Multistep Consolidated Phototherapy Mediated by a NIR-activated

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Biological and Medical Applications of Materials and Interfaces

Multistep Consolidated Phototherapy Mediated by a NIR-activated Photosensitizer Yudong Xue, Jipeng Li, Guoliang Yang, Zhiyong Liu, Huifang Zhou, and Weian Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10605 • Publication Date (Web): 21 Aug 2019 Downloaded from pubs.acs.org on August 22, 2019

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ACS Applied Materials & Interfaces

Multistep Consolidated Phototherapy Mediated by a NIR-activated Photosensitizer Yudong Xue,a,†Jipeng Li,b,† Guoliang Yang,a Zhiyong Liu,a Huifang Zhou,b,* Weian Zhanga,* a. Shanghai

Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, China.

b. Shanghai

Key Laboratory of Orbital Diseases and Ocular Oncology, Department of

Ophthalmology, Shanghai Ninth People’s Hospital, Shanghai 200011, China.

KEYWORDS: multistep phototherapy, controllable release, heptamethine cyanine, NIR-activatable, photocleavable linker

ABSTRACT

The multi-functional effect of a single molecule for therapeutic functionalities on a single theranostic nano-system has a great significance to enhance the accuracy of diagnosis and improve the efficacy of therapy. Herein, a biocompatible multistep photo-therapeutic system (Ppa-Cy7-PEG-biotin) that contains a photosensitizer pyropheophorbide A (Ppa) with the covalent conjunction of a near-infrared (NIR) cyanine dye (Cy7) was successfully fabricated 1

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and functionalized with biotin for flexible specific tumor targeting phototherapy. These theranostic micelles will disaggregate after NIR irradiation via the photodegradation of cyanine accompanied by the photothermal conversion and the optically-controlled release for the restoration of photodynamic function of Ppa. Consecutive promoted treatments of photosensitive molecules greatly prolonged the tumor retention time and treatment efficiency, performing a multistep anti-tumor effect both in vitro and in vivo. Distinguishing from the simple phototherapeutic configurations which only act on the superficial areas of tumors at mild doses, the multistep therapy can be competent for broadly damaging to the superficial and deeper regions of tumors at the same dose. Therefore, as opposed to the simple phototherapeutic configurations, this strategy presents a photoactivation-based multistep phototheranostic platform with enormous potential in enhanced combined phototherapy of cancer.

INTRODUCTION Phototherapy as a class of minimal invasive, safe and effective treatments covers two major strategies: photodynamic therapy (PDT) and photothermal therapy (PTT), which exhibit significantly reduced side effects and improved tissue specificity in comparison with conventional cancer treatments.1-7 PDT normally depends on the photosensitizers where they transfer energy light radiation to the ambient oxygen for generating reactive oxygen species (ROS), such as singlet oxygen (1O2) to destroy tumor tissues.8-13 While PTT usually untilizes the photothermal reagents which can give rise to the thermal ablation of target tumor sites via 2


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the strong absorbance of near-infrared (NIR) light for generating heat.14-19 In recent years, according to clinical observation, that's not enough to significantly provide better outcomes with any single therapeutic therapy. Therefore, the development of combination therapy has already aroused people's great attention, which could dramatically improve therapeutic efficiency, prevent the local recurrence and minimize the side effects.20-25 In particular, for achieving a synergistic effect for phototherapy, the combination of PDT and PTT has been widely studied and lots of nanoparticles have been developed.26-32 Although these agents for the combination therapy have been reported with encouraging treatment effects, most of them have been developed by a simple way of physical encapsulation, while they embody more disadvantages such as, low embedding efficiency, poor stability and repeatability between different batches under physiological conditions, potential premature leakage in the process of preparation and transportation and high-risk of nonspecific phototoxicity.33-40 In addition, the complexity and uncertainty of the interaction mechanism of phototherapeutic combination systems arising from imprecise loading of phototherapeutic components maybe induce some negative effects on each other, containing the intrinsic biosecurity and biodegradability and the unpredictable resistance of among the combined effects of phototherapeutic systems. Therefore, the development of smart and integrative activated-phototherapy system for multistep therapy with both photodynamic and photothermal functions can effortlessly and securely control the degree of associated treatment, facilitate further penetration of drugs in the tumor tissue and maximize the therapeutic effect with precise progressive controlled release and treatment other than random combination therapy.

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Near-infrared (NIR) photocleavable and controllable molecules with less photo-damage, less scattering, and deeper penetration into tissues or extracellular matrices have recently attracted increasing interest for drug delivery, cell adhesion, etc.41-44 Heptamethine cyanine dyes with large molar extinction coefficients and broad wavelength tunability have gained popularity because of their superior near-infrared absorbing capabilities,45-48 convenient chemical modification,49-51 and multifunctional contrast agents for fluorescence imaging ultrasound imaging and photoacoustic imaging.36,

52-55

Furthermore, cyanine dyes could

effectively generate the photothermal effect, which release the absorbed near-infrared light energy in the form of non-radiative decay, causing a local temperature increase.56-59 However, everything has two sides and the side effect of cyanine dyes is unavoidable: it has been proved that the ROS contribute to the photodegradation with the photofading of cyanine dyes.60-62 Videlicet, cyanine dyes will be degraded by the singlet oxygen induced by themselves and then lose all their functions.63-65 Although, cyanine dyes has certain limitations in use, but we can precisely transform this inevitable flaw into a reliable advantage in another one way. As far as we know, this flaw of cyanine was rarely taken advantage of the release and activation for drug delivery. In this work, it is smart to use this defect of heptamethine cyanine (Cy7) as a node to design the well-structured, stable quantitative and near-infrared photodegradative nanomicelle

(Ppa-Cy7-PEG-biotin,

PCB)

for

realizing

imaging

guided

multistep

photothermal and photodynamic therapy of targeting cancer (Scheme 1), where hydrophobic pyropheophorbide A (Ppa) as an outstanding photosensitizer was incorporated for PDT. PEG-biotin, a targeting ligand, was used to provide both water-solubility and 4


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biocompatibility for specifically active targeting of tumor cells due to the presence of biotin.66-67 This phototherapy strategy exhibits the following advantages compared to previous conventional combined phototherapies: 1) Cy7 as a NIR-controlled linker can be photocleavable under the irradiation of 808 nm light source, while it also can produce considerable photothermal effect and fluorescence imaging guidance ability. 2) The Ppa-conjugated amphiphilic PCB contains both photosensitizer and photothermal agent with Cy7 as a bridging agent, and can be assembled into spherical micelles in aqueous solution, which can take advantages of the NIR-controlled release of Ppa with excellent penetrating ability and high sensitivity in situ of tumor. 3) The photodynamic effect of Ppa can be well restrained by Cy7 under normal conditions, but it can be activated by photocleavable Cy7 under the irradiation of 808 nm light source, when the micelles were effectively enriched in the tumor tissue through the active targeting and enhanced permeability and retention (EPR) effect. Therefore, the biocompatible multidimensional PCB with stable and quantifiable molecular structure exhibit the ability of multistep treatments with superior light controlled PSs internalization and release accompanied by a noticeable photoactivation effect. It could be great potential for the application in optimal combination therapy, gradually consolidating tumor treatment and preventing tumor recurrence.

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Scheme 1. Schematic illustration of the property of Ppa-Cy7-PEG-biotin (PCB) for NIR light activated tumor-targeting multistep phototherapy.

RESULTS AND DISCUSSION Synthesis and characterization of NIR-activated PCB micelles PCB was synthesized from Ppa and HO-Cy7-PEG-biotin (Scheme S1). For the PEGylation of heptamethine cyanine, according to the synthesis method of previous literature,68-69 the 1H NMR spectrum of dihydroxy heptamethine cyanine (HO-Cy7-OH) is shown in Figure S1. The proton peak signals at δ = 8.24, 6.44, 2.77, and 1.92 ppm belong to the protons from the cyclohexene and the heptamethine bridge of the cyanine matrix, respectively, which indicate that the HO-Cy7-OH had been successfully synthesized. Furthermore, in order to link biotin-functionalized PEG (N3-PEG-biotin) with cyanine by click reaction, the asymmetrical 6


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heptamethine cyanine (HO-Cy7-yne) containing a hydroxyl group and an alkyne group was synthesized by the esterification with the propargyl-3-carboxy-propionate (Figure S2). The 1H

NMR spectrum of HO-Cy7-yne (Figure S3) shows the complex peak cleaving and signal

overlap of asymmetric cyanine. The most obvious phenomenon could be discovered that the protons of the heptamethine bridge have transformed from two groups of signals to four groups, in which the chemical environments of the 8th and 2th proton peak signals have small changes, and the chemical shifts are similar to symmetry structures (δ = 5.89 and 8.05 ppm). While the 7th and 1th proton peak signals were de-shielded after the reaction and the chemical shifts shifted towards low field (δ = 6.93 and 8.55 ppm). These results indicated that we successfully obtained HO-Cy7-yne. Then, we started to obtain the amphiphilic compound PCB by covalent attachment using cyanine as a binding site. Briefly, the click reaction was used to introduce N3-PEG-biotin to one side of HO-Cy7-yne. The 1H NMR spectrum of HO-Cy7-PEG-biotin is shown in Figure S4. The characteristic peaks in the high field region confirm the presence of PEG segments. The characteristic peak signals of the protons (1, 2, 8, 9) on the heptamethine bridge and the protons on the triazole ring (3) can be clearly observed by amplifying signal peak. Moreover, the 1H NMR spectrum of PCB shown in Figure S5 revealed that the characteristic peak signals of Ppa appear for Ppa conjugation with HO-Cy7-PEG-biotin. In addition, the molecular weight of PEG-biotin, HO-Cy7-PEG-biotin and PCB were measured by GPC, respectively. As is demonstrated in the Figure S6, the GPC curves of aforementioned samples were gradually moved to the higher molecular weight region. And the number-averaged molecular weights and polydispersity index (PDI) of N3-PEG-biotin, 7



HO-Cy7-PEG-biotin and PCB are 1830 g/mol (PDI = 1.17), 2980 g/mol (PDI = 1.24) and 3820 g/mol (PDI = 1.08), respectively. Thus, based on the results of 1H-NMR and GPC, PCB were successfully synthesized. In the same way, we also successfully prepared Ppa-Cy7-PEG (PC) without biotin (Figure S7-8). The Ultraviolet-visible (UV-Vis) absorption spectra (Figure S9A) indicated that both the characteristic absorption peaks of Ppa and Cy7 appeared in the PCB. And the fluorescence emission of Ppa from PCB was almost completely quenched, which indicated Cy7 could effectively inhibit the photoactivity of Ppa by the quenching effect (Figure S9B). Meanwhile, it could be found that the absorbance of PCB at 700-850 nm declined sharply after 808 nm irradiation owing to the photobleaching of Cy7 at NIR region, and the fluorescence of Ppa from PCB remarkably recovered, owing to the attenuate quenching effect of Cy7 on Ppa with the photodegradation of Cy7. In marked contrast, 670 nm irradiation has minimal influence on the photophysical properties of PCB. PCB could be self-assembled into micelles in aqueous solution with an integrated effect of hydrophilic-hydrophobic interactions by using the dialysis method. As shown in Figure S10, the critical micelle concentration (CMC) values of PCB and PC measued by pyrene fluorescence were 3.122 × 10-3 mg/mL and 3.454 × 10-3 mg/mL, respectively. And their surface charges measued by the zeta potential were -3.9 mV and -14.3 mV, respectively. Transmission electron microscopy (TEM) imaging shows that PCB can spontaneously form uniform spherical micellar morphology (Figure 1A). Through the measurement of DLS, we could find that PCB micelles can be well dispersed in various solutions including aqueous media, pure DMEM, and FBS solution with excellent colloidal stability. The hydrodynamic sizes for the PCB micelles in water were determined to be 94.1 nm by DLS (Figure S11). 8


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PCB micelles in 10% FBS media which is widely performed for imitating the physiological conditions, overall exhibited wider stability as their hydrodynamic sizes only slightly increased from 94.1 nm to 110.6 nm. Meanwhile, all the micelles maintained excellent stability independently. No significant aggregates were appeared and the particle sizes had no obvious changes during ten days (Figure S12). Furthermore, all average sizes of micelles remained invariant over time in aqueous solution and were also extremely stable in other solutions. In order to notarize the photosensitive behavior of PCB micelles with optical radiation, after exposure with 670 nm light or 808 nm light, the average sizes of the micelles increased separately from 91.9 nm to 122.2 nm or 325.2 nm, respectively (Figure 1B). Moreover, as illustrated in Figure S13, PCB micelles were destroyed and some irregular aggregates were formed after the 808 nm irradiation. These results demonstrated the excellent stability of the micelles in various physiological solutions and the NIR-controlled degradation of the micelles for drug delivery. The release of Ppa from micelles were studied by using a dialysis membrane tubing with different pretreatments of irradiations. From Figure 1C, it was observed that the formed PCB micelles were highly stable in phosphate buffered saline (PBS) at physiological temperature, without showing significant release of Ppa and less than 8% total Ppa was released even after 2 days. Importantly, we could find that a relatively swift and violent release under the treatment with the NIR laser irradiation during the first 10 h, and nearly 80% Ppa from PCB micelles were released in this period. However, 670 nm irradiation only gave rise to little effect of the release process of Ppa beacause of the inhibition of Ppa’s photoactivity by Cy7. These results indicated that the cyanine with the light-triggered responsivity from PCB 9



micelles could greatly accelerate the dissociation of micelles and enhance the specific release of Ppa under the irradiation. Furthermore, the generations of 1O2 (Figure S14) with different modes of light pretreatment also confirmed that NIR laser irradiation can effectively promote the release and emancipate the photoactivity of Ppa, which verified the previous results of DLS and release. For the purpose of demonstrating the photothermal conversion ability of micelles, PCB micelles with different concentrations in aqueous media were exposed to an 808 nm NIR laser (0.75 W/cm2) for 6 min. Figure 1D and S15 obviously demonstrate the concentration-dependent photothermal performance of PCB micelles under 808 nm irradiation, and the temperature of solution could be effectively promoted from 25 to 70 °C with the concentration from 0 to 600 μg/mL. By contrast, pure water as a control can’t trigger the increase of temperature with less than 2 °C under the same experimental conditions.

(A)

(B)

(C)

(D)

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Figure 1. (A) TEM images of PCB micelles (scale bar, 100 nm). (B) Optically-controlled size distributions of PCB micelles after 670 nm or 808 nm irradiation. (C) Release behavior of Ppa from PCB in PBS (pH = 7.4) at 37 ℃ after different pretreatments of irradiations (D) Corresponding photothermal images of the aqueous solution and PCB micelles with different concentrations (0 (H2O), 37.5, 75, 150, 300 and 600 μg/mL) upon irradiation with 808 nm laser (0.75 W/cm2).

Cell uptake and intracellular release behavior of PCB micelles For the verification of facilitating cellular uptakes of micelles, CLSM was utilized to investigate the intracellular distribution and cellular uptake of micelles by using HepG2 cells. Intracellular red fluorescence of Cy7 as an indicator can be directly assessed by CLSM. HepG2 cells were cultured with PC and PCB micelles with a Ppa concentration of 40 μg/mL for 12 h, respectively and the blue fluorescence of came from the nuclear stain Hochest 33342. As shown in Figure 2A, the fluorescence image of Cy7 could be discerned obviously in the cell cytoplasm when exposed to the PC micelles or PCB micelles within 2 h and the fluorescence intensity of Cy7 showed a great enhancement with incubation time increased. Moreover, with the biotin as a cancer targeting unit for HepG2 cells, PCB micelles could be selectively internalized by receptor-positive HepG2 cells compared to the non-biotinylated PC micelles. The observations suggested that PCB micelles were more facilely internalized through predominant endocytic process, and the biotin targeting ligand amplified and accelerated the endocytosis of PC micelles for comparison with PC micelles. Furthermore, intracellular Ppa release and internalization with different irradiations were further evaluated 11



by CLSM (Figure 2B). In the absence of irradiation, PCB showed a fairly weak fluorescent signal of Ppa. Even with 670 nm irradiation, there were only a slight increase of the fluorescence intensity, but the red fluorescence of Ppa was widely improved throughout the cytoplasm after the pretreatment of 808 nm irradiation, which demonstrated that Ppa was can be controllably released and activated with 808 irradiation due to the disassembly micelles caused by the degradation of cyanine and the reactivation of Ppa. As shown in Figure 2C, the results of flow cytometry also exhibited similar intracellular trends of fluorescence intensity to that of CLSM, indicating that the NIR-triggered release and activation of Ppa from PBC. Cy7

Merge

(B)

PCB

PCB+670 PCB+808

Hochest

Hochest

Merge

PCB 2h

Ppa

PC 2h

(A)

(C) PC 12h

Control PCB PCB+670 PCB+808

PCB 12h

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Ppa

Figure 2. (A) Confocal fluorescence images of PC and PCB internalized by HepG2 cells for 2 h and 12 h, respectively. The images from left to right were the cell nucleus stained with Hochest (Ex/Em = 405/450 nm), Cy7 fluorescence (Ex/Em = 660/780 nm) and merge of images (Scale bar: 30 µm). (B) Intracellular release behavior of Ppa after incubation with 12


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PCB under different irradiations (Scale bar: 30 µm). Red fluorescence is the fluorescence of Ppa (Ex/Em = 405/700 nm). (C) Flow cytometry analysis of release and activation of Ppa from PCB after different pretreaments of irradiations.

In vitro cytotoxicity of PCB micelles To explore the potential cytotoxicity of PC and PCB micelles, the MTT assay was subsequently applied to evaluate the relative cell viabilities of HepG2 cells and A549 cells with or without irradiation after incubated with PC micelles or PCB micelles at various concentrations for 24 h. As depicted in Figure 3A, the results show that no significant cytotoxicity of PC micelles and PCB micelles were exhibited for both HepG2 cells and A549 cells even with a high Ppa concentration up to 40 μg/mL under dark conditions, and all the relative viabilities of cells remained 90%. After that, to further verify the capability of cell damage caused by PC micelles and PCB micelles, the phototoxicity of PC micelles and PCB micelles in different concentrations was investigated in the presence of laser irradiation with different combinations and regulations for wavelengths, which include the laser irradiation of PDT at 670 nm (20 mW/cm2) for 6 min, the laser irradiation of PTT at 808 nm (0.5 W/cm2) for 6 min, the laser irradiation of PDT+PTT and the laser irradiation of PTT+PDT. For the single treatment of PDT or PTT, it could merely kill partial cells in varying degrees. Contrastingly, the multistep treatments were proved to damage tumor cells more effectively but the sequence of phototherapy combination had a further effect on the therapeutic efficiency which the combination of PTT+PDT was more efficient than PDT+PTT due to high photodynamic efficiency of the released and activated Ppa after 808 nm irradiation 13



caused by the photodegradation behavior of cyanine (Figure 3B and S16). In addition, for comparison between PC and PCB, a further drop in the cell viability was detected against both HepG2 cells and A549 cells whether the single treatments and multistep treatments of PCB or not. It demonstrated that the biotin-receptor mediated more intracellular uptake of PCB, and adequately bring about the higher phototoxicity, which was in accord with the above results of cell uptake.

(A)

(B)

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Figure 3. (A) Cytotoxicity of HepG2 and A549 cells after the incubation with PC or PCB micelles at different concentrations without irradiation. (B) Cytotoxicity of HepG2 cells after the incubation with PC or PCB under different wavelength combinations of irradiation (670 nm, 808 nm, 670+808 nm and 808+670 nm). The statistical data are presented as mean ± SD (n = 5).

In vivo optical imaging and multistep phototherapy of PCB micelles For purpose of evaluating in vivo real-time distribution and tumor accumulation of PCB micelles, the fluorescence imaging was performed at different points in time after intravenous injection of PCB micelles. The fluorescence signals distributed extensively throughout the body after 1 hour post-injection. However, the fluorescence signals continuously strengthened in tumor site and reached to a peak value after intravenous injection 24 hours (Figure 4A and S17), which reflected the effective enrichment of micelles in tumor and embodied fast and efficient tumor-targeting of this phototheranostic micelles. Furthermore, due to the strong NIR absorption and high tumor accumulation of PCB micelles, after 24 h intravenous post-injection with PCB micelles, HepG2 tumor bearing mice were exposed with 808 nm laser and the temperature of tumor site rapidly increased from ≈ 37 °C to ≈ 67 °C within 5 min (Figure 4B). And there was no significant change of the temperature at the tumor site without PCB injection.

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(A)

(B)

Figure 4. (A) In vivo tumor-targeting fluorescence imaging at 0.5 h, 1 h, 3 h, 6 h, 12 h, 24 h and 48 h post tail intravenous injection of PCB (Ex/Em = 750/830). (B) IR thermal images of HepG2 tumor-bearing mice under different 808 nm laser irradiation time (1 W/cm2) after tail intravenous injection of saline and PCB.

Thus, encouraged by the efficient accumulation of PCB micelles in tumor, the feasibility of PCB micelles with tail intravenous injection in multistep antitumor therapy in vivo was studied. Saline and saline with laser irradiation were employed as the controls. The mice for the PDT group were irradiated with 670 nm laser irradiation (0.3 W/cm2) for 10 min. While for the PTT, the mice were irradiated with 808 nm laser (1 W/cm2) to remain the tumor site at certain temperature for 6 min. Saline and PCB micelles without laser irradiation could not 16


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inhibit the tumor growth. The tumor growth of PDT group was only partially inhibited and after only PTT treatment, the tumor growth was inhibited in the first 7 days. However, whatever after either the single PDT or PTT treatment, a mild regrowth of the tumors was observed later on and it indicated that the tumor growth was hardly inhibited unless increasing the drug dose or the amount of irradiation, resulting in the danger of excessive treatment. By comparison, both PDT+PTT and PTT+PDT groups could significantly inhibit the growth of tumors after treatments (Figure 5A and S19B). This multistep therapy was also directly confirmed to hold great therapeutic effect by the tumor weights (Figure 5B and S19C and representative tumor images (Figure 5C). Nevertheless, these two therapeutic efficacies showed a difference in the tumor suppression, which implied that the sequence of phototherapy combination was an important key point for multistep therapy and PTT+PDT treatment had an advantage for the promotion of integrative collaborative treatment. Similar results also were found in the treatment process of PC micelles which could more effectively eliminate tumor tissues with synergist effects rather than PDT+PTT. It might be because low photodynamic efficiency of the unreleased and unactivated Ppa without 808 nm and the degradation of cyanine by the singlet oxygen from Ppa could be predicted that if the treatment mode of PDT+PTT for anticancer was employed, which could reduce to the utilization efficiency of dual photosensitive reagents for collaborative treatment. Moreover, by contrast, PCB micelles revealed a better elimination of the tumor than PC because of the affinity of biotin with cancer cells. Then, no significant body weight loss or noticeable

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abnormality was dicovered in all groups with treatments of mice under irradiation (Figure S18 and S19A), which means no significant physiological toxicity of PC or PCB. Besides, the hematoxylin and eosin (H&E) staining assay was introduced to further assess the therapeutic effects after various treatments. Based on H&E staining results (Figure 5D), apoptosis and necrosis of tumor cells could be only observed in some small extends of tumor sites for PDT or PTT groups. The majority of cells were still alive in the deeper regions after only PDT or PTT treatment. Therefore, neither PDT nor PTT could completely and effectively destroy tumors, especially in the deep parts of tumors. Fortunately, the multistep therapy resulted in high degrees of tumor cell necrosis and apoptosis even in deep tumor tissues, which displayed an intensive multistep effect in accord with the antitumor data. In addition, there was no apparent damage or inflammation revealing in the major organs of mice receiving all sorts of treatments (Figure S20).

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(A)

(B)

(C)

(D)

Figure 5. In vivo multistep antitumor therapy after tail intravenous injection of saline, PC or PCB. (A) Relative tumor volume curves of tumor-bearing nude mice treated with PCB micelles after various treatments (the statistical data are presented as mean ± SD (n = 4)). (B) The final tumor weight of tumors collected from various treatment groups treated with PCB after treatments. (C) Representative tumor images after various treatments. (D) Histological examination of tumor sites after various treatments (scale bar = 100 μm).

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CONCLUSIONS In summary, we have successfully established a novel, NIR manipulative and tumor-targeting multifunctional theranostic agent (PCB), which could be realized a synergistic effect in multistep phototherapy of cancer with achieving encouraging therapeutic outcomes. In this system, the obtained biotin-targeting nanomicelles could promote intracellular uptake of photosensitizer (Ppa) to provide more reliable photodynamic antitumor efficiency, which could be further enhanced with the photodegradation behavior and photothermal heating of cyanine under the irradiation. It could remarkably accelerate to enhance the delivery of photosensitizers through the photodegradation of cyanine in the case of photothermal therapy. Ppa was covalently anchored on the hydrophilic cyanine (Cy7-PEG-biotin) and thus could hardly be released and activated from their carrier without irradiation. However, Ppa could be released and activated with the fracture of cyanine after PTT, while photodynamic effect of Ppa would be roundly activated and enhanced. In vivo fluorescence and IR thermal imaging are conducted, which proves the efficient and uniform enrichment of PCB micelles in tumor site. Multistep PTT and PDT achieved a remarkable synergistic effect in anti-tumor growth, with more favorable and powerful multistep of PTT+PDT treatment. It would be of great promise for cancer phototheranostics which afford consolidated synergistic therapeutic efficacy without improving the risk of excessive treament and be beneficial in keeping cancer from recurring.

ASSOCIATED CONTENT

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Supporting Information: < Experimental details and methods, the synthetic route of PCB, 1H

NMR spectra of synthetic compounds, UV-Vis absorption and fluorescence emission

spectra, GPC curves, the CMC and zeta potential of PC and PCB micelles, the stability of PC and PCB micelles in various solutions, the TEM image of PCB after 808 nm irradiation, the generation of 1O2 of PCB micelles, the photothermal heating curves of PCB micelles, the average fluorescence intensity values of PCB micelles for tumor regions, in vivo antitumor efficacy and safety evaluation of PC micelles,

Histological examination of major organs.>

This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (H. Zhou). *E-mail: [email protected] (W. Zhang). Author Contributions † These two authors contributed equally. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (No. 21574039, 21875063 and 81770960), the Shanghai International Cooperation Program 21



(19440710600), the Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20152228), the Science and Technology Commission of Shanghai (17DZ2260100) and the National Key Research and Development Plan (2018YFC1106100).

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Multistep Consolidated Phototherapy Mediated by a NIR-activated

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