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Sep 6, 2016 - •S Supporting Information. ABSTRACT: Real-time tracking of a drug delivery system and its therapeutic effects in vivo are crucial to d...
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Real-Time Imaging Tracking of a Dual Fluorescent Drug Delivery System Based on Zinc Phthalocyanine-Incorporated Hydrogel Xia Dong,†,# Chang Wei,†,# Hongli Chen,‡ Jingwen Qin,‡ Jie Liang,† Deling Kong,† Tianjun Liu,† and Feng Lv*,† †

Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300192, PR China ‡ School of Life Science and Technology, Xinxiang Medical University, Xinxiang, Henan, 453003, PR China S Supporting Information *

ABSTRACT: Real-time tracking of a drug delivery system and its therapeutic effects in vivo are crucial to designing a novel pharmaceutical system and revealing the mechanism of drug therapy. Multispectral fluorescence imaging can locate the drug and carrier simultaneously without interference. This advanced method enables the tracking of a drug delivery system. Herein, a doxorubicin (Dox) loaded zinc phthalocyanine incorporated hydrogel was developed as a dual fluorescent drug delivery system to monitor the release of the drug and the degradation of the carrier. An injectable thermosensitive hydrogel based on a four-arm poly(ethylene glycol) (PEG)−poly(ε-Caprolactone) (PCL) copolymer was prepared and characterized with a zinc phthalocyanine core as the drug carrier. The hydrogel degradation and drug delivery in vivo were tracked by a multispectral fluorescence imaging system in nude mice bearing hepatic tumors. Moreover, the real-time tumor inhibition progress was tracked in vivo for 18 days by bioluminescence imaging. A multispectral analysis can separate the fluorescence signals from the drug and carrier in the Dox loaded hydrogel and provide their location in the tumor tissue. The drug release and hydrogel degradation can be drastically tracked respectively without mutual interference. The fluorescence imaging results reveal improved tumor inhibitory effects of the Dox loaded hydrogel. Optical imaging allows for visible tracking of the entire drug delivery process. The Dox loaded phthalocyanine incorporated thermosensitive hydrogel is a potential visible drug delivery system for tumor therapy. KEYWORDS: imaging tracking, multispectral fluorescence, hydrogel degradation, drug release, zinc phthalocyanine

1. INTRODUCTION

release tends to be detected in a blood sample by HPLC or radioactivity measurement.7,8 For the assessment of the carrier degradation, in vitro or ex vivo assays include the gravimetric or volume determination of periodic samples or their physicochemical properties measurement of molecular weight, mechanical properties, morphology, and viscosity.9−11 The main drawback of these methods is that in vitro or ex vivo assays cannot objectively reflect the real-time process of the drug release and the status of the implanted carrier in vivo because of the complex biotissue systems and environmental forces. With the development of medical imaging, real-time imaging tracking of the drug delivery system in vivo is quite a challenge. Traditional imaging modalities, such as radionuclide imaging, computed tomography (CT), single photon emission computed tomography (SPECT), and magnetic resonance imaging

Hydrogels are three-dimensional polymeric networks that are widely applied as tissue engineering scaffolds, drug carriers, and surgical implants in tissue repair and drug delivery systems.1,2 They are biocompatible and soft in nature with large quantities of water or biological fluids, affording their excellent permeability for transport of nutrients and metabolites. Among various hydrogels, the injectable hydrogels bring about the special advantage of minimal surgical damage. Following gelation after injection, the hydrogels can become drug delivery deposits for therapy or cell-growing scaffolds for tissue regeneration.3,4 All of these features make the injectable hydrogels attractive as drug delivery systems. The drug loaded injectable hydrogels have contributed to advancements in tumor therapy.5,6 For a drug delivery system, the monitoring of drug delivery and carrier degradation is crucial to the design of a suitable carrier for therapy. To determine the release of the drug or the degradation of the carrier from a drug delivery system, an in vitro or ex vivo evaluation is commonly performed. The drug © XXXX American Chemical Society

Received: July 18, 2016 Accepted: September 1, 2016

A

DOI: 10.1021/acsbiomaterials.6b00403 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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PCL copolymer as the polymer backbone. Using nude mice bearing luciferase expressed hepatic tumor as models, not only was the fluorescent drug delivery system tracked in vivo by multispectral fluorescence imaging system, but also the therapeutic effects can be monitored in real-time by bioluminescence imaging along with the sustained delivery of the drug. From the implantation of the drug delivery system to the tumor therapy, the whole process can be visualized by optical imaging. Moreover, the relationship between the drug delivery and the tumor therapy will be further illustrated according to the imaging location and tracking. The Dox loaded zinc phthalocyanine conjugated hydrogel could be a potential visible drug delivery system for tumor therapy.

(MRI), have been considered for tracking or monitoring the drug release or carrier degradation in vivo.12−14 However, it is difficult to simultaneously track the drug release and the material degradation in the case of small exogenous markers. In many cases, a single method can only track either the drug or the carrier in a drug delivery system. In comparison with conventional in vivo imaging skills, fluorescence imaging techniques are advantageous because individual animals can be tracked over an extended period of time with high sensitivity, low radiation, and noninvasion.15,16 In vivo fluorescence imaging has been applied in the fields of tumor diagnosis,17,18 long-term detection of glucose,19,20 inflammation monitoring,21,22 tissue engineering regrowth,23 and materials degradation and drug release tracking.24−26 More importantly, fluorescence imaging can be serviced for the monitoring of two or more fluorescence signals from the different drugs and carriers by the unmixing of fluorescence spectra.25,27 For instance, dual fluorescent N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were reported for passive tumor targeting with pH-sensitive drug release to simultaneously monitor the drug and carrier in vivo.25 The body fate of the polymer and the model drug can be separated noninvasively by dual fluorescent labels. Multispectral fluorescence imaging can locate the drug and carrier simultaneously without any interferences. This advanced method gives important potential for the tracking of a drug delivery system. In our prior report, fluorescence drug delivery systems with porphyrin incorporated hydrogels have been investigated using rhodamine as the model drug by subcutaneous injections.28 The drug delivery processes have been successfully tracked with visible fluorescence imaging. However, on the basis of the difference in the microenvironment of the tumor and the subcutaneous tissue, a drug delivery system with therapeutic effects was further tracked by an intratumoral implantation with significant clinical potential. To allow fluorescence tracking in vivo, dual fluorescence drug delivery systems need to be further developed with a fluorescent carrier and another fluorescent drug. Zinc phthalocyanine conjugated polymer is a suitable fluorescent carrier for fluorescence imaging in vivo because of its intense fluorescence in the near-infrared region. Zinc phthalocyanines have recently been reported as optical probes both in vitro and in vivo because they are low light scattering and have low autofluorescence from biological tissues in the near-infrared region. We developed several zinc phthalocyanine fluorescent probes for tumor imaging.29,30 The results demonstrated that zinc phthalocyanine conjugated compounds have favorable imaging effects with good biocompatibility. Zinc phthalocyanine labeled polyethylene glycol was prepared in our lab to track and monitor the in vivo process of polyethylene glycol,31 suggesting that zinc phthalocyanine labeled polyethylene glycol has good optical stability and high emission ability in the near-infrared region. Zinc phthalocyanines could become a novel label to track the carrier in vivo because of their beneficial fluorescence emission. As doxorubicin (Dox) is an antitumor drug with fluorescence, it is optimal to be applied for the tracking of the drug release by fluorescence imaging.32,33 In this paper, a dual fluorescence drug delivery system was designed with a Dox loaded zinc phthalocyanine conjugated hydrogel. A thermosensitive fluorescence hydrogel (poly(ε-Caprolactone) (PCL)− poly(ethylene glycol) (PEG)−zinc phthalocyanine (PHA)− PEG−PCL) was synthesized by the ring-opening polymerization based on the zinc phthalocyanine as a core and PEG and

2. EXPERIMENTAL SECTION 2.1. Materials. 4-Chlorophthalonitrile was provided by Alpha Chemical (Shijiazhuang) Limited Company, China. Doxorubicin hydrochloride (Dox) was purchased from Dakub Meilun Biology Technology Co., Ltd. (Dalian, China). Poly(ethylene glycol) (PEG, Mn = 1000, Merck) was vacuum-dried at 60 °C for 12 h before use. εCaprolactone (ε-CL, Aladdin, China) was purified by vacuum distillation. Chloral hydrate (>99.0, pharmaceutical grade) was provided from Yulong Algae Co., Ltd.(Qingdao, China). Stannous octoate (Sn(Oct)2, Aladdin, China) and other reagents were all analytic reagent (AR) grade. Nude mice (7 weeks old, 20−25 g) were used. All animal procedures were conducted following the protocol approved by the Institutional Laboratory Animal Ethics Committee and the Institutional Animal Care and Use Committee (IACUC), Peking Union Medical College, People’s Republic of China. All animal experiments were performed in compliance with the Guiding Principles for the Care and Use of Laboratory Animals, Peking Union Medical College, People’s Republic of China. Animals were housed in cages with free access to food and water 2.2. Synthesis and Characterization of PCL−PEG−PHA− PEG−PCL Copolymer. Zinc phthalocyanine conjugated PEG was synthesized by the substitution reaction of 4-chlorophthalonitrile and PEG.31 Then, zinc phthalocyanine conjugated PEG (1 g) and ε-CL (1 g) were polymerized under the catalysis of Sn(Oct)2 (0.1 g) in a polymerization tube under vacuum at 120 °C for 24 h. The mixture was dissolved in dichloromethane and then precipitated with cold petroleum ether, filtrated, and dried to provide the PCL−PEG−PHA− PEG−PCL copolymer. 1 H NMR spectra were recorded on a VARIAN INOVA instrument at 500 MHz using CDCl3 as solvent and TMS as an internal reference. Infrared spectra were recorded by FTIR on a Nicolet 2000 instrument from 4000 to 400 nm −1 with KBr plates. Gel permeation chromatography (GPC) on an Agilent 110 HPLC was used to determine the molecular weight and polydispersity of the copolymer. The sample was dissolved in DMF to a concentration of 2 mg mL−1. The thermal properties of the copolymer were analyzed under a nitrogen atmosphere at a heating and cooling rate of 5 °C min−1 from −10 to 80 °C by differential scanning calorimetry (DSC) (Q 2000, TA Instruments, USA). A multimode microplate spectrum photometer (Varioskan TM Flash, ThermoFisher Scientific, USA) was used to scan UV−vis and fluorescence spectra of the PCL−PEG−PHA−PEG− PCL copolymer in an aqueous solution at a concentration of 2 mg mL−1. 2.3. Sol−Gel−Sol Phase Transition, Fluorescence Imaging in Vitro. The sol−gel−sol phase transition of the hydrogel was determined using the tube-inversion method. The optical photo was taken with a concentration of 40% at different conditions from 10 to 60 °C with a heating rate of 1 °C min−1. The gel and sol statuses were defined as “no flow” and “flow” by inverting the tube horizontally, respectively. After gelation, the hydrogel was taken for fluorescence imaging in vitro by an in vivo imaging system (Maestro EX, CRI, USA). In addition, multispectral imaging for tracking the carrier and B

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Figure 1. Synthetic route for the PCL−PEG−PHA−PEG−PCL copolymer. drug was carried out by the Maestro CRI in vivo imaging system with dual excitation wavelengths of 523 and 605 nm. 2.4. In Vitro Drug Release from the Hydrogel by HPLC. The Dox loaded hydrogel was prepared by the mixing of Dox and the polymer solution at room temperature. The in vitro drug-release profile of Dox from the hydrogel was performed in PBS at pH 7.4 or 5.5 and 37 °C by HPLC (Waters2695, USA). At preset time points, 2 mL of the extra fluid was taken to measure Dox absorption and the same volume of fresh PBS was added to maintain the volume. The concentration of released Dox was calculated from a standard curve of known absorption of 485 nm. All experiments were performed in triplicate. 2.5. Multispectral Fluorescence Imaging Tracking of the Dox Loaded Hydrogel and Bioluminescence Imaging Monitoring for Tumor Therapy. Nude mice were randomly assigned to the experimental groups and control group (n = 3 for each group). Fluorescence imaging of the hydrogel in vivo was first evaluated by a subcutaneous injection. In the experimental group, the copolymer aqueous solution (100 μL, 40%) was injected into the subcutaneous tissue of the back of each mouse. After gelation, the mice were anesthetized by an intraperitoneal injection of chloral hydrate, and then, in vivo fluorescence imaging was performed using a CRI imaging system with an exposure time of 200 ms at an excitation wavelength of 605 nm. Next, nude mice bearing tumors were modeled for imaging tracking of the Dox loaded hydrogel and its tumor therapy. Luciferase expressed hepatic cells Bel-7402 (1 × 106) in 0.1 mL of normal saline (NS) were injected into the armpit region of Balb/c nude mice, which were divided into different treatment groups (3 mice/group) when the tumor volume reached approximately 100 mm3. Then, free Dox, the hydrogel, and the Dox loaded hydrogel were injected by an

intratumoral injection, respectively. At predetermined time points, the mice were anesthetized by an intraperitoneal injection of chloral hydrate and then fluorescence and bioluminescence imaging were performed after administration from 0 to 18 days. Multispectral imaging for tracking the carrier and drug was carried out by the Maestro CRI in vivo imaging system with dual excitation wavelengths of 523 and 605 nm. The hydrogel and Dox can be unmixed with green and red to separate the spectral species from the cube file. Moreover, the single component of the hydrogel and the drug was calculated quantitatively by the Maestro software. The tumor therapy was monitored for 3 day periods by bioluminescence imaging (IVIS Lumina system, Xenogen, USA). At the end of the imaging, anesthetized mice were sacrificed and organ tissues including heart, liver, spleen, lung, and kidney from the hydrogel group and the control group were dissected and fixed in 4% formaldehyde of neutral phosphate-buffered saline. Then, the samples were processed routinely to paraffin, sectioned to 5 μm-thick slices after fixation, stained with hematoxylin and eosin (H&E), and examined by optical microscopy.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of the Copolymer. The PCL−PEG−PHA−PEG−PCL copolymer was synthesized with ε-caprolactone and zinc phthalocyanine conjugated PEG by the ring-opening copolymerization, as shown in Figure 1. On the basis of the core of the four-armed zinc phthalocyanine conjugated PEG, a star copolymer forms an amphipathic structure. Similar to the structure of the four arm porphyrin PCL−PEG copolymer,28,34 zinc phthalocyanine was conjugated as the core at the polymer backbone instead of C

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Figure 2. Sol−gel−sol phase transition of PCL−PEG−PHA−PEG−PCL hydrogel: (A) a sol state at 20 °C, (B) a gel state at 37 °C, and (C) a precipitate at 50 °C.

Figure 3. UV−vis and fluorescence spectra of the PCL−PEG−PHA−PEG−PCL copolymer in aqueous solution at a concentration of 2 mg mL−1: (A) UV−vis spectrum from 450 to 800 nm; (B) fluorescence spectra with an excitation wavelength of 630 nm.

PCL was caused by crystallization at 10−20 °C and that of PEG at −5−0 °C. The thermal characteristics are silimar to the PCL−PEG−PCL copolymer or the porphyrin conjugated PCL−PEG−PCL copolymer.34 The link of zinc phthalocyanine alone brings a slight temperature change of the endothermic and exothermic processes. 3.2. Sol−Gel−Sol Phase Transition and Optical Characterization. The basic requirements of the thermosensitive hydrogel are its injectability and location ability with a sol state at room temperature and a gel state at body temperature. The PCL−PEG−PHA−PEG−PCL copolymer is amphiphilic in nature because of the combination of hydrophilic PEG block and hydrophobic PCL and phthalocyanine blocks. The sol− gel−sol transition is presented in Figure 2. The aqueous solutions of the PCL−PEG−PHA−PEG−PCL copolymer undergo a sol−gel−sol phase transition as the temperature increases. Like other PCL−PEG−PCL hydrogels, the PCL− PEG−PHA−PEG−PCL hydrogel can turn into a nonflowing gel at physiological temperature from an injectable flowing sol at room temperature.35 When the temperature is further increased, the nonflowing gel transforms into a precipitate with the gel−sol phase transition. Because the gel−sol phase transition temperature is higher than body temperature, the hydrogel state can be retained until it degrades gradually at body temperature. The two typical intense and sharp Q-bands in the nearinfrared area from 600 to 700 nm signify the nonaggregated state of the PCL−PEG−PHA−PEG−PCL copolymer (Figure 3). Generally, the aqueous solution of phthalocyanine has only a wide peak at the Q-band because of the cofacial aggregation of phthalocyanine in water. The fluorescence spectrum of the PCL−PEG−PHA−PEG−PCL copolymer is presented in Figure 3. From the graph, it can be observed that the PCL− PEG−PHA−PEG−PCL copolymer appears as a peak at 675 nm with an excitation wavelength of 630 nm. It is suitable for the excitation and emission light in the near-infrared region to

as the end group. When the polymer material is completely degraded, the phthalocyanine molecules can be dissociated from the polymer. It is advantageous that the identification of phthalocyanine molecules can reflect and track the degradation of the copolymer materials. The chemical structures of the PCL−PEG−PHA−PEG− PCL copolymer were characterized by 1H NMR and FT-IR spectra. From the FT-IR spectrum of PCL−PEG−PHA− PEG−PCL shown in Figure S1, the peaks of OH, CH2, and C− O of PEG can be obviously observed at 3500, 2900−2800, and 1100 cm−1 in each spectrum. In the spectrum of the phthalocyanine conjugated PEG, the peak of CN at 1655 cm−1 and the benzene ring at 850 cm−1 can signify the existence of zinc phthalocyanine molecules, whereas the minor signals of zinc phthalocyanine disappeared in the spectrum of the PCL− PEG−PHA−PEG−PCL copolymer from the shielding of the long chain of PEG and PCL. 1H NMR spectroscopy was also performed to verify the structure of the PCL−PEG−PHA− PEG−PCL copolymer (Figure S2). The characteristic peaks of PCL, PEG, and phthalocyanine can be observed. The methylene protons of −(CH 2 ) 3 −, −OCCH 2 −, and −CH2OOC− are attributed to the signals of PCL units at 1.35, 1.60, 2.25, and 4.00 ppm, respectively. The PEG unit was affirmed by the characteristic peak of −CH2CHO− at 3.60 ppm. Although the Ar−H of phthalocyanine only offered minor signals at 7.96, 8.00, and 8.03 because of the shielding from the long chain of PEG and PCL, it still showed the successful synthesis of the PCL−PEG−PHA−PEG−PCL copolymer. Additionally, GPC measurement offered a mean molecular weight (Mn) of 4900 and a PDI (Mw/Mn) of 1.15 (Figure S3). To obtain the thermal characteristics of the PCL−PEG− PHA−PEG−PCL copolymer, DSC analysis was performed as shown in Figure S4. During the heating process, two endothermic peaks appeared in the range of 20−30 and 40− 45 °C, signifying the melting signal of PEG and PCL, respectively. In the cooling stage, the exothermic peak of D

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33% of the initial drug is released rapidly during the first 48 h, which is sililar to the trend for the water-soluble drug in the drug loaded hydrogel. Generally, a thermosensitive hydrogel has a multipore network structure with large quantities of water, promoting the dispersion of the water-soluble drug. With the increasing balance of the drug content around the drug loaded hydrogel, the release rate becomes obviously low. The released drug can reach 43% after 6 days. Because Dox is an acidsensitive drug, the drug release was affected by the pH value. At a neutral pH, the drug release only reached 25% after 6 days with a slower release. Due to microenvironments with different pH values, the drug delivery at the tumor region or other biotissues will be different, signifying the evaluation of the drug delivery is more objective with clinical significance with an intratumoral injection. In practical use, this drug loaded hydrogel can withstand long-term storage without the spontaneous release of drug contents before the injection because it is a mixed solution state of drug and polymer without gelation. Even so, it is best to use it as soon as possible after preparation due to the simple and fast loading process of the drug loaded hydrogel. 3.4. Fluorescence Imaging Tracking of Drug Release and Hydrogel Degradation. Fluorescent imaging has beneficial potential in clinical applications although it is a challenge to detect the whole-body fluorescence imaging of humans with comparably acceptable results in signal quality. The successful translation of fluorescence imaging to humans mainly relies on having an available system and method to scan the whole or large areas of the body. Additionally, the penetration depth is of vital importance to fluorescence imaging for humans. One study demonstrated a fluorescence imaging technique that has been successfully applied for wholebody fluorescence imaging to adult humans, suggesting the potential and feasibility for clinical detection.36 The fluorescence imaging tracking of drug release and material degradation is an important application direction. The fluorescence imaging of the PCL−PEG−PHA−PEG− PCL hydrogel in vitro and in vivo subcutaneously can be

facilitate in vivo fluorescence imaging. Due to the disaggregation of molecules, the fluorescence emission intensity of the PCL−PEG−PHA−PEG−PCL solution formed by the conjugation of phthalocyanine and polymer was stronger than that of phthalocyanine and a small molecule. The phthalocyanine polymer can protect the phthalocyanine by the spatial effect of the polymer, thereby preventing self-quenching of the fluorescence, even if the PCL−PEG−PHA−PEG−PCL solution turns into the solid gel from the sol state. 3.3. Drug Release in Vitro. To evaluate the drug release from the hydrogel in vitro, we immerse the Dox-loaded hydrogel in PBS at a pH of 5.5 over 7 days and recorded the release of Dox by HPLC at regular intervals (Figure 4). The

Figure 4. Drug release from hydrogel in vitro in PBS at pH of 5.5 and 7.4. The concentration of released Dox was calculated from a standard curve of known absorption of 485 nm. The results were expressed as mean ± SD (n = 3).

weak acid microenvironment in the tumor is the appropriate condition for the evaluation of the drug release. Approximately

Figure 5. Fluorescence imaging of phthalocyanine-incorporated hydrogel in vivo by subcutaneous injection with an exposure time of 200 ms at an excitation wavelength of 605 nm (inset: fluorescence imaging in vitro of the hydrogel in a tube). E

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Figure 6. Multispectral fluorescence imaging of mixed fluorescence of the drug and hydrogel with green and red: (A) mixed fluorescence of the drug and the hydrogel; (B) unmixed fluorescence of the drug; (C) unmixed fluorescence of the hydrogel.

Figure 7. Bioluminescence imaging and multispectral fluorescence imaging for the drug loaded hydrogel of mixed fluorescence of the drug and the hydrogel with green and red, representing one of three in each group: (A) bioluminescence imaging of the tumor; (B) mixed fluorescence of the drug and the hydrogel; (C) unmixed fluorescence of the hydrogel; (D) unmixed fluorescence of the drug.

Figure 8. Fluorescence imaging tracking of drug release after direct Dox injection (A) vs Dox loaded hydrogel injection (B) with rainbow color, with one representative of three in each group. The fluorescence signals of the drug were recorded with an excitation wavelength of 523 nm.

F

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ACS Biomaterials Science & Engineering detected by the in vivo imaging system as shown in Figure 5. This feature of satisfactory fluorescence can be used for the tracking and monitoring of the implants in vivo. To investigate the drug delivery system in vivo objectively, the Dox loaded hydrogel was injected intratumorally into the hepatic tumor. The visible drug delivery system can be tracked and monitored wholly by fluorescence imaging and bioluminescence imaging. Either the drug delivery system or the location of the tumor can be clearly observed through optical imaging. Tumor growth or inhibition can be reflected by the luminescent signals, while the drug and carrier can be identified by multispectral fluorescence, respectively. Multispectral fluorescence imaging can track several drugs or materials at the same time, which is unique compared to other imaging methods. For tracking the drug release and materials degradation in a drug delivery system, dual fluorescence labeling is relative to the quality of fluorescence imaging. Because Dox can emit the fluorescence in a wide range of the spectrum, it is important to develop a fluorescence carrier without fluorescence interference. The phthalocyanine moiety provides an obvious difference to Dox in the fluorescence emission. Because the multispectral analysis spectrum is different for each specific fluorescent material, a multicolor composite image can be generated to distinguish and separate difference labels from the drug and carrier (Figure 6). The Dox loaded hydrogel can be located in the tumor area with yellow, signifying the overlap of Dox and the hydrogel. By splitting the image, we can clearly distinguish between the drug with green and the material with red (Figure 7). Moreover, the material degradation and drug release can be quantitatively analyzed by fluorescence imaging, respectively. It can be assumed that the decay of fluorescence reflects the material degradation and drug release, respectively. In our prior report, fluorescence drug delivery systems with porphyrin incorporated hydrogels have been investigated using rhodamine or Dox as the model drug by subcutaneous injections.28,37 The drug delivery processes have been successfully tracked with visible fluorescence imaging. On the basis of the differences between the microenvironment of the tumor and the subcutaneous tissue, the Dox loaded hydrogel was further tracked for the tumor therapy by an intratumoral implantation with significant clinical potential in the present paper. As shown in Figure 8, a sustained decrease in the fluorescence signal was observed from the Dox loaded hydrogel over time after implantation, while the free Dox was rapidly distributed in less than 3 days without persistent retention at the administration site. The hydrogel significantly enhanced the persistent retention of Dox, and the decay of the fluorescence intensity directly reflected the drug release process. A qualitative comparison of the drug release in Figure 9 indicated that the release can reach 90% after 6 days with a sustained process. In the first 2 days, a rapid release reached more than 60%. From then on, the release rate gives a steadily sustained decrease through day 6 with 9% fluorescence signal. On the ninth day, no obvious fluorescence signal remained, signifying the complete release of the drug. Compared to the in vitro drug release, it causes a more rapid release in vivo by intratumoral implantation. This observed difference in the drug release was a result of the different bioenvironmental factors that affect the diffusion and permeability of the Dox loaded hydrogel in vitro and in vivo. Overall, the in vivo results revealed a reasonable trend and correlation with the in vitro release. As a phthalocyanine moiety was conjugated as the core of the hydrogel backbone, the degradation of the hydrogel can be

Figure 9. Quantitative analysis of drug release after direct Dox injection (red) vs Dox loaded hydrogel injection (black) by fluorescence imaging. The single component of the drug was calculated quantitatively by the Maestro software. The results were expressed as mean ± SD (n = 3).

analyzed qualitatively and quantitatively by measuring the relative fluorescence intensity of phthalocyanine with respect to the time of implantation. To track the in vivo erosion process of the hydrogel, the fluorescence signal from the hydrogel was further monitored for 18 days. As shown in the fluorescence imaging in Figure 10, a progressive decrease in the fluorescence signals was observed from the hydrogel over the time after implantation. Because the degradation of the hydrogel is a slow process for metabolism, the fluorescence signal shows an increasing decay for 18 days. Due to the absorption and permeation, the fluorescence of the hydrogel shrinks rapidly in the first 2 days. Then, with the invasion of enzymes and other biomolecules, the degradation of the hydrogel is accelerated gradually, leading to the increasing decrease of fluorescence intensities. It gave a similar degradation mechanism as that of other PEG and PCL implants.38,39 A qualitative comparison of the images further demonstrated the hydrogel degradation process in Figure 11. The fluorescence signals decrease by 27% within the first 2 days. With continued degradation over 18 days, it retains the fluorescence signal of approximately 23% compared to the original fluorescence. The hydrogel can be noninvasively tracked from the decay of the total fluorescence signals in real time. The fluorescent hydrogel for tracking in vivo generally can be a different fluorescence compound formed by conjugation or entrapment. Compared to other fluorescent dyes labeled at the polymer end, the fluorescence tag at the core of the polymer backbone can ensure the fluorescence efficiency without the early selective breakage of the fluorescent tag. Similar to the porphyrin conjugated hydrogel,28,34 the phthalocyanine conjugated polymer can be tracked long-term until the material has completely degraded. 3.5. In Vivo Bioluminescence Imaging for Tumor Therapy. Over a period of 18 days following the implantation of the drug delivery system, in vivo bioluminescence imaging was performed to track and monitor tumor inhibition by luciferase expression simultaneously. As shown in Figure 12, bioluminescence imaging revealed that the Dox loaded hydrogel can promote sustained tumor inhibition efficiently with a dynamic process. Compared to the Dox loaded hydrogel group, the free group caused some inhibition of tumor progression, while rapid tumor growth was observed in the control and the hydrogel group. Quantitative analysis of imaging further illustrated the therapeutic efficacy of the Dox loaded hydrogel (Figure 13). In the control group and the G

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Figure 10. Hydrogel degradation from the hydrogel with rainbow color by fluorescence imaging, with one representative of three in each group. The fluorescence signals of the hydrogel were recorded with an excitation wavelength of 605 nm.

Figure 13. Tumor inhibition rate of drug therapy by bioluminescence imaging. Bioluminescence imaging was recorded after administration from 0 to 18 days. The results were expressed as mean ± SD (n = 3).

Figure 11. Quantitative analysis of hydrogel degradation by fluorescence imaging. The single component of the drug was calculated quantitatively by the Maestro software. The results were expressed as mean ± SD (n = 3).

hydrogel group, the tumor can increasingly grow 2- or 3-fold after 18 days. The tumor growth is inhibited in the Dox loaded

Figure 12. In vivo bioluminescence imaging for tumor therapy, with one representative of three in each group. Luciferase expressed hepatic cells Bel7402 (1 × 106) in 0.1 mL of normal saline (NS) were injected into the armpit region of Balb/c nude mice, which were divided into four different groups including control, Dox, hydrogel, and Dox loaded hydrogel. H

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hydrogel group and free Dox group only after administration for 6 days.Therefore, increased inhibition is observed with the sustained release of Dox from the hydrogel, and the tumor volume can decrease 40%. However, it cannot inhibit the tumor growth further in the free Dox group with the rapid metabolism of Dox. Even so, tumor growth still can be inhibited finally after 18 days because of the efficacy of Dox. The imaging comparison revealed the better tumor inhibitory effect of the Dox loaded hydrogel than free Dox at the same concentration because the hydrogel retains Dox in the tumor longer with a sustained release. In vivo imaging tracked the real-time process of the tumor inhibition. The tumor inhibition was dependent on the release of the drug, suggesting the importance of the drug delivery system with the sustained and controlled release. For in vivo implants, the biocompatibility and safety of the hydrogel might be a concern. The H&E staining analysis suggested that the hydrogel did not bring obvious histopathological changes or abnormal damage compared to the control group (Figure S5). It confirms the good biocompatibility and safety of this implanted hydrogel in vivo.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.6b00403. Characterization of the PCL−PEG−PHA−PEG−PCL copolymer and in vivo biocompatibility of the hydrogel by H&E staining (PDF)

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REFERENCES

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4. CONCLUSIONS In summary, a dual fluorescent drug delivery system was successfully designed for the tracking of drug delivery and tumor therapy by fluorescence imaging and bioluminescence imaging. An injectable thermosensitive phthalocyanine incorporated hydrogel based on a four-arm PEG−PCL copolymer was prepared and characterized as the drug carrier. The Dox loaded hydrogel was tracked by imaging for tumor therapy using nude mice bearing luciferase expressed hepatic tumors as models. The fluorescent drug delivery system was tracked in vivo by a multispectral fluorescence imaging system, and the tumor therapy effect can be real-time monitored by bioluminescence imaging along with the delivery of the drug, allowing for the complete monitoring of the drug delivery system by optical imaging.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: 86-22-87893236. E-mail: [email protected]. Author Contributions #

X.D. and C.W. contributed equally to this work and should be considered cofirst authors. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 31200732, 81601595), the Natural Science Foundation of Tianjin, China (Nos. 14JCYBJC17400, 16JCYBJC27800), and the Program for Innovative Research Team in Peking Union Medical College. I

DOI: 10.1021/acsbiomaterials.6b00403 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsbiomaterials.6b00403 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX