Enhanced Fluorescence Emission and Singlet Oxygen Generation of

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Enhanced Fluorescence Emission and Singlet Oxygen Generation of Photosensitizers Embedded in Injectable Hydrogels for Imaging-Guided Photodynamic Cancer Therapy Liu-Yuan Xia, Xiaodong Zhang, Meng Cao, Zhan Chen, and Fu-Gen Wu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00725 • Publication Date (Web): 18 Aug 2017 Downloaded from http://pubs.acs.org on August 19, 2017

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Enhanced Fluorescence Emission and Singlet Oxygen Generation of Photosensitizers Embedded in Injectable Hydrogels for Imaging-Guided Photodynamic Cancer Therapy Liu-Yuan Xia,†,║ Xiaodong Zhang,†,║ Meng Cao,† Zhan Chen,‡ and Fu-Gen Wu*,†

†

State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering,

Southeast University, 2 Sipailou Road, Nanjing 210096, P. R. China ‡

Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor,

Michigan 48109, United States

*Corresponding Author: E-mail: [email protected]

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ABSTRACT Benefiting from their inherent localized and controlled release properties, hydrogels are ideal delivery systems for therapeutic drugs or nanoparticles. In particular, the applications of

hydrogels for the delivery and release of photo-responsive drugs or nanoparticles are receiving increasing attention. However, the effect of the hydrogel matrix on the fluorescence emission and singlet oxygen generation efficiency of the embedded photosensitizers (PSs) has not been clarified. Herein, meso-tetrakis (1-methylpyridinium-4-yl) porphyrin (TMPyP) as a water-soluble PS is encapsulated into an injectable hydrogel formed by glycol chitosan and dibenzaldehyde-terminated telechelic poly(ethylene glycol). Compared to free TMPyP, the TMPyP encapsulated in the hydrogel exhibits three distinct advantages: (1) More singlet oxygen was generated under the same laser irradiation condition; (2) Much longer tumor retention was observed due to the low fluidity of the hydrogel; (3) The fluorescence intensity of TMPyP was significantly enhanced in the hydrogel due to its decreased self-quenching effect. These excellent characteristics lead to remarkable anticancer efficacy and superior fluorescence emission property of the TMPyP–hydrogel system, promoting the development of imaging-guided photodynamic therapy.

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1. INTRODUCTION Photodynamic therapy (PDT), as a highly promising treatment modality for numerous malignant and nonmalignant diseases,1–5 takes advantage of photosensitizers (PSs) which can produce reactive oxygen species (ROS) when exposed to light in the presence of oxygen.6–9 ROS are highly reactive and likely to contribute to the increased levels of endogenous DNA10 and protein (including enzyme)11 damages in cancer cells, eventually causing the destruction of target tissue. PDT has attracted much attention in the last decades and has been regarded as a powerful tool in oncology for its merits over conventional treatment options. The advantages of using PDT include the relatively low cost, minimal invasion, low systematic toxicity, low mutagenic potential, rapid effect in single treatment, and effectiveness of repeated treatments at the same site without the total-dose limitation.12,13 However, clinical use of most existing PSs has been severely limited by their low efficacy due to their insufficient water solubility, severe self-quenching effect, and/or low tumor selectivity.14–19 Thus, much effort has been devoted to overcoming these limitations.20–23 On the other hand, as three-dimensional polymeric and hydrophilic networks, hydrogels are able to imbibe large amounts of water and have been widely used in various fields, such as materials for protein separation, matrices for cell-encapsulation, and devices for the controlled release of drugs.24–31 Apart from the large water-absorbing capacity, hydrogels may also minimize irritation to the surrounding tissue of the disease focus.25,32–37 Additionally, as excellent drug delivery vehicles, hydrogels have excellent characteristics including biodegradability,24,38–40 low toxicity,38,41–45 and non-immunogenicity.46–48 Compared with conventional drug carriers, hydrogel-based drug delivery systems have

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several distinct advantages in PDT. First, a hydrogel matrix is usually composed of compounds with diverse polarities, making them suitable for encapsulating various molecules and nanoparticles, including PSs and other functional agents. Second, thanks to the low fluidity of the hydrogels, the PSs in the hydrogel matrix can stay at the tumor site for a long time period with low unwanted diffusion, which can therefore reduce the side effects of PDT.49 Considering these reasons, Conde et al. designed a prophylactic hydrogel patch which can combine gene therapy, drug therapy, and phototherapy. The system cannot only complete tumor remission when applied to non-resected tumors, but also prevent the tumor recurrence when applied following the tumor resection.37 Xing et al. demonstrated that a gold-biomineralization-triggered, injectable, and self-healing hydrogel can be used for photothermal therapy (PTT) due to the locally formed AuNPs under light irradiation.9 After encapsulating water-soluble PSs, the system provides combined PDT and PTT treatment, realizing the purpose of “one injection, multiple treatment”. Although many efforts have been devoted to utilizing hydrogels for PDT in recent years, the influence of the hydrogel medium on the properties of the encapsulated PSs still needs to be investigated. Herein, meso-tetrakis (1-methylpyridinium-4-yl) porphyrin (TMPyP) was selected as a model of water-soluble PSs and encapsulated into an injectable hydrogel, which was prepared by using the Schiff base linkages formed between the aldehyde groups of dibenzaldehyde-terminated dialdehyde-functionalized

telechelic PEG,

poly(ethylene

DF-PEG)

glycol)

synthesized

by

(or

termed

as

esterification

of

hydroxyl-terminated PEG with 4-formylbenzoic acid and the amine groups of glycol chitosan.50,51 TMPyP has excellent properties for PDT, including high chemical purity, good

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water-solubility, negligible dark toxicity, high photochemical reactivity, amphiphilicity for tissue penetration, and fast clearance from the body. The hydrogel has been proved to be injectable with self-healing ability,51 which means that it can be injected without gel fragmentation and can be integrated as bulk gels at the target site with a needle. Moreover, the gelation time of this kind of hydrogel is appropriate, which can protect the embedded drugs from diffusing out of the target sites or clogging in the needle during delivery. Besides, since the hydrogel components (glycol chitosan and DF-PEG) and the formed hydrogel have low toxicity,52–54 the hydrogel has been widely applied in the biomedical field including tissue engineering55 and drug delivery.26 To prepare TMPyP-loaded hydrogel, we simply mixed TMPyP aqueous solution and the precursor solutions (glycol chitosan and DF-PEG) of the injectable hydrogel without introducing organic solvent or chemical conjugation. For the first time, we found that TMPyP in the hydrogel matrix could emit stronger fluorescence and produce more toxic single oxygen (1O2) than that in aqueous solution. These improved properties of TMPyP may be attributed to the high viscosity of the hydrogel medium, which may suppress the rotation of TMPyP molecules and the collision-induced quenching between TMPyP molecules. The high singlet oxygen yield of TMPyP and the prolonged tumor retention of the hydrogel make the TMPyP-loaded hydrogel an attractive candidate in PDT. Through intratumoral administration, PDT using TMPyP-loaded hydrogel could ablate the tumor more efficiently than that only using free TMPyP (Scheme 1).

Scheme 1. Schematic illustrating the synthetic route of TMPyP-loaded hydrogel and in vivo anticancer application.

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2. EXPERIMENTAL SECTION Materials. Poly(ethylene glycol) (PEG, M.W. ~6000 Da), 4-formylbenzoic acid, N,N’-dicyclohexylcarbodiimide

(DCC),

4-(dimethylamino)

pyridine

(DMAP),

tetrahydrofuran (THF), diethyl ether, and ethylenediaminetetraacetic acid (EDTA) were purchased from Aladdin (China). Glycol chitosan, 2,2,6,6-tetramethylpiperidine (TEMP), and meso-tetrakis (1-methylpyridinium-4-yl) porphyrin (TMPyP) were obtained from Sigma Aldrich. 5,10,15,20-Tetrakis(4-sulfonatophenyl)porphyrin (TPPS) was ordered from the Tokyo Chemical Industry. All the reagents were used without any further purification. All solutions were prepared with deionized water (18.2 MΩ cm) purified by a Milli-Q system (Millipore). Synthesis of Difunctionalized PEG (DF-PEG). DF-PEG was synthesized according to a

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previous report. Briefly, PEG (9.78 g, 1.63 mmol), 4-formylbenzoic acid (0.98 g, 6.52 mmol), and DMAP (0.050 g, 0.41 mmol) were dissolved in 100 mL of anhydrous THF, followed by the addition of DCC (1.68 g, 8.15 mmol) under a nitrogen atmosphere. The mixture was stirred at room temperature for 18 h, and the white precipitation was collected by filtration. The final product was obtained after repeated dissolution in THF and precipitation in diethyl ether for three times and drying. Hydrogel Preparation. According to the previous study, an appropriate formula of the self-healing hydrogel was suggested to be 1.5% (w/w) glycol chitosan and 1% (w/w) telechelic DF-PEG.51 In our experiments, glycol chitosan (30 mg) and DF-PEG (20 mg) were dissolved in the TMPyP aqueous solution so that TMPyP could be well dispersed within the hydrogel. Characterization. The morphology and pore size of TMPyP-loaded hydrogel were presented by scanning electron microscopy (SEM). After complete gelation, the TMPyP-loaded hydrogel (0.1 mg/mL) was frozen in liquid nitrogen for 10 min. Then after further lyophilization, the sample was imaged using a scanning electron microscope (SEM, ULTRA Plus, Zeiss, Germany). Fourier transform infrared (FTIR) spectra of glycol chitosan, DF-PEG, and freeze-dried hydrogel were recorded on a Thermo Scientific Nicolet iS50 FTIR spectrometer. Fluorescence spectra of free TMPyP solution and TMPyP-loaded hydrogel were characterized by a spectrofluorophotometer (RF-5301PC, Shimadzu, Japan). Rheological Test. Dynamic shear rotation measurements were used to characterize the viscoelastic properties of the cross-linked hydrogel. These rheological measurements at rotational shear deformation were carried out with a MCR302 rheometer (Anton Paar GmbH)

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using parallel smooth plates of 40 mm diameter. Mechanical spectra were recorded in a constant strain mode over the frequency range of 1–103 Hz (rad/s) at 25 °C. Singlet Oxygen (1O2) Measurement. The 1O2 generation of different samples were measured using the singlet oxygen sensor green (SOSG) kit. First, the SOSG reagent (1 µL, 100 mM) was added into 2 mL of free TMPyP solution and TMPyP-loaded hydrogel before gelation (5 µg/mL TMPyP in both samples), respectively. Then, both samples were irradiated by a laser (532 nm) at a power density of 10 mW/cm2.The 1O2 generation of the above two samples was recorded using a fluorescence spectrometer every 5 min within the duration of irradiation for 40 min. Electron Paramagnetic Resonance (EPR) Studies. The generation of 1O2 was also verified by EPR experiments using TEMP as a trapping reagent. The EPR experiments were performed at room temperature on an EPR equipment EMX-10/12 (Bruker, Germany). The reaction solution (prepared with 0.05 M potassium phosphate buffer, pH = 7.8) contained the following reagents: 0.1 mg/mL TMPyP, 1.5% (w/w) glycol chitosan, 1% (w/w) DF-PEG, 0.05 M TEMP, and 10–4 M EDTA. The mixture was exposed to a Viewlex projector lamp and the signal of singlet oxygen was recorded after irradiation for 0, 3, and 5 min, respectively. Cell Culture. Mouse uterine cervical carcinoma (U14) cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium, supplemented with 10% FBS and 100 IU/mL penicillin-streptomycin at 37°C in a humid atmosphere (5% CO2). MTT Assay. To test the cytotoxicity of TMPyP-loaded hydrogel and control groups (free TMPyP solution and the hydrogel) in vitro, U14 cells were seeded into 96-well plates at a density of 5 × 103 cells per well. TMPyP-loaded hydrogel and the hydrogel were extracted

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using RPMI 1640 medium for 24 h to obtain the leachates. Sequential dilutions of the stock solution were prepared to vary the concentration of the leachates. The concentration of TMPyP in all three groups was fixed to 0.1 mg/mL. After incubation for 24 h, dilutions at different concentrations (0.5%, 1%, 2%, 5%, 10%, and 20%; n = 3) were added separately to each well (100 µL per well). Then, after further incubation for 24 h, 10 µL of MTT solution (5 mg/mL) was added to each well. After incubation for another 4 h at 37 oC, 150 µL of DMSO was added to each well after removal of the original culture medium containing MTT. Finally, the optical density (OD) at 570 nm was measured by a microplate reader (Multiskan FC, Thermo-Scientific, USA). Cell viability was calculated as follows: Cell viability (%) = (ODSample – ODBlank) / (ODControl – ODBlank) × 100% In Vitro Release Behavior of the TMPyP-Loaded Hydrogel. Release of TMPyP from hydrogels with different TMPyP concentrations (50, 100, or 200 µg/mL) was determined in phosphate buffered saline (PBS). For each concentration, 1 mL of TMPyP-loaded hydrogel (below) and 1 mL of PBS solution (above) were placed in a 4 mL Eppendorf tube in an incubator at 25 oC. At different time intervals, 100 µL of the supernatant was taken out for the measurement of the absorbance at 450 nm, and then put back to the original solution. In Vitro PDT. To study the phototoxicity of TMPyP-loded hydrogel, U14 cells were seeded at a density of 5 × 104 cells per well in an 8-well culture plate and incubated for 24 h. TMPyP-loaded hydrogels prepared in RPMI 1640 culture medium were gelatinated with calcein-AM (10 µM) and PI (50 µM) at the TMPyP concentration of 4 and 8 µg/mL, respectively. All values presented above were final concentrations. Free TMPyP solutions (TMPyP: 4 and 8 µg/mL) were prepared by directly dissolving TMPyP molecules in RPMI

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1640 medium with calcein-AM (10 µM) and PI (50 µM). Followed by PBS washing, U14 cells were separately treated with hydrogel, free TMPyP solutions, or TMPyP-loaded hydrogels for 30 min. Afterwards, cells were irradiated with a 532 nm laser at a power density of 10 mW/cm2 for 5 min. After further incubation for 3 h, the cells were observed using a confocal microscope (TCS SP8, Leica, Germany) with a 63× oil immersion objective. Confocal fluorescence images were acquired at calcein-AM channel (Ex: 488 nm, Em: 500–550 nm) and PI channel (Ex: 552 nm, Em: 605–655 nm), respectively. Mice Cultivation. Female BALB/c-nu mice (18 ± 2 g) aged 4 weeks were purchased from Yangzhou University Medical Center (Yangzhou, China) and used under protocols approved by Southeast University Laboratory Animal Center. After being acclimatized for at least 7 days, U14 tumors were inoculated by subcutaneous injection of 1 × 107 U14 cells (suspended in 100 µL of PBS) onto the dorsal side of nude mice. Intratumoral Retention Experiments. The intratumoral retention time of the TMPyP-loaded hydrogel was investigated by in vivo fluorescence imaging. U14 tumor-bearing nude mice were intratumorally administered with 100 µL of free TMPyP solution (0.1 mg/mL), hydrogel, and TMPyP-loaded hydrogel (TMPyP dose: 0.1 mg/mL), respectively. Under deep anesthesia by continuous inhalation of a mixture of oxygen with isoflurane (5%), the treated mice were imaged by a Cri Maestroin and PerkinElmer in vivo imaging system with an excitation wavelength of 540 nm and an emission wavelength of 670 nm at different time points. In Vivo PDT. U14 tumor-bearing mice were cultivated as previously described. While the tumor volume approached ~50 mm3, mice were randomly divided into 4 groups for different

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treatments: (1) Intratumorally injected with 100 µL of PBS, (2) intratumorally injected with 100 µL of free TMPyP solution (0.1 mg/mL), (3) intratumorally injected with 100 µL of hydrogel, and (4) intratumorally injected with 100 µL of TMPyP-loaded hydrogel (TMPyP dose: 0.1 mg/mL). Photoactivation (532 nm, 10 mW/cm2, 30 min) was carried out 10 min after the completion of the administration on half of the mice in each group for two consecutive days (one treatment per day). The other half of mice left in each group without photoactivation were set as the control groups. Tumor volumes were monitored every day for 7 days. Tumor volumes were calculated as width2 × length/2. Histopathological Study. 7 days after in vivo PDT, U14 tumor-bearing nude mice were sacrificed and the tumors and major organs (heart, liver, spleen, lung, and kidneys) were collected and fixed in 4% paraformaldehyde, followed by making paraffin sections. Hematoxylin and eosin (H&E) staining of the above sections was performed following standard protocols.

3. RESULTS AND DISCUSSION Characterization. The hydrogel formed by the cross-linking of glycol chitosan and DF-PEG through Schiff base chemistry was colorless under white light. With the presence of TMPyP, the resultant hydrogel became red, which was similar with the color of the TMPyP solution (Figure 1a). The as-prepared hydrogel and TMPyP-loaded hydrogel showed a similar well-defined porous morphology as revealed by scanning electron microscopy (SEM) (Figure 1b). The interconnection between the pores could be ascribed to the network formed by the cross-linking between glycol chitosan and DF-PEG. Besides, the successful fabrication of the

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injectable hydrogel was confirmed by Fourier transform infrared spectroscopy (FTIR). Figure 1c shows the FTIR spectra of glycol chitosan, DF-PEG, and freeze-dried hydrogel (TMPyP-free). In the FTIR spectrum of DF-PEG, the absorption peak at ~2738 cm–1 was attributed to the C–H stretching vibration of aldehyde groups. Besides, the C=O stretching vibration from the aldehyde groups at ~1715 cm–1 could also be clearly observed, indicating that DF-PEG was successfully synthesized. The peak corresponding to the C=O stretching vibration of aldehyde groups was absent in the FTIR spectrum of the hydrogel, suggesting that the coupling reaction occurred between the amine groups of glycol chitosan and the aldehyde groups of DF-PEG to form the hydrogel network. To characterize the viscoelastic properties of the hydrogel itself and TMPyP-containing hydrogel, we have also carried out the rheological measurements. Both of the two hydrogels have similar viscoelastic properties. As shown in Figure 1d, the apparent viscosity decreased sharply with the increasing shear rate, suggesting that the hydrogels showed shear thinning behavior. It might be explained by the fact that shear causes the disruption of the molecular aggregates with high viscosity.56 Thus under a large compressive strain through the needle injection, the gel could become a sol due to the formation of more fragile intercluster tanglements. Upon strain removal, the sol could convert back to the gel state immediately, revealing the injectable property of the hydrogel. Besides, the changes of the storage modulus (G’) and loss modulus (G’’) of the TMPyP-free hydrogel and TMPyP-containing hydrogel as a function of frequency (ω) at 37 oC were also investigated (Figure 1e and 1f). For both of the two hydrogels, the G’ value was considerably higher than the G’’ value over the entire range of the frequency investigated and both G’ and G’’ exhibited a relatively pronounced plateau.

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This mechanical spectrum is characteristic for a well-developed network possessing high mechanical strength.57 With good mechanical stability, the hydrogels would be degraded more slowly,58 prolonging the intratumoral retention time.

Figure 1. (a) Photographs of hydrogel, TMPyP in PBS (TMPyP concentration: 100 µg/mL), and TMPyP in hydrogel before and after gelation (TMPyP concentration: 100 µg/mL). (b) Typical SEM images of hydrogel (left) and TMPyP-loaded hydrogel (right). (c) FTIR spectra of glycol chitosan, DF-PEG, and the hydrogel. (d) Shear rate-dependent viscosity changes of PBS, TMPyP-containing PBS, hydrogel, and TMPyP-loaded hydrogel. Frequency-dependent (at a strain of 0.5%) shear rheology of the TMPyP-free hydrogel (e) and TMPyP-loaded hydrogel (f).

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Next, we collected the UV-vis absorption spectra of TMPyP in PBS and in hydrogel (Figure 2a). It can be seen that the two samples exhibit a similar spectral pattern, with the peaks locating at ~520, ~555, ~588, and ~642 nm. Since the peak intensity at 520 nm is the highest, we will choose a laser with the excitation wavelength of 532 nm (which is close to 520 nm) for the following irradiation experiments. The fluorescence properties of the TMPyP-loaded hydrogel were also studied (Figure 2b and 2c). At the same concentration of 100 µg/mL, the fluorescence emission intensity of TMPyP in hydrogel (pH = 7.4) showed a ~2-fold increase as compared with that of TMPyP in phosphate buffer saline (PBS, pH = 7.4) (Figure 2b). To further evaluate the fluorescence properties of the TMPyP-loaded hydrogel, fluorescence spectra with different concentrations of TMPyP were collected (Figure 2c). When the samples were excited at 532 nm, the FL emission intensity (at 632 nm) first increased sharply at the concentration lower than 100 µg/mL, and then reached the maximum at the concentration of ~250 µg/mL. When the concentration increased to > 500 µg/mL, the FL intensity decreased strikingly. We hypothesize that this phonomenon is due to the aggregation-caused quenching (ACQ) or self-quenching effect of the fluorescent TMPyP molecules, which means high fluorophore (i.e., TMPyP) concentrations can result in quenching due to a variety of interactions, such as radiative and non-radiative transfer, and excimer formation. For many aromatic compounds, the formation of sandwich-shaped excimers and exciplexed aided by strong intermolecular π–π stacking interactions is a general phenomenon, which would cause the ubiquitous concentration-quenching effect. Additionally, the intrinsic injectable property and red fluorescence emission of TMPyP-loaded hydrogel

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were confirmed by the photograph pictured under 365 nm UV irradiation (Figure 2d). Singlet Oxygen Generation. . Next, we detected the generation of singlet oxygen (1O2) from TMPyP in PBS and hydrogel under 532 nm irradiation by using the Singlet Oxygen Sensor Green (SOSG) kit. The fluorescence emission intensity of this SOSG probe increases with the 1O2 concentration.59 The 1O2 generation of TMPyP in PBS and hydrogel was measured immediately after irradiation under a 532 nm laser every 5 min within the duration of 40 min (Figure 2e). It was shown that the 1O2 generation rate of TMPyP in hydrogel was significantly faster than that of TMPyP in PBS. Specifically, at the end of the laser irradiation (40 min), the total 1O2 generation amount of TMPyP in hydrogel was approximately twice as large as that of TMPyP in PBS. The above results indicated that TMPyP-loaded hydrogel possessed a much higher 1O2 production efficiency than free TMPyP solution at the same TMPyP concentration. The generation of more 1O2 for TMPyP in hydrogel than TMPyP in PBS was also verified by the electron paramagnetic resonance (EPR) results (Figure S1 and S2 in the Supporting Information). The stronger PL emission and higher 1O2 generation of the PSs in hydrogel as compared with those in PBS may be caused by the high viscosity of the hydrogel medium. It has been reported that the fluorescent molecules in a low viscous environment (such as water) have a higher possibility to collide with each other, forming aggregates via π–π interaction, resulting in a higher degree of self-quenching.60 While in a highly viscous environment, they are hard to rotate. In this case, the three dimensional structure of the hydrogel helps to stabilize TMPyP molecules within the hydrogel matrix, which reduces the rotation and intermolecular collision of the PS molecules, leading to the enhancement of FL intensity and ROS generation. To verify this, another water-soluble PS,

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TPPS, was used. The FL intensity enhancement of SOSG was also observed when TPPS was encapsulated into the hydrogel (Figure 2f), further confirming that the high viscosity of the hydrogel medium could cause the stronger PL emission and higher 1O2 generation of the PSs in hydrogel, regardless of the type of the water-soluble PSs.

Figure 2. (a) UV-vis absorption spectra of TMPyP in PBS and in hydrogel. (b) Fluorescence emission spectra of TMPyP (100 µg/mL) in PBS and hydrogel under 532 nm excitation. (c) Fluorescence emission intensities (at 632 nm) of different concentrations of TMPyP in PBS and hydrogel. (d) Photographs of the hand-written letters “SEU” (“SEU” is the abbreviation of Southeast University) using TMPyP-loaded hydrogel on a slide (illuminated under white light and 365 nm UV light, respectively). Fluorescence intensity change of SOSG for TMPyP (e) and TPPS (f) in PBS and hydrogel as a function of irradiation time.

In Vitro Anticancer Activity. To evaluate the in vitro anticancer efficacy of hydrogel,

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TMPyP in PBS, and TMPyP in hydrogel, U14 cell line was selected. The cells after various treatments were analyzed by the live/dead staining assay using calcein acetoxymethyl ester (calcein-AM) (to stain live cells green) and propidium iodide (to stain dead cells red). Figure 3a and 3b show the confocal imaging results and the corresponding quantification of the dead cell ratios, respectively. In the hydrogel group, almost all the U14 cells were alive after the laser irradiation treatment, indicating the good cytocompatibility of the hydrogel matrix. The U14 cells incubated with TMPyP solution exhibited considerably less cell death than those incubated with TMPyP-loaded hydrogel at the same concentration of TMPyP. The above results clearly demonstrated the markedly improved anticancer performance for TMPyP dispersed in hydrogel as compared with that dispersed in PBS. To evaluate the biocompatibility of TMPyP-loaded hydrogel, we thus assessed the dark cytotoxicity of different samples towards U14 cells. The results shown in Figure 3c indicated that the leachates from TMPyP-loaded hydrogel exhibited negligible cytotoxicity. We have also investigated the release profiles of TMPyP from the hydrogel as a function of incubation time (Figure S3 in the Supporting Information). Typically, the release mechanism from a biodegradable hydrogel is diffusion-controlled at an initial stage and then a combination of diffusion and degradation at a later stage.61 In our system, we observed a three-stage cumulative release profile of TMPyP from the TMPyP-loaded hydrogel, depending on the concentration of TMPyP (50, 100, and 200 µg/mL): an initial rapid release (the first stage) followed by a slower release (the second stage) and an accelerated release thereafter (the third stage). After 24 h, the total release of the PS from the hydrogel (TMPyP: 100 µg/mL) was 54%. 17


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Figure 3. (a) Confocal images of calcein-AM (green)- and PI (red)-stained U14 cells 1 h after incubation with different samples (I–VI) for 30 min and treatment with laser irradiation (532 nm, 10 mW/cm2) for 5 min. Scale bar = 25 µm. (b) Percentages of cell death obtained by the calcein-AM/PI staining results of different groups (I–VI) as shown in (a). (c) Dark cytotoxicity of sequential dilutions at different concentrations (0.5%, 1%, 2%, 5%, 10%, and 20%) of stock solutions from three groups. The three groups are leachates extracted from TMPyP-loaded hydrogel (100 µg/mL), leachates from unloaded hydrogel, and free TMPyP solution (100 µg/mL), respectively.

In Vivo Tumor Imaging and Anticancer Activity. On the basis of the in vitro results, we

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then studied the feasibility of using TMPyP-loaded hydrogel for in vivo fluorescence imaging. Two groups of U14 tumor-bearing nude mice were intratumorally administered with free TMPyP and TMPyP-loaded hydrogel (with the same TMPyP concentration of 0.1 mg/mL), respectively, and then imaged by an animal imaging system. The fluorescence intensities of free TMPyP in the tumor region showed a descending tendency after injection and no signal could be detected at 1 h (Figure 4a). In contrast, TMPyP-load hydrogel exhibited remarkably strong fluorescence intensity (Figure 4b) and slow fluorescence decay (Figure 4a). Thus, the TMPyP-loaded hydrogel could achieve excellent in vivo fluorescence imaging and prolonged retention at the tumor site. Encouraged by the superior in vitro therapeutic effect and the prolonged retention of TMPyP-loaded hydrogel, we then conducted the in vivo imaging-guided PDT on the U14 subcutaneous tumor model. All mice were randomly divided into four groups, and intratumorally treated with PBS solution, hydrogel, free TMPyP (0.1 mg/mL), and TMPyP-loaded hydrogel (dose: TMPyP 0.1 mg/mL), respectively. The PDT efficiency was assessed by measuring tumor volumes with a caliper every day. Surprisingly, the tumor growth for mice treated with TMPyP-loaded hydrogel was effectively inhibited after PDT without noticeable regrowth within a period of 7 days. In contrast, other groups exhibited little therapeutic efficacy (Figure 4c,d). Specifically, the average tumor volume of mice exposed to a 532 nm laser irradiation was significantly reduced with a V/V0 of 10.3 ± 1.9 in the free TMPyP-treated group while the ratio in the TMPyP-loaded hydrogel-treated group was even smaller at 2.4 ± 1.2. The above results indicated that the TMPyP-loaded hydrogel group produced drastically inhibitory effects against the tumor growth and decreased its propagation considerably. Besides, hematoxylin and eosin (H&E)

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staining of tumor slices from TMPyP-loaded hydrogel-treated group showed that most cells were severely damaged with few intact tumor cells in view, whereas the tumors of the other groups were only partially or scarcely damaged (Figure 4e). Systemic toxicity was further evaluated by analyzing the tissue slices from mice administered with hydrogel, free TMPyP, and TMPyP-loaded hydrogel, respectively (Figure S4, Supporting Information). In the TMPyP-loaded hydrogel-treated group, no obvious inflammation, cell necrosis, or apoptosis were observed in the main organs including heart, liver, spleen, lung, and kidneys, indicating the excellent biocompatibility of TMPyP-loaded hydrogel.

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Figure 4. (a) In vivo fluorescense imaging of U14 tumor-bearing mice after administration of the TMPyP-loaded hydrogel and free TMPyP solution over time, respectively. All images were acquired on a Cri Maestroin and PerkinElmer in vivo imaging system (Ex: 540 nm, Em: 670 nm). (b) Photographs of free TMPyP solution and TMPyP-loaded hydrogel under 540 nm excitation. (c) Representative tumor images from different groups taken at the 7th day. (d) 21


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Tumor growth curves of mice in different groups after various treatments. (e) H&E-stained tumor slices in mice treated with hydrogel, free TMPyP solution, and TMPyP-loaded hydrogel, respectively, after 7 days of PDT (Scale bar = 100 µm).

4. CONCLUSIONS In conclusion, we developed an injectable TMPyP-loaded hydrogel and realized a significantly enhanced imaging-guided anticancer performance as compared to the hydrogel-free counterpart. With the assistance of the hydrogel matrix, the encapsulated TMPyP molecules have a lower chance to collide with each other, which leads to the remarkable decrease of the self-quenching of PSs and significantly enhances the fluorescence emission and singlet oxygen generation. Both the in vitro and in vivo experiments confirmed the superior anticancer efficacy of the TMPyP-loaded hydrogel as compared to the TMPyP solution. In addition, the in vitro and in vivo tests both demonstrated that the TMPyP-loaded hydrogel was very safe, ensuring its potential clinical applications. Overall, the present work takes full advantage of the hydrogel matrix and successfully improves both the fluorescence emission and singlet oxygen generation of the PSs by using hydrogels. Such a simple and effective method to modulate the properties of PSs has not been uncovered before. We believe that the present work will provide an in-depth understanding on the interaction mechanism between PSs and hydrogels, which may promote the development of novel imaging-guided PDT systems using injectable hydrogels.

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ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: EPR experiment, in vitro release behavior, histopathological study. AUTHOR INFORMATION

Corresponding Authors *E-mail: [email protected] Author Contributions ║

These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (21673037), Graduate Students’ Scientific Research Innovation Project of Jiangsu Province Ordinary University (SJLX16_0054), Fundamental Research Funds for the Central Universities

(2242015R30016),

Six

Talents

Peak

Project

23


in

Jiangsu

Province

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(2015-SWYY-003), and Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. ZC acknowledges the support from the University of Michigan for his sabbatical.

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Enhanced Fluorescence Emission and Singlet Oxygen Generation of

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