Cascade-Promoted Photo-Chemotherapy against Resistant Cancers

Article pubs.acs.org/cm

Cite This: Chem. Mater. 2018, 30, 3486−3498

Cascade-Promoted Photo-Chemotherapy against Resistant Cancers by Enzyme-Responsive Polyprodrug Nanoplatforms Wenhui Wang,†,‡ Guohai Liang,†,‡ Wenjia Zhang,†,‡ Da Xing,*,†,‡ and Xianglong Hu*,†,‡ †

MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China ‡ College of Biophotonics, South China Normal University, Guangzhou 510631, China

Downloaded via KAOHSIUNG MEDICAL UNIV on June 30, 2018 at 17:28:56 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: Cisplatin has long been the first-line treatment for a variety of solid tumors. However, the poor pharmacokinetics and intrinsic or acquired drug resistance are the main challenges in cancer therapy. Herein, endogenous enzymeresponsive cisplatin polyprodrug nanoplatforms were developed for cascade-promoted photo-chemotherapy against drugresistant cancers. The polyprodrug nanoplatforms, ICG/Poly(Pt), were fabricated from the indocyanine green (ICG) photosensitizer and cisplatin polyprodrug amphiphiles, PEG-b-P(Ptco-GFLG)-b-PEG, consisting of repeated enzyme-degradable GFLG peptides and cisplatin prodrug units in the hydrophobic block and hydrophilic PEG chains, exhibiting ∼24.7 wt % cisplatin loading. Upon cellular uptake in lysosomes, cathepsin B could partially degrade the nanoplatforms into cisplatin prodrug, and then 808 nm laser irradiation would excite ICG to afford reactive oxygen species (ROS) and local hyperthermia, thus launching the phototherapy. Furthermore, the concurrent photodynamic and photothermal process could damage lysosomes to accelerate the cytosolic movement of the cisplatin prodrug away from lysosomes, which was followed by GSH reduction into active cisplatin to initiate cascade chemotherapy. In addition, the polyprodrug nanoplatforms provided dual-model photoacoustic and fluorescence imaging to guide the therapeutic treatments. In vitro and in vivo explorations proved that ICG/Poly(Pt) could significantly inhibit the cisplatin-resistant A549/DDP cancers. The well-defined polyprodrug nanoplatforms exhibited great potential for imaging-guided cascade treatments of resistant cancers in intelligent biomedicine.

■

INTRODUCTION Cisplatin can disrupt DNA structure in cancer cell nuclei, which has been widely used in the treatment of various solid cancers, including testicular, ovarian, bladder, head and neck, breast, and lung cancers,1,2 producing significant improvement in the survival rate and quality of life of afflicted persons. However, the further application of platinum (Pt) agents is frequently limited by the severe dose-dependent side effects (e.g., nephrotoxicity and neurotoxicity)3 and, in particular, the emergence of cisplatin resistance in cancers.4−6 Some cancer cells are intrinsically resistant, while others acquire resistance after several initial treatments.7 The initial sensitivity of cancer cells to cisplatin is high, but the majority of cancer patients will eventually relapse with the development of cisplatin-resistant cancers.8 Therefore, it is imperative to address the resistance issues and enhance the therapeutic efficacy.9,10 In view of the severe drug resistance of cisplatin, many strategies have been put forward to improve the therapeutic index of cisplatin. One strategy to combat cisplatin resistance is to develop Pt(IV) prodrug delivery systems,11−17 which could enhance the cancer selectivity and reduce the adverse effects simultaneously. Pt(IV)-based prodrugs could be reduced and activated by © 2018 American Chemical Society

endogenous reductive species, such as glutathione (GSH) and ascorbic acid, forming cytotoxic Pt(II) complexes in cancer cells.18−22 Moreover, drug delivery nanocarriers could protect the cargo molecules from metabolic degradation within the hepatorenal tract, prolong the blood circulation lifetime, and facilitate the accumulation of lesions, thus enhancing the therapeutic efficiency with few side effects. Various smart delivery systems that can respond to external stimuli (such as light, magnetic field, ultrasound, and hyperthermia) or internal stimuli (such as reduction/oxidation, pH, and enzyme) showed even better performances.23−26 However, conventional strategies based on a similar anticancer mechanism with cisplatin often suffered other limitations, including low drug loading, a high risk of multidrug resistance, and the final compromised anticancer activities,27 resulting in limited success in the circumvention of platinum resistance. Recently, synergistic therapy has attracted much attention for potentially overcoming drug resistance in cancer cells.28,29 Received: March 18, 2018 Revised: May 3, 2018 Published: May 3, 2018 3486

DOI: 10.1021/acs.chemmater.8b01149 Chem. Mater. 2018, 30, 3486−3498

Article

Chemistry of Materials Scheme 1. Illustration of ICG-Loaded Enzyme-Responsive Polyprodrug Nanoplatformsa

a ICG/Poly(Pt) was composed of ICG and cisplatin polyprodrug amphiphiles tethered with repeating degradable GFLG peptides, PEG-b-P(Pt-coGFLG)-b-PEG. Upon cellular uptake, cathepsin B in the lysosomes partially degrades ICG/Poly(Pt) into free ICG and the cisplatin prodrugs. Then 808 nm laser irradiation affords ROS and heat for phototherapy based on light-absorbing ICG, which can promote lysosome damage and escape of cisplatin prodrugs into the reductive cytosol, followed by reduction into the cisplatin parent drug to activate cascade chemotherapy. For in vivo theranostics, photoacoustic (PA) and fluorescence (FL) dual-model imaging was performed for the guided therapy.

■

RESULTS AND DISCUSSION Fabrication and Characterization of Cisplatin Polyprodrug Nanoplatforms. Enzyme-responsive polyprodrug amphiphiles, PEG-b-P(Pt-co-GFLG)-b-PEG, were synthesized from condensation polymerization of diamine-tethered GFLG peptides and dicarboxyl-functionalized cisplatin prodrugs and subsequent PEG decoration (Scheme S1). PEG-b-P(Pt-co-GFLG)-bPEG was characterized by 1H nuclear magnetic resonance (NMR) spectroscopy, which revealed a relatively high cisplatin loading content, up to ∼24.7 wt %. The aqueous self-assembly of PEGb-P(Pt-co-GFLG)-b-PEG afforded cisplatin polyprodrug micelles, noted as Poly(Pt), exhibiting a spherical morphology as determined by transmission electron microscopy (TEM) analysis, and a diameter of ∼200 nm determined by dynamic light scattering (DLS) (Figure 1A). Furthermore, PEG-b-P(Pt-co-GFLG)-bPEG could encapsulate ICG to afford ICG-loaded polyprodrug micelles, ICG/Poly(Pt), with an ICG loading of ∼16.2 wt %. The formed ICG/Poly(Pt) had a uniform spherical morphology with a diameter of ∼170 nm (Figure 1B). The TEM image of ICG/Poly(Pt) appeared to be darker than that of Poly(Pt), which could be ascribed to the presence of ICG with a high electron density. The ζ potential values of Poly(Pt) and ICG/ Poly(Pt) were demonstrated to be slightly negative, −10.6 and −6.2 mV, respectively, which favored minimizatioon of nonspecific protein absorption in blood circulation. Moreover, when the compounds were stored at room temperature for ≤4 weeks, almost constant diameters of ICG/Poly(Pt) and Poly(Pt) were observed, indicating the excellent stability of cisplatin polyprodrug micelles (Figure S2). On the other hand, compared with that of free ICG, the absorption of ICG/Poly(Pt) was obviously red-shifted and

The integration of alternative therapeutic approaches with different drugs or diverse working mechanisms provided opportunities to overcome the drug resistance of cancer cells.30−32 Additionally, a well-demonstrated polyprodrug strategy exhibited distinct properties, featuring repeating prodrug units, high drug loading, responsive prodrug cleavage, and concurrent active drug release, which demonstrated great potency in the hierarchical self-assembly,33 microstructure-modulated theranostic application,34 and membrane modulation of polyprodrug-gated vesicles.35 The polyprodrug strategy has also been investigated to develop polymeric platforms with diverse morphologies, drug types, and responsive signals in biomedicine.36−47 Inspired by this, the rational design of a cisplatin polyprodrug with other therapeutic modalities has the potential to combat drug resistance in the treatment of persistent cancers. In this work, endogenous enzyme-responsive cisplatin polyprodrug nanoplatforms, ICG/Poly(Pt), were developed from the co-assembly of ICG and cisplatin polyprodrug amphiphiles, PEG-b-P(Pt-co-GFLG)-b-PEG, composed of repeating cathepsin B-degradable GFLG peptides and cisplatin prodrug units in the middle block and hydrophilic PEG block. Upon cellular uptake, cathepsin B in lysosomes partially degrades the nanoparticles to release free ICG and cisplatin prodrugs. Then 808 nm laser irradiation affords ROS and heat to launch the phototherapy based on the light-absorbing ICG, which can damage the lysosome and promote the cytosolic delivery of cisplatin prodrugs into the reductive cytosolic milieu, followed by reduction to the cisplatin parent drug to activate cascade chemotherapy. For in vivo theranostics, photoacoustic (PA) and fluorescence dual-model imaging was performed for the guided therapy (Scheme 1). 3487


Article

Chemistry of Materials

Figure 1. Physicochemical characterization of the ICG-loaded polyprodrug nanoplatforms, ICG/Poly(Pt). As a control, ICG-free Poly(Pt) aqueous dispersions were fabricated from the self-assembly of PEG-b-P(Pt-co-GFLG)-b-PEG. TEM images and hydrodynamic diameter distribution recorded for (A) Poly(Pt) and (B) ICG/Poly(Pt). (C) UV absorption spectra of free ICG, Poly(Pt), and ICG/Poly(Pt). (D) Relative absorbance intensities recorded at 785 nm for the water dispersion of free ICG and ICG/Poly(Pt) at room temperature for different durations. Left and right inset photographs are of the samples at 0 and 4 weeks, respectively. (E) In vitro PA images of ICG/Poly(Pt) at different contents. (F) Linear relationship between PA signal intensity and the content of ICG/Poly(Pt).

protease that displays increased levels in the cytoplasm of various cancer cells, such as colorectal cancer, malignant glioma, breast cancer, lung cancer, prostate cancer, and melanoma,50 which was demonstrated to degrade GFLG sequence efficiently.51−56 The presence of repeating GFLG peptide sequence and cisplatin prodrugs in the backbone of PEG-b-P(Pt-coGFLG)-b-PEG allowed the enzyme and redox dual-responsive degradation as well as determination of a controlled release profile of cisplatin prodrugs (Scheme 1). To determine whether ICG/Poly(Pt) could respond to enzyme and reductive stimuli, the morphology and size changes of ICG/Poly(Pt) were examined upon incubation with or without papain and GSH by TEM and DLS analysis. Papain has activity similar to that of lysosomal cathepsin B, which can degrade GFLG peptide efficiently.57 No detectable aggregation or significant morphology and size change was observed upon incubation at pH 7.4 without papain (Figure 2A), whereas the polyprodrug nanoplatforms could degrade into small fragments after incubation at pH 5.5 with papain (Figure 2B). If the inhibitor of papain was present, the degradation was inhibited remarkably (Figure 2C). To mimic the endocytosis and intracellular trafficking pathway (Scheme 1), undergoing lysosomal trapping (acidic and enzymatic milieu) and subsequent lysosomal escape into the reductive cytosolic milieu (high GSH content), the dispersion of ICG/Poly(Pt) was first treated with papain at pH 5.5, followed by incubation with GSH at pH 7.4, and almost no residue was observed via TEM analysis (Figure 2D), indicating that the GFLG cleavable sites and ester bonds linked with cisplatin were possibly cleaved sequentially by papain under simulated acidic lysosomal conditions in the reductive cytosolic milieu. This result was further confirmed by a DLS test (Figure 2E). The cascade treatment with enzyme and GSH could degrade the nanosized aggregates of ICG/Poly(Pt) into almost molecularly dispersed species. Moreover, elevated GSH levels have been found in cisplatin-resistant cell lines,58 and high levels of

became wider because of the probable stacking effect of ICG in the hydrophobic core (Figure 1C). The absorbance intensity at 808 nm was around the maximum absorption peak, while the absorption of water and tissue was limited at 808 nm, making ICG/Poly(Pt) potentially superior for deep-penetrating light treatment. In addition, the stability of ICG/Poly(Pt) and free ICG in the dark was evaluated by their ultraviolet−visible (UV−vis) absorption (Figure 1D). The absorbance intensity of ICG/Poly(Pt) remained at ∼90% of the initial value at 1 week and ∼80% at 4 weeks, while that of free ICG decreased to ∼33 and ∼13% at 1 and 4 weeks, respectively. The color of the ICG solution disappeared almost completely at 4 weeks, suggesting severe ICG degradation, whereas the change in the color of the ICG/Poly(Pt) solution was inconspicuous. The polymeric envelope significantly isolated ICG from the surrounding robust environment; thus, the stability of ICG was improved greatly upon its being encapsulated by the cisplatin polyprodrug amphiphiles, PEG-b-P(Pt-co-GFLG)-b-PEG. Additionally, the strong near-infrared (NIR) absorption of ICG/Poly(Pt) implied that the polyprodrug nanoplatforms could potentially be employed as a novel PA agent.48 In vitro PA analysis was performed to evaluate this potency. ICG/Poly(Pt) and free ICG at various mass concentrations were embedded in agar gel cylinders to produce PA imaging phantoms on a multispectral optical tomography (MSOT) imaging system. As presented in Figure 1E, ICG/ Poly(Pt) and free ICG both exhibit mass concentrationdependent PA signals, and the sample of ICG/Poly(Pt) was much brighter than free ICG at the same ICG content, exhibiting a much stronger PA intensity. These results verified that ICG/Poly(Pt) would be an ideal contrast agent for PA imaging, exhibiting PA signals stronger than those of free ICG counterpart due to the potential photothermally enhanced PA signals and relatively high stability of ICG in ICG/Poly(Pt) (Figure 1F).49 Endogenous Enzyme-Responsive Degradation of Polyprodrug Nanoplatforms. Cathepsin B is a cysteine 3488


Article


Figure 2. Cascade enzymatic and reductive degradation as well as in vitro drug release properties of ICG/Poly(Pt). TEM images recorded for ICG/ Poly(Pt) after different treatments: (A) pH 7.4 without papain, which is like the physiological condition, (B) ICG/Poly(Pt) treated at pH 5.5 with papain, simulating the acidic and enzymatic condition of lysosomes, (C) ICG/Poly(Pt) treated at pH 5.5 with papain in the presence of a papain inhibitor, and (D) sample B followed by GSH treatment at pH 7.4, mimicking the lysosomal escape into the reductive cytosolic environment. (E) Hydrodynamic diameter distribution of ICG/Poly(Pt) after different treatments. (F) In vitro Pt release under different treatment conditions.

Figure 3. Cellular uptake and intracellular trafficking of ICG/Poly(Pt). CLSM images of A549 cells upon incubation with ICG/Poly(Pt) in the dark (left) or under 808 nm laser irradiation (right, 5 min, 1.0 W cm−2). The scale bar is 20 μm.

37 °C) suggested that the prepared drug-delivering vehicles would be stable in physiological blood circulation with reduced drug leakage and potential side effects.57 Cellular Uptake and Intracellular Trafficking. The cellular uptake and intracellular localization of ICG/Poly(Pt) against A549 lung cancer cells were observed at diverse incubation durations by confocal laser scanning microscopy (CLSM) imaging. Representative images suggested that ICG/Poly(Pt) nanoplatforms were quickly internalized by A549 cells upon incubation for 4 h (Figure 3). The merged fluorescence images from ICG and Lysotracker Green channels further demonstrated that the nanoparticles were mainly located in lysosomes at 4 h, exhibiting a co-localization ratio of >85%. When the incubation time was further extended to 12 h, the value was still >70%. This suggested the long-term trapping of nanoparticles in lysosomes, which was favorable for the enzymatic degradation of polyprodrug nanoplatforms into cisplatin prodrug species and free ICG release in lysosomes. Notably, the presence of ICG in the polyprodrug nanoplatforms allowed the use of 808 nm laser irradiation to examine

GSH provide favorable conditions for sufficient degradation of cisplatin prodrugs.59 On these bases, Poly(Pt) was expected to release cytotoxic parent cisplatin much faster in cancer cells than in normal cells. Furthermore, in vitro enzyme-responsive controlled release of Pt from ICG/Poly(Pt) was investigated under comparable conditions. As shown in Figure 2F, there was limited leakage of Pt from ICG/Poly(Pt) in the absence of papain at pH 7.4, whereas in the presence of papain, obvious Pt release could be observed during prolonged enzyme treatment at pH 5.5, which was attributed to the high activity of papain in an acidic environment. As a control, when the enzyme was pretreated with antipain hydrochloride, an efficient inhibitor of papain,60 there was much less Pt release under either acidic or neutral conditions. The combined pretreatment with papain and subsequent treatment with GSH could also produce obvious drug release. These results suggested that the cisplatin polyprodrug could specifically respond to papain to mediate controlled release of cisplatin prodrugs. Additionally, the high stability of the polyprodrug nanoplatforms in PBS (pH 7.4, 3489


Article


Figure 4. Photoinduced hyperthermia and ROS formation to disrupt lysosomes. (A) Temperature elevation and (B) singlet oxygen generation induced by ICG/Poly(Pt) upon laser irradiation. (C) Observation of lysosomal disruption for A549 cells after treatment with ICG/Poly(Pt) at various incubation times after laser irradiation (808 nm, 5 min, 1.0 W cm−2) using AO staining (p < 0.01). The scale bar is 40 μm.

generation, primarily singlet oxygen (1O2),65 which can cause serious damage to subcellular organelles.66−69 To validate the potential generation of ROS from ICG for photodynamic therapy, the dispersion of ICG/Poly(Pt) was irradiated with an 808 nm laser light. The ROS state was monitored by nonfluorescent 2,7-dichlorodihydrofluorescein (DCFH), which can be oxidized by ROS to generate fluorescent 2,7-dichlorofluorescein (DCF), and it was employed as a standard probe to detect ROS.70 Moreover, the presence of cellular peroxidases is favorable for the oxidation of DCFH to DCF. The fluorogenic intensity of DCF in the group of ICG/Poly(Pt) significantly increased, much higher than that of free ICG and other control groups along with the extended irradiation time (Figure 4B). This result indicated that ICG/Poly(Pt) had a ROS yield that was higher than that of free ICG,23,71 implying that it was highly preferable to damage lysosomal membranes via the well-accepted photochemical internalization effect of ROS.67 Encouraged by the results presented above, we further explored the disruption ability of ICG/Poly(Pt) on lysosomes under 808 nm laser irradiation by acridine orange (AO) staining. AO is a metachromatic fluorophore that can emit green fluorescence in the cytoplasm, while the green fluorescence transformed into red fluorescence upon it being trapped in lysosomes.72 In the absence of 808 nm laser irradiation, CLSM images showed that the acidic lysosomes in A549 cells treated with PBS for 8 h displayed remarkable red fluorescence for both PBS and ICG/ Poly(Pt), which agreed well with the lysosome co-localization analysis described above (Figure 4C). It demonstrated that the lysosomes remained intact and laser irradiation itself is inoffensive to lysosomes. However, upon laser irradiation, the red fluorescence from AO decreased remarkably in the presence of ICG/Poly(Pt) for 4 h and almost vanished at 8 h, indicating irreversible disruption of the lysosomal membrane. Thus, the hyperthermia and ROS produced by ICG/Poly(Pt) could dramatically destroy the lysosomes, which favored the translocation of released Pt(IV) prodrug fragments into cytoplasm for reduction-activated cascade chemotherapy to maximize the therapeutic efficiency (Scheme 1).72,73 In Vitro Cytotoxicity Evaluation. To evaluate the in vitro cytotoxicity of ICG/Poly(Pt), non-cisplatin-resistant A549 cells (Figure S5) and cisplatin-resistant A549/DDP cells (Figure 5)

the effect of NIR laser irradiation (Figure 1C). The laser irradiation based on ICG/Poly(Pt) obviously facilitated the outdiffusion of fluorescent pixels of Lysotracker and ICG, which even entered the cell nucleus, whereas only laser irradiation did not destroy the lysosomes of cancer cells in the absence of ICG/Poly(Pt) (Figure S4). We envisaged that the laserinduced hyperthermia and ROS based on light-absorbing ICG could disrupt ICG/Poly(Pt) into Pt(IV) prodrug fragments, which would be further reduced into cisplatin by GSH in cytoplasm, and further increased the level of accumulation of the drug inside cancer cells for the enhanced intracellular permeability and fluidity (Scheme 1).61 Then a great deal of Pt(II) complex would enter the cell nuclei and destroyed the DNA structure, eventually resulting in cell apoptosis. Moreover, the hyperthermia itself could cause severe cell damage, including loss of the nucleus, cell shrinkages, and coagulation. Taking these factors into consideration, we were not surprised that laser irradiation significantly increased the level of uptake of ICG/Poly(Pt) by A549 cells. Photothermally and Photodynamically Induced Lysosomal Disruption. The production potency of hyperthermia and ROS for ICG/Poly(Pt) was interrogated in detail. The aqueous dispersions of Poly(Pt), ICG/Poly(Pt), and free ICG were exposed to laser irradiation for different durations (808 nm, 1 W/cm2), and the dispersion temperature was monitored concurrently (Figure 4A). After a total irradiation time of 10 min, the absolute temperature increases of ICG/Poly(Pt) and free ICG were observed to be ∼17.8 and ∼15.5 °C, respectively, while that of the control sample (PBS) was only ∼2.6 °C. Notably, ICG/Poly(Pt) showed an increase in temperature that was larger than that of free ICG under laser irradiation, similar to the case of previously reported ICG-loaded lipid nanoparticles.62 The ICG encapsulated in Poly(Pt) had a condensed concentration that was higher than that of free ICG, and the excitation thermal radiation was also entrapped in the enclosure of nanoplatforms,61 resulting in a higher photothermal conversion efficiency and less heat dissipation upon laser irradiation.63 Notably, the photothermal effect of ICG-loaded nanoparticles was demonstrated to damage lysosomes to remarkably promote cytosolic delivery of cargoes.64 Upon irradiation, photosensitizers can transmit the absorbed energy to surrounding oxygen molecules, resulting in ROS 3490


Article


Figure 5. In vitro cytotoxicity of ICG/Poly(Pt). (A) Cell apoptosis and necrosis analysis of cisplatin-resistant A549/DDP cells with a flow cytometer after different treatments. (B) Statistical analysis of the apoptotic and necrotic cell percentage in panel A. (C) Cell viability of A549/DDP cells determined by a MTT assay (808 nm, 1 W/cm2, 5 min). (D) Quantitative evaluation of cell survival after incubation of samples with or without laser irradiation. (E) Fluorescence images of cathepsin B positive A549 cells and cathepsin B negative L-O2 cells upon incubation with ICG/Poly(Pt). Viable cells were stained with green Calcein-AM, while dead and late apoptotic cells were stained with red PI (p < 0.01). The scale bar is 40 μm.

ICG/Poly(Pt) cooperated well with light irradiation in killing cancer cells; thus, it was meaningful to improve the local therapeutic efficacy while reducing their systemic toxicity. The therapeutic effect of laser irradiation toward different groups was interrogated by MTT assays against two cell lines (Figure 5D and Figure S5D). ICG/Poly(Pt) and laser irradiation did lead to much stronger inhibition, while the effect in other groups was not obvious. Overall, in vitro results clearly pointed out the conclusion that NIR laser light-activated combination therapeutic effects of ICG/Poly(Pt) could induce the highest ratio of cancer cell death. To visually evaluate the in vitro therapeutic effect of ICG/Poly(Pt), a live/dead double assay was performed in the presence and absence of laser irradiation (Figure S5E). The group treated with ICG/Poly(Pt) and laser irradiation exhibited intense homogeneous red fluorescence, indicating severe cell death induced by cascade photo-chemotherapy.62,74 In contrast, other groups displayed much a low ratio of red to green fluorescent cells, suggesting unconsidered cell death. Furthermore, considering the presence of cathepsin B-responsive GFLG repeating peptides in the polyprodrug nanoplatforms, two kinds of cell lines with diverse expression levels of

were examined in parallel. Flow cytometry assays (FACS) were used for quantitative analysis of the cancer cell killing effect. The apoptotic and necrotic cells were stained with Alexa Flour 488-annexin V and propidium iodide (PI), respectively. Upon treatment with PBS, free ICG, cisplatin, Poly(Pt), and ICG/ Poly(Pt) with or without laser irradiation, low apoptotic and necrotic rates were observed for the cells treated with PBS and free ICG. The cells treated with ICG/Poly(Pt) and laser irradiation showed a remarkable increase in the level of cell apoptosis, much higher than those of cisplatin or Poly(Pt) (Figure 5A and Figure S5A). Statistical analysis suggested that ∼94.82 and ∼85.06% of A549/DDP and A549 cells were apoptotic, respectively, upon treatment with ICG/Poly(Pt) and laser irradiation (Figure 5B and Figure S5B), indicating that the synergistic therapy was mainly caused by cell apoptosis rather than necrosis. A similar trend was observed by MTT assays, because the proposed cascade photo-chemotherapy was responsible for the improved efficiency of ICG/Poly(Pt) (Figure 5C and Figure S5C). Moreover, in comparison with other treatment groups, the group treated with ICG/Poly(Pt) and laser irradiation showed much more significant cytotoxicity, which indicated that 3491


Article


Figure 6. Evaluation of combating cisplatin resistance by ICG/Poly(Pt). Cell viability of (A) cisplatin-sensitive A549 cells and (B) cisplatin-resistant A549/DDP cells against ICG/Poly(Pt) and cisplatin upon incubation for 24 h with or without laser irradiation. (C) IC50 values determined for cisplatin and ICG/Poly(Pt) against two cell lines. (D) Representative images of A549/DDP MCCS upon their treatment with PBS, cisplatin, and ICG/Poly(Pt) with laser irradiation (808 nm, 1 W/cm2, 5 min). (E) Growth statistics of A549/DDP MCCS after different treatments (n = 3).

cathepsin B were evaluated upon incubation with ICG/ Poly(Pt) and laser irradiation. A549 lung cancer cells, which are known to express a high level of cathepsin B, were treated with 100 μM ICG/Poly(Pt) for 36 h with or without laser irradiation before being stained with Calcein-AM/PI. CLSM images showed that red fluorescent cells indicated that some cells were killed after incubation with ICG/Poly(Pt), whereas much more extensive cell death was observed for ICG/Poly(Pt) with laser irradiation. On the other hand, control studies using L-O2 cells, a normal human hepatic cell line expressing a low level of cathepsin B, showed only limited cell death with the same treatments as described above (Figure 5E). These results suggested that the cleavage sites in the backbone of cisplatin polyprodrug amphiphiles endowed ICG/Poly(Pt) with a higher cytotoxicity toward cathepsin B-overexpressing cancer cells, which is beneficial for enhancing the therapeutic efficacy with reduced side effects. Inhibition of Drug Resistance. To verify whether the polyprodrug nanoplatforms could overcome the drug resistance of cancer cells, the in vitro cytotoxicity of cisplatin and ICG/ Poly(Pt) toward A549 cells and A549/DDP cells was further examined by MTT assays (Figure 6A,B). The IC50 values of cisplatin for A549 and A549/DDP cells were determined to be ∼24.7 and ∼57.9 μM, respectively, exhibiting a resistance index of ∼2.3 (Figure 6C). In contrast, ICG/Poly(Pt) and irradiation gave IC50 values of ∼21.6 and ∼42.2 μM toward A549 and A549/DDP cells, respectively, and the resistance index was just ∼1.9, which indicated that the resistance of A549/DDP cells to ICG/Poly(Pt) was lower than that to cisplatin. In other words, ICG/Poly(Pt) nanoplatforms were able to overcome the cisplatin resistance to some extent against A549/DDP lung cancer cells. In addition, to further evaluate the inhibitory effect of ICG/ Poly(Pt) against cisplatin-resistant A549/DDP cells, multicellular cancer spheroids (MCCS) were incubated with cisplatin or ICG/ Poly(Pt) with laser irradiation for 24 h at a Pt content of 100 μM. The culture medium was removed and refreshed, and the MCCS were allowed to grow in fresh medium for an additional 7 days.

Representative images of the MCCS displayed no significant inhibitory effect for the group treated with cisplatin, and the diameter of the MCCS increased from ∼250 to ∼340 μm over time because of the cisplatin resistance of A549/DDP cells (Figure 6D,E). In sharp contrast, the group of ICG/Poly(Pt) with laser irradiation exhibited distinct inhibition potency against the MCCS, revealing a decrease in the size of MCCS from ∼240 to ∼110 μm. The improved therapeutic efficacy of ICG/Poly(Pt) with laser irradiation could be ascribed to the highly efficient cascade photochemotherapeutic damage.75 These results demonstrated that ICG/Poly(Pt) could effectively overcome the drug resistance of cancer cells by increasing the effective drug delivery and specific activation in cancer cells based on the proposed cascade photo-chemotherapy. In Vivo Dual-Mode Imaging. Before in vivo evaluation, the serum stability of ICG/Poly(Pt) was examined upon incubation with 40 mg/mL BSA for 48 h, displaying an unobvious size change and remarkable simulated physiological stability, which was favorable for prolonged blood circulation (Figure S3). Upon intravenous injection, an important issue is whether ICG/Poly(Pt) could accumulate in xenograft cancer efficiently. The inherent fluorescence of ICG was beneficial for the observation and evaluation of biodistribution. The fluorescent images of mice were recorded upon intravenous injection with ICG/ Poly(Pt) and free ICG (Figure 7A). It was observed that the fluorescence signal of cancers appeared slightly at 1 h upon intravenous injection with ICG/Poly(Pt). After 4 h, the cancer region became very distinguishable from other tissues, and the fluorescence contrast of cancer was still robust 12 and 24 h post-injection, indicating effective accumulation and prolonged retention in cancer sites. Whereas for free ICG, a fast spread of fluorescence signals in the whole body was detected, and the fluorescence faded quickly with little contrast in the cancer region during the whole observation process because of the fast clearance of ICG from the body. Ex vivo fluorescence imaging and semiquantitative analysis further confirmed the obvious fluorescence signal in the cancer sites of mice treated with 3492


Article


Figure 7. In vivo biodistribution and dual-mode fluorescence and PA imaging. (A) In vivo fluorescent imaging of A549/DDP cancerous mice upon treatment with free ICG and ICG/Poly(Pt) at different time intervals. (B) Typical fluorescent images and (C) quantification of major organs and cancers collected from mice treated with free ICG and ICG/Poly(Pt) 24 h post-injection (p < 0.05). (D) Blood circulation profiles of ICG/Poly(Pt) and free ICG. (E) In vivo PA imaging of cancer sites for A549/DDP cancerous mice upon intravenous injection. (F) Relative PA signal intensities at the cancer sites in panel E.

ICG/Poly(Pt), while for the mouse receiving free ICG, the fluorescence signal was mainly located in the liver (Figure 7B,C). By quantitatively measuring the fluorescence signal in the blood samples, we calculated the blood circulation half-life of ICG/ Poly(Pt) to be ∼4.2 h, remarkably longer than that of free ICG (∼0.08 h) (Figure 7D). It accounted for the gradual accumulation of ICG/Poly(Pt) within cancers through the EPR effect. On the other hand, the dynamic accumulation and metabolism of polyprodrug nanoplatforms in cancers were monitored by PA imaging (Figure 7E). Before injection of ICG/Poly(Pt), a weak PA background signal was obtained, which corresponded to the endogenous light-absorbing hemoglobin molecules in blood. However, upon intravenous injection with ICG/Poly(Pt), the PA images showed a gradually enhanced signal at the cancer sites, indicating that ICG/Poly(Pt) accumulated in cancer cells via the well-accepted EPR effect. Along with the time extension, the nanoparticles diffused throughout the cancer tissue. A maximum signal was obtained 3 h post-injection, up to ∼3.2-fold compared with the background; thus, the overall contrast of the cancer region was enhanced significantly. Subsequently, the weakened PA signal revealed the metabolism of nanoparticles. In contrast, the PA signal from the free ICG group was much weaker and its intensity decreased rapidly, which further confirmed that Poly(Pt) could prevent the degradation of ICG and enhance its PA signal intensity. The PA images suggested that

ICG/Poly(Pt) nanoplatforms could preferentially accumulate at the cancer sites. At 24 h post-injection, the PA intensity of the ICG/Poly(Pt) group was determined to be ∼3-fold greater than that of free ICG (Figure 7F). The proper size and high stability of ICG/Poly(Pt) nanoplatforms endowed them with superior penetration through the defective blood vessels and subsequent retention in cancer sites. The enhanced contrast of the cancer region would facilitate the guidance of subsequent therapeutic treatments. In Vivo Cancer Inhibition by the Cisplatin Polyprodrug Nanoplatforms. To demonstrate the in vivo hyperthermia induced by ICG/Poly(Pt), the temperature of the cancer sites was recorded upon laser irradiation 24 h postinjection (Figure 8A). The photothermal images revealed that ICG/Poly(Pt) caused a significant temperature increase (ΔT) of ∼26 °C in the cancer region being irradiated (808 nm, 1 W/cm3), whereas other groups treated with PBS with laser, free ICG with laser, or ICG/Poly(Pt) with interval irradiation had much lower ΔT values [∼12 °C (Figure 8B)]. These results demonstrated that ICG/Poly(Pt) dispersions were able to generate potent hyperthermia (>45 °C) to induce cancer destruction in vivo, benefiting from the enhanced cancer accumulation and the deep penetration of 808 nm light. Notably, PA imaging indicated that the level of accumulation of ICG/Poly(Pt) in the tumor was most significant ∼3−6 h post-injection, which was 3493


Article


Figure 8. In vivo inhibition evaluation of ICG/Poly(Pt) on resistant cancers. (A) Thermal images of A549/DDP cancerous mice under 808 nm laser irradiation (1 W/cm2, 5 min) upon intravenous injection with PBS, free ICG, and ICG/Poly(Pt). (B) Quantitative analysis of the temperature changes of cancer sites. (C) Growth curves of A549/DDP cancer in different treatment groups. (D) Survival curves of mice after cascade photochemotherapy. (E) Typical H&E-stained slices of cancer tissues collected from different treatment groups (p < 0.05). The scale bar is 200 μm.

∼2.4-fold greater than the tumor PA intensity at 3 h versus 24 h post-injection (Figure 7F). However, the photothermal imaging of tumor-bearing mice 24 h post-injection indicated the obvious temperature increase at tumor sites, which was efficient enough for tumor inhibition (Figure 8A,B); thus, these results demonstrated that the accumulation of ICG/Poly(Pt) in the tumor was significant for long-term tumor treatments, even though laser irradiation was performed 24 h post-injection. Collectively, a high therapeutic efficiency would be predictable if the laser irradiation were performed 3−6 h post-injection. Finally, in vivo cancer inhibition was examined upon intravenous injection with ICG/Poly(Pt) into A549/DDP cancerous mice, and then the cancers were irradiated at 1.0 W cm−2 for 5 min 24 h post-injection. It was found that the cancer volumes increased by ∼6-fold within 21 days for the groups treated with PBS, laser irradiation alone, and even ICG with laser irradiation, indicating limited inhibition of cancer growth was caused by these treatments (Figure 8C). The groups treated with cisplatin and Poly(Pt) resulted in the delay of cancer growth to some extent; however, the cancers still grew by ∼3.8-fold within 21 days. In contrast, the group treated with ICG/Poly(Pt) and subsequent laser irradiation exhibited obvious cancer inhibition, and some cancers were even completely eradicated (Figure S6A,B). The irradiation significantly amplified the cancer inhibition of ICG/ Poly(Pt) based on the photo-chemotherapy. First, the encapsulated ICG induced local hyperthermia and ROS generation under NIR laser irradiation, which could cause severe damage to

cancer cells by the robust phototherapy. Furthermore, the photothermal and photodynamic process could damage lysosomes and accelerate the lysosomal escape of cisplatin prodrug moieties from lysosomes into the cytosolic milieu, which was further activated for subsequent cascade chemotherapy. Thus, the polyprodrug nanoplatforms could amplify the therapeutic efficacy dramatically for cooperative photo-chemotherapy. Furthermore, the mice in the group treated with ICG/Poly(Pt) and laser irradiation had the longest median survival time of 60 days; the mice in other groups exhibited a significantly lower survival ratio (Figure 8D). Finally, H&E-stained cancer sections confirmed the efficient apoptosis and necrosis of cancer cells (Figure 8E). The body weight of mice did not significantly change in different treatment groups, suggesting unobvious systematic toxicity (Figure S6C). In addition, there were no obvious pathological changes of the main organs in the treatment groups (Figure S7), which further indicated that current polyprodrug nanoplatforms had minimal in vivo side effects.

■

CONCLUSIONS In summary, cisplatin polyprodrug nanoplatforms, ICG/Poly(Pt), have been readily developed for cascade photo-chemotherapy to combat cisplatin-resistant cancers. The stability of ICG in ICG/Poly(Pt) was remarkably improved by the encapsulation and protection of cisplatin polyprodrug amphiphiles, PEG-bP(Pt-co-GFLG)-b-PEG, which were tethered with repeating enzyme-degradable GFLG peptides and cisplatin prodrug units. 3494


Article


cells were exposed to the laser at an energy density of 1 W/cm2 for 5 min. After being stained with Lysotracker Green for an additional 0.5 h, the cells were washed with PBS and imaged immediately. Lysosomal Disruption. To observe the disruption of lysosomes, AO was used as an intracellular indicator of acidic organelle integrity in A549 cells. A549 cells on a confocal dish were treated with 100 μM ICG/Poly(Pt) for 3 h. Then the cells were washed with PBS and incubated in fresh medium, followed by a 5 min irradiation at 808 nm (1.0 W cm−2). After being incubated for 4 and 8 h, the cells were washed and stained with 3 μM AO for 15 min. Finally, the cells were washed three times with PBS before being subjected to CLSM observation. In Vitro Inhibition of Cancer Cells. A549 cells were plated onto a glass bottom Petri dish at a density of 30000 cells/plate in 0.5 mL of culture medium for 12 h. Subsequently, cells in two groups were treated with PBS, free ICG, Poly(Pt), and ICG/Poly(Pt) (100 μM). Eight hours later, one group was subjected to laser irradiation (808 nm, 1 W/cm2, 5 min). After another 24 h incubation, cells were washed with PBS and stained with Calcein-AM and PI. Cell apoptosis was observed via CLSM ∼0.5 h later. In normal live cells, phosphatidylserine (PS) is located on the cytoplasmic surface of the cell membrane. However, in apoptotic cells, PS is translocated from the inner to the outer leaflet of the plasma membrane, thus exposing PS to the external cellular environment.77 The human anticoagulant, Annexin V, is a 35−36 kDa Ca2+-dependent phospholipid-binding protein that has a high affinity for PS.78 FITClabeled Annexin V can identify apoptotic cells by binding to PS exposed on the outer leaflet.79 Furthermore, propidium iodide (PI) cannot permeate live cells and apoptotic cells but stains dead cells with red fluorescence, binding tightly to the nucleic acids in the cell. After being stained with Annexin V and PI, apoptotic cells show green fluorescence, dead cells show red and green fluorescence, and live cells show little or no fluorescence. These populations can easily be distinguished using a flow cytometer.80 Herein, the cytotoxicity was also evaluated by the quantification of apoptotic A549 cells and A549/DDP cells. Cells were seeded into six-well plates at a density of 1 × 105 cells/well and treated with different samples in a similar profile; then the cells were harvested, and the Alexa Fluor 488 Annexin V/PI Cell Apoptosis Kit was used to detect and quantify the apoptosis with the BD Accuri C6 flow cytometer. Annexin V positive, PI negative cells were scored as apoptotic. Doubly stained cells were considered as necrotic/late apoptotic cells. Ten thousand events were collected for each sample, and cells without any treatment were used as a control. In Vivo Imaging and Biodistribution Analysis. Cancerous mice were established by subcutaneous inoculation of an A549/DDP cell suspension (5 × 106 cells/mouse) into the right flank region of 4-week-old athymic female nude mice. The A549/DDP cancerous mice were randomly divided into two groups. Mice in groups 1 and 2 were intravenously injected with free ICG and ICG/Poly(Pt) (2 mg of ICG/kg), respectively. Photoacoustic imaging was performed 0, 3, 6, 12, and 24 h post-injection. The incident energy density on the cancer surface was set to ∼10 mJ/cm2. Fluorescence semiquantitative analysis of ICG was conducted at different time points after injection using the ex/in vivo imaging system (CRi maestro) with a 704 nm excitation wavelength and a 735 nm filter to collect the fluorescence signals from ICG. The mice after injection at 24 h were sacrificed, and the heart, liver, spleen, lung, kidneys, and cancerous tissue were collected for imaging and semiquantitative biodistribution analysis. For in vivo photothermal imaging, ICG/Poly(Pt) (2 mg of ICG/kg) or free ICG (2 mg of ICG/kg) was intravenously injected into the A549/DDP cancerous mice. Thermal imaging was performed by an infrared thermal imaging camera when the cancer cells were exposed to an 808 nm laser with a power density of 1 W/cm2. Blood Circulation. Blood circulation was measured by drawing 10 μL blood from the tail vein of the A549/DDP cancerous mice after intravenous injection of free ICG and ICG/Poly(Pt) (2 mg of ICG/kg). The blood samples were dissolved in 180 μL of lysis buffer (1% sodium dodecyl sulfate, 1% Triton X-100, and 40 mM Tris-acetate),

Considerable accumulation of ICG/Poly(Pt) in cancerous cells was demonstrated, which was potentially favorable for longterm light treatments in extended cancer therapy. ICG/Poly(Pt) could be efficiently degraded into cisplatin prodrugs in lysosomes by cathepsin B that was overexpressed in many cancer cells. Furthermore, upon 808 nm laser irradiation, the encapsulated ICG could induce local hyperthermia as well as ROS formation, which not only caused photothermal and photodynamic injury but also destroyed the lysosomes to accelerate the cytosolic delivery of cisplatin prodrugs. The prodrug species were subsequently reduced to toxic cisplatin under reductive cytosolic conditions, thus further activating the cascade chemotherapy. Both in vitro and in vivo evaluations demonstrated that ICG/ Poly(Pt)-based cascade photo-chemotherapy could amplify the therapeutic efficacy and had a superior anticancer effect compared with the effect of an individual treatment. As a proof of concept, it was systematically verified to combat the cisplatinresistant A549/DDP cancers. The integrated polyprodrug nanoplatforms are promising for the treatment of persistent cancers for future intelligent cancer theranostics.

■

MATERIALS AND METHODS

Synthesis of Enzyme-Degradable Cisplatin Polyprodrug Amphiphiles. Platinum complex Pt(NH3)2Cl2(OOCCH2CH2COOH (84.9 mg, 0.16 mmol),76 EDC (71.39 mg, 0.397 mmol), and DMAP (8.09 mg, 0.01324 mmol) were dissolved in dried dimethylformamide (DMF) and stirred at 20 °C for 5 h. Then the GFLG peptide (57.55 mg, 0.78 mmol) in 500 μL of dried DMF was dropped into the mixture and the mixture stirred at 40 °C for 24 h. Subsequently, PEG (55.62 mg, 0.0278 mmol, molecular weight of 2000) in 400 μL of dried methylbenzene was dropped into the mixture, and stirring was continued for 48 h. The clear yellow solution was added with an excess of ether, and the precipitate was isolated and washed with ether repeatedly. The obtained solid was dissolved and precipitated three times to afford a light yellow solid, PEG-b-P(Pt-co-GFLG)-b-PEG. 1H NMR (DMSO-d6, 400 MHz): δ 1.49 [t, −CH(CH3)2], 4.25 (t, −CH2−), 3.81 (t, −CH2−), 7.14−8.32 (t, −CH2−), 0.6−1.01 (d, −CH3), 1.70 (s, −NH3), 9.04 (s, −NH−). The loading content of cisplatin was determined to be ∼24.7 wt % by ICP-MS analysis of the Pt content. All characteristic resonance peaks were found in the 1H NMR spectrum of PEG-b-P(Ptco-GFLG)-b-PEG (Figure S1). In Vitro Drug Release. Because the enzyme papain has activity similar to that of lysosomal cathepsin B, we studied the release of the drug from an ICG/Poly(Pt) dispersion in the presence or absence of papain. The concentration of papain in McIlvaine’s buffer [50 mM citrate/0.1 M phosphate and 2 mM EDTA (pH 5.4)] was determined by UV−vis spectroscopy at 480 nm. Glutathione in McIlvaine’s buffer (10 mM) was mixed with the enzyme and preincubated for 5 min at 37 °C. The enzyme-responsive ICG/Poly(Pt) nanoplatforms were then added and reacted with papain (50 × 10−9 M) at different pH values (5.5 and 7.4) in the presence or absence of a papain inhibitor (50 × 10−6 M). Upon treatment with the enzyme with various durations (e.g., 6, 12, 18, 24, and 36 h), the supernatant was collected to determine the content of released Pt by ICP-MS. Detection of Reactive Oxygen Species. DCFH was employed to evaluate ROS generation. ICG/Poly(Pt), Poly(Pt), and free ICG were mixed with DCFH (20 μg/mL) in water (water as a control). Then the solutions were irradiated with a NIR laser (808 nm, 1.0 W cm−2); 200 μL of the solution was removed every minute to measure the fluorescence of DCF. Cellular Uptake and Intracellular Trafficking. Cancer cells were seeded on a glass bottom Petri dish at a density of 10000 cells/plate in 0.5 mL of culture medium for 12 h before being exposed to the designed treatments and then examined with CLSM. Lysotracker Green was used to label lysosomes, and ICG was used to label the ICG/Poly(Pt). Free ICG and ICG/Poly(Pt) (100 μM) were added to the culture milieu. After being incubated for 3.5, 7.5, and 11.5 h, the 3495


Article

Chemistry of Materials and the fluorescence intensity was recorded (excitation wavelength of 785 nm, emission wavelength of 805 nm; n = 3). In Vivo Anticancer Efficacy. A549/DDP cancerous mice were randomly divided into six groups (n = 5): (1) laser irradiation only, (2) PBS, (3) cisplatin solution, (4) free ICG with laser irradiation, (5) Poly(Pt), and (6) ICG/Poly(Pt) with laser irradiation. The applied dosages were set to 5 mg of cisplatin/kg and 2 mg of ICG/kg. For groups 2−6, intravenous injection was performed every 3 days, with a total of five injections. Cancer volumes and body weights were monitored. Cancer volumes were calculated as (width2 × length)/2. All mice were sacrificed 21 days after the first treatment, and the excised cancerous tissue was weighed. The survival of the mice was monitored and recorded accordingly. Examination of Histology. Three weeks after the chemophototherapy, the mice were sacrificed and major organs (heart, liver, spleen, lung, and kidney) and cancerous tissues were excised, fixed in a 4% paraformaldehyde solution, and then processed routinely into paraffin. The sliced tissues were stained with hematoxylin and eosin (H&E) and examined with an inverted florescence microscope system (Nikon Ti-S).

■

Theranostic Nanoplatform for Bimodal Imaging Guided Combination Cancer Therapy. Adv. Funct. Mater. 2016, 26, 2207−2217. (4) Min, Y.; Mao, C. Q.; Chen, S.; Ma, G.; Wang, J.; Liu, Y. Combating the drug resistance of cisplatin using a platinum prodrug based delivery system. Angew. Chem., Int. Ed. 2012, 51, 6742−6747. (5) Ang, W. H.; Khalaila, I.; Allardyce, C. S.; Juillerat-Jeanneret, L.; Dyson, P. J. Rational Design of Platinum(IV) Compounds to Overcome Glutathione-S-Transferase Mediated Drug Resistance. J. Am. Chem. Soc. 2005, 127, 1382−1383. (6) Lee, R. F. S.; Riedel, T.; Escrig, S.; Maclachlan, C.; Knott, G. W.; Davey, C. A.; Johnsson, K.; Meibom, A.; Dyson, P. J. Differences in cisplatin distribution in sensitive and resistant ovarian cancer cells: a TEM/NanoSIMS study. Metallomics 2017, 9, 1413−1420. (7) Burger, H.; Loos, W. J.; Eechoute, K.; Verweij, J.; Mathijssen, R. H. J.; Wiemer, E. A. C. Drug transporters of platinum-based anticancer agents and their clinical significance. Drug Resist. Updates 2011, 14, 22−34. (8) Koberle, B.; Tomicic, M. T.; Usanova, S.; Kaina, B. Cisplatin resistance: preclinical findings and clinical implications. Biochim. Biophys. Acta, Rev. Cancer 2010, 1806, 172−182. (9) Mackiewicz, N.; Nicolas, J.; Handke, N.; Noiray, M.; Mougin, J.; Daveu, C.; Lakkireddy, H. R.; Bazile, D.; Couvreur, P. Precise Engineering of Multifunctional PEGylated Polyester Nanoparticles for Cancer Cell Targeting and Imaging. Chem. Mater. 2014, 26, 1834− 1847. (10) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-responsive nanocarriers for drug delivery. Nat. Mater. 2013, 12, 991−1003. (11) Xiao, H. H.; Song, H. Q.; Zhang, Y.; Qi, R. G.; Wang, R.; Xie, Z. G.; Huang, Y. B.; Li, Y. X.; Wu, Y.; Jing, X. B. The use of polymeric platinum(IV) prodrugs to deliver multinuclear platinum(II) drugs with reduced systemic toxicity and enhanced antitumor efficacy. Biomaterials 2012, 33, 8657−8669. (12) Xiao, H. H.; Noble, G. T.; Stefanick, J. F.; Qi, R. G.; Kiziltepe, T.; Jing, X. B.; Bilgicer, B. Photosensitive Pt(IV)-azide prodrug-loaded nanoparticles exhibit controlled drug release and enhanced efficacy in vivo. J. Controlled Release 2014, 173, 11−17. (13) Yang, Q.; Qi, R. G.; Cai, J.; Kang, X.; Sun, S.; Xiao, H. H.; Jing, X. B.; Li, W. L.; Wang, Z. H. Biodegradable polymer-platinum drug conjugates to overcome platinum drug resistance. RSC Adv. 2015, 5, 83343−83349. (14) Xiao, H. H.; Stefanick, J. F.; Jia, X. Y.; Jing, X. B.; Kiziltepe, T.; Zhang, Y.; Bilgicer, B. Micellar nanoparticle formation via electrostatic interactions for delivering multinuclear platinum(II) drugs. Chem. Commun. 2013, 49, 4809−4811. (15) Xiao, H. H.; Song, H. Q.; Yang, Q.; Cai, H. D.; Qi, R. G.; Yan, L. S.; Liu, S.; Zheng, Y. H.; Huang, Y. B.; Liu, T. J.; Jing, X. B. A prodrug strategy to deliver cisplatin(IV) and paclitaxel in nanomicelles to improve efficacy and tolerance. Biomaterials 2012, 33, 6507−6519. (16) Du, R.; Xiao, H. H.; Guo, G. N.; Jiang, B.; Yan, X.; Li, W. L.; Yang, X. G.; Zhang, Y.; Li, Y. X.; Jing, X. B. Nanoparticle delivery of photosensitive Pt(IV) drugs for circumventing cisplatin cellular pathway and on-demand drug release. Colloids Surf., B 2014, 123, 734−741. (17) Min, Y. Z.; Li, J. M.; Liu, F.; Yeow, E. K. L.; Xing, B. G. NearInfrared Light-Mediated Photoactivation of a Platinum Antitumor Prodrug and Simultaneous Cellular Apoptosis Imaging by Upconversion-Luminescent Nanoparticles. Angew. Chem., Int. Ed. 2014, 53, 1012−1016. (18) Miller, M. A.; Zheng, Y. R.; Gadde, S.; Pfirschke, C.; Zope, H.; Engblom, C.; Kohler, R. H.; Iwamoto, Y.; Yang, K. S.; Askevold, B.; Kolishetti, N.; Pittet, M.; Lippard, S. J.; Farokhzad, O. C.; Weissleder, R. Tumour-associated macrophages act as a slow-release reservoir of nano-therapeutic Pt(IV) pro-drug. Nat. Commun. 2015, 6, 8692. (19) Johnstone, T. C.; Suntharalingam, K.; Lippard, S. J. The Next Generation of Platinum Drugs: Targeted Pt(II) Agents, Nanoparticle Delivery, and Pt(IV) Prodrugs. Chem. Rev. 2016, 116, 3436−3486. (20) Zheng, Y. R.; Suntharalingam, K.; Johnstone, T. C.; Lippard, S. J. Encapsulation of Pt(IV) prodrugs within a Pt(II) cage for drug delivery. Chem. Sci. 2015, 6, 1189−1193.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.8b01149. Additional expermental details, synthetic routes (Scheme S1), 1H NMR analysis, DLS analysis of storage stability and serum stability, CLSM images for evaluating the effect of NIR irradiation only, cell viability test toward non-cisplatin-resistant A549 cells, in vivo evaluation, and H&E staining images of major organs (Figures S1−S7) (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Da Xing: 0000-0002-5098-0487 Xianglong Hu: 0000-0001-9202-1543 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the National Natural Scientific Foundation of China (NSFC, 21674040 and 81630046), the Natural Science Foundation for Distinguished Young Scholars of Guangdong Province (2016A030306013), the Guangdong Program for Support of Top-notch Young Professionals (2015TQ01R604), the Scientific Research Projects of Guangzhou (201607010328 and 201805010002), and the Science and Technology Planning Project of Guangdong Province (2015B020233016 and 2014B020215003).

■

REFERENCES

(1) Jamieson, E. R.; Lippard, S. J. Structure, Recognition, and Processing of Cisplatin-DNA Adducts. Chem. Rev. 1999, 99, 2467− 2498. (2) Lebwohl, D.; Canetta, R. Clinical development of platinum complexes in cancer therapy: an historical perspective and an update. Eur. J. Cancer 1998, 34, 1522−1534. (3) Feng, L.; Gao, M.; Tao, D.; Chen, Q.; Wang, H.; Dong, Z.; Chen, M.; Liu, Z. Cisplatin-Prodrug-Constructed Liposomes as a Versatile 3496


Article

Chemistry of Materials (21) Zheng, Y. R.; Suntharalingam, K.; Johnstone, T. C.; Yoo, H.; Lin, W.; Brooks, J. G.; Lippard, S. J. Pt(IV) Prodrugs Designed to Bind Non-Covalently to Human Serum Albumin for Drug Delivery. J. Am. Chem. Soc. 2014, 136, 8790−8798. (22) Zhang, Z. Y.; Wu, Y. J.; Kuang, G. Z.; Liu, S.; Zhou, D. F.; Chen, X. S.; Jing, X. B.; Huang, Y. B. Pt(IV) prodrug-backboned micelle and DCA loaded nanofibers for enhanced local cancer treatment. J. Mater. Chem. B 2017, 5, 2115−2125. (23) Zhao, P.; Zheng, M.; Luo, Z.; Gong, P.; Gao, G.; Sheng, Z.; Zheng, C.; Ma, Y.; Cai, L. NIR-driven Smart Theranostic Nanomedicine for On-demand Drug Release and Synergistic Antitumour Therapy. Sci. Rep. 2015, 5, 14258. (24) Kim, H.; Lee, D.; Kim, J.; Kim, T. I.; Kim, W. J. Photothermally triggered cytosolic drug delivery via endosome disruption using a functionalized reduced graphene oxide. ACS Nano 2013, 7, 6735− 6746. (25) Garcia-Fernandez, L.; Herbivo, C.; Arranz, V. S.; Warther, D.; Donato, L.; Specht, A.; del Campo, A. Dual photosensitive polymers with wavelength-selective photoresponse. Adv. Mater. 2014, 26, 5012− 5017. (26) Oliveira, H.; Perez-Andres, E.; Thevenot, J.; Sandre, O.; Berra, E.; Lecommandoux, S. Magnetic field triggered drug release from polymersomes for cancer therapeutics. J. Controlled Release 2013, 169, 165−170. (27) Bhirde, A. A.; Patel, V.; Gavard, J.; Zhang, G.; Sousa, A. A.; Masedunskas, A.; Leapman, R. D.; Weigert, R.; Gutkind, J. S.; Rusling, J. F. Targeted killing of cancer cells in vivo and in vitro with EGFdirected carbon nanotube-based drug delivery. ACS Nano 2009, 3, 307−316. (28) Song, G. S.; Chao, Y.; Chen, Y. Y.; Liang, C.; Yi, X.; Yang, G. B.; Yang, K.; Cheng, L.; Zhang, Q.; Liu, Z. All-in-One Theranostic Nanoplatform Based on Hollow TaOx for Chelator-Free Labeling Imaging, Drug Delivery, and Synergistically Enhanced Radiotherapy. Adv. Funct. Mater. 2016, 26, 8243−8254. (29) Chen, Y. Y.; Song, G. S.; Dong, Z. L.; Yi, X.; Chao, Y.; Liang, C.; Yang, K.; Cheng, L.; Liu, Z. Drug-Loaded Mesoporous Tantalum Oxide Nanoparticles for Enhanced Synergetic Chemoradiotherapy with Reduced Systemic Toxicity. Small 2017, 13, 1602869. (30) Lane, D. Designer combination therapy for cancer. Nat. Biotechnol. 2006, 24, 163−164. (31) Sun, T. M.; Du, J. Z.; Yao, Y. D.; Mao, C. Q.; Dou, S.; Huang, S. Y.; Zhang, P. Z.; Leong, K. W.; Song, E. W.; Wang, J. Simultaneous delivery of siRNA and paclitaxel via a ″two-in-one″ micelleplex promotes synergistic tumor suppression. ACS Nano 2011, 5, 1483− 1494. (32) Cong, Y.; Xiao, H.; Xiong, H.; Wang, Z.; Ding, J.; Li, C.; Chen, X.; Liang, X. J.; Zhou, D.; Huang, Y. Dual Drug Backboned Shattering Polymeric Theranostic Nanomedicine for Synergistic Eradication of Patient-Derived Lung Cancer. Adv. Mater. 2018, 30, 1706220. (33) Hu, X. L.; Hu, J. M.; Tian, J.; Ge, Z. S.; Zhang, G. Y.; Luo, K. F.; Liu, S. Y. Polyprodrug Amphiphiles: Hierarchical Assemblies for Shape-Regulated Cellular Internalization, Trafficking, and Drug Delivery. J. Am. Chem. Soc. 2013, 135, 17617−17629. (34) Hu, X. L.; Liu, G. H.; Li, Y.; Wang, X. R.; Liu, S. Y. CellPenetrating Hyperbranched Polyprodrug Amphiphiles for Synergistic Reductive Milieu-Triggered Drug Release and Enhanced Magnetic Resonance Signals. J. Am. Chem. Soc. 2015, 137, 362−368. (35) Hu, X. L.; Zhai, S. D.; Liu, G. H.; Xing, D.; Liang, H. J.; Liu, S. Y. Concurrent Drug Unplugging and Permeabilization of PolyprodrugGated Crosslinked Vesicles for Cancer Combination Chemotherapy. Adv. Mater. 2018, 30, 1706307. (36) Asafo-Adjei, T. A.; Dziubla, T. D.; Puleo, D. A. Synthesis and Characterization of a Simvastatin Polyprodrug for Potential Tissue Regeneration Applications. Tissue Eng., Part A 2014, 20, S92. (37) Yuan, Y. Y.; Liu, J.; Liu, B. Conjugated-Polyelectrolyte-Based Polyprodrug: Targeted and Image-Guided Photodynamic and Chemotherapy with On-Demand Drug Release upon Irradiation with a Single Light Source. Angew. Chem., Int. Ed. 2014, 53, 7163−7168.

(38) 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 nanoplatform for targeted cancer therapy. Biomaterials 2016, 76, 238− 249. (39) Chen, D.; Huang, Y.; Xu, S. T.; Jiang, H. Y.; Wu, J. L.; Jin, X.; Zhu, X. Y. Self-Assembled Polyprodrug Amphiphile for Subcutaneous Xenograft Tumor Inhibition with Prolonged Acting Time In Vivo. Macromol. Biosci. 2017, 17, 1700174. (40) Guo, Y. F.; Hao, C. Y.; Wang, X. K.; Zhao, Y. N.; Han, M. H.; Wang, M. C.; Wang, X. T. Well-defined podophyllotoxin polyprodrug brushes: preparation via RAFT polymerization and evaluation as drug carriers. Polym. Chem. 2017, 8, 901−909. (41) Hu, X. L.; Liu, S. Y. Charge-conversional polyprodrug amphiphiles for intracellular dual-responsive drug delivery. J. Controlled Release 2017, 259, e144. (42) Pang, Q.; Zheng, X. W.; Luo, Y.; Ma, L.; Gao, C. Y. A photocleavable polyprodrug-loaded wound dressing with UV-responsive antibacterial property. J. Mater. Chem. B 2017, 5, 8975−8982. (43) Sun, P.; Chen, D.; Deng, H. P.; Wang, N.; Huang, P.; Jin, X.; Zhu, X. Y. ″Bottom-up″ Construction of Multi-Polyprodrug-Arm Hyperbranched Amphiphiles for Cancer Therapy. Bioconjugate Chem. 2017, 28, 1470−1480. (44) Xu, X. D.; Saw, P. E.; Tao, W.; Li, Y. J.; Ji, X. Y.; Bhasin, S.; Liu, Y. L.; Ayyash, D.; Rasmussen, J.; Huo, M.; Shi, J. J.; Farokhzad, O. C. ROS-Responsive Polyprodrug Nanoparticles for Triggered Drug Delivery and Effective Cancer Therapy. Adv. Mater. 2017, 29, 1700141. (45) Zhu, K. N.; Liu, G. H.; Hu, J. M.; Liu, S. Y. Near-Infrared LightActivated Photochemical Internalization of Reduction-Responsive Polyprodrug Vesicles for Synergistic Photodynamic Therapy and Chemotherapy. Biomacromolecules 2017, 18, 2571−2582. (46) Li, Y.; Li, Y.; Ji, W.; Lu, Z.; Liu, L.; Shi, Y.; Ma, G.; Zhang, X. Positively Charged Polyprodrug Amphiphiles with Enhanced Drug Loading and Reactive Oxygen Species-Responsive Release Ability for Traceable Synergistic Therapy. J. Am. Chem. Soc. 2018, 140, 4164− 4171. (47) Tan, J. J.; Deng, Z. Y.; Liu, G. H.; Hu, J. M.; Liu, S. Y. Antiinflammatory polymersomes of redox-responsive polyprodrug amphiphiles with inflammation-triggered indomethacin release characteristics. Biomaterials 2018, DOI: 10.1016/j.biomaterials.2018.03.035. (48) Pu, K. Y.; Shuhendler, A. J.; Jokerst, J. V.; Mei, J. G.; Gambhir, S. S.; Bao, Z. N.; Rao, J. H. Semiconducting polymer nanoparticles as photoacoustic molecular imaging probes in living mice. Nat. Nanotechnol. 2014, 9, 233−239. (49) Chen, Y. S.; Frey, W.; Walker, C.; Aglyamov, S.; Emelianov, S. Sensitivity enhanced nanothermal sensors for photoacoustic temperature mapping. J. Biophotonics 2013, 6, 534−542. (50) Shim, M. K.; Yoon, H. Y.; Ryu, J. H.; Koo, H.; Lee, S.; Park, J. H.; Kim, J. H.; Lee, S.; Pomper, M. G.; Kwon, I. C.; Kim, K. Cathepsin B-Specific Metabolic Precursor for In Vivo Tumor-Specific Fluorescence Imaging. Angew. Chem., Int. Ed. 2016, 55, 14698−14703. (51) Lee, G. Y.; Qian, W. P.; Wang, L. Y.; Wang, Y. A.; Staley, C. A.; Satpathy, M.; Nie, S. M.; Mao, H.; Yang, L. L. Theranostic Nanoparticles with Controlled Release of Gemcitabine for Targeted Therapy and MRI of Pancreatic Cancer. ACS Nano 2013, 7, 2078− 2089. (52) Lock, L. L.; Cheetham, A. G.; Zhang, P.; Cui, H. Design and construction of supramolecular nanobeacons for enzyme detection. ACS Nano 2013, 7, 4924−4932. (53) Lee, J. S.; Groothuis, T.; Cusan, C.; Mink, D.; Feijen, J. Lysosomally cleavable peptide-containing polymersomes modified with anti-EGFR antibody for systemic cancer chemotherapy. Biomaterials 2011, 32, 9144−9153. (54) Schneider, G. F.; Subr, V.; Ulbrich, K.; Decher, G. Multifunctional Cytotoxic Stealth Nanoparticles. A Model Approach with Potential for Cancer Therapy. Nano Lett. 2009, 9, 636−642. (55) Gondi, C. S.; Rao, J. S. Cathepsin B as a cancer target. Expert Opin. Ther. Targets 2013, 17, 281−291. 3497


Article

Chemistry of Materials (56) Zhong, Y. J.; Shao, L. H.; Li, Y. Cathepsin B-cleavable doxorubicin prodrugs for targeted cancer therapy (Review). Int. J. Oncol. 2013, 42, 373−383. (57) Li, N.; Li, N.; Yi, Q.; Luo, K.; Guo, C.; Pan, D.; Gu, Z. Amphiphilic peptide dendritic copolymer-doxorubicin nanoscale conjugate self-assembled to enzyme-responsive anti-cancer agent. Biomaterials 2014, 35, 9529−9545. (58) Meijer, C.; Mulder, N. H.; Timmer-Bosscha, H.; Sluiter, W. J.; Meersma, G. J.; de Vries, E. G. Relationship of cellular glutathione to the cytotoxicity and resistance of seven platinum compounds. Cancer Res. 1992, 52, 6885−6889. (59) Yuan, Y.; Kwok, R. T. K.; Tang, B. Z.; Liu, B. Targeted Theranostic Platinum(IV) Prodrug with a Built-In AggregationInduced Emission Light-Up Apoptosis Sensor for Noninvasive Early Evaluation of Its Therapeutic Responses in Situ. J. Am. Chem. Soc. 2014, 136, 2546−2554. (60) Li, J. M.; Liu, F.; Shao, Q.; Min, Y. Z.; Costa, M.; Yeow, E. K. L.; Xing, B. G. Enzyme-Responsive Cell-Penetrating Peptide Conjugated Mesoporous Silica Quantum Dot Nanocarriers for Controlled Release of Nucleus-Targeted Drug Molecules and Real-Time Intracellular Fluorescence Imaging of Tumor Cells. Adv. Healthcare Mater. 2014, 3, 1230−1239. (61) Zheng, M. B.; Yue, C. X.; Ma, Y. F.; Gong, P.; Zhao, P. F.; Zheng, C. F.; Sheng, Z. H.; Zhang, P. F.; Wang, Z. H.; Cai, L. T. Single-Step Assembly of DOX/ICG Loaded Lipid-Polymer Nanoparticles for Highly Effective Chemo-photothermal Combination Therapy. ACS Nano 2013, 7, 2056−2067. (62) Sheng, Z. H.; Hu, D. H.; Zheng, M. B.; Zhao, P. F.; Liu, H. L.; Gao, D. Y.; Gong, P.; Gao, G. H.; Zhang, P. F.; Ma, Y. F.; Cai, L. T. Smart Human Serum Albumin-Indocyanine Green Nanoparticles Generated by Programmed Assembly for Dual-Modal Imaging-Guided Cancer Synergistic Phototherapy. ACS Nano 2014, 8, 12310−12322. (63) Wu, Y. Y.; Gao, D. Y.; Zhang, P. F.; Chen, C.; Sheng, Z. H.; Hu, D. H.; Zheng, M. B.; Gong, P.; Li, C. S.; Gao, G. H.; Cai, L. T. Theranostic iron oxide nanoparticles protected by NIR-emitting multidentate-ligands for multimodal imaging-guided photothermal therapy. Nanomedicine 2016, 12, 567−567. (64) Wang, Y. Y.; Deng, Y. B.; Luo, H. H.; Zhu, A. J.; Ke, H. T.; Yang, H.; Chen, H. B. Light-Responsive Nanoparticles for Highly Efficient Cytoplasmic Delivery of Anticancer Agents. ACS Nano 2017, 11, 12134−12144. (65) Tu, J.; Wang, T. X.; Shi, W.; Wu, G. S.; Tian, X. H.; Wang, Y. H.; Ge, D. T.; Ren, L. Multifunctional ZnPc-loaded mesoporous silica nanoparticles for enhancement of photodynamic therapy efficacy by endolysosomal escape. Biomaterials 2012, 33, 7903−7914. (66) Trachootham, D.; Alexandre, J.; Huang, P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discovery 2009, 8, 579−591. (67) Wan, Z. H.; Mao, H. J.; Guo, M.; Li, Y. L.; Zhu, A. J.; Yang, H.; He, H.; Shen, J. K.; Zhou, L. J.; Jiang, Z.; Ge, C. C.; Chen, X. Y.; Yang, X. L.; Liu, G.; Chen, H. B. Highly Efficient Hierarchical Micelles Integrating Photothermal Therapy and Singlet Oxygen-Synergized Chemotherapy for Cancer Eradication. Theranostics 2014, 4, 399−411. (68) Varkouhi, A. K.; Scholte, M.; Storm, G.; Haisma, H. J. Endosomal escape pathways for delivery of biologicals. J. Controlled Release 2011, 151, 220−228. (69) Guo, Z. Q.; Zou, Y. L.; He, H.; Rao, J. M.; Ji, S. S.; Cui, X. N.; Ke, H. T.; Deng, Y. B.; Yang, H.; Chen, C. Y.; Zhao, Y. L.; Chen, H. B. Bifunctional Platinated Nanoparticles for Photoinduced Tumor Ablation. Adv. Mater. 2016, 28, 10155−10164. (70) Khdair, A.; Gerard, B.; Handa, H.; Mao, G.; Shekhar, M. P.; Panyam, J. Surfactant-polymer nanoparticles enhance the effectiveness of anticancer photodynamic therapy. Mol. Pharmaceutics 2008, 5, 795− 807. (71) Wan, Z.; Mao, H.; Guo, M.; Li, Y.; Zhu, A.; Yang, H.; He, H.; Shen, J.; Zhou, L.; Jiang, Z.; Ge, C.; Chen, X.; Yang, X.; Liu, G.; Chen, H. Highly efficient hierarchical micelles integrating photothermal therapy and singlet oxygen-synergized chemotherapy for cancer eradication. Theranostics 2014, 4, 399−411.

(72) Yang, H.; Mao, H. J.; Wan, Z. H.; Zhu, A. J.; Guo, M.; Li, Y. L.; Li, X. M.; Wan, J. L.; Yang, X. L.; Shuai, X. T.; Chen, H. B. Micelles assembled with carbocyanine dyes for theranostic near-infrared fluorescent cancer imaging and photothermal therapy. Biomaterials 2013, 34, 9124−9133. (73) Zhang, Z. J.; Wang, L. M.; Wang, J.; Jiang, X. M.; Li, X. H.; Hu, Z. J.; Ji, Y. H.; Wu, X. C.; Chen, C. Y. Mesoporous Silica-Coated Gold Nanorods as a Light-Mediated Multifunctional Theranostic Platform for Cancer Treatment. Adv. Mater. 2012, 24, 1418−1423. (74) Sun, C. Y.; Cao, Z. Y.; Zhang, X. J.; Sun, R.; Yu, C. S.; Yang, X. Z. Cascade-amplifying synergistic effects of chemo-photodynamic therapy using ROS-responsive polymeric nanocarriers. Theranostics 2018, 8, 2939−2953. (75) Li, J.; Han, Y.; Chen, Q.; Shi, H.; ur Rehman, S.; Siddiq, M.; Ge, Z.; Liu, S. Dual endogenous stimuli-responsive polyplex micelles as smart two-step delivery nanocarriers for deep tumor tissue penetration and combating drug resistance of cisplatin. J. Mater. Chem. B 2014, 2, 1813−1824. (76) Yang, J.; Liu, W.; Sui, M.; Tang, J.; Shen, Y. Platinum (IV)coordinate polymers as intracellular reduction-responsive backbonetype conjugates for cancer drug delivery. Biomaterials 2011, 32, 9136− 9143. (77) van Engeland, M.; Nieland, L. J. W.; Ramaekers, F. C. S.; Schutte, B.; Reutelingsperger, C. P. M. Annexin V-affinity assay: A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 1998, 31, 1−9. (78) Andree, H. A.; Reutelingsperger, C. P.; Hauptmann, R.; Hemker, H. C.; Hermens, W. T.; Willems, G. M. Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers. J. Biol. Chem. 1990, 265, 4923−4928. (79) Koopman, G.; Reutelingsperger, C.; Kuijten, G.; Keehnen, R.; Pals, S.; van Oers, M. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994, 84, 1415−1420. (80) Vermes, I.; Haanen, C.; Steffensnakken, H.; Reutellingsperger, C. A Novel Assay for Apoptosis - Flow Cytometric Detection of Phosphatidylserine Expression on Early Apoptotic Cells Using Fluorescein-Labeled Annexin-V. J. Immunol. Methods 1995, 184, 39− 51.

3498


Cascade-Promoted Photo-Chemotherapy against Resistant Cancers

Recommend Documents