Acid-Triggered Nanoexpansion Polymeric Micelles for Enhanced

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Acid-Triggered Nanoexpansion Polymeric Micelles for Enhanced Photodynamic Therapy Sheng Zhong,†,‡ Chao Chen,†,§ Guoliang Yang,‡ Yucheng Zhu,‡ Hongliang Cao,*,‡ Beijian Xu,‡ Yaoqin Luo,‡ Yun Gao,‡ and Weian Zhang*,‡ Shanghai Key Laboratory of Functional Materials Chemistry, School of Materials Science and Engineering, and §State Key Laboratory of Bioreactor Engineering, Biomedical Nanotechnology Center, School of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China

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‡

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ABSTRACT: Photodynamic therapy (PDT) as a noninvasive and selective treatment technology has presented great potential in cancer prevention and precision medicine, but its therapeutic efficacy is still greatly inhibited by the limitations of photosensitizers (PSs) in the microenvironment such as the aggregation caused quenching (ACQ) of PSs. Herein, we proposed an “acidtriggered nanoexpansion” method to further reduce the aggregation of photosensitizers by constructing acetal-based polymeric micelles. A pH-responsive amphiphilic block copolymer, POEGMA-b-[PTTMA-co-PTPPC6MA] was synthesized by reversible addition−fragmentation chain transfer (RAFT) polymerization and self-assembled into spherical micelles. In the normal physiological environment, the micelles were stable and had good biocompatibility. Upon entry into the acidic microenvironment of the tumor, the acid-responsive hydrophobic 2, 4, 6trimethoxybenzaldehyde in the micelles hydrolyzed and generated a hydrophilic diol moiety. Although the hydrophility of the micellar core was increased, the assembled structure of block copolymers was not dissociated but expanded. The responsive expansion of the micelles could allow the photosensitizers to well-disperse in the core, whereas more tumor-dissolved oxygen entered the micelles. This phenomenon could provide a better nanoenvironment for photosensitizers to reduce the ACQ of the photosensitizers, leading to more singlet oxygen (1O2) produced under the laser irradiation (650 nm). Both in vitro and in vivo studies have demonstrated that the remarkable photodynamic therapeutic efficacy of acid-responsive micelles could be realized. Thus, the acid-triggered nanoexpansion method might provide more possibilities to develop efficient platforms for treating cancers. KEYWORDS: acid-triggered nanoexpansion, photodynamic therapy, porphyrin, RAFT polymerization, aggregation caused quenching (ACQ)

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limited by π−π interaction and their hydrophobic characteristics, which cause the quenching of the electronic excited state, thereby resulting in the limited 1O2 quantum yield.21 Meanwhile, the inadequate oxygen in the tumor environment will also limit the production of singlet oxygen.22,23 In order to overcome these challenges, typically, metal−organic frameworks (MOFs) were constructed to prevent the aggregation of PSs,24 and a porphyrin/polyhedral oligomeric silsesquioxane (POSS) alternating copolymer was also designed to decrease the aggregation between porphyrin units.25 Additionally, MnO2 was utilized to react with endogenous H2O2 in tumors to produce O2 and further regulate hypoxic tumor microenvironment.26 Recently, stimuli-responsive drug delivery systems (especially, endogenous stimuli such as redox species,27−30 enzymes,31−35 and pH gradient36) offer a great promise in

INTRODUCTION Photodynamic therapy (PDT) plays an important role in the field of cancer treatment.1 The general process of photodynamic therapy involves photosensitizers (PSs) activated under light irradiation with a specific wavelength to generate a large amount of reactive oxygen species (ROS), ultimately causing irreversible damage of diseased tissues or cells.2−4 In the past decades, singlet oxygen (1O2) has been widely studied as a common ROS.5−10 It is well-known that amphiphilic block copolymers have been widely used in drug delivery systems for PDT because of their high drug-loading efficiency and more controllable drug release.11−13 The small molecular photosensitizers could be encapsulated in assembled micelles formed from amphiphilic block copolymers or quantitatively covalently conjugated to the polymer chain to form release system.14,15 Although porphyrins and their derivatives as mostly used PSs can be well delivered to tumor sites by block copolymers, they are still inhibited by some limitations in the tumor microenvironment such as the ACQ of photosensitizers and the hypoxic environment of tumors.14,16−20 Traditional PSs are © XXXX American Chemical Society

Received: July 18, 2019 Accepted: August 26, 2019

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DOI: 10.1021/acsami.9b12620 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scheme 1. Schematic Representation of the Acid Response Process of PM Copolymer and the Formation of PM Micelles and the Mechanism of Photodynamic Therapy

polymers containing cyclic benzylidene acetals have good biocompatibility; (ii) the cyclic benzylidene acetal contains a hydrophobic aromatic ring which will help form micelles, whereas most hydrophobic drugs contain a cyclic ring or an aromatic ring, and the aromatic group has a strong π−π interaction with these drug molecules, which would result in higher drug-loading rates and drug-encapsulation rates; (iii) the cyclic benzylidene acetal is rapidly cleaved into a polar diol moiety under the acidic tumor microenvironment, resulting in hydrophobic−hydrophilic transformation. It is worth noting that the hydrophobic−hydrophilic transition resulted from the cyclic benzylidene acetal in the acid environment would lead to the swelling of the photosensitizer micelles, and further provide the potential to alleviate the aggregation of photosensitizers while to promote more oxygen into the micelles. To the best of our knowledge, there is currently no report on the acidtriggered nanoexpansion strategy based on acetal moieties for enhance photodynamic therapy. Herein, we design and construct an acetal-based polymeric photosensitizer platform (Scheme 1) to enhance the efficacy of photodynamic therapy by an “acid-triggered nanoexpansion” method. The block copolymer POEGMA-b-[PTPPC6MA-coPTTMA] (PM) was synthesized through the copolymerization of porphyrin-based momoner (TPPC6MA) and acetal-based monomer (TTMA) by using hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) as RAFT chain transfer agent, and PM was further self-assembled into spherical micelles. The block copolymer has the following advantages: (i) the pH-sensitive acetal moieties of TTMA units undergo a hydrophobic to hydrophilic transition in tumor intracellular acidic microenvironment (pH 5.0−6.5) that causes the nanoparticles to swell, thereby reducing the aggregation of porphyrin units and providing a better nanoenvironment for photosensitizers; (ii) the micelle prepared from this block copolymer would have low

improving drug release efficiency, controlling drug release and reducing side effects. It is known that the physiological pH in normal tissues and blood in the human body is approximately 7.4, the intracellular endo/lysosomal pH ranges from 4.0 to 6.5, and the extracellular pH in tumor tissues is about 6.8.37−40 Thus, this pH gradient presented in the tumor microenvironment offers a great potential for specific targeting of tumors and controlled release drugs at tumor sites by designing some specific pH-responsive moieties in release systems, which has been widely reported in previous studies.41−44 The pHresponsive nanocarriers cover a wide range of categories, e.g., the vehicle is inherently sensitive to pH,45 reversible crosslinking via metal−ligand coordination,46 and pH-sensitive linkers (e.g., hydrazone, acetal47). As a typical pH-responsive moiety, cyclic benzylidene acetal has also been used in the construction of drug delivery systems for cancer therapies. For example, Zhao et al. synthesized a comblike amphiphilic copolymer with a cyclic benzylidene acetal functionalized skeleton, which could release curcumin in cells as an effective nanocarrier because of its ability to trigger hydrophobic− hydrophilic transition in acid condition.48 Wang et al. constructed a pH-responsive amphiphilic diblock copolymer containing a cyclobenzyl acetal, which self-assembled into welldefined vesicles for the intracellular delivery of hydrophobic drug Nile red and hydrophilic doxorubicin hydrochloride (DOX·HCl).49 Zhong et al. synthesized an asymmetric triblock copolymer based on a pH-responsive cyclic benzylidene acetal moiety, which self-assembled into a “chimeric” polymeric group for delivery and release of granzyme B apoptosis protein and hydrophilic DOX·HCl.50 Similar to other typical acidresponsive linkages such as hydrazine,51−53 acetal,54,55 oxime bonds,56 and cis-acotinyl,57,58 the hydrophobic cyclobenzylidene acetal structure rapidly cleaves under mild acidic conditions to form new hydrophilic structures. In addition, it has the following characteristics in a nanomedicine system: (i) B

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Figure 1. (a) Acid-responsive process of the PM micelle. (b) UV/vis absorption spectra of PM copolymer in THF and PM micelles in water. (c) Hydrolysis kinetics of PM micelles at different pH values (5.0, 6.8, and 7.4) by measuring the change in UV/vis absorbance at 292 nm. (d) UV/vis absorption spectra of PM micelles in PBS (pH 5.0) at different times. (e−g) DLS size distribution of PM micelles (PBS, pH 5.0) at 0, 12, and 36 h; data are given as the mean ± SD (n = 3); inset: TEM images of PM micelles; scale bar: 200 nm (e, f), 500 nm (g).

to the higher molecular weight region in comparison with that of the POEGMA homopolymer. In addition, the synthesized polymers have a relatively narrow polydispersity index (PDI) (Table S1), revealing that the RAFT polymerization was wellcontrolled. pH-Triggered Response of the Copolymer Micelles. The self-assembly behavior of PC and PM was further studied, and the micelles of these copolymer were prepared by a typical nanoprecipitation method.61 Typically, pyrene is used as a fluorescence probe to measure the critical micelle concentration (CMC) of the amphiphilic copolymer PC and PM in phosphate buffer saline (PBS). As a result, the CMC of PM and PC could be measured to be 1.26 and 0.63 μg/mL, respectively (Figure S7). The CMC of PC is smaller than that of PM, since the hydrophobic chain of PC is longer than that of PM. In addition, from the UV/vis absorption spectrum of the assembled PM micelles (Figure 1b), it was observed that the spectrum exhibited a certain degree of red shift, and the absorption intensity at 650 nm was also slightly improved. The self-assembled morphologies of PC and PM in PBS (pH 7.4) were observed by transmission electron microscope (TEM) (Figure S8), and the corresponding hydrodynamic diameters (Dh) were 158 and 174 nm, as measured by dynamic light scattering (DLS) (Figure S9a, S9b), respectively. These results showed that these micelles had a similar Dh, which was consistent with the result of TEM. As a result, it can be observed that the typical core−shell spherical micelles (about 100 nm) are formed in Figure S8. Careful observation reveals that there are some small black spots around the micelles in the image. This may be because that the salt particles are formed after the PBS solution was air-dried, and presented as small

phototoxicity in a normal physiological environment and be beneficial for blood circulation.

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RESULTS AND DISCUSSION Synthesis of Amphiphilic Block Copolymer (PM and PC). In our previous work, we have synthesized porphyrincontaining methyl acrylate monomer via three-step reactions (Scheme S1, Figure S1).59 The acetal-based monomer, 5methyl-2-(2,4,6-trimethoxyphenyl)-[1,3]-5-dioxanylmethyl methacrylate (TTMA), was synthesized according to a previous report by Grinstaff (Scheme S2, Figure S2).60 Synthesis of amphiphilic block copolymers PC and PM is through a two-step reaction. First, the hydrophilic block POEMGA was synthesized using 4-cyano-4-(ethylsulfa-nylthiocarbonyl) sulfanyl pentanoic acid (CESPA) as a chain transfer agent (CTA) (Scheme S3, Figure S3). The degree of polymerization (DP) of the homopolymer POEGMA could be calculated about 10. Then, POEGMA was further applied as the macro-RAFT agent for the subsequent RAFT polymerization of TPPC6MA to prepare amphiphilic block copolymer POEGMA-b-PTPPC6MA (PC) (Scheme S4, Figure S4), and POEGMA was also utilized for RAFT copolymerization of TPPC6MA with TTMA to produce amphiphilic block copolymers PM (Scheme S5, Figure S5). The results of amphiphilic block copolymers are summarized in Table S1. The DP of TPPC6MA (DP = 3) and TTMA (DP = 10) in the block copolymers was calculated by comparing δ 3.36 ppm (protons of OEGMA) to that δ2.78 ppm (protons of TPPC6MA) and 6.09 ppm (protons of TTMA) (Figure S4). Similarly, the DP of PC was also calculated as listed in Table S1. The GPC curves of these polymers were shown in Figure S6, and it could be seen that the traces of PC and PM shifted C

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ACS Applied Materials & Interfaces black spots in the TEM image when the micelles were prepared and stored in PBS solution. According to previous reports, the acetal moiety is relatively stable in neutral or alkaline media but rapidly hydrolyzed in an acidic environment.48,49,60 In this work, the acid-response process of PM micelles is illustrated in Figure 1a. To determine the extent of hydrolysis of the cyclobenzylidene acetal moieties under various pH environments (pH 5.0, 6.8 and 7.4), the absorbance at 292 nm (the characteristic absorbance of the hydrolyzed small molecule, 2, 4, 6-trimethoxybenzaldehyde) in UV/vis spectroscopy was recorded. Figure 1c shows that the rate of hydrolysis of the acetal in the PM micelles is highly dependent on the pH, e.g., the stronger the acidity of the micelles, the faster the hydrolysis rate, which is consistent with previous reports.48,49 Typically, PM micelles remained stable for long time in the normal physiological environment (pH 7.4), and there was almost no hydrolysis. However, the micelles exhibited a rapid structural change in the acidic conditions, especially for the fast hydrolysis at pH 5.0. Additionally, the hydrolysis degree of acetal moieties was dependent on the time and it nearly completely hydrolyzed at pH 5.0 after 24 h (Figure 1c). In contrast, the UV/vis absorption intensity of PC micelles in PBS (pH 5.0) did not change with time, indicating that the micelles did not have acid-responsive characteristic (Figure S10). Afterward, the size and morphological changes of the micelles in response to the hydrolysis of the cyclobenzylidene acetal were evaluated by DLS and TEM. As shown in Figure 1e, the Dh of the micelles (pH 5.0) gradually increased and the unimodal distribution of the hydrodynamic diameter also became broad with a small shoulder peak during the first 12 h, indicating the occurrence of the hydrolysis and the micelles were swollen. With the incubation time up to 36 h, it was found that the size of the PM micelles became much larger, and the hydrodynamic diameter changed from a single peak to a bimodal distribution with a quite small peak. Correspondingly, the polydispersity index increased sharply from 0.16 (0 h) to 0.52 (36 h), indicating the formation of hydrophilic diol moieties. Furthermore, it could be seen that the size of PM micelles remained constant after about 24 h. In contrast, the Dh of PM polymeric micelles did not change significantly after 36 h of incubation in PBS (pH 7.4) (Figure S9a), because the cyclic benzylidene acetal moiety is quite stable in the neutral. The TEM images corresponding to the above processes were shown in the insets of Figure 1e, 1f and 1g. The size of micelles gradually increased with incubation time at pH 5.0 and most micelles were swollen at 36 h, but their morphologies did not change significantly. This means that the PM micelles were only swollen but they still could keep spherical assembled structures when the cyclic benzylidene acetal moieties were hydrolyzed. Additionally, for the control samples of PC micelles, they could remain stable in a weakly acidic environment. In addition, we explored the effect of the acetal structure on the photophysical properties of micelles after hydrolysis. It could be seen that there was no different UV/vis absorption (300−700 nm) for two samples in PBS (pH 5.0) (Figure S11b). However, the fluorescence intensity of PM micelles is significantly improved with the hydrolysis time (Figure 2a). Here, the hydrolysis of the acetal groups greatly improved the hydrophilicity of the core, where the porphyrin units can be well dispersed. For the control samples of PC, the fluorescence intensity did not increase but reduce due to the self-

Figure 2. Fluorescence emission spectra of (a) PM, (b) PC micelles at different time intervals in a buffer solution (pH 5.0). (c) UV/vis spectra at 420 nm after 10 s of irradiation of DBPF with PM micelles in a buffer solution (pH 5.0 and 7.4) for different times (1 and 12 h). (d) UV/vis spectra at 420 nm after 10 s of irradiation of DBPF with PC micelles in a buffer solution (pH 5.0 and 7.4) for different times (1 and 12 h).

aggregation of the hydrophobic porphyrin units in the micelles (Figure 2b).62 In addition, PM below the CMC could not form micelles, so it could be identified as broken micelles. To compare the effects of the broken micelles and expanded micelles on the photosensitizer ACQ under acidic environment, we measured the fluorescence intensity of PM in acidic environments above CMC and below CMC, and compared the fluorescence intensity of the same concentration of free photosensitizers in organic solvent. When the concentration of PM and free meso-tetraphenylporphyrin (TPP) was 4 × 10−5 mg/mL (CMC), the fluorescence intensity of TPP in THF was also significantly higher than that of PM micelles in PBS (pH 5.0) at 0 h. However, the fluorescence intensity of TPP in the organic solvent remained stable after 12 h, and the fluorescence intensity of PM in PBS (pH 5.0) increased with time (Figure S13b). It could be seen from the above results that the fluorescence intensity of the expanded PM micelles in the acidic environment increased with time but the broken micelles decreased, the fluorescence intensity of expanded PM micelles in PBS (pH 5.0) would be close to that of the free TPP in THF. It could be explained that the swollen PM micelles reduced the ACQ of the photosensitizers and enhanced the fluorescence intensity compared with the broken micelles. Singlet Oxygen (1O2) Generation. The yield of 1O2 is a very important parameter for evaluating the efficacy of photodynamic therapy. As a singlet oxygen scavenger, 1,3diphenylisobenzofuran (DPBF) was often used to detect the D

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which is consistent with the above results of the 1O2 detected by DPBF. On the basis of the above results, it is reasonable to propose that hydrolysis of the acid-responsive cyclic benzyl acetal structure in the weakly acidic microenvironment of the tumor is beneficial for photodynamic therapy of the tumors. Thereby, the antitumor efficacy of these micelles against HepG2 cells was evaluated. First, the biocompatibility of PC and PM micelles were evaluated by MTT assay. The experimental results showed that the survival rate of HepG2 cells without laser irradiation was higher than 90% (Figure 4b), indicating that the two micelles have good biocompatibility. Then, we evaluated the cell viability treated by the two micelles with irradiation to determine the efficacy of PDT. As shown in Figure 4c, the phototoxicity of two micelles was increased with the concentration of porphyrin. When the concentration of porphyrin in PM and PC micelles was higher than 25 μg/mL, the phototoxicity of PM micelles was significantly higher than that of PC. As we expected, the acid-responsive cyclic benzyl acetal structure in the acidic tumor microenvironment could effectively enhance the efficacy of PDT. In Vivo Tumor Fluorescence Imaging and PDT Effects. Encouraged by the improved antitumor efficacy of PM micelles versus PC micelles exposed to laser irradiation in HepG2 cells, the in vivo antitumor efficacy of these samples was explored. The distribution of PM micelles at different time was first determined by fluorescence imaging of tumors in mice. As shown in Figure 4d, it could be clearly observed that because of the enhanced permeability and retention (EPR) effect, the porphyrin fluorescence signal of the PM micelles increased over time in the tumor region. After 48 h of injection of PM micelles into the tail vein of the mice, the mice were euthanized, and tumors and organs were collected for fluorescence imaging (Figure S15). It could be observed that the porphyrin fluorescence signal in the normal organs was significantly weaker than tumor, demonstrating that PM micelles had an effective accumulation in the tumor region. To study the phototherapeutic effect of each micelle in vivo, we performed the following five treatment methods in the HepG2 tumor-bearing nude mouse model: (a) PBS, (b) PC micelles (no laser), (c) PC micelles with laser (PC micelles + L), (d) PM micelles (no laser), and (e) PM micelles with laser (PM micelles + L). On the basis of the results of previous in vivo tumor fluorescence imaging, the tumors of mice were irradiated with 650 nm laser for 30 min within 24 h after administration. After treatment, the tumor size and body weight of all mice were recorded every other day for 2 weeks. As a result, the mice treated with PBS (pH 7.4) and the PC and PM micelles groups without any illumination showed similar maximum tumor growth. However, the size of the tumors with irradiation was decreased and the phototherapeutic efficacy of PM micelles was better than that of PC micelles (Figure 5a, b). Furthermore, representative photographs of tumors excised from mice (Figure 5c) demonstrated that the mice treated with PM micelles after illumination could provide superior antitumor efficacy over PC micelles. In addition, the body weight of all mice remained stable during the treatment, indicating that these systemic agents did not have major systemic toxicity and that mice grew well (Figure 5d). Standard H&E staining was further histologically applied to evaluate the therapeutic effect of each treatment group on tumors. It was worth noting that the H&E-stained tumor tissue of mice treated with PM micelles

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O2 produced by these micelles and the UV/vis absorption spectrum of DPBF was monitored at intervals of 10 s with a laser of 650 nm. The yield of singlet oxygen was evaluated by observing the absorbance attenuation of up to 420 nm. It could be seen that PM, PC and free TPP had a similar singlet oxygen yield in THF (Figure S14). However, the singlet oxygen yield of PM micelles increased with the increase of acidity in PBS (pH 5.0 and 7.4), and their singlet oxygen yield in PBS (pH 5.0) was significantly higher than those in pH 7.4. The singlet oxygen yield of PC did not change, because they are quite stable at pH 5.0−7.4 (Figure 2d). At the same time, we also studied the singlet oxygen yield of the micelle at the same pH with different standing time. It was found that only the singlet oxygen yield of PM micelles increased with the extension of the standing time, further illustrating the acid-responsive polymers could greatly promote singlet oxygen yield. Cellular Uptake of Micelles. To evaluate intracellular uptake of PC and PM micelles, the micelles were added to HepG2 cells and incubated together for different time. The fluorescence intensities of intracellular porphyrins of the two samples were observed to increase with incubation time by confocal laser scanning microscopy (CLSM) (Figure 3),

Figure 3. HepG2 cell uptake of PM and PC micelles at different times (4 and 24 h). Scale bar: 50 μm.

demonstrating that the uptake of PC and PM micelles was time-dependent. Furthermore, because the swelling of PM micelles in the acidic environment of tumor cells reduced the ACQ of porphyrins, the intensity of fluorescence of PM micelles was stronger than that of PC micelles for the same incubation time (Figure 3). To evaluate the effect of PM micelles on the amount of ROS produced in cells, the probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFHDA) was utilized to reveal the level of ROS (1O2). It could be observed from Figure 4a that there was almost no fluorescence in the cells with PBS (pH 7.4) under the 650 nm laser irradiation, but cells treated with PC and PM micelles showed significant green fluorescence, demonstrating that ROS was produced within cells treated by PC and PM micelles after laser irradiation. Moreover, acid-responsive PM micelles could generate significantly stronger fluorescence than PC micelles, E

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Figure 4. (a) CLSM images of intracellular 1O2 generation. Scale bar: 50 μm. (b) Cell viability of HepG2 cells with different concentration of porphyrin without irradiation. Data are given as the mean ± SD (n = 5) (c) Cell viability of HepG2 cells with different concentration of porphyrin after laser irradiation. Data are given as the mean ± SD (n = 5), *p > 0.05, **p < 0.05, and ***p < 0.001, determined by a Student’s t test. (d) Fluorescence images of mice injected with PM micelles at different times.

moiety is rapidly cleaved to form a polar diol, which undergoes a hydrophobic to hydrophilic transition. The results of DLS and TEM verified the acid-responsive expansion process of micelles, which provides the possibility to reduce the aggregation of the photosensitizers. Meanwhile, in vitro and in vivo studies have been confirmed that the response expansion of the micelles can make porphyrin produce more singlet oxygen to enhance the antitumor effect, and the fluorescence intensity of the photosensitizer is improved to some extent. Therefore, the phenomenon that the micelles expand under acidic conditions could develop new ideas for simultaneously reducing the ACQ of the photosensitizers and providing a better microenvironment for photosensitizers.

under laser irradiation caused more severe tumor cell apoptosis than PC micelles, further verifying that PM micelles containing acid-responsive moieties had more excellent phototherapic effects (Figure 5e). After 14 days of PDT, the histological morphology of the main normal organs (i.e, heart, liver, spleen, lung and kidney) of the mice in each treatment group was studied by H&E staining. From the results of Figure S16, it could be seen that there were no obvious tissue damage or pathological change in the tissues and organs of the mice in each treatment group, which proved that it had good biocompatibility and future therapeutic prospects.

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CONCLUSIONS In summary, we have proposed a novel “acid-triggered nanoexpansion” method for enhancing the efficiency of photodynamic therapy. Spherical micelles have been obtained by the self-assembly of the copolymer, POEGMA-b[PTPPC6MA-co-PTTMA]. After the micelles enter the acidic microenvironment of the tumor, the cyclic benzylidene acetal

EXPERIMENTAL SECTION

Synthesis of Amphiphilic Block Copolymer, PM. Typically, POEGMA (200 mg, 42.11 μmol), TPPC6MA (150 mg, 188.0 μmol), TTMA (95 mg, 260.5 μmol), AIBN (1.82 mg, 11.1 μmol), and 3 mL of THF were added into a dry polymerization tube. The mixture solution was degassed and the polymerization tube then was sealed

F

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Experimental details and additional data (Figures S1− S16, Schemes S1−S5, Table S1) (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Email: [email protected] (H.C.). *Email: [email protected] (W.Z.). ORCID

Hongliang Cao: 0000-0002-9342-7980 Weian Zhang: 0000-0002-1717-597X Author Contributions †

S.Z. and C.C. contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Shanghai Natural Science Foundation (18ZR1408300) and the National Natural Science Foundation of China (21574039 and 21875063).

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Figure 5. (a) Relative tumor volume of the mice after different treatments (n = 4).(b) Tumor weights of mice after different treatments on the 14th day (n = 4), *p > 0.05, **p < 0.05, and ***p < 0.001, determined by a Student’s t test. (c) Photographs of excised tumors from tumor-bearing mice after received various treatments on the 14th day (n = 4). (d) Body weight changes of mice after different treatments (n = 4). (e) H&E- stained tumor after different treatments on the 14th day (n = 4). Scale bar: 100 μm.

under vacuum. The polymerization tube was heated at 70 °C and stirred for 48 h, and then precipitated three times with an excess of ice diethyl ether. The product (181 mg, 35%) was characterized by 1H NMR (Figure S4). Self-Assembly of Block Copolymer (PM and PC). Typically, 10 mg of the copolymer was first dissolved in 10 mL of THF and copolymer solution (1 mL) was then dropwise added into 4.0 mL of PBS (10 mM, pH 7.4) over 1 h. After being further stirred for 1 h, the micelles were formed in the mixture solution. In addition, the solution was stirred overnight and the resulting solution was dialyzed against PBS (pH 7.4) for 3 days to remove THF. Cellular Uptake Assays. CLSM is used to detect the uptake of drugs by HepG2 (liver hepatocellular carcinoma) cells. Similarly, cell plating was first performed in a Petri dish, 10,000 cells per dish and 2 mL of medium was added for culture. After 24 h of incubation, PC and PM micelles containing the drug (porphyrin) at a concentration of 25 μg/mL were mixed with the medium and added to a 6-well plate at 2 mL/well. After the solution was cultured 4 and 24 h, the medium was removed and the HepG2 cells were washed 3 times with PBS (pH 7.4), and then 4% paraformaldehyde solution was added to the culture dish for 20 min. Thereafter, the cells were washed again to ensure that the excess paraformaldehyde was removed and DAPI was added to the culture dish for nuclear staining for 3 min. Finally, excess DAPI was removed and the HepG2 cells in the culture dish were imaged using a laser confocal microscope.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b12620. G

DOI: 10.1021/acsami.9b12620 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsami.9b12620 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX