored to the surface of FA-PEG-PDBO-BPT micelles as the targeting motif. As shown in Scheme 1C, after systematic administration of the mitochondria-targeting photosensitizer 5(3-hydroxy-p-(4-trimethylammonium)butoxyphenyl)-10,15,20triphenylporphyrin chlorine (MTPP) loaded self-assembled prodrug micelles (FA-PEG-PDBO-BPT@MTPP), they were first accumulated at the tumor site through the enhanced permeability and retention (EPR) effect, and the hydrophilic PEG outer shell was expected to improve the blood circulation of micelles. Then, the prodrug micelles were effectively internalized by tumor cells and trapped in endosome by folate receptor-mediated endocytosis. The weakly acidic environment of endo-/lysosomes could subsequently induce the protonation of PDEA, leading to the escape of prodrug micelles from endo-/lysosome to cytoplasm and destabilization of micelles. Next, the high concentration of GSH in cytoplasm would B
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Figure 1. (A) 1H NMR spectra (400 MHz, 298 K) of final copolymer FA-PEG-PDBO-BPT. (B) GPC traces of PDEA, bPEG-BBO-BDO, bPEGPDBO-BOH, and FA-PEG-PDBO-BPT, respectively. (C) 1H NMR (400 MHz, 298 K) and (D) ESI-MS spectra of the mitochondria-targeting photosensitizer MTPP. (E) TEM image of the FA-PEG-PDBO-BPT@MTPP micelles before (a) and after respective treatments with pH 5.0 (b), 10 mM GSH (c), and pH 5.0 plus 10 mM GSH (d) for 2 h. Scale bars: 100 nm for panels a, c and d and 500 nm for panel b, respectively. (F) Absorption spectra of ABDA in the presence of micelles@MTPP treated with pH 5.0 plus 10 mM GSH for 2 h under laser irradiation over different periods of time. (G) 1O2 generation by micelles@MTPP treated without/with laser and pH 5.0 plus 10 mM GSH after irradiation for different times, respectively.
residues, which was determined by gel-permeation chromatography (GPC), 1H NMR spectroscopy, and MALDI-TOF (Figure 1A,B and Figures S1 and S3 and Table S1 in the Supporting Information). Moreover, GPC, NMR, and FTIR evidently confirmed the successful stepwise conjugations/ functionalization of copolymer (Figure 1B and Figures S1 and S2 and Table S1). In addition, average molecular weights of various intermediate products were calculated from 1H NMR, MALDI-TOF, and GPC, which all showed excellent consistency with the theoretical values (Table S1). Therefore, these results suggested that FA-PEG-PDBO-BPT copolymer was successfully synthesized with an average molecular weight of 14880, and the loading content of CPT was calculated as 19.58%. The prodrug micelles were prepared with an oil-in-water emulsion approach.27 It exhibited a relatively low critical micelle concentration (cmc) of 13.7 mg/L (Figure S4), which was sufficient to overcome the dilution effect in blood circulation and maintain its stability, thus improving its drug delivery performance for tumor therapy in vivo. A positively charged
rapidly cleave the disulfide bonds, and the removal of CPT would convert the hydrophobic BOH residues back to hydrophilic (Scheme 1B), eventually disintegrating the micelles. In addition, the released MTPP would specifically accumulate in the mitochondria, causing mitochondrial damage and then initiating the mitochondria-mediated apoptotic pathways upon light irradiation. Therefore, we hypothesized that the cascaderesponsive disassemble micellar drug delivery system with dualtargeting capability was a promising alternative for tumor therapy, as it could significantly improve the bioavailability of a chemotherapeutic and effectively suppress tumor growth with reduced side effects in vivo.
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RESULTS AND DISCUSSION The prodrug polymer FA-PEG-PDBO-BPT was synthesized by the ring-opening copolymerization reaction using bPEG as the initiator (Scheme 1 A). Briefly, it was comprised of PEG (Mw, 5000), PDBO blocks possessed eight units of the BBO residues and 1 unit of PDEA, and BPT segments possessed 12 units of 5-benzyloxy-1,3-dioxan-2-one (BDO) and nine units of CPT C
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Figure 2. pH-responsive-protonation, redox-responsive drug release profile of FA-PEG-PDBO-BPT@MTPP micelles. (A) 1H NMR spectra (400 MHz, 298 K) of FA-PEG-PDBO-BPT copolymer in D2O before (a1) and after (a2) incubating with pH 5.0 buffer for 4 h. (B) ζ potential of FAPEG-PDBO-BPT micelles in PBS with various pH values. (C) Schematic illustration of the protonation process of FA-PEG-PDBO-BPT@MTPP micelles. (D) Fluorescence emission spectra (λex = 367 nm) of CPT release from FA-PEG-PDBO-BPT micelles after treatments with various concentrations of GSH. (E) HPLC curve of free CPT and FA-PEG-PDBO-BPT micelles incubated in 10 mM GSH MeOH/H2O (4:1, v/v) solution for 2 and 4 h, respectively. (F) Drug release profile of CPT (solid lines) and MTPP (dash lines) from FA-PEG-PDBO-BPT@MTPP micelles after treatments with various concentrations of GSH.
spherical structure (Figure 1E(a)), and its average hydrodynamic diameter was determined as approximately 90 nm via dynamic laser scattering (DLS, Figure S9), which was suitable for drug delivery in vivo because they could extravasate at tumor sites due to the EPR effect. From a clinical perspective, an excellent nanocarrier for tumor therapy should not only be structurally stable with a consistent size and morphology under blood circulation but also disassemble at tumor sites and release cargo responding to certain stimuli. The stability of micelles in serum was first investigated by respective dissolving bare micelles and micelles@MTPP in 10% fetal bovine serum (FBS) and measuring its size change by DLS (Figure S10). There was no significant size change for both bare micelles and micelles@ MTPP after incubation at 37 °C for 6 days. It suggested that the micelles possessed a desirable stability profile for further investigation, which might be caused by the shielding effect of external PEG shell, in turn reducing nonspecific uptake
porphyrin derivative MTPP (mitochondria-targeting photosensitizer) was synthesized and loaded into micelles. The synthesis procedure of MTPP was shown in Figure S5, and the related various characterizations including 1H NMR, MS, HPLC, and absorption spectra for the compound were depicted in Figure 1C,D and Figures S6 and S7. It was implied that MTPP was successfully synthesized with high purity. As shown in Figure S8, the MTPP loaded micelles drug delivery system (FA-PEG-PDBO-BPT@MTPP, abbreviated as micelles@ MTPP) exhibits spectral properties similar to those of MTPP (Figure S7), suggesting that the MTPP molecules were successfully encapsulated into the micelles. Quantitatively, the drug loading content (DLC) and drug loading efficiency (DLE) of MTPP were determined to be 15.8% and 48.7%, respectively. The morphology and structure of micelles@MTPP were characterized by transmission electron microscopy (TEM).28 The micelles were highly monodispersive with a well-defined D
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Chemistry of Materials clearance.29 The morphologic changes of micelles@MTPP were then investigated by incubating micelles with various tumor-mimetic environments and observed by TEM. As shown in Figure 1E, the micelles were swelled when exposed to pH 5.0 (Figure 1E(b)), and collapsed under treatment with GSH (Figure 1E(c)); moreover, the combinational treatments with weak acid (pH 5.0) and GSH induced the final disassembly of micelles (Figure 1E(d)). These results were also consistently confirmed by DLS measurements (Figure S9). The phenomena could be explained in that the protonation of hydrophobic PDEA segments triggered by low pH would cause micelles swelling, while the removal of CPT caused by GSH cleavage would convert the BOH from hydrophobic to hydrophilic and induce the fragmentation and disassembly of the micelles. The results suggested again that FA-PEG-PDBO-BPT micelles could be used as a potential stimuli-responsive disassemble drug carrier for tumor therapy. To reveal the ROS generation efficiency of micelles@MTPP under pH 7.4 (mitochondrion-mimetic pH), a water-soluble 9,10-anthracenediylbis(methylene)dimalonic acid (ABDA) was employed as the indicator to measure the ROS production by monitoring the decrease of ABDA absorbance at 401 nm.30 As depicted in Figure 1F, the characteristic absorption peak of ABDA in micelles@MTPP dispersion treated with pH 5.0 and GSH (simulation lysosomes and cytoplasm in tumor cells) at 401 nm dramatically decreased with irradiation time (from 0 to 20 min) and finally reached a nadir after 20 min. In contrast, the characteristic absorption band of ABDA changed very little upon pH 7.4 (simulation blood environment) despite laser irradiation (Figure S11). Moreover, the quantitative analysis indicated that micelles@MTPP treated with pH 5.0 plus GSH could effectively generate ROS upon laser irradiation, which were 6.6- and 7-fold more efficient than that achieved at pH 7.4 without laser and plus laser for 20 min, respectively (Figure 1G). The reasons could be explained by MTPP being encapsulated inside the hydrophobic core of FA-PEG-PDBOBPT micelles at neutral conditions (pH 7.4); thus the aggregation of MTPP quenched its phototoxicity through homofluorescence resonance energy transfer (homo-FRET) between MTPP molecules;20 On the contrary, the co-work between GSH and pH 5.0 induced the complete micelles disassembly and MTPP deaggregation as shown in TEM (Figure 1E), thus resulting in the destruction of homo-FRET and activation of phototoxicity. These results indicated that the synthesized photosensitizers MTPP with mitochondria-targeting ability efficiently produced ROS under irradiation. Moreover, the micelles@MTPP display quenched phototoxicity in the blood circulation and effective phototoxicity in tumor cells, and the MTPP loaded micelles were promising photosensitizer nanocarriers for drug delivery in vitro. To reveal the pH-responsive protonation of micelles, the FAPEG-PDBO-BPT micelles were treated with acid stimuli to observe their ionization and ζ potential changes. 1H NMR was first employed to investigate the process of protonation. As shown in Figure 2A, the resonances of hydrophobic PDEA segments were not observed in the spectrum of FA-PEGPDBO-BPT copolymer in D2O (pH 7.4), as PDEA units were encapsulated inside of the micelles and could not be detected by 1H NMR analysis (Figure 2A(a1)). However, after incubation with pH 5.0 aqueous solution for 4 h, the hydrophobic PDEA became positively charged hydrophilic residues due to the protonation of the tertiary amine groups, and then the resonances of the transformed PDEA motifs,
together with PEG, were therefore detected by 1H NMR spectroscopy in D2O (Figure 2A, panel a2 vs panel a1). The result suggests that the protonation of micelles was highly sensitive to low pH stimuli. In addition, this protonation and hydrophobic−hydrophilic transformation of PDEA were indicated by the ζ potentials of the micelles at different pH values (Figure 2B). At physiological environment (pH ∼ 7.4), the micelles were apparently negatively charged due to the conjugation of anionic FA molecules. However, the ζ potential of the micelles changed gradually from negative (−12 mV) to positive (14 mV) as the pH value decreased (from 7.4 to 4.5), indicating the protonation of the tertiary amine groups of PDEA, which was illustrated in Figure 2C. Besides, the pKa of micelles was calculated as 6.4 through potentiometric titration (Figure S12).31 Thus, these data reflected again the pHresponsive-protonation ability of the micelles. Redox-responsive drug release profile of prodrug micelles was investigated by treating with various concentrations of GSH. It was observed that the characteristic absorption peaks of CPT (orange arrow) were found in the absorption spectrum of prodrug micelles (Figure S13), indicating CPT prodrug was successfully introduced to the polymer backbone. Actually, the absorption intensity of CPT in prodrug micelles (equivalent CPT concentration) was obviously weaker than free CPT, owing to the π−π interaction of CPT conjugated in the micellar core,32,33 whereas it increased significantly to almost the same level of free CPT after treatment with GSH, implying that the FA-PEG-PDBO-BPT drug delivery system could effectively release CPT prodrug in response to GSH. Besides, the fluorescence intensity of CPT in prodrug micelles was also steadily enhanced along with increasing GSH concentrations (from 0 to 10 mM), and the micelles incubated with 10 mM exhibited almost the same intensity as that of free CPT (Figure 2D), implying the nearly complete CPT release from prodrug micelles. Meanwhile, the redox-responsive release behavior was further confirmed by high performance liquid chromatography (HPLC). After exposure to GSH (10 mM) for 2 h, a new retention time peak of free CPT molecules (dashed box) appeared in the retention curve of prodrug micelles (Figure 2E). With the incubation time extending to 5 h, the CPT peak was further enhanced whereas the prodrug micelles peak decreased, which suggested that GSH were responsible for the cleaving of disulfide bond and CPT release, and the amount of prodrug release was positively correlated with incubation time. Moreover, the quantitative analysis was employed to further investigate the drug release profile of cascade-responsive disassemble micelles. As shown in Figure 2F, the control group (no GSH) exhibited a negligible CPT and MTPP leakage (both below 7%) within 60 h, indicating the good stability of the micelles. In contrast, when exposed to 2 mM GSH and 10 mM GSH, around 36% and 80% CPT (solid lines) were released from prodrug micelles after treatment for 60 h, respectively. The result again suggested the strong positive impacts of GSH concentration and incubation time on the drug release. Interestingly, 26% and 50% of MTPP (dash lines) were also released from the drug delivery system after treatment with 2 mM GSH and 10 mM GSH for 60 h, respectively. More importantly, the drug delivery system treated with 10 mM GSH exhibited the nearly complete drug release both for CPT (91%) and MTPP (97%) when the pH value was tuned down to 5.0. It could be explained that the removal of CPT caused by GSH cleavage would convert the BOH residues from hydrophobic to hydrophilic, thereby inducing the subsequent fragmentation E
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Figure 3. (A) Cytotoxicity assay of HeLa cells treated with irradiation (20 min), MTPP (5 μM), CPT (15 μg/mL), FA-PEG-PDBO-BPT (abbreviated as micelles, 76.6 μg/mL, equivalent CPT concentration of 15 μg/mL), FA-PEG-PDBO-BPT@MTPP micelles (abbreviated as micelles@MTPP, 76.6 μg/mL), and micelles@MTPP plus irradiation (20 min) for 12 and 24 h, respectively. Error bars present as mean ± SD (n = 4); **, p < 0.01. (B) Concentration-dependent cytotoxicity of HeLa cells treated with micelles@MTPP plus/without laser irradiation (20 min) over 24 h. Cellular uptake and intracellular distribution of micelles: HeLa cells incubated with free MTPP, FA-PEG-PDBO-BPT@MTPP micelles with/ without laser irradiation (665, 100 mW/cm2, 10 min) for 12 and 24 h and detected by (C) CLSM and (D) FCM, respectively. Nuclei were labeled with DAPI (blue), MTPP exhibited red fluorescence, and cytoskeleton was stained with Alexa 488-phalloidin (green). Scale bar: 50 μm.
have cytotoxicity levels similar to that of free CPT after incubation for 12 and 24 h, whereas the cells treated with micelles@MTPP and irradiation (665 nm, 100 mW/cm2, 20 min) showed the strongest cytotoxicity among all treatment groups regardless of the treatment time (p < 0.01). Besides, micelles@MTPP drug delivery system also exhibited dosedependent and laser-dependent cytotoxicity for HeLa cells as well (Figure 3B). The reason could be explained in that free CPT could enter cytoplasm through the diffusion pathway, whereas the cellular entry of prodrug micelles depends on endocytosis, endo-/lysosomal escape, and the subsequent redox-responsive drug release, the latter being generally more time-consuming than diffusion. Notably, similar cytotoxicity caused by both prodrug micelles and micelles@MTPP (without irradiation) compared to free CPT are attributed to its cellular targeting and reduction-sensitive linker, which improves the drug delivery efficacy and facilitates CPT release in cells. In addition, the hydrophobicity of free CPT also reduced its cytotoxicity in these circumstances. Upon exposure to laser irradiation (20 min), the large amount of ROS produced by MTPP therefore induced the more severe cytotoxicity against HeLa cells. Moreover, both MTPP and micelles@MTPP groups exhibited laser-dependent growth inhibition of HeLa cells compared to control (Figures 3A and S14), which further
and reassembly of micelles. Thus, MTPP could partly be released under the reassembly process of polymer micelles. After exposure to an acidic solution environment of pH 5.0, the protonation of PDEA could also result in the conversion of a hydrophilic state and induce the final disassembly of micelles as well as complete drug release. In addition, the charge repulsion between cationic MTPP and protonated PDEA may also contribute to the completed MTPP release. These results were all consistently confirmed by TEM (Figure 1E, panel a vs panels b−d). Considering the acidic environment in endo-/lysosome and high GSH concentration in the cytoplasm of tumor cells, these results indicate that the cascade-responsive FA-PEGPDBO-BPT micelle was a promising platform for tumor therapy in vivo. To investigate whether the FA-PEG-PDBO-BPT drug delivery system could enhance antitumor activity of CPT, CCK-8 assay was used to assess its cytotoxicity against HeLa cells.34 As shown in Figure 3A, the cells treated with MTPP (5 μM), CPT (15 μg/mL), FA-PEG-PDBO-BPT prodrug micelles (76.6 μg/mL, equivalent CPT concentration of 15 μg/mL), and micelles@MTPP (76.6 μg/mL) with or without irradiation all exhibited potent time-dependent cytotoxicity compared to the control group. Meanwhile, only FA-PEG-PDBO-BPT micelles and micelles@MTPP (without irradiation) groups F
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Figure 4. Subcellular distribution, mitochondria targeting, and intracellular release of drug loaded prodrug micelles. HeLa cells were incubated with FA-PEG-PDBO-BPT@MTPP micelles for different times and then stained with LysoTracker Green (A) or Rh123 (B) for imaging lysosome escape and mitochondria targeting. HeLa cells were incubated with free CPT, FA-PEG-PDBO-BPT@MTPP micelles with/without laser irradiation (10 min) for 6 h and detected by (C) CLSM and (D) FCM, respectively. Nuclei were labeled with Reddot2 (red), CPT exhibited blue fluorescence, MTPP exhibited red fluorescence, and cytoskeleton was stained with Alexa 488-phalloidin (green). Mitochondrial membrane depolarization of HeLa staining JC-1 probe were detected by CLSM (E) and FCM (F). HeLa cells were incubated with micelles@MTPP (76.6 μg/mL, equivalent to 5 μM MTPP) for 12 h with laser irradiation for various times (0, 5, 10, and 20 min). “J-aggre” and “J-mono” are the abbreviations for “aggregation state of JC-1 dye” and “monomer state of JC-1 dye”, respectively. Scale bars were 20, 50, and 20 μm for A−C and E, respectively. G
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the micelles@MTPP were first found in the peripheral area of cells after incubation for 1 h, then all dispersed inside cells after 3 h, and even some of them entered into the mitochondria, indicated by the moderately overlapped red fluorescence of micelles@MTPP and green fluorescence of rhodamine 123 (Rh123, a mitochondria-specific fluorescent probe).42 All these observations suggested that the micelles@MTPP had been successfully internalized by HeLa cells and the released MTPP from micelles@MTPP system trends to distribute in mitochondria. After 8 h of incubation, the red fluorescence overlapped completely with the green and showed the significantly yellow fluorescence in those mitochondria (PCC = 0.924), indicating the completed micelles disassembly and MTPP release. Moreover, the results confirmed again the excellent mitochondria-targeting capability of cationic porphyrin MTPP. To further reveal the intracellular micelles disassembly and drug release, HeLa cells were incubated with micelles@MTPP to monitor the colocalization between CPT and MTPP that were either conjugated or trapped in the hydrophobic core of micelles. As shown in Figure 4C,D, micelles@MTPP could effectively deliver hydrophobic drugs (e.g., CPT) to tumor cells via FA-receptor-mediated endocytosis, which was revealed by previous investigations (Figure 3C,D and Figure S15). Moreover, both blue fluorescence of CPT and red dotted fluorescence of MTPP were separated from the micelles@ MTPP after incubation for 6 h (white arrow, Figure 4C), showing the significant decrease of the overlapping regions between blue and red fluorescence, which suggested that the micelles disassembly and drug release had been initiated by low pH and GSH. As the incubation time extended to 12 h, the separation tendency became more obvious and the released CPT then entered the nuclei, whereas MTPP distributed in mitochondria (Figure S16 and Figure 4B).43 This result may provide additional evidence for the intracellular micelles disassembly and drug release. The reasons could be explained as follow: after escaping from lysosomes, the high concentration of GSH in the cytoplasm induced CPT release via breaking of the disulfid linkage, and the hydrophobic− hydrophilic transformation caused by pH-responsive protonation and redox-responsive CPT removal eventually resulted in the complete micelles disassembly as well as drug release. Moreover, the electrostatic repulsion between cationic MTPP and protonated PDEA could further accelerate MTPP release. Eventually, the released CPT was enriched in the nuclei while the cationic porphyrin MTPP was preferably located in mitochondria. To thoroughly investigate the antitumor effect of MTPP in a drug delivery system, dichlorofluorescein diacetate (DCF-DA) was used as indicator to monitor the intracellular ROS generation of HeLa cells that were treated with MTPP and micelles@MTPP individually under irradiation. DCF-DA is nonfluorescent but can be easily oxidized by ROS to afford highly fluorescent dichlorofluorescein.44 It was observed that both free MTPP and micelles@MTPP produced negligible ROS compared to control, revealing by a negligible green fluorescence (Figure S17A). Contrastingly, in both cases, a large amount of ROS was generated when exposed to irradiation (665 nm, 100 mW/cm2, 10 min). Moreover, the amount of ROS generated by micelles@MTPP was significantly higher than that of MTPP based on the relative quantitative analysis (p < 0.01, Figure S17B). It was suggested that the FAPEG-PDBO-BPT system effectively delivered MTPP to tumor
confirmed the highly effective ROS generation capability of MTPP. The results indicated that micelles@MTPP drug delivery system effectively improved the bioavailability of CPT and significantly enhanced cytotoxicity in vitro. The cellular-targeting capability of the micelles-based system was first investigated by incubating HeLa cells with micelles@ MTPP and observing its endocytosis and distribution using confocal laser scanning microscopy (CLSM). As shown in Figure 3C, only minimal free MTPP was taken in by HeLa cells regardless of the incubation time, evidenced by the relatively weak red fluorescence, whereas the micelles@MTPP with/ without irradiation both exhibited a much higher intracellular red fluorescence compared to MTPP after incubation for 12 h, and the red fluorescence of MTPP in cytoplasm was even higher after 24 h, implying that micelles@MTPP was effectively endocytosed by HeLa cells despite laser irradiation. These results were also consistently confirmed by FCM (Figure 3D). Notably, the low cell internalization efficiency of free MTPP was mainly attributed to its poor water solubility, which was consistent with previous study.35 Therefore, micelles@MTPP could significantly enhance uptake efficiency and deliver hydrophobic photodynamic drugs to tumor cells. To further verify the FA-receptor-mediated endocytosis pathway, HUVEC cells (FA-receptor-negative cell line) and HeLa cells blocked FA receptor were employed as negative control to evaluate its internalization. As show in Figure S15A, the normal HeLa cells (FA-receptor-positive cell line) could take in micelles@MTPP with high efficiency, whereas the uptake of micelles was significantly reduced in the two negative groups, which was confirmed by the relative FCM results (Figure S15B). These results demonstrate again that the enhanced uptake of the micelles@MTPP in HeLa cells was achieved by FA-receptormediated endocytosis, which was consistent with our previous works.36,37 After being endocytosed by HeLa cells, the micelles were generally trapped in endo-/lysosome.38 In order to protect bioactive substances (e.g., antitumor drugs) from deactivation caused by low acidic environment and various enzymes in endo-/lysosomes, the successful escape from endo-/lysosome was important for drug delivery systems.39,40 Herein the endo-/ lysosomal escape capability of FA-PEG-PDBO-BPT was investigated using a co-staining experiment and observed by CLSM. LysoTracker Green, a widely used lysosome probe, could be specially marked the acidic lysosome with green fluorescence. As shown in Figure 4A, the dotted red fluorescence of micelles@MTPP were first found at the rim of cells after incubation for 1 h, which is a sign of imminent endocytosis. After incubation for 3 h, the micelles@MTPP were then transferred into lysosomes, evidenced by the well-matched yellow fluorescence that resulted from the overlapping red (micelles) and green fluorescence (lysosomes). Moreover, the Pearson correlation coefficient (PCC, indicating the degree of colocalization between red and green fluorescence) was calculated to be 0.893, which confirmed again the distinct colocalization between micelles@MTPP and lysosomes.41 After extending the incubation to 8 h, the red fluorescence was transferred from lysosomes to cytoplasm (PCC = 0.143), implying that the protonation of PDEA triggered by the low pH in lysosomes facilitated the successful escape of micelles from lysosomes to cytoplasm. Meanwhile, the mitochondrial-targeting capability of FAPEG-PDBO-BPT system was further investigated using costaining assay and observed by CLSM. As shown in Figure 4B, H
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Figure 5. (A) Apoptosis of HeLa cells induced by PBS (control), MTPP, CPT, micelles, and micelles@MTPP without/plus laser irradiation (10 min) after 12 h of incubation determined using Annexin V-FITC/PI staining. (B) Mitochondria apoptotic pathway-associated proteins in HeLa cells were examined by Western blotting after incubation with MTPP, CPT, FA-PEG-PDBO-BPT micelles and micelles@MTPP in the absence/presence of laser irradiation (10 min) for 24 h. Quantitative analysis of proteins expressions (C, Bax/Bcl-2; D, Cytochrome C; E, cleaved caspase 9; F, cleaved caspase 3) via Image J software. Error bars present as mean ± SD (n = 4); *, p < 0.05; **, p < 0.01.
PEG-PDBO-BPT micelles, and micelles@MTPP with or without irradiation. The related apoptosis levels were evaluated using Annexin V-FITC/PI labeling assay by FCM. As shown in Figure 5A, both FA-PEG-PDBO-BPT micelles and micelles@ MTPP induced more severe apoptosis than free MTPP or CPT, suggesting again the enhanced antitumor efficiency of the FA-PEG-PDBO-BPT@MTPP drug delivery system. Moreover, the micelles@MTPP treatment groups caused the most severe apoptosis among all groups when exposed to irradiation (p < 0.01, Figure S18). Furthermore, micelles@MTPP also exhibited an irradiation time-dependent apoptosis pattern in HeLa cells (Figure S19), and the apoptosis level of HeLa cells gradually increased as the irradiation time increased (from 0 to 30 min). These data directly revealed again that the micelles@MTPP drug delivery system could induce cancer cells apoptosis with high efficiency when combined with laser irradiation. On the other hand, considering the mitochondria damage and cell apoptosis caused by micelles@MTPP under irradiation, the expression of proteins associated with cell apoptosis and mitochondrial apoptotic pathway were investigated by Westernblot assay. It was known that pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 play key roles in the early apoptotic process and may associate with the mitochondrial apoptotic pathway,48 thus widely used as important indicators for cell apoptosis detection. In addition, the damage of mitochondria is frequently caused by the remarkable release of cytochrome c and then the mitochondrial apoptotic pathway is initiated through the cascade activation of caspase-9 and caspase-3.49,50 Therefore, the expression of the above-mentioned protein should be investigated for comprehensive antitumor evaluation.
cells and locally generated a large amount of ROS under irradiation for tumor therapy. As revealed by the above investigations (Figure 4B), the released MTPP could be specifically enriched in mitochondria; therefore, the mitochondrial damage caused by ROS should be investigated for the evaluation of antitumor efficiency. A membrane-permeable JC-1 dye was used to monitor the damaging of mitochondria and the change of mitochondrial membrane potential.45 JC-1 could preferentially aggregate in the normal mitochondria with high membrane potential and emit red fluorescence, whereas dispersion in cytoplasm existed as monomer formation and exhibited green fluorescence.46 Therefore, the red/green ratio was widely used as a reference to evaluate the damaged state of mitochondria. It was observed that the normal mitochondria in the control group were marked with punctate red and green fluorescence (Figure 4E). However, HeLa cells treated with the micelles@MTPP system exhibited decreased red fluorescence and increased green fluorescence when exposed to irradiation, ascribing to the collapse of mitochondrial membrane potential.47 Moreover, the magnitude of the fluorescence change was positively correlated to irradiation time. These results were also consistently confirmed by the subsequent FCM results (Figure 4F). These data indicated that the ROS generated by MTPP significantly induced the mitochondria damage. Although the previous cytotoxicity assays had revealed the enhanced antitumor activity of the FA-PEG-PDBO-BPT drug delivery system, the apoptosis caused by the micelles system in HeLa cells should also be further studied. On the one hand, HeLa cells were respectively treated with MTPP, CPT, FAI
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Figure 6. In vivo antitumor effects of micelles. (A) Photographs of the tumors extracted from the mice after treatments with saline (control), MTPP, CPT, FA-PEG-PDBO-BPT micelles, and micelles@MTPP plus/without irradiation for 0, 7, and 20 days, respectively. Scale bar: 1 cm. (B) Relative tumor volumes and (C) survival rates of mice after various administrations. (D) Morphological changes and apoptosis analysis of tumor tissues by H&E staining and TUNEL assay. Scale bar: 100 μm. (E) Representative H&E staining of liver extracted from the mice after various treatments. Scale bar: 100 μm. (F) CPT distribution in tumor and main organs in tumor-bearing mice after intravenous injection CPT and prodrug micelles FA-PEGPDBO-BPT for 24 h. Error bars present as mean ± SD (n = 6); **, p < 0.01.
As shown in Figure 5B, CPT, FA-PEG-PDBO-BPT micelles, and micelles@MTPP groups induced the significant upregulation of Bax and down-regulation of Bcl-2 compared to control and free MTPP (Figure 5C), owing to the enhanced drug delivery efficiency and potent cell damage caused by free drug or prodrug micelles. Moreover, micelles and micelles@ MTPP without irradiation also caused the dramatic upregulation of cytochrome c, cleaved caspase-9, and cleaved caspase-3 (Figure 5D−F). It was attributed to the previously revealed fact that the released CPT from prodrug micelles induced the mitochondrial hyperpolarization and damage by inhibiting cellular respiration.51 Furthermore, when exposed to irradiation, Bax/Bcl-2, cytochrome c, cleaved caspase-9, and cleaved caspase-3 protein levels were significantly elevated both in MTPP- and micelles@MTPP-treated cells except for those free CPT and micelles-treated cells (Figure 5D−F). More importantly, micelles@MTPP caused the highest expression of the above proteins among all the treatment groups (p < 0.01), indicating that laser irradiation significantly enhanced mitochondrial damage and tumor cell apoptosis. These results
consistently demonstrated that the micelles@MTPP drug delivery system combined with laser irradiation significantly improved antitumor efficiency in vitro. To evaluate the antitumor efficacy of the micelles@MTPP drug delivery system in vivo, a tumor (HeLa cells)-bearing mouse model was established and then intravenously injected with different drug formulations at an equivalent CPT dose of 3 (mg/kg)/day. The administration was performed twice per week and continued for 21 days. It was observed that all treatment groups exhibited various extents of tumor suppression compared to control (Figure 6A,B). Only MTPP and micelles@MTPP treatment groups showed the significant difference in tumor suppression between the irritated and nonirritated conditions, which was confirmed by the quantitative analysis of tumor volumes, implying the antitumor effect of photodynamic therapy. Moreover, both micelles and micelles@MTPP demonstrated much higher tumor inhibition than MTPP and CPT even without laser irradiation (p < 0.01), indicating the enhanced antitumor efficiency of the FA-PEGPDBO-BPT drug delivery system. Importantly, the micelles@ J
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logical and histological features of major tissues of the tumorbearing mice after different administrations were examined and observed by microscope using H&E staining. As expected, compared to the control group, both free CPT and CPT plus irradiation groups caused serious liver damage, indicated by the decreasing hepatic cells and infiltration of inflammatory cells (Figure 6E).54,55 In contrast, negligible liver damage was displayed in micelles and micelles@MTPP groups with/ without irradiation groups. Furthermore, there was no obvious damage to the other tissues (heart, kidney, spleen, and lung) of mice treated with micelles or micelles@MTPP without/plus irradiation (Figure S22). These results confirmed again that the FA-PEG-PDBO-BPT drug delivery system significantly reduced the side effects of CPT. Finally, we examined the biodistribution of the FA-PEGPDBO-BPT drug delivery system in major organs of mice. As shown in Figure 6F, sole CPT administration resulted in very low accumulation in tumor after treatments for 24 h, whereas the dose was primarily deposited in liver, lung, and spleen, thus inducing severe liver damage and body weight loss (Figure 6E and Figure S21). In contrast to free CPT, the concentration of CPT was remarkably higher in the tumor (about 4-fold) and lower in the main organs especially for liver after treatment with FA-PEG-PDBO-BPT micelles for 24 h, indicating that this drug delivery system could effectively deliver CPT to tumor tissues via the EPR effect and FA-receptor-mediated endocytosis. Moreover, the lower accumulation of micelles in liver was probably due to the shielding of PEG, which reduced the uptake by the reticuloendothelial system (RES). In addition, it should be noted that CPT was largely deposited in lung and liver tissues, caused by the unavoidable uptake by the RES and embolization of lung capillaries due to drug precipitation (hydrophobic CPT) in the bloodstream.55 The in vitro and in vivo data suggested that the FA-PEG-PDBO-BPT drug delivery system combined with laser irradiation effectively delivered CPT and MTPP into tumors through dual-targeting strategy, thus dramatically suppressing tumor growth while efficiently reducing side effects as well.
MTPP with irradiation group induced the strongest tumor suppression among all treatment groups (p < 0.01). The result of final tumor tissue weight also exhibited a similar suppression tendency of tumor growth (Figure S20). Additionally, the survival time of mice treated with the laser-irritated micelles@ MTPP group was obviously longer than other groups (Figure 6C). The reasons could be explained as follows: (1) FA-PEGPDBO-BPT micelles with appropriate sizes were first accumulated in tumor tissues via EPR effect and then effectively internalized by tumor cells via FA-receptor-mediated endocytosis; (2) the micelles@MTPP entrapped in endo-/lysosome were then protonated in response to the locally low pH environment, leading to the escaping of micelles@MTPP from endo-/lysosome to cytoplasm; (3) the high concentration of GSH in cytoplasm would rapidly cleave the disulfide bonds and induce CPT release, and moreover, the disruption to the hydrophilic−hydrophobic balance by pH-responsive protonation and redox-responsive CPT removal induced complete micelles disassembly as well as MTPP release; (4) the released MTPP could be specifically accumulated in mitochondria, and the ROS produced by MTPP locally caused the loss of membrane potential and activated the mitochondria apoptotic pathway under laser irradiation. These results demonstrated that combining the micelles@MTPP drug delivery system with laser irradiation could more effectively suppress the tumor growth in vivo. The appearance and evaluation of in situ apoptosis at the tumor site after treatment with the above drug formulations were assessed by the terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) assay and hematoxylin and eosin (H&E) staining. As shown in Figure 6D, both micelles and micelles@MTPP induced more severe apoptosis (magenta color in tumor tissues) than MTPP and CPT without irradiation, which provided directy evidence for their enhanced pro-apoptotic capability in vivo. The hydrophobic and nontargeting properties of CPT were another reason that may explain the relatively weaker cell apoptosis. Meanwhile, additional irradiation significantly enhanced the apoptotic level of tumor tissues treated with both MTPP and micelles@MTPP. Furthermore, the laser-irradiated micelles@ MTPP treatment group induced the most severe cell apoptosis among all groups, evidenced by a large number of the magenta dots in the TUNEL assay and cells separation/shrinkage in H&E observation (Figure 6D). Moreover, H&E assay also revealed apoptosis results similar to those of the TUNLE results. These results confirmed again that FA-PEG-PDBOBPT micelles could effectively deliver CPT and MTPP inside tumor cells and kill tumor cells with high efficiency in vivo. Although the micelles@MTPP system could effectively cause cell apoptosis and inhibit tumor growth, the biosafety of the micelles@MTPP system in vivo should be further investigated. We first periodically monitored the changes in body weight of the tumor-bearing mice after different treatments, since it may reflect the systematic toxicity of different drug formations. It was observed that the mice treated with both free CPT and CPT plus irradiation resulted in rapid body weight loss in the post-treatment period (Figure S21, p < 0.01), which was due to the poor water-solubility and high toxic side effects of CPT.52,53 On the contrary, the weight steadily increased with the feeding time after treatments for mice treated with micelles and micelles@MTPP with/without irradiation, indicating that this FA-PEG-PDBO-BPT drug delivery system showed negligible systemic toxicity for tumor therapy. Moreover, the morpho-
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CONCLUSION In summary, we have designed and developed a caspaseresponsive disassemble micellar drug delivery system with dualtargeting (both cellular and subcellular targeting) loaded in a novel mitochondrion-targeting photosensitizer MTPP for tumor therapy. The comprehensive in vitro and in vivo characterizations demonstrated that the FA-PEG-PDBO-BPT system could significantly enhance uptake efficiency and effectively deliver CPT and MTPP to tumor cells; the subsequent hydrophobic−hydrophilic transformation caused by pH-responsive protonation in lysosomes and redoxresponsive CPT removal in cytoplasm induced complete micelles disassembly as well as drug release. Furthermore, the released MTPP could selectively target mitochondria and activate a mitochondrial apoptotic pathway upon light irradiation as a result of ROS generation, leading to the dramatically higher antitumor effect and lower systematic toxicity in vivo. This study provides promising disassemble nanocarriers for drug delivery, which holds great potential in the field of targeted tumor therapy.
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EXPERIMENTAL SECTION
Materials. α-Bromo-γ-butyrolactone (BBO), 5-hydroxy-2-phenyl1,3-dioxane, benzyl bromide, ethyl-3-(3-(dimethylamino)propyl) K
DOI: 10.1021/acs.chemmater.7b02513 Chem. Mater. XXXX, XXX, XXX−XXX
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32H, -OCH2CH2CH-), 3.57 (m, 455H, -OCH2CH2-), 3.4−3.5 (d, 12H, -OCH2CHCH2-), 1.37 (s, 9H, -CH3). FTIR: 2884, 1774, 1111 cm−1. The 1H NMR, FTIR, and GPC spectra of bPEG-BBO-BDO were shown in Figures S1d, S2c, and 1B, respectively, with accompanying data in Table S1. Synthesis of PDEA Copolymer. A dried 100 mL roundbottomed flask was initially degassed by freeze−pump−thaw cycles. Dried 2-ethanolamine (0.121 mL, 2 mmol) and 20 mL of anhydrous THF were added in mixture via a dry syringe. Potassium naphthalene (2 mmol) in THF was then added slowly under stirring at room temperature for 30 min. Subsequently, the freshly anhydrous DEA (30 mmol) was added quickly and initiated the anionic polymerization reaction. After stirring for 5 h, 8 mL of methanol was added to terminate the reaction. The oily polymer (PDEA; Mw, 2500) was purified and obtained by repeated precipitation in cold n-hexane (88% yield). The molecular weight of PDEA was calculated as 2200 with narrow PDI (1.32) by GPC. 1H NMR (400 MHz, CDCl3, ppm): 4.14 (d, 2H, -COOCH2CH2-), 2.67−2.7 (m, 2H, -COOCH2CH2N-), 2.48−2.54 (m, 4H, -NCH2CH3), 1.87 (d, 2H, -CH2CCOOCH3), 0.97 (s, 6H, -CH3). FTIR: 1740, 1648, 1517 cm−1. The 1H NMR, FTIR, and GPC spectra of PDEA were shown in Figures S1e, S2d, and 1B, respectively, with accompanying data in Table S1. Synthesis of bPEG-PDBO-BOH Copolymer. Briefly, DIPEA (1.93 mmol) and potassium iodide (1.68 mmol) were added in degassed DCM (30 mL) solution containing PDEA (1 mmol) under nitrogen at 0 °C, and bPEG-BBO-BDO (2.78 mg, 0.02 mmol) was added to this solution under stirring at 0 °C for 2 h. After stirring for 6 h at room temperature, the reaction solution was extracted thrice by water and then bPEG-PDBO-BDO was obtained by precipitation into diethyl ether (69% yield). 1H NMR (400 MHz, CDCl3, ppm): 7.2− 7.28 (m, 60H, aromatic), 4.68−4.6 (d, 24H, PhCH2), 4.47−4.3 (d, 8H, -OOCCHCH2), 4.26−4.1 (d, 48H, -OCH2CHCH2-), 4.03 (d, 26H, -CH2 in PDEA chain), 3.85−3.63 (m, 32H, -OCH2CH2CH-), 3.57 (m, 455H, -OCH2CH2-), 3.48−3.36 (d, 12H, -OCH2CHCH2-), 2.87 (m, 26H, COOCH2CH2N in PDEA chain), 2.5 (m, 54H, -NCH2CH3 in PDEA chain), 1.99 (d, 26H, -CH2CCOOCH3 in PDEA chain), 1.37 (s, 9H, -CH3), 0.98 (s, 78H, -CH3 in PDEA chain). The 1H NMR spectrum of bPEG-PDBO-BDO was shown in Figure S1f. bPEG-PDBO-BDO (1 g) was hydrogenated in a mixed solution of methanol (25 mL) and THF (75 mL) with 10% Pd/C and activated 20% Pd (OH)2/C as catalyst under the hydrogen pressure of 2.5 MPa. The reaction mixture, in a Parr bottle, was evacuated and purged with H2 three times. The reaction temperature was kept at 30 °C for 24 h. Palladium residues were filtered off. The reaction mixture was evaporated to give an oily product bPEG-PDBO-BOH. The molecular weight of bPEG-PDBO-BOH was calculated as 9400 with narrow PDI (1.18) by GPC. 1H NMR (400 MHz, CDCl3, ppm): 4.4−4.3 (d, 8H, -OOCCHCH2), 4.27−4.08 (d, 48H, -OCH2CHCH2), 4.03 (d, 26H, -CH2 in PDEA chain), 3.8−3.63 (m, 32H, -OCH2CH2CH-), 3.57 (m, 455H, -OCH2CH2-), 3.47−3.4 (d, 12H, -OCH2CHCH2), 2.87 (m, 26H, COOCH2CH2N in PDEA chain), 2.5 (m, 54H, -NCH2CH3 in PDEA chain), 1.99 (d, 26H, -CH2CCOOCH3 in PDEA chain), 1.37(s, 9H, -CH3), 0.98 (s, 78H, -CH3 in PDEA chain). The typical peaks of the benzyl group and PhCH2 at 7.28−7.2 and 4.68−4.6 ppm disappeared (red dotted frame) in 1H NMR spectra of bPEGPDBO-BOH. The 1H NMR and GPC spectra of bPEG-PDBO-BOH were shown in Figures S1g and 1B, respectively, with accompanying data in Table S1. Synthesis of CPT-S-S-OH Prodrug. CPT (4.31 mmol) and triphosgene (1.59 mmol) were dissolved in anhydrous DCM (110 mL) under nitrogen. The tubes were placed into an ice−water bath for 10 min; DMAP (13.8 mmol) suspended in anhydrous DCM (8 mL) was then added dropwise to this solution under stirring at 0 °C for 30 min. Subsequently, DTDE (43 mmol) in anhydrous THF (15 mL) was added to the above reaction mixture and stirred overnight at room temperature. The mixture was respectively washed with 0.1 M HCl aqueous solution, brine, and water. The organic layer was separated and dried over anhydrous MgSO4. The solvent was removed in vacuo, and the residue was purified by recrystallization from chloroform/ methanol (3/10, v/v; 61% yield). ESI-MS (m/z (M+)): calcd, 528.5;
carbodiimidehydrochloride (EDC·HCl) palladium hydroxide on carbon (Pd(OH)2/C), palladium on carbon (Pd/C), N-hydroxysuccinimide (NHS), folic acid (FA), dicyclohexylcarbodiimide (DCC), and 9,10-anthracenediylbis(methylene) dimalonic acid (ABDA) were purchased from Sigma-Aldrich (Beijing, China). L-Carnitin was purchased from Alfa Aesar (Tianjin, China). 2,2′-Dithiodiethanol (DTDE) was supplied by Tokyo Kasei Kogyo Co., Ltd. (TCI, China). Camptothecin (CPT), triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), 4-nitrophenyl chloroformate, 2-ethanolamine, 5-(4aminophenyl)-10,15,20-triphenylporphine, 1-hydroxybenzotriazole (HOAT), 2-(N,N-diethylamino)ethyl methacrylate (DEA), pyridine, N,N-dimethylformamide (DMF), trifluoroacetic acid (TFA), dichloromethane (DCM), triphosgene, and 4-(dimethylamino)pyridine (DMAP) were purchased from J&K Scientific Ltd. Tetrahydrofuran (THF), methanol, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and sodium hydride (NaH) were supplied by Aladdin Industrial Co., Ltd. tBoc amine PEG hydroxyl (bPEG−OH; Mw, 5000) was purchased from JenKem Technology Co., Ltd. (Beijing, China). Diethyl ether and ethyl acetate were bought from Chuandong Chemical Co. Ltd. (Chongqing, China). Primary antibodies including mouse anti-β-actin, rabbit anti-Bax, rabbit anti-Bcl-2, and rabbit anti-cytochrome c were obtained from Boster Biological Technology Co., Ltd. (Wuhan, China); rabbit anti-cleaved caspase-9 and rabbit anti-cleaved caspase-3 were purchased from Cell Signaling Technology, Inc. Secondary antibody of horseradish peroxidase-conjugated goat IgG was purchased from Beyotime (Jiangsu, China). Other chemicals were provided by Oriental Chemicals Co., Ltd. (Chongqing, China). All the reagents were analytical grade. Synthesis of 5-Benzyloxy-1,3-dioxan-2-one. BDO was synthesized according to previous literature procedure.56 In brief, the precursor product of 2-benzyloxy-1,3-propanediol (BPD) was first synthesized. Sodium hydride (0.125 mol) was dissolved in dried THF (400 mL) under ice−water bath condition; 5-hydroxy-2-phenyl-1,3dioxane (0.1 mol) was then added into the above solution and stirred at 0 °C for 30 min. Then, benzyl bromide (0.12 mol) was added into the mixture solution and stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure and then refluxed in 3% HCl aqueous for 2 h. The mixture was quenched by saturated Na2CO3 solution and then extracted with ethyl acetate. BPD was obtained by vacuum distillation. 1H NMR (400 MHz, CDCl3, ppm): 7.14−7.3 (m, 5H, aromatic), 4.57 (s, 2H, PhCH2-), 3.61−3.72 (m, 4H, -CH2-), 3.49 (m, 1H, -OCH-), 2.23 (s, 2H, CH2OH). The 1H NMR spectrum of BPD was shown in Figure S1a. Next, BPD (2.1 g) and ethyl chloroformate (0.1 mol) were dissolved in dried THF (100 mL). The solution was stirred at 0 °C for 30 min under nitrogen environment. TEA (0.11 mol) was added dropwise into the mixture solution under stirring at room temperature for 4 h. The resultant product BDO was obtained as solid precipitate by filtration and vacuum drying (65% yield). The residue was recrystallized from THF/diethyl ether. 1H NMR (400 MHz, CDCl3, ppm): 7.25−7.32 (m, 5H, aromatic), 4.58 (s, 2H, PhCH2-), 4.34−4.43 (m, 4H, -OCH2CH-), 3.81−3.83 (p, 1H, -OCH-). 13C NMR (CDCl3, ppm): 64.09, 67.67, 68.8, 74.84, 75.1, 75.35, 125.82, 126.37, 126.76, 134.65, 145.86. FTIR: 1737 cm−1 (CO). The 1H NMR, 13C NMR and FTIR spectra of BDO were shown in Figures S1b,l and S2b, respectively. Synthesis of bPEG-BBO-BDO Copolymer. bPEG-BBO-BDO was synthesized by ring-opening copolymerization of BDO and BBO using bPEG as the initiator. Briefly, bPEG−OH (400 mg, 0.08 mmol), BBO (165 mg, 1 mmol), BDO (347 mg, 1.66 mmol), and DBU (20 μL, 0.13 mmol) were suspended in dry chloroform (8 mL) under nitrogen. The mixture solution was degassed by three freeze−pump− thaw cycles and stirred at 30 °C for 48 h under nitrogen; the reaction mixture was respectively precipitated into cold methanol and cold diethyl ether for three times. The product was harvested as a viscous residue by vacuum−rotary evaporation. The molecular weight of bPEG-BBO-BDO copolymer had a Mn of 7900 and a PDI of 1.21 calculated by GPC. 1H NMR (400 MHz, CDCl3, ppm): 7.26−7.29 (m, 60H, aromatic), 4.52−4.67 (d, 24H, PhCH2), 4.36−4.4 (d, 8H, -OOCCHCH2), 4.1−4.2 (d, 48H, -OCH2CHCH2-), 3.65−3.83 (m, L
DOI: 10.1021/acs.chemmater.7b02513 Chem. Mater. XXXX, XXX, XXX−XXX
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Chemistry of Materials found, 529.35. 1H NMR (400 MHz, CDCl3, ppm): 8.37−8.29 (m, 1H), 8.2 (d, 1H), 7.9 (dd, 1H), 7.81 (dd, 1H), 7.69−7.55 (m, 1H), 7.41 (d, 1H), 5.66 (dd, 1H), 5.34 (m, 1H), 4.36−4.29 (m, 2H), 3.84− 3.81 (m, 2H), 2.92−2.81 (m, 4H), 2.23 (m, 1H), 2.12 (m, 1H), 1.03 (t, 3H). FTIR: 1746, 1663, 1457, 1396, 1259, 1157 cm−1. The 1H NMR, FTIR, and ESI-MS spectra of CPT-S-S-OH were shown in Figures S1h,m and S2e, respectively. Synthesis of PEG-PDBO-BPT Copolymer. CPT-S-S-OH (0.962 mmol) was combined with DIPEA (3.846 mmol) and pyridine (77.4 μL) in 8 mL of anhydrous DCM in 20 mL Schleck flasks, and 4nitrophenyl chloroformate (2.89 mmol) in anhydrous DCM was added dropwise at 0 °C and stirred for 4 h at room temperature. The reaction mixture was concentrated in vacuo and then dissolved in 3 mL of DMF. Next, TEA (0.8 mL) and bPEG-PDBO-BOH (721 mg) in DMF (3 mL) were added to the mixture, and the reaction was continued to be stirred for 24 h. The crude product was obtained by precipitation into diethyl ether, the solvent was removed in vacuum, and the resulting residue was re-dissolved in DCM and purified by column chromatography on silica gel using DCM/MeOH (13:1) (78% yield, and the conjugation efficiency of CPT was 18.3%). The removal of Boc protection groups from bPEG-PDBO-BPT was according to our previous literature.57 1H NMR (400 MHz, CDCl3, ppm): 8.21 (m, 9H, -CH- in CPT chain), 8.05 (d, 9H, -CH- in CPT chain), 7.87 (dd, 9H, -CH- in CPT chain), 7.78 (dd, 9H, -CH- in CPT chain), 7.69 (m, 9H, -CH- in CPT chain), 7.61 (d, 9H, -CH- in CPT chain), 5.67−5.7 (dd, 18H, -CH2- in CPT chain), 5.2−5.3 (m, 18H, -CH- in CPT chain), 4.57 (d, 8H, -OOCCHCH2), 4.45−4.1 (d, 48H, -OCH2CHCH2), 4.01 (d, 26H, -CH2- in PDEA chain), 3.69−3.88 (m, 32H, -OCH2CH2CH-), 3.57 (m, 455H, -OCH2CH2-), 3.46 (d, 12H, -OCH2CHCH2), 2.66 (m, 26H, COOCH2CH2N in PDEA chain), 2.4−2.58 (m, 54H, NCH2CH3 in PDEA chain), 1.96 (d, 26H, -CH2CCOOCH3 in PDEA chain), 1.86 (m, 18H, -CH- in CPT), 1.83 (m, 9H, -CH- in PDEA chain), 1.19 (t, 27H, -CH3 in CPT), 0.98 (s, 78H, -CH3 in PDEA). The 1H NMR spectrum of PEG-PDBO-BPT was shown in Figure S1i. Synthesis of FA-PEG-PDBO-BPT copolymer. FA (1 mmol) was combined with DCC (0.55 mmol) and NHS (0.55 mmol) in 30 mL of DMF solution. After stirring at room temperature for 2 h, 0.5 mmol of PEG-PDBO-BPT was added to the reaction mixture. The mixture solution was filtrated, and the residue in DMF was added dropwise to water with 4 times volume and dialyzed in a dialysis bag (MWCO, 3500 Da) for 4 days to remove DMF. The final product was harvested by lyophilization (48.4% yield). The molecular weight of FA-PEGPDBO-BPT was respectively calculated as 13600 with narrow PDI (1.24) by GPC and as 13266 by MALDI-TOF. 1H NMR (400 MHz, CDCl3, ppm): 8.35 (m, the proton of folate), 7.25 (m, the proton of folate), 6.89 (m, the proton of folate), 8.21 (m, 9H, -CH- in CPT chain), 8.06 (d, 9H, -CH- in CPT chain), 7.89 (dd, 9H, -CH- in CPT chain), 7.78 (dd, 9H, -CH- in CPT chain), 7.69 (m, 9H, -CH- in CPT chain), 7.61 (d, 9H, -CH- in CPT chain), 5.67−5.7 (dd, 18H, -CH2- in CPT chain), 5.23−5.26 (m, 18H, -CH- in CPT chain), 4.57(d, 8H, -OOCCHCH2), 4.42−4.16 (d, 48H, -OCH2CHCH2), 4.01 (d, 26H, -CH2- in PDEA chain), 3.69−3.8 (m, 32H, -OCH2CH2CH-), 3.57 (m, 455H, -OCH2CH2-), 3.46 (d, 12H, -OCH2CHCH2), 2.66 (m, 26H, COOCH2CH2N in PDEA chain), 2.49−2.51 (m, 54H, NCH2CH3 in PDEA chain), 1.96 (d, 26H, -CH2CCOOCH3 in PDEA chain), 1.86 (m, 18H, -CH- in CPT), 1.83 (m, 9H, -CH- in PDEA chain), 1.19 (t, 27H, -CH3 in CPT), 0.98(s, 78H, -CH3 in PDEA). The relatively high integral of FA proton possibly attributes to the additional electrostatic absorption between FA and PEG-PDBO-BPT molecules. A similar phenomenon was also observed in related previous works.58,59 FTIR: 2926, 2857, 1745, 1625, 1576, 1110 cm−1. The 1H NMR, FTIR, GPC, and MALDI-TOF spectra of FA-PEG-PDBO-BPT were shown in Figures S1j,k, S2f, 1B, and S3, respectively, with accompanying data in Table S1. Synthesis of the Mitochondria-Targeting Photosensitizer 5(3-Hydroxy-p-(4-trimethylammonium)butoxyphenyl)10,15,20-triphenylporphyrin Chlorine. The synthesis route of MTPP was depicted as Figure S4. Briefly, L-carnitin (174.1 mg) was suspended in 2 mL of anhydrous ethanol; HOAT (147 mg) and EDC·
HCl (207 mg) suspended in anhydrous DCM (8 mL) were added to this solution and stirred at room temperature for 30 min. 5-(4Aminophenyl)-10,15 20-triphenylporphyrin (139 mg) was added to this solution, and the pH was adjusted to basicity with TEA (200 μL). The reaction was stirred for another 4 h; then water with 4 times volume was added to the solution. The resulting precipitate was isolated by filtration with a spin-dryer and dried in vacuo (89% yield). The purity of MTPP (99%) was determined by high performance liquid chromatography (HPLC) with a C18 column (Bioband HP-120 C18, 4.6 mm × 150 mm, 5 μm) and using a linear gradient of acetonitrile and DI water containing 0.1% TFA. ESI-MS (m/z (M+)): calcd, 774.5; found (M+ Cl−), 803.78. 1H NMR (400 MHz, CDCl3, ppm): 8.83 (s, 8H), 8.21 (d, 6H), 7.99 (d, 2H), 7.7−7.86 (s, 9H), 7.06 (d, 2H), 4.03 (s, 2H), 3.53 (s, 2H), 3.25 (d, 11H), 2.11 (s, 2H). The 1 H NMR spectrum, mass spectrum, and HPLC spectrum of MTPP were shown in Figures 1C,D and S5, respectively. Preparation of Micelles and MTPP Loaded Micelles (FA-PEGPDBO-BPT@MTPP). The formation of micelles or MTPP loaded micelles were achieved by an evaporation method as reported previously.20 A 20 mg amount of the polymer prodrug combined with/without 10 mg of MTPP were dissolved in 2 mL of DMSO. After stirring for 4 h, the mixture solution was added dropwise to deionized water (10 mL) under magnetic stirring at room temperature for 24 h. Then, the dispersion was dialyzed against deionized water (MWCO, 3.4 kDa) for 48 h to remove DMSO and then freeze-dried. The content of CPT in FA-PEG-PDBO-BPT was quantified by UV/vis spectroscopy based on the standard curve of CPT at λ = 365 nm. The UV absorbance of the MTPP loaded micelles solution at 421 nm (MTPP) was measured as well, and the drug loading content was determined by a linear standard curve of MTPP. Drug loading content (DLC) and drug loading efficiency (DLE) were calculated according to the following formulas:
DLC/(wt %) =
DLE/% =
amount of loaded drug × 100 weight of compolymer
amount of loaded drug × 100 weight of drug in feed
Material Characterization. Molecular weights and polydispersity index of the copolymers were measured by a gel-permeation chromatograph (GPC) equipped with PL gel, 5 mm MIXED-C columns (Waters 1424 detector, Agilent Technologies, USA). The eluent used was THF, and the flow rate was 1.0 mL min−1 at 40 °C. 1 H NMR spectra were recorded on a Bruker Avance 500 MHz NMR spectrometer (NMR, Swiss). Matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry (Autoflex speed, Bruker Daltonic Inc., USA), mass spectrometry (Waters Acquity SQ Detector UPLC-MS), and Fourier transforms infrared spectroscopy (FTIR; model 6300, BioRad Co. Ltd., USA) were employed to monitor and analyze the functionalization procedures of the copolymer. Saturated MALDI matrix solutions were prepared in MeOH/water solutions (80/20, v/v) containing 0.1% TFA. Sinapinic acid was used as the matrix. The morphology of the FA-PEG-PDBOBPT micelle was characterized by transmission electron microscopy (TEM; LIBRA 200 CS, Carl Zeiss Co., Germany). The size and size distribution of micelles in aqueous solution were measured by ζ potential measurement (Nano ZS90 Zetasizer, Malvern Instruments Co. Ltd., U.K.) equipped with DLS. HPLC was performed on a C18 column (Bioband HP-120, 4.6 mm × 150 mm, 5 μm) using a linear gradient of acetonitrile and DI water containing 0.1% TFA as a mobile phase with the flow rate of 0.6 mL/min at 29 °C. Critical Micelle Concentration. Pyrene was used as the fluorescence probe to investigate the cmc of FA-PEG-PDBO-BPT micelle. Pyrene was first dissolved in acetone and then diluted 6 × 10−6 M with distilled water. Subsequently, acetone was remove by evaporation under nitrogen airflow protection. Then FA-PEG-PDBOBPT polymer was dispersed in the above solution with different concentrations from 1.0 × 10−5 to 1 mg/mL. The final concentration of pyrene was adjusted to 6 × 10−7 M. The samples were equilibrated M
DOI: 10.1021/acs.chemmater.7b02513 Chem. Mater. XXXX, XXX, XXX−XXX
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μM MTPP) and a fluorescent dye (1 μM LysoTracker or 2 μM Rhodamine 123 Green) at 37 °C under 5% CO2 for 1, 3, and 8 h, respectively. The cells were subsequently visualized and analyzed by CLSM. Targeting Efficiency and Endocytosis Level of Micelle in Vitro. Targeting efficiency and endocytosis level assays of FA-PEGPDBO-BPT micelles were carried out according to our previous literature.27 Briefly, HeLa and HUVEC cells were seeded on confocal microscopy dishes/6-well plates at 1 × 105 cells density and then cultured with micelles@MTPP (76.6 μg/mL) for 4 h. Besides, HeLa cells preincubated with free FA (5 mg/mL) for 2 h before coincubation with micelles, which acts as negative control. These cell samples were then fixed and stained with DAPI and Alexa 488 and then visualized by CLSM. In addition, the above cell samples were also directly collected via centrifugation and then analyzed by FCM. Measurement of Intracellular ROS Accumulation. The generation of intracellular ROS was measured by CLSM using DCFDA as the fluorescence sensor. HeLa cells were respectively incubated with MTPP and FA-PEG-PDBO-BPT@MTPP micelles for 12 h; then the medium was replaced with fresh medium containing DCF-DA. After incubation for another 20 min, laser irradiation (665 nm, 100 mW/cm2) was performed subsequently for 10 min. The cells were washed with PBS buffer and imaged by CLSM. FCM Analysis of Cells Apoptosis Induced by micelles@ MTPP. HeLa cells were seeded on confocal microscopy dishes and allowed to grow to 60−70% confluence. Afterward, cells were treated with CPT (15 μg/mL), MTPP (5 μM), FA-PEG-PDBO-BPT (76.6 μg/mL, equivalent of 15 μg/mL CPT), and FA-PEG-PDBO-BPT@ MTPP micelles (76.6 μg/mL, the same dosage as those of CPT and MTPP) for 12 h with laser irradiation for various time intervals (0, 5, 10, and 20 min). HeLa cells were washed with PBS and harvested through centrifugation. The collected cells were then suspended into cell binding solution and co-stained with Annexin V-FITC (5 μL) and PI (10 μL) under dark environment for 10 min. The final cell samples were analyzed by FCM (BD Biosciences). Western-Blot Analysis. Mitochondria apoptotic pathway-associated proteins in HeLa cells treated with various formulations were examined by Western blotting. Briefly, HeLa cells were seeded on 6well plates and then treated with CPT (15 μg/mL), MTPP (5 μM), FA-PEG-PDBO-BPT micelles (76.6 μg/mL), and micelles@MTPP (76.6 μg/mL, the same dosage as those of CPT and MTPP) for 24 h with or without laser (665 nm, 100 mW/cm2, 10 min). Subsequently, cell samples were washed with PBS and solubilized with lysis buffer; then the total proteins were harvested by centrifugation (12000 rpm × 10 min, 4 °C). BCA protein assay kit (Beyotime) was used to determine the protein concentration. The protein samples were separated by 15% SDS-PAGE and transferred to PVDF membranes. After incubation with 5% skim milk in TBST for 1 h, the membranes were then incubated with primary antibodies at 4 °C for another 12 h. Next, the membranes were incubated with specific secondary antibody conjugated with horseradish peroxidase (1:8000 dilution) at room temperature for 1 h. Finally, the protein bands were visualized by enhanced chemiluminescence (ECL) detection reagents and captured using an Azure c300 Imaging System (Azure Biosystems). Densitometry data analysis was performed with ImageJ software 1.45f. In Vivo Antitumor Efficacy of FA-PEG-PDBO-BPT@MTPP. Male nude mice (4−6 weeks old) were provided by Beijing Institution for Drug Control, China. All animal experiments were strictly performed according to guidelines of the Institutional Animal Care and Use Committee of China. Subcutaneous HeLa xenograft mouse model was used to evaluate the therapeutic efficacy of different drug formulations. When tumor volume reached 50−100 mm3, balb/c nude mice were randomly divided to nine groups and intravenously administrated with saline (control), MTPP, CPT, FA-PEG-PDBOBPT micelles, and micelles@MTPP with or without radiation (665 nm, 100 mW/cm2, 10 min) at the dose of 3 mg/kg CPT equivalent (n = 6). The treatment was given two times a week and continued for 21 days. The body weights and tumor volumes of mice were recorded per 2 days. Tumor volume (V) was calculated using the following formula: V = the longest dimension × (the shortest dimension)2/2. The
at room temperature under dark environment for 24 h. The excitation spectra were recorded and analyzed by a fluorescence spectrophotometer (LS50B, PerkinElmer, USA) at wavelengths ranging from 300 to 380 nm. The emission wavelength was set at 390 nm. Finally, the intensity ratios (I339 /I333 ) vs the logarithm of the polymer concentrations were plotted. Measurement of ROS Level in solution. A chemical oxidation method based on 9,10-anthracenediylbis(methylene) dimalonic acid (ABDA) was employed to monitor the 1O2 generation from the MTPP loaded micelles (micelles@MTPP). Water-soluble ABDA exhibits photobleaching when oxidized by 1O2 to its endoperoxide, which causes the decrease in ABDA absorption at 401 nm. Micelles@ MTPP (10.3 mM) were combined with ABDA (10 mM) in 1.5 mL of PBS solution. After stirring in the dark at room temperature for 0.5 h, the mixture was then irradiated by near-infrared laser (665 nm, 100 mW/cm2) over different periods of time. The absorbance of the ABDA was recorded by a UV/vis/near-IR spectrometer (Lambda 900, PerkinElmer instruments, USA). The control experiment was also carried out using 10 mM ABDA solution containing no micelles@ MTPP. Drug Release Behavior. Briefly, FA-PEG-PDBO-BPT@MTPP micelles (3 mg) suspended in 1 mL of PBS (pH 7.4) were then transferred to a dialysis bag (MWCO, 3.4 kDa). The dialysis tubes were subsequently immersed into a glass tube containing 30 mL of PBS (pH 7.4), PBS with 2 mM of glutathione, and PBS with 10 mM of GSH and then incubated with shaking in the dark at 37 °C. At predetermined time intervals, 0.6 mL of incubation medium was taken out for analysis and replaced with the same volume of fresh medium. The total released drugs were measured by a UV/vis/near-IR spectrometer (Lambda 900, PerkinElmer) at wavelength 365 nm. Cell Culture and Cytotoxicity Evaluation. Human cervical cancer cells (HeLa) and human umbilical vein endothelial cells (HUVEC) were incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) and 1% (w/v) penicillin (100 U/mL)/streptomycin (100 μg/ mL) with 5% CO2 at 37 °C. The in vitro anticancer effect of polymer prodrugs was evaluated against HeLa cells by CCK8 assay. HeLa cells were incubated with CPT (15 μg/mL), MTPP (5 μM), FA-PEG-PDBO-BPT (76.6 μg/ mL, equivalent of 15 μg/mL CPT), and FA-PEG-PDBO-BPT@ MTPP micelles (76.6 μg/mL, the same dosage as those of CPT and MTPP) in 24-well plates for 12 and 24 h with or without laser irradiation (665 nm, 100 mW/cm2, 20 min). Then, the culture solution was removed and replaced with 200 μL of fresh medium and 20 μL of CCK-8 reagent for each well, and the cell samples were incubated for another 1.5 h at 37 °C. The mixture solution was measured with a spectrophotometric microplate reader (Bio-Rad 680, USA) at 450 nm. In Vitro Cellular Uptake and Subcellular Location of Micelles. The cellular uptake of FA-PEG-PDBO-BPT@MTPP micelles in HeLa cells was analyzed and visualized by confocal laser scanning microscopy (CLSM) and flow cytometry (FCM), respectively. HeLa cells were seeded on confocal microscopy dishes/ 6-well plates at 1 × 105 cells density. After cell confluence reached around 60−70%, HeLa cells were respectively treated with MTPP and micelles@MTPP with/without laser irradiation (665 nm, 100 mW/ cm2, 10 min) for 12 and 24 h. On the one hand, HeLa cells were fixed with 4% paraformaldehyde for 30 min, then respectively stained with Alexa 488-phalloidin (Cytoskeleton actin maker, excitation/emission = 495 nm/518 nm) and DAPI (the nucleus marker), followed by imaging via CLSM (LSM 510 META Olympus, Japan). On the other hand, HeLa cells were washed with PBS and harvested by centrifugation (2000 rpm × 10 min). The collected cells were then suspended into cell binding solution (Beyotime) and then analyzed using FCM (BD Biosciences). The visualization of the subcellular localization of micelles@MTPP endocytosed by HeLa cells was also detected by CLSM as well. Similarly, HeLa cells were seeded on confocal microscopy dishes and allowed to grow to 60−70% confluence. Afterward, cells were resuspended in DMEM medium containing the micelle sample (5 N
DOI: 10.1021/acs.chemmater.7b02513 Chem. Mater. XXXX, XXX, XXX−XXX
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survival rate of mice in each group was monitored for another 20 days after the last dosing. H&E and TUNEL Assays. The mice were sacrificed after treatment with the aforementioned formulations for 21 days. The tumor and primary tissues (heart, lung, kidney, spleen, and liver) were collected, fixed, embedded in paraffin, and sliced. After deparaffnization with xylene, the tissue sections were stained with hematoxylin and eosin (H&E) for histological analysis, or detected with the In Situ Cell Death Detection Kit (Beyotime) for in situ TUNEL assay, and eventually visualized by CLSM (LSM 510 META Olympus). Biodistribution Study. To assess the distribution of micelle formulation on tissues, HeLa cell tumor-bearing nude mice were injected intravenously with CPT and FA-PEG-PDBO-BPT prodrug micelles at a dose of 3 mg/kg CPT. After administration of 24 h, the mice were killed and major tissues were excised, washed, and homogenized in 0.5 mL of DMSO, followed by centrifugation at 15000 rpm for 15 min. The content of CPT inside each tissue was measured by fluorescence spectrophotometry and a standard curve. The distribution of micelle in tissue/organs was expressed as μg(amount of micelle)/g(weight of tissues or organs). Statistical Analysis. We performed the statistical analysis with software of OriginPro (version 9.0) through Student’s t test and oneway analysis of variance (ANOVA). All data were expressed as means ± standard deviation (SD). The confidence levels of 95% and 99% were regarded as significant differences.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.7b02513. NMR, ESI-MS, MALDI-TOF, and FTIR spectra of copolymer synthesis; cmc characterizations; HPLC curve of MTPP; various absorption spectra; absorption and emission spectra of MTPP loaded micelles; stability of micelles in serum; particle size distributions (DLS); cell viability assays; endocytosis efficiency of micelles; 3D reconstructed image and video screen shot of FA-PEGPDBO-BPT@MTPP within HeLa cells; generation and quantification analyses; irradiation time-dependent apoptosis; final weights of tumors; mouse weight survey; histological examination of major tissues; and molecular weight parameters of various synthesis intermediate products (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Kaiyong Cai: 0000-0001-9029-680X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the National Key R&D Program of China (Grant No. 2016YFC1100300), the National Natural Science Foundation of China (Grant No. 21274169), and the Innovation Team in University of Chongqing Municipal Government (Grant No. CXTDX 201601002).
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REFERENCES
(1) Xu, X. D.; Cheng, Y. J.; Wu, J.; Cheng, H.; Cheng, S. X.; Zhuo, R. X.; Zhang, X. Z. Smart and Hyper-Fast Responsive Polyprodrug O
DOI: 10.1021/acs.chemmater.7b02513 Chem. Mater. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.chemmater.7b02513 Chem. Mater. XXXX, XXX, XXX−XXX