Acid-Responsive Polymeric Doxorubicin Prodrug Nanoparticles

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Acid-Responsive Polymeric Doxorubicin Prodrug Nanoparticles Encapsulating a Near-Infrared Dye for Combined Photothermal-Chemotherapy Yuanyuan Zhang, Cathleen Teh, Menghuan Li, Chung Yen Ang, Si Yu Tan, Qiuyu Qu, Vladimir Korzh, and Yanli Zhao Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.6b02896 • Publication Date (Web): 06 Sep 2016 Downloaded from http://pubs.acs.org on September 11, 2016

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Chemistry of Materials

Acid-Responsive

Polymeric

Encapsulating

a

Doxorubicin

Near-Infrared

Prodrug Dye

for

Nanoparticles Combined

Photothermal-Chemotherapy

Yuanyuan Zhang,†,‡,∆ Cathleen Teh,§,∆ Menghuan Li,|| Chung Yen Ang,† Si Yu Tan,† Qiuyu Qu,† Vladimir Korzh,*,§ and Yanli Zhao*,†,||

†

Division of Chemistry and Biological Chemistry, School of Physical and Mathematical

Sciences, Nanyang Technological University, 21 Nanyang Link, 637371, Singapore ‡

School of Chemical Engineering, Huaihai Institute of Technology, Lianyungang 222005, P.

R. China §

Zebrafish Translational Unit, Institute of Molecular and Cell Biology, 61 Biopolis Drive,

138673, Singapore ||

School of Materials Science and Engineering, Nanyang Technological University, 639798,

Singapore

Abstract: Combination therapy with high spatial and temporal resolution is highly promising for efficient medical treatment of cancer. In this study, doxorubicin (DOX) conjugated amphiphilic block copolymer with a terminal folic acid moiety was prepared, which could self-assemble into nanoparticles by encapsulating organic near-infrared (NIR) absorbing dye IR825 for combined photothermal-chemotherapy. The resulting PDOX/IR825 nanoparticles showed excellent colloidal stability and monodispersity in aqueous solution. Specifically, the

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conjugated DOX could be released quickly in weak acidic environment for chemotherapy due to the cleavage of acid-labile hydrazone bond. Meanwhile, the encapsulated dye could convert the NIR light energy into heat with high efficiency, making the self-assembled nanoparticles an effective platform for photothermal therapy. Confocal microscopy observations and flow cytometry analysis confirmed that the PDOX/IR825 nanoparticles could be efficiently endocytosed by HeLa cells and deliver DOX into the nuclei of cancer cells. The in vitro cell viability assays indicated that both DOX-sensitive HeLa cells and DOX-resistant A2780/DOXR cells were completely killed by the treatment of PDOX/IR825 under NIR light irradiation. Significant tumor regression was also observed in the zebrafish liver hyperplasia model upon combinational therapy provided from the PDOX/IR825 nanoparticles. Hence, the PDOX/IR825 nanoparticles exhibited a great potential in site-specific combined photothermal-chemotherapy of tumor.

INTRODUCTION Various self-assembled polymeric nanosystems have recently been developed as promising vehicles to deliver therapeutic agents for the treatment of cancer.1-3 These nanocarriers have drawn increasing interests owing to enhanced drug solubility and bioavailability, prolonged circulation time, reduced side effects, and passive accumulation at the tumor sites.4 Despite of traditional delivery methods by loading drugs inside polymeric nanosystems, there is an alternative approach where the polymeric prodrugs are prepared by anchoring the drug molecules on the polymer substrate via cleavable covalent bonds.5,6


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As compared with conventional drug loading method, polymeric prodrugs are particularly advantageous in some aspects, since they could significantly improve drug loading and minimize undesired release of drugs during the circulation for enhanced therapeutic efficiency. The polymeric prodrug nanocarriers effectively combine the advantages from the stability of prodrugs and long circulation time of nanocarriers. From a theoretical perspective for effective therapy, the drug molecules would only be released from prodrug nanocarriers when triggered by environmental stimuli, such as pH change,7-9 redox,10,11 enzyme action,12 as well as light irradiation,13 on account of the cleavage of the covalent bonds under these conditions. It is known that the pH value in endosomes (pH 5.0−6.5) and lysosomes (pH 4.5−5.0) of cancer cells is more acidic than the physiological pH of 7.4.14 Thus, various pH-sensitive polymeric nanoparticles have been developed for the delivery of doxorubicin (DOX),14,15 one of the most widely used clinical anticancer drugs. For the preparation of pH-responsive polymeric DOX prodrugs, many of the research efforts have been paid to the utilization of hydrazone bond, because the hydrazone bond can be efficiently cleaved under the acidic endo/lysosomal environment.16-18 DOX could be easily conjugated through the formation of hydrazone linkage between the carbonyl group of DOX and hydrazide group of some polymer substrates. More importantly, drug conjugated amphiphilic block copolymers could self-assemble to form nanoparticles in aqueous solution, facilitating the co-delivery of a second therapeutic agent into the tumor sites by encapsulating it in the hydrophobic cores. Near-infrared (NIR) light mediated photothermal therapy is another emerging modality for anticancer treatment, which employs heat generated from absorbed NIR light to



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destruct cancer cells with high efficiency.19-21 It is well known that cells are sensitive to the increase of ambient temperature, and thus local hyperthermia (above 42 °C) generated by light irradiation would kill cancer cells with a negligible damage to the surrounding normal tissue and cellular components. Currently, the most widely studied NIR-absorbing species for photothermal therapy include gold nanostructures,22,23 copper sulfide nanoparticles,24 carbon nanomaterials,25,26 and conjugated polymers.27,28 Nevertheless, non-biodegradable nature and potential toxicity of these materials severely inhibit their clinical translation potential.29 In this regard, NIR-absorbing dyes, particularly the family of indocyanine derivatives that have excellent biodegradability and photothermal conversion efficiency, are more promising candidates for photothermal therapy. It should be noted that a direct systematic administration of these dyes including indocyanine green (ICG),30-32 cypate,33,34 IR780,35,36 and IR82537,38 for photothermal therapy was strictly restricted, due to their poor aqueous stability or solubility. To address these issues, these dyes are often incorporated into nanocarriers to improve their aqueous solubility as well as stability, prolong their blood circulation time, and facilitate their tumor-specific delivery.39,40 While chemotherapy is the dominant approach for clinical treatment of cancer, the concept of combining chemotherapy with other therapeutic modalities such as photothermal therapy has also been put forward to ensure therapeutic efficacy for cancer. Combined photothermal-chemotherapy is capable of overcoming the limitations of single therapeutic approach, which may generate additional or synergistic effects to improve therapeutic outcome. For instance, poly(lactic-co-glycolic acid) nanoparticles were used to entrap ICG and

DOX

simultaneously.41,42

The

co-loading

of

cypate

and

DOX

inside


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pH/reduction-responsive vesicles showed superiority as a therapeutic platform for synergistic therapy.43 In another recent study, IR825, a newly developed NIR-absorbing dye with improved photostability and excellent photothermal conversion efficiency, was first complexed with cationic polyethylenimine and then coated with polyethylene glycol (PEG).44 The obtained J-aggregates were eventually loaded with DOX to exhibit combined therapeutic effect under the NIR laser irradiation. Nevertheless, current combinational therapeutic systems are achieved by simply loading anticancer drugs and dyes inside polymeric nanoparticles either simultaneously or separately, presenting excessive uncertainty to the loading amount of therapeutic agents. As a result, it is highly advantageous to develop polymeric prodrugs for the encapsulation of photothermal dyes, since this approach not only simplifies the drug loading process, but also reduces the premature drug release during the circulation. However, targeting ligand functionalized polymeric prodrugs for the delivery of NIR-absorbing dyes to the tumor sites have not been reported so far to the best of our knowledge. Based on the above considerations, we first prepared an amphiphilic polymeric prodrug (PDOX) via reversible addition−fragmentation chain-transfer (RAFT) polymerization of monomethoxy oilgo(ethylene glycol) methacrylate (OEGMA),45 followed by the functionalization of targeting ligands, the removal of the protection groups and eventually the conjugation of DOX molecule (Scheme 1). Then, dye IR825 was loaded into the self-assembled polymeric prodrug nanoparticles with high loading efficiency. The multifunctional therapeutic system (PDOX/IR825) integrates the targeting ligand, DOX prodrug and photothermal agent into one nanocarrier. Other important features of this



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therapeutic system are: (1) DOX was conjugated to the polymer backbone via acid-labile hydrazone bond and could only be released in weakly acidic cellular environment, (2) NIR-absorbing dye IR825 having high light-to-heat conversion efficiency was loaded into the polymeric DOX prodrug nanoparticles, and (3) folic acid was functionalized on the polymer as the targeting ligand, and the as-prepared polymeric nanoparticles could effectively accumulate in the solid tumor having overexpressed folate receptors. The therapeutic effect of this multifunctional system was evaluated using cancer cells in vitro and further validated in the zebrafish liver hyperplasia model in vivo. The results indicate that the NIR-absorbing dye loaded polymeric prodrug nanoparticles are a promising multifunctional platform for combined photothermal-chemotherapy.

Scheme 1. Schematic illustration of IR825 loaded polymeric DOX prodrug nanoparticles for targeted and combined photothermal-chemotherapy.


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Scheme 2. Synthesis of folic acid functionalized amphiphilic polymeric prodrug (PDOX) via RAFT polymerization and post-functionalization.

RESULTS AND DISCUSSION Synthesis and Characterization. The amphiphilic polymeric PDOX prodrug was prepared via a multiple synthetic process (Scheme 2). Firstly, the hydrophilic macroRAFT agent POEGMA was prepared by RAFT polymerization of OEGMA. A typical 1H nuclear magnetic resonance (NMR) spectrum of POEGMA is shown in Figure S1 (Supporting Information), and all the characteristic peaks including δ 3.37 ppm (terminal methoxy protons) and δ 3.64 ppm (PEG ethylene protons) can be observed. In addition, the signal at δ 1.24 was assigned to dodecyl methylene protons of the POEGMA. By comparing with the methoxy peak area at δ 7 ACS Paragon Plus Environment


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3.37 ppm, the degree of polymerization was determined to be about 22. Gel permeation chromatography (GPC) analysis revealed that the number-average molecular weight (Mn) of the POEGMA was 9.1 kg mol-1, which was close to the theoretical value (Mn(th) = 9.7 kg mol-1) and the value determined by 1H NMR (Mn(NMR) = 9.9 kg mol-1) (Table S1 in the Supporting Information). The molecular weight distribution (Mw/Mn) of the POEGMA was determined to be 1.16, indicating that the RAFT polymerization of OEGMA was achieved in a controlled manner. The RAFT block copolymerization of methacrylamide tert-butyl carbazate (MABH) was carried out using POEGMA as the macroRAFT agent. The 1H NMR spectrum of resulting PMABH-b-POEGMA copolymer contains a series of peaks around δ 3.37 and 3.64 attributing to PEG protons as well as signals at 1.40−1.49 assigned to the tert-butyl protons on MABH (Figure S1, Supporting Information). Compared with Fourier transform infrared (FT-IR) spectrum of POEGMA (Figure S2, Supporting Information), a new peak with strong intensity at around 1674 cm−1 was observed, which was attributed to the stretching vibration of newly introduced C=O bond from the hydrazide group. Consistently, after the copolymerization with the monomer MABH, the characteristic curves of POEGMA entirely left-shifted to high molecular weight region without obvious residuals (Figure 1a), indicating that the RAFT polymerization could serve as an efficient method to prepare the PMABH-b-POEGMA copolymer. The Mn of PMABH-b-POEGMA determined by GPC increased to 12.1 kg mol−1 with the Mw/Mn value of 1.18. These results indicate the successful copolymerization of MABH for the formation of the block copolymer PMABH-b-POEGMA.


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nanoparticles nanoparticles

Figure 1. (a) GPC traces of POEGMA homopolymer (Mn = 9.1 kg mol-1, Mw/Mn = 1.16), and PMABH-b-POEGMA diblock copolymer (Mn = 12.1 kg mol-1, Mw/Mn = 1.18). (b) UV–Vis– NIR spectra of IR825 in methanol, PDOX and PDOX/IR825 suspensions in phosphate buffer (pH = 7.4). (c) Photos of PDOX (left) and PDOX/IR825 nanoparticles (right) in phosphate buffer (pH = 7.4). (d) Size distribution of PDOX and PDOX/IR825 nanoparticles determined by DLS. (e) TEM image of PDOX/IR825 nanoparticles. Scale bar: 200 nm. (f) Changes of the diameter of PDOX/IR825 nanoparticles upon time in phosphate buffer (pH = 7.4) at 25 °C monitored by DLS.

In order to enhance the targeting selectivity of therapeutic agents, the aminated folic acid was conjugated with the terminal carboxylic group of PMABH-b-POEGMA via an amidation reaction. The successful conjugation of folic acid was confirmed by UV–Vis absorption spectrum. The absorption spectrum of PMABH-b-POEGMA-FA displayed a new peak around 355 nm as compared with the absorption spectrum of its precursor, which could be 9 ACS Paragon Plus Environment


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assigned to the conjugated folic acid (Figure S3, Supporting Information). Using the UV-Vis standard curve method at 355 nm, the coupling yield with folic acid was determined to be 45%. After treating PMABH-b-POEGMA-FA with trifluoroacetic acid (TFA), the FT-IR spectrum of the obtained PNHNH2-b-POEGMA-FA revealed that the stretching vibration of the ester C=O group at 1726 cm−1 reduced significantly (Figure S2, Supporting Information), indicating the cleavage of the tert-butoxycarbonyl (BOC) protecting group. As compared with PMABH-b-POEGMA, the

1

H NMR spectrum of PNHNH2-b-POEGMA-FA showed a

complete disappearance of signals at δ 1.46 assigned to tert-butyl protons of the BOC group (Figure S1, Supporting Information), demonstrating that almost all the BOC protecting group was removed. In addition, the amplified peaks at 8.67, 8.12, 7.38 and 6.57 ppm corresponding to the resonances of the protons in the folate moiety further confirm the successful conjugation of the targeting ligand. Finally, acid-labile PDOX prodrug was obtained by treating the deprotected PNHNH2-b-POEGMA-FA with DOX·HCl in anhydrous methanol with a catalytic amount of TFA. After the conjugation, a broad absorption peak was appeared in the range of 400−600 nm for the methanol solution of PDOX (Figure S4, Supporting Information), which was consistent with the UV–Vis absorption characteristics of free DOX. The successful conjugation of DOX was also verified by 1H NMR spectrum (Figure S1, Supporting Information), in which the signals of the DOX aromatic protons were clearly observed at δ 8.02, 7.88 and 7.63 ppm. The conjugation degree of DOX was determined using fluorescence spectroscopy after the treatment of PDOX with HCl solution to completely cleave the hydrazone linkage.46,47 It was found that the conjugated DOX content in the prodrug was as


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high as 20 wt%. High drug content is beneficial for minimizing the total dose of therapeutic agents required for effective cancer treatment, which could potentially reduce undesired side effects.48 Self-Assembly Behavior. To prepare amphiphilic prodrug polymers for the self-assembly, the strategy of selectively coupling hydrophobic drugs to one block of doubly hydrophilic block copolymers have been extensively exploited.49,50 After the conjugation of the hydrophobic DOX on PNHNH2-b-POEGMA-FA, the resultant PDOX prodrug consisting of a hydrophilic POEGMA and a hydrophobic DOX-containing block was capable of undergoing the self-assembly in aqueous solution. The suspension of the PDOX prodrug exhibited red color with the absorption peak around 490 nm, which was attributed to the DOX content (Figure 1b,c). The morphology and size of the self-assembled prodrug nanoparticles were investigated by transmission electron microscopy (TEM) and dynamic light scattering (DLS). As shown in Figure S5, the TEM image of PDOX nanoparticles revealed a spherical morphology with monodispersed size distribution at around 40 nm, which was consistent with the DLS analysis (Figure 1d). The IR825 dye could be encapsulated into the hydrophobic domains of prodrug nanoparticles by hydrophobic interactions. After the encapsulation of IR825 inside the core, the average size of obtained PDOX/IR825 nanoparticles increased to 150 nm (Figure 1e). Consistently, the size of PDOX/IR825 nanoparticles determined by DLS right-shifted after the dye loading, while the monodispersity was not affected (Figure 1d). Since IR825 was encapsulated inside the core, the increase of the nanoparticle size should be attributed to the increased hydrophobic core volume after the dye loading.



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As shown in Figure 1c, the successful encapsulation of IR825 by PDOX nanoparticles resulted in a color change of the suspensions from light red to brown. The absorption spectrum of free IR825 in methanol showed a sharp absorption peak at 826 nm. On the other hand, PDOX/IR825 nanoparticles in phosphate buffer (pH = 7.4) exhibited a broadened and red-shifted absorption peak at 842 nm. To determine the loading content of the photothermal dye, the PDOX/IR825 suspension was freeze-dried, dissolved in MeOH, and analyzed with UV−Vis−NIR spectroscopy. The loading content of IR825 was found to be 18 wt%. Colloidal stability of the dye-loaded nanoparticles was also studied. After storing at 25 oC in phosphate buffer at pH 7.4 for 72 h, the PDOX/IR825 nanoparticle suspension remained clear without obvious precipitation. In addition, the diameter of the nanoparticles monitored by DLS only increased slowly upon time (Figure 1f), and the monodispersity did not change too much (Figure S6, Supporting Information), indicating favorable colloidal stability at physiological conditions. However, when treated with acetate buffer at pH 5.0, the size distribution curve displayed a bimodal distribution with a small peak around 60 nm and a large peak around 120 nm in 4 h. After the incubation for 48 h, it was notable that the size of degraded nanoparticles further increased to over 600 nm. The size change of the PDOX/IR825 nanoparticles was initiated by the cleavage of acid-labile hydrazone bond when exposed to the acidic environment, resulting in the dissociation of the nanostructure. To break down the hydrazone linkages completely, PDOX/IR825 nanoparticles were treated with 0.1 M HCl at 25 oC overnight. As shown in Figure S7 in the Supporting Information, the IR825 dye was precipitated due to the loss of solubilization effect after the total dissociation of PDOX nanocarriers.


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Figure 2. (a) pH-Dependent release of DOX from the PDOX prodrug nanoparticles at 37 oC. (b) UV−Vis−NIR absorbance spectra of PDOX/IR825 suspensions and ICG solutions before and after 5 min of 808 nm laser irradiation (0.8 W cm−2). Inserted images are the photos of PDOX/IR825 nanoparticle suspensions (the first and third) and ICG solutions (the second and fourth) before and after the laser irradiation. (c) Heating curves of phosphate buffer (pH = 7.4), PDOX nanoparticles (1 mg mL−1), and different concentrations of PDOX/IR825 nanoparticles (0.05, 0.1 and 0.25 mg mL−1) suspended in phosphate buffer (pH = 7.4) under 808 nm laser irradiation at a power density of 0.8 W cm−2. (d) Temperature increments of PDOX/IR825 nanoparticle suspension (0.25 mg mL−1) under 808 nm laser irradiation at the power density of 0.8 W cm−2 for 5 cycles (5 min of irradiation for each cycle).



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Drug Release and Photothermal Effects. Considering the acid-liable nature of hydrazone linkage, the acidic tumor microenvironment could readily initiate the release of conjugated DOX from PDOX. Thus, PDOX prodrug nanoparticles were incubated at 37 oC in acidic buffers (pH 5.0 and 6.0), and the physiological condition of pH 7.4 was used as the control. The release results are shown in Figure 2a. Under mild acidic conditions of pH 6.0 and 5.0, corresponding percentages of released DOX after continuous incubation for 60 h were approximately 67 and 60%, respectively. In contrast, less than 10 % of DOX was released at pH 7.4 after the same incubation time. These results indicate that PDOX prodrug nanoparticles were stable under physiological conditions and also capable of releasing DOX in an accelerated manner when exposed to weak acidic environment, which are preferable for the delivery of anticancer drugs to slightly acidic intracellular microenvironment of cancer cells. The relatively high IR825 loading capacity also facilitates the utilization of the PDOX/IR825 for effective photothermal therapy. The photostability of PDOX/IR825 was first investigated, while ICG, an extensively studied photothermal dye, was used as the control. After 5 min of NIR laser irradiation (808 nm, 0.8 W cm−2), the green color of ICG in phosphate buffer (pH 7.4) turned into nearly colorless. On the contrary, no significant change was observed for the color of PDOX/IR825 (Figure 2b and Figure S8 in the Supporting Information). Consistently, the variation of the absorption spectrum of PDOX/IR825 after laser irradiation was much smaller in comparison to ICG under the same illumination conditions, where the NIR absorbance of ICG decreased to almost zero. IR825 is a heptamethine dye containing a rigid cyclohexenyl ring that could significantly improve the


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photostability as compared with ICG.51 In addition, the protection by the polymeric nanoparticles is another reason for the high photostability of IR825. To evaluate the photothermal efficiency, the suspensions of PDOX/IR825 nanoparticles at various concentrations were exposed to 808 nm laser at a power density of 0.8 W cm−2, and in the meanwhile, the temperature changes of the suspensions upon time were monitored using thermal imager. As displayed in Figure 2c, the temperature increases in phosphate buffer and PDOX nanoparticle suspension under the NIR irradiation were almost negligible, while the PDOX/IR825 nanoparticle suspension exhibited an evident concentration-dependent temperature increase. The fastest and greatest temperature increase was obtained at the highest concentration of PDOX/IR825 (0.25 mg mL−1), of which the temperature increased by 38 oC after 5 min irradiation. This rapid temperature increase can confer irreversible damage to cancer cells and thermally destroy the tumor tissues. In addition, no significant loss of the photothermal conversion capability was observed after five cycles of repeated laser-induced photothermal heating (Figure 2d). On account of the excellent photothermal stability of PDOX/IR825, it could be used as a robust photothermal heater for continuous and repetitive photothermal treatment of tumors. Intracellular Uptake and DOX Release. Efficient delivery of therapeutic agents into the cytoplasm or nucleus is crucial for the successful treatment of cancer. Taking advantage of intrinsic red fluorescence of DOX, the cellular uptake of PDOX nanoparticles and intracellular release of DOX using HeLa cells at different incubation times were evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry. In the CLSM study, three types of fluorescence were detected and collected, which were blue color of Hoechst 33342



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(used for staining cell nucleus), red color of DOX and green color of Lysotracker Green (used for the staining of lysosomes). As expected, free DOX was internalized quickly by HeLa cells, and strong red fluorescence was observed in the cytoplasm and nucleic regions after 4 h incubation (Figure 3a). This observation is because that free DOX could be transported through the cell membrane via passive diffusion and then interact rapidly with the DNA in the nuclei. In contrast, the DOX fluorescence in those cells treated with PDOX nanoparticles for 4 h was significantly weaker and overlapped largely with the lysosomes (green). On account of slight acidity in lysosomal compartments (pH 4.5−5.0), DOX from PDOX could be released gradually inside cells. The cellular uptake of PDOX and the DOX release after the internalization were found to be time-dependent, from which the accumulated DOX fluorescence in cytoplasm and nuclei eventually reached a very high level at the incubation time of 24 h (Figure 3a). Significant DOX fluorescence located in the cell nucleus confirmed that DOX was successfully released from acid-labile PDOX prodrug nanoparticles into the cytoplasm and then interacted with the DNA in the nucleus, since the PDOX nanoparticles themselves could not pass the nuclear pore complex due to the large size of nanoparticles. To quantify the cellular uptake, flow cytometric analysis was then performed using HeLa cells treated with free DOX and PDOX prodrug nanoparticles at different incubation time. Free DOX could enter HeLa cells very quickly, and the DOX fluorescence intensity in the cells after 1 h incubation was much stronger than that of cells treated with PDOX nanoparticles (Figure 3b). As the incubation time increased, the fluorescence intensity of the cells treated with PDOX nanoparticles began to increase even faster than the cells treated with free DOX, and they reached a comparable level at 24 h. Collectively, the CLSM and flow


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cytometry results demonstrate that the PDOX nanoparticles were efficiently endocytosed by HeLa cells and capable of releasing DOX continuously inside cells.

Figure 3. Intracellular uptake of free DOX and PDOX nanoparticles. (a) CLSM images of HeLa cells incubated with free DOX and PDOX prodrug nanoparticles for different period of time at an equivalent DOX concentration of 4 µg mL−1. Images from left panel to right present cell nuclei stained by Hoechst 33342 (blue), DOX fluorescence in cells (red), lysosomes stained by Lysotracker Green (green) and overlay of the three images, respectively. All images were acquired in the same scale bar (20 µm). (b) Flow cytometric analysis of the DOX fluorescence intensity in HeLa cells incubated with free DOX and PDOX prodrug nanoparticles for different period of time at an equivalent DOX concentration of 1 µg mL−1. Control group refers to the cells without any treatment.



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To confirm the targeting role of folic acid ligand on the PDOX nanoparticles, the cellular uptake of polymeric DOX prodrug nanoparticles without folic acid (PDOX(FA–)) by HeLa cells was also evaluated using flow cytometry (Figure S9, Supporting Information). After 4 and 24 h of incubation, decreased fluorescence intensity of PDOX(FA–) treated cells was observed as compared to the PDOX treated cells, indicating that the folic acid conjugation could enhance the cellular uptake of the PDOX by HeLa cells via the folate-receptor mediated endocytosis.

Figure 4. (a) Relative viabilities of HeLa and A2780/DOXR cells incubated with PDOX/IR825 nanoparticles (30 µg mL−1, equivalent DOX concentration of 5 µg mL−1) after 808 nm laser irradiation at different power densities for 5 min. The cell viability values were all normalized to the control of untreated cells. (b) Relative viabilities of HeLa and 18 ACS Paragon Plus Environment

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A2780/DOXR cells after incubated with PDOX/IR825 nanoparticles (30 µg mL−1) under different irradiation durations at the power density of 1.0 W cm−2. (c,d) Relative viabilities of (c) HeLa and (d) A2780/DOXR cells after incubation with different concentrations of free DOX and PDOX/IR825 without and with the laser irradiation at the power density of 1.2 W cm−2. Error bars were based on the standard error of mean (n = 4). N.S.: no significance, *p < 0.05, **p < 0.01, and ***p < 0.001 based on Student’s t test).

In Vitro Antitumor Study. To evaluate the therapeutic efficacy of PDOX/IR825 on cancer cells in vitro. DOX-sensitive HeLa cells and DOX-resistant A2780/DOXR cells were adopted, as these cell lines are over-expressed with folate receptors. The folate receptors could strongly interact with the folic acid targeting moiety on the nanoparticles for the targeted delivery of therapeutic agents.52 The cells were treated with PDOX/IR825 nanoparticle suspensions under different laser irradiation conditions for 5 min. The relative viabilities of cells were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Laser power and illumination duration are two critical parameters that influence the efficacy of photothermal therapy. As shown in Figure 4a, an increase in laser power density improves the killing efficiency of treated cancer cells. With irradiation time fixed at 5 min, most HeLa and A2780/DOXR cells were destroyed when the 808 nm laser power density exceeded 1.2 W cm−2. Likewise, the duration of laser irradiation is important to the successful treatment of cancer cells. With laser power density fixed at 1.0 W cm−2, the irradiation duration for 5 min or longer significantly enhanced the killing efficiency of treated cancer cells (Figure 4b).



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Anticancer effect of free DOX and PDOX/IR825 with a combinational therapeutic capability was then investigated using cancer cells. Both free DOX and PDOX/IR825 showed concentration-dependent cytotoxicity against tested cancer cells (Figure 4c,d). To facilitate the comparison between different therapeutic agents, the concentration of PDOX/IR825 was expressed as the equivalent concentration of free DOX. When the tested concentration was above 5 µg mL−1 (HeLa) or 2 µg mL−1 (A2789/DOXR), the therapeutic activity of PDOX/IR825 for these two cell lines showed a significant enhancement through photothermal damage after the laser irradiation. For the treatment of HeLa cells, free DOX showed better antitumor effects than PDOX/IR825 at the same DOX dose level when the NIR irradiation was excluded. As shown in Table S2 in the Supporting Information, the half maximal inhibitory concentration value (IC50) for PDOX/IR825 against HeLa cells without the laser irradiation was 6.7 µg mL−1, while IC50 for free DOX was 3.9 µg mL−1. These observations may be explained by the sustained acid-triggered release of DOX from prodrug nanoparticles, which resulted in the delayed and abated therapeutic efficacy. Under the irradiation of NIR laser, the IC50 value of PDOX/IR825 decreased to 2.0 µg mL−1, which was attributed to additional photothermal effect. The treatment of DOX-resistant A2789/DOXR cells with free DOX or PDOX/IR825 all showed low therapeutic efficacy. The IC50 of PDOX/IR825 was 12.6 µg mL−1, while the IC50 of free DOX already exceeded the concentration range. Encouragingly, under the laser irradiation, the IC50 of PDOX/IR825 for the A2780/DOXR cells decreased to 1.7 µg mL−1, which was even lower than that (2.0 µg mL−1) on HeLa cells. These results demonstrate that A2780/DOXR cells were more susceptible to the temperature increase as compared with HeLa cells under the conditions measured. Thus, combined


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photothermal-chemotherapy provided by PDOX/IR825 is highly effective to both DOX-sensitive HeLa cells and DOX-resistant A2780/DOXR cells.

In Vivo Combinational Therapeutic Efficacy. Encouraged by the excellent therapeutic effects of PDOX/IR825 in vitro, we further studied its antitumor performance on solid tumors using transgenic zebrafish based liver tumor model. The scheme of the combined photothermal-chemotherapy on liver tumor-bearing transgenic zebrafish larva was shown in Figure 5a. Strong expression of tagged enhanced green fluorescent protein (EGFP)-krasv12 in the liver tumor of transgenic zebrafish enables non-invasive and live imaging of the developing tumor by fluorescence microscopy or confocal laser microscopy,53,54 which is a straightforward approach to evaluate the antitumor efficacy of therapeutic agents.55 Since the expression of EGFP-krasv12 oncogene is induced by the mifepristone added inside the larvae media, the process is much easier to perform and control than that in rodents. In this study, liver tumor-bearing larvae were divided into four groups. Group 1 was injected with water as the control. Group 2 was injected with free DOX, while Groups 3 and 4 were injected with PDOX/IR825 nanoparticles without and with NIR laser illumination, respectively. Imaging of tumors was acquired by confocal imaging and the volume of each tumor was then assessed before and after 24 h treatment. Representative 3D fluorescent images of each group are shown in Figure 5b, while tumor volume measurements of all samples using the Image J are shown in Figure S10 (Supporting Information). It could be observed that a reduction in tumor size was detected in all groups injected with therapeutic agents (free DOX and PDOX/IR825 nanoparticles). Similar to in vitro antitumor study, the most efficient tumor regression was observed in the combinational treatment group of PDOX/IR825 with NIR laser irradiation,



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where the reduction in tumor volume was accompanied by fluorescent intensity decrease of green fluorescent protein (GFP). The fluorescent intensity decrease is a result of NIR induced local hyperthermia that damages cell membranes and degrades cellular proteins (including GFP) during the antitumor process.

Figure 5. In vivo antitumor activity on solid tumors using transgenic zebrafish based liver tumor models. (a) Scheme of combined photothermal-chemotherapy of live tumor-bearing transgenic zebrafish larva treated with PDOX/IR825 nanoparticle suspensions under 808 nm laser irradiation. (b) Representative 3D fluorescent images of four liver tumor groups. Liver tumor bearing zebrafish larvae were injected with H2O, free DOX, and PDOX/IR825 without


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and with the laser irradiation, respectively. The irradiated groups were exposed to 5 min of 808 nm laser irradiation with the power density of 1.2 W cm−2. (c) Changes in the relative tumor volume in each group measured after 24 h post-treatment.

To quantify the therapeutic efficacy of each treatment group, the percentage change in tumor size in each group was normalized to their initial size. As shown in Figure 5c, rapid uncontrolled growth occurred in H2O injected control, while the tumor regression was observed in other treatment groups. Free DOX group showed better curative effects than PDOX/IR825 without the laser irradiation, which was in good agreement with in vitro MTT results. As compared to the groups treated with free DOX and PDOX/IR825 without the laser irradiation, the tumor regression from PDOX/IR825 injected group with the laser irradiation was significantly enhanced, where the tumor volume decreased by 50% over 1 day. These results collectively demonstrate that the combination of prodrug chemotherapy with photothermal therapy significantly improves the overall anticancer efficacy on zebrafish liver hyperplasia models.

CONCLUSIONS In summary, we have developed a multifunctional platform based on polymeric DOX prodrug nanoparticles encapsulating IR825 dye for combined photothermal-chemotherapy. It was found that the PDOX/IR825 nanoparticles were stable under normal physiological pH, while sustained DOX release was achieved under mild acidic conditions. In addition, the encapsulated IR825 dye could efficiently convert the NIR light into heat upon NIR laser



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irradiation, making the self-assembled platform capable of repeatable photothermal therapy. In vitro study confirmed that PDOX/IR825 showed higher therapeutic efficacy on both HeLa and A2780/DOXR cells under NIR laser irradiation through the combinational photothermal-chemotherapy. Moreover, PDOX/IR825 nanoparticles under NIR laser irradiation exhibited superior therapeutic efficacy on zebrafish liver hyperplasia models in vivo. Taken together, the present results might provide a promising approach for safe and effective site-specific combinational anticancer therapy.

MATERIALS AND METHODS Materials. OEGMA (Mn = 500 g mol-1, Sigma-Aldrich) was passed through a basic alumina column to remove inhibitors prior to the use. Azobisisobutyronitrile (AIBN), DOXˑHCl, N-hydroxysuccinimide

(NHS),

diisopropylcarbodiimide,

2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT), trifluoroacetic acid (TFA) and all solvents were purchased from Sigma-Aldrich and used without any purification. Aminated folic acid (FA-NH2), cationic heptamethine indocyanine dye IR825, and MABH were synthesized according to previously reported procedures.56-58 Instruments. 1H NMR spectra were recorded on a Bruker BBFO-400 spectrometer using deuterated reagents as solvents. FT-IR spectroscopy was carried out on a Shimadzu Prestige-21 spectrophotometer. The number-average molecular weight Mn and molecular weight distribution Mw/Mn were determined using a Waters 150C GPC with the refractive detector at 30 °C, where DMF was used as the eluent. TEM images were obtained on JEM-1400 (JEOL) at an acceleration voltage of 100 kV. The UV−Vis−NIR absorption and


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fluorescence emission spectra were performed on Shimadzu UV-3600 and Shimadzu RF5301PC spectrophotometer, respectively. Hydrodynamic diameters were determined by a Malvern Zetasizer Nano-S DLS system at 25 oC. CLSM images were acquired by a Leica TCS confocal microscope with a Nikon Eclipse TE2000-S objective. Flow cytometry experiments were conducted by BD LSRFortessa X-20 cell analyzer. Synthesis of MacroRAFT Agent POEGMA. OEGMA (2.77 mL, 6 mmol), DDMAT (72.9 mg, 0.2 mmol) and AIBN (4.11 mg, 0.025 mmol) were dissolved in 1,4-dioxane (12.0 mL) in a Schlenk tube to give a ratio of [OEGMA]:[DDMAT]:[AIBN] = 30:1:0.125. The tube was sealed and the light yellow solution was degassed by purging with nitrogen for approximately 20 min. The polymerization was carried out under nitrogen atmosphere at 70 °C for 6 h. The polymerization was stopped upon air exposure and the polymer was purified by the precipitation into excess cold diethyl ether for three times. The resultant vicious oil was dried under vacuum at room temperature, and denoted as POEGMA (2.17 g, yield: 70 %, Mn(GPC) = 9.1 kg mol−1, Mw/Mn = 1.16). Synthesis of Block Copolymer by Chain Extension. The diblock copolymer was polymerized by RAFT polymerization of MABH using POEGMA as the macroRAFT agent. Typically, monomer MABH (0.954 g, 4.8 mmol), POEGMA (0.858 g, 0.06 mmol), AIBN (1.9 mg, 0.012 mmol) and 1,4-dioxane (4.0 mL) were transferred into the Schlenk tube and the resulting solution was degassed by purging with nitrogen for approximately 20 min. After the polymerization under nitrogen atmosphere at 70 oC for 12 h, the copolymer was purified by the precipitation into excess cold diethyl ether for two times. The resultant block



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copolymer

was

dried

under

vacuum

at

room

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temperature,

and

denoted

as

PMABH-b-POEGMA (1.22 g, yield: 38 %, Mn(GPC) = 12.1 kg mol−1, Mw/Mn = 1.18). Folic Acid Modification and Deprotection. Initially, the α-terminal carboxylic group of PMABH-b-POEGMA

was

activated

using

carbodiimide

coupling.

Typically,

PMABH-b-POEGMA (1.0 g, 0.05 mmol), NHS (0.058 g, 0.5 mmol), diisopropylcarbodiimide (0.077 mL, 0.5 mmol), and TFA (0.035 mL, 0.25 mmol) were dissolved in anhydrous DMF (15 mL), and the resulting solution was allowed to stir at room temperature for 48 h. The NHS activated PMABH-b-POEGMA was collected by the precipitation in cold diethyl ether and dried under vacuum. For the folic acid modification, the above-activated PMABH-b-POEGMA (0.50 g, 0.025 mmol), FA-NH2 (0.0966 g, 0.2 mmol), and a catalytic amount of TFA were dissolved in anhydrous DMF (8.0 mL), and the reaction was carried out at room temperature for 48 h. After the removal of the solvent under vacuum, the obtained product was re-dissolved in DMSO and dialyzed (molecular weight cut-off (MWCO) = 3500 Da) against DMSO for two days. Subsequently, the mixture was further dialyzed against deionized water for another two days. Finally, folic acid modified polymer PMABH-b-POEGMA-FA was obtained after lyophilization. The presence of the conjugated folic acid was confirmed using UV–Vis spectrum at 355 nm. To remove the protection group of BOC, TFA (4.0 mL) was added to the solution of PMABH-b-POEGMA-FA (0.2 g) in dichloromethane (4.0 mL), and the reaction mixture was stirred at room temperature for 3 h. After the removal of solvent under vacuum, the product was re-dissolved in THF and precipitated into excess hexane. The resulting deprotected


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copolymer was dried and denoted as PNHNH2-b-POEGMA-FA (1.22 g, yield: 38 %, Mn(GPC) = 11.8 kg mol−1, Mw/Mn = 1.15). Synthesis of DOX Prodrug. For the DOX conjugation, PNHNH2-b-POEGMA-FA (50 mg) and DOXˑHCl (25 mg) were dissolved in anhydrous methanol (3 mL), followed by the addition of a catalytic amount of TFA (2 µL). After stirring at room temperature in the dark for 36 h, the product was purified by extensive dialysis against methanol (MWCO = 3500 Da) for one day. After the removal of the solvent, polymeric prodrug denoted as PDOX (45 mg, yield: 60%) was obtained as dark red powder. To determine DOX conjugating content, 2.0 mg of PDOX was treated with 1.0 M HCl at room temperature overnight. After the dilution with deionized water, the amount of conjugated DOX was determined using fluorescence spectrum (excitation wavelength 480 nm, emission wavelength 560 nm) Formation of IR825 Loaded PDOX Nanoparticles. The IR825 loaded PDOX nanoparticles were prepared by a solvent exchange method. At first, IR825 (3.0 mg), Et3N (3 µL) and as-synthesized PDOX (10.0 mg) were dissolved in methanol (1.0 mL). The weak alkaline Et3N was added to facilitate the dye loading, since the dye becomes more hydrophobic in the subsequent self-assembled system. After stirring at room temperature for 4 h, phosphate buffer (4.0 mL, pH 7.4) was added dropwise. Then, the resulting dispersion was transferred into a dialysis tube (MWCO = 3500 Da) and dialyzed against phosphate buffer (pH = 7.4) for 24 h, yielding IR825 loaded nanoparticles denoted as PDOX/IR825. To determine the loading content of IR825, aliquots of the PDOX/IR825 suspension were treated with lyophilization to remove water. Finally, the obtained solid product was re-dissolved in CH3OH and analyzed



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with UV–Vis spectroscopy (absorption at 825 nm) using a standard curve method. The pure PDOX nanoparticles without encapsulated IR825 were also prepared using a similar method.

In Vitro Drug Release. The triggered release of conjugated DOX from PDOX prodrug nanoparticles under acidic environment was investigated using the dialysis method. Briefly, the PDOX suspension in phosphate buffer (3.0 mL, pH 7.4) was firstly placed into a dialysis bag (MWCO = 3500 Da) and dialyzed against buffer medium (40 mL) at pH 5.0, 6.0 or 7.4 in the dark at 37 oC. At the designed time intervals, the release medium (2.0 mL) was taken out and its fluorescence emission intensity at 560 nm under the excitation wavelength of 480 nm was determined by the fluorescence spectrometer, while a same amount (2.0 mL) of fresh buffer medium was added back to compensate the volume loss. The concentration of DOX presented in the dialysate was calculated. The releasing experiments were repeated three times independently, and the mean value was obtained as the final result. Cellular Uptake Study. Human cervical carcinoma HeLa cells were cultured with Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 U mL-1) and streptomycin (100 µg mL-1) under 5% CO2 atmosphere at 37 °C. In the CLSM study, HeLa cells were seeded in a 6-well tissue culture plate (2 mL medium) at a density of 2.0 × 105 cells per well. After culturing at 37 oC and 5% CO2 for 24 h, an appropriate amount of PDOX nanoparticle suspension with an equivalent DOX concentration of 5 µg mL-1 was added into the cell culture medium. After further incubation for 1, 4 and 24 h respectively, the culture medium was removed and the cells were washed three times with phosphate buffered saline (PBS). In addition, cells treated with free DOX were incubated as a control. Finally, the cells were stained alive with Hoechst 33342 (nucleus staining, blue color)


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and Lysotracker Green (lysosome staining, green color), and mounted for observations with CLSM. For the flow cytometric study, HeLa cells were also seeded in a 6-well tissue culture plate (2 mL medium) at a density of 2.0 × 105 cells per well. After culturing for 24 h, free DOX or PDOX nanoparticles were added in the culture medium, all of which the equivalent DOX concentrations were maintained at 1 µg mL-1 for every well. After the incubation for 1, 4 or 24 h, the cells were washed with PBS for two times and treated with trypsin (0.5 mL) for 2 min. Subsequently, fresh medium (0.5 mL) was added to each culture well, and the cells were collected via centrifugation at 2000 rpm for 3 min. After one more round of washing with PBS (0.5 mL) and centrifugation, the cells were re-suspended in PBS (1 mL), and subjected to the flow cytometry analysis.

In Vitro Cytotoxicity Study. In this study, DOX-sensitive HeLa cells were incubated in DMEM, while DOX-resistant ovarian carcinoma A2780/DOXR cells were seeded in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with the same content of FBS and antibiotics. Briefly, the cells were seeded in 96-well plates (100 µL medium) and incubated overnight. When the cell confluence reached around 60-70%, the medium was replaced with fresh one (90 µL), and then PDOX/IR825 suspensions (10 µL each) with various concentrations were added. After 4 h of incubation, the cultural medium was replaced and the cells were irradiated with 808 nm laser, while the cell groups without any treatment were used as controls. After the incubation for 20 h, the MTT solution (10 µL, 5 mg L-1) was added in the medium. The cells were incubated for another 4 h, and then the medium was



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replaced by DMSO (150 µL) to dissolve the resultant purple crystals. Finally, optical densities of the samples were measured using a microplate reader (infinite M200, TECAN) at 490 nm.

In Vivo Antitumor Efficacy Studies in Zebrafish Liver Hyperplasia Model. Transgenic zebrafish embryos expressing enhanced green fluorescent protein EGFP-krasv12 oncogene were treated with mifepristone to induce liver hyperplasia as reported in literature.53 The tumor belongs to the category of genetically engineered zebrafish tumor model that can express enhanced green fluorescence after the treatment with mifepristone. Briefly, after 4 days of induction with 1 µM mifepristone, liver tumor-bearing zebrafish larvae were selected for use in antitumor studies. Liver tumor-bearing larvae were mounted in 1% low melting agarose, with the liver and heart facing upward for direct injection of samples into the liver. Confocal microscopic images of the liver tumors were acquired using an upright Zeiss Axiovert 200M laser scanning microscope (LSM Meta 510, Carl Zeiss), while the detection of EGFP and tracing agent was enabled with two excitation laser lines (30 mW Ar and 1 mW HeNe) and emission band-pass filters of 505 nm/530 nm and 560 nm/615 nm that visualize EGFP-krasv12 and internalized fluorescence tracer, respectively. All mounted zebrafish larvae were imaged immediately after the injection of the freshly prepared therapeutic agents, while the larvae injected with water were used as a control. After imaging, one group injected with PDOX/IR825 was immediately exposed to 5 min of 808 nm laser irradiation with the power density of 1.2 W cm−2. Then, all the larvae were imaged again 1 day later. All images were obtained under the same acquisition settings. Quantification of liver tumor volume was evaluated using the Volumest plugin in Image J.


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ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website. 1

H NMR spectra, UV–Vis absorption spectra, TEM image, photos of nanoparticle

suspensions, DLS data, flow cytometry analysis, tumor volume measurements in each sample group, and table of IC50 values. (PDF) AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]. Author Contributions ∆

These authors (Y.Z. and C.T.) contributed equally to this work.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We thank the NTU-A*STAR Silicon Technologies Centre of Excellence under the program grant No. 11235100003 and the NTU-Northwestern Institute for Nanomedicine. V.K. and C.T. were supported by Institute of Molecular and Cell Biology institutional grant from the Agency for Science, Technology, and Research (A*STAR) of Singapore.

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Acid-Responsive Polymeric Doxorubicin Prodrug Nanoparticles

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