Biomaterials xxx (2014) 1e10

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Theranostic porphyrin dyad nanoparticles for magnetic resonance imaging guided photodynamic therapy Xiaolong Liang a, Xiaoda Li b, Lijia Jing b, Xiuli Yue b, Zhifei Dai a, * a b

Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 April 2014 Accepted 21 April 2014 Available online xxx

Photodynamic therapy (PDT) is a site-specific treatment of cancer involving the administration of a photosensitizer (PS) followed by the local light activation. Besides efficient PSs, image guidance is essential for precise and safe light delivery to the targeting site, thus improving the therapeutic effectiveness. Herein, we report the fabrication of theranostic porphyrin dyad nanoparticles (TPD NPs) for magnetic resonance imaging (MRI)-guided PDT cancer therapy, where the inner metal free porphyrin functions as a photosensitizer for PDT while the outer Mn-porphyrin serve as an MRI contrast agent. Covalent attachment of porphyrins to TPD NPs avoids premature release during systemic circulation. In addition, TPD NPs (～60 nm) could passively accumulate in tumors and be avidly taken up by tumor cells. The PDT and MRI capabilities of TPD NPs can be conveniently modulated by varying the molar ratio of metal free porphyrin/Mn-porphyrin. At the optimal molar ratio of 40.1%, the total drug loading content is up to 49.8%, 31.3% for metal free porphyrin and 18.5% for Mn-porphyrin. The laser light ablated the tumor completely within 7 days in the presence of TPD NPs and the tumor growth inhibition was 100%. The relaxivities were determined to be 20.58 s
1 mM
1 for TPD NPs, about four times as much as that of Mnporphyrin (5.16 s
1 mM
1). After 24 h intravenous injection of TPD NPs, MRI images showed that the whole tumor area remained much brighter than surrounding healthy tissue, allowing to guide the laser light to the desired tumor site for photodynamic ablation. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Porphyrin Theranostic Nanoparticles Photodynamic therapy Magnetic resonance imaging

1. Introduction In recent decades, photodynamic therapy (PDT) has become one of the most promising noninvasive and safe treatment protocols for cancer therapy [1e4]. As a highly localized therapy, PDT involves focusing the light outside the body on photosensitizers (PSs) in the disease tissue in vivo to produce reactive oxygen species (ROS) to cause an effective and selective destruction of diseased tissues without damaging to the surrounding healthy ones [5e7]. However, most of clinically used photosensitizers are hydrophobic and strongly aggregate in aqueous media [8,9]. This aggregation significantly reduces their photosensitizing efficacy since only monomeric species are appreciably photoactive [9]. To improve the PSs delivery and effectiveness, liposomes have been widely applied to accommodate PSs [9,10]. But, the unavoidable premature release

* Corresponding author. Tel./fax: þ86 010 82529377. E-mail addresses: [email protected], [email protected] (Z. Dai). URL: http://bme.pku.edu.cn/~daizhifei

from liposomes results in reduced efficacy of treatment because the release of the PS drugs is not a prerequisite for PDT action unlike conventional chemotherapy [11,12]. In addition, the drug loading contents are generally less than 10% in liposomes. Attacking this problem head on, nanocarriers with a high PS loading efficiency but no premature release are much sought after. Besides, aiming for the improvement of the PDT efficacy and safety, the location and size of the tumors and the distribution of PSs must be identified before PDT treatment, and the therapeutic effectiveness has to be assessed after therapy. All these tasks could be carried out by integrating contrast-enhanced diagnostic imaging capability with PDT. Therefore, the imaging guided PDT was investigated as one of the most exciting strategies for cancer treatments [3,13e16]. The nanoparticles combining PSs and imaging functions are becoming an attractive choice for the image-guided PDT. Among various diagnostic imaging modalities, magnetic resonance imaging (MRI) is currently one of the most powerful diagnostic tools due to its capability to provide threedimensional topographical data with high spatial resolution and timely feedback information of disease tissue in vivo [17e23]. In

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addition, the utilization of contrast agents can improve the sensitivity of MRI greatly. Paramagnetic Gd3þ complexes have been the predominant T1 contrast agents in clinical MR imaging due to their high contrast ability [24e28]. Yet, the renal toxicities of the Gd3þ-based contrast agents have raised our concerns [29e 32]. Iron oxide NPs are the first clinically used T2 contrast agents [33]. But, a negative contrast induced by iron oxide NPs may be confused with areas of innate hypointensities [34]. As another early important family of T1 paramagnetic contrast agents, Mn complexes have attracted intensive interests recently [35]. Mn porphyrins have shown great potential for the use of T1 contrast agents in cancer imaging due to the excellent stability, high longitudinal relaxivity and tumorous “preferential uptake” property [36]. Especially, a water soluble metalloporphyrin of Mn(III)-meso-tetrakis(4-sulfonatophenyl) porphyrin (Mn-TPPS4) has been reported as a stable and cell-permeable contrast agent with comparable longitudinal (T1) magnetic resonance relaxivity with that of the commercial MRI contrast agents Gd-DTPA [37e 41] and favoring to localize in tumors [42,43]. However, like the other paramagnetic metal complexes, Mn porphyrins have short half-lives in vivo, conducing to poor imaging outcomes [44]. Nanotechnology is evolving with the goal to achieve high MR contrast by keeping the target specificity intact. With this aim in mind, the nanoparticles incorporating Mn complexes are gaining tremendous attention. This paper reported, as a proof of concept, on a versatile theranostic porphyrin dyad nanoparticles (TPD NPs) for the MRI guided PDT by loading two types of porphyrin derivatives: metal free porphyrin as a photosensitizer for PDT of cancer and Mnporphyrin as a T1 contrast agent for MRI (Fig. 1). We synthesized a porphyrin grafted lipid (PGL) of 4,40 -(2-((4-(dihexadecylamino)4-oxobutanoyloxy)methyl)-2-((4-oxo-4-(4-(10,15,20triphenylporphyrinyl-5-(4-Aminophenyl))butanoyloxy)methyl) propane-1,3-diyl) bis (oxy) bis (4-oxobutanoic acid) according to our reported method [45]. Then, TPD NPs were fabricated from this porphyrin grafted lipid with carboxyl groups through a self-

assembly process using the conventional baghamn method [45], followed by the covalent attachment of Mn(III)-5-(4aminophenyl)-10,15,20-tri(4sulfonatophenyl)porphyrin (NH2Mn-TPPS3) to the surface of the nanoparticles through the formation of stable amide bonds. Both MRI contrast behavior and PDT ablation of cancer cells for the obtained TPD NPs were evaluated in vitro and in vivo. 2. Materials and methods 2.1. Materials Pyrrole, benzaldehyde, AlCl3, manganese acetate were purchased from Sigmae Aldrich. N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC$HCl) N-hydroxysuccinimide (NHS) and N, N-Dimethylformamide (DMF) are commercially available and were used without further purification. MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] is a product of Sigma. Calcein-AM was purchased from ALEXIS corporation and Deuterium oxide (99.9 atom % D) from Isotec Inc. Deionized (DI) water was obtained by a Milli-Q Water Purification system. 2.2. Synthesis of NH2-TPPS3 and NH2-Mn-TPPS3 NH2-TPPS3 and NH2-Mn-TPPS3 were prepared according to the literature [46]. Briefly, 5,10,15,20-Tetraphenyl porphyrin was first synthesized from benzaldehyde and pyrrole with AlCl3 as catalyst, followed by nitration with fuming nitric acid and subsequent reduction, and reaction with concentrated sulfuric acid to get NH2TPPS3. Mn-porphyrin of NH2-Mn-TPPS3 was easily obtained by metallizing with manganese acetate. 2.3. Fabrication of PGL NPs 3-5 mg of PGL was dissolved in 2e5 mL of CHCl3, which was then removed by a nitrogen stream to form a thin film on the wall of vial. The film was then dried under vacuum overnight. Then, 2e5 mL of PBS was added to the vial, followed by incubation in a 60  C water bath for 30 min. The mixture was vortexed for 20 min followed by a 10e15 min sonication with a probe-type sonicator until a clear and transparent solution was obtained. 2.4. Preparation of TPD NPs To the solution of PGL NPs was added EDC. After 10 min of stirring, NHS was added to the solution and the mixture was stirred for 20 min at room temperature. Then, the solution of Mn-porphyrin derivative with amino group was added and the mixture was further stirred for 16 h at room temperature. The used molar ratio of PGL : EDC: NHS was 1:2.5:2.5. A series of TPD NPs were prepared by choosing various Mn-porphyrin/PGL molar ratios. After reaction, the solution was dialyzed against PBS solution using a 12,000 Da membrane (Millipore) for 3e4 days with frequent replacement of the PBS solution to remove excessive EDC, NHS and unconjugated Mn-porphyrin. 2.5. Nanoparticle characterization UVeVis spectra were performed on a Varian 4000 UVeVis spectrophotometer. Fluorescence emission spectra were tested using a Varian Cary Eclipse fluorescence spectrophotometer. Dynamic light scattering (DLS) measurements were performed by a 90Plus/BI-MAS instrument (Brookhaven Instruments Co., U.S.A). The TEM sample was prepared by immersing a 300-mesh Formvar-coated copper grid into the PGL NPs and TPD NPs suspension (0.2 mM). After 10 min of incubation, samples were blotted away and the grids were negatively stained with freshly prepared and sterile-filtered 4% (w/v) uranyl acetate aqueous solution for 5 min at room temperature. The grid thus obtained was then washed with distilled water and dried in air. Finally, TEM images were obtained using H-7650 with a tungsten filament at an accelerating voltage of 100 kV. 2.6. Singlet oxygen detection by chemical method

Fig. 1. Schematic illustration for the formation of theranostic porphyrin dyad nanoparticles.

Detection of singlet oxygen was carried out chemically as described in the literature [11,12,45], disodium salt of 9,10-anthracenedipropionic acid (ADPA) was used as a singlet oxygen sensor, which could be bleached to its corresponding endoperoxide by singlet oxygen. Decrease in optical density of ADPA at 378 nm was monitored spectrophotometrically and recording as a function of irradiated time. Briefly, 3 mL of 4.88 mM PGL NPs or TPD NPs in D2O was mixed with 150 mL of ADPA in D2O (7.5 mM). Blank group with ADPA alone dispersed in D2O were used as a reference. The above mixture was irradiated with a laser source of 650  5 nm, and their optical densities at 378 nm were recorded every 2 or 5 min by a UV/Vis spectrophotometer.

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X. Liang et al. / Biomaterials xxx (2014) 1e10 2.7. In vitro studies with tumor cells Hela cells (ATCC) and HUVECs cells were routinely cultured in RPMI-1640 medium (GIBCO) with 10% fetal bovine serum (FBS). Unless otherwise mentioned, all the experiments were performed at room temperature. 2.7.1. Uptake and imaging The Hela cells were trypsinized and resuspended in culture medium with a concentration of 8  105 cells/mL. Next, 2 mL of medium was combined with 0.10 mL of cell suspension on 35 mm culture plates, which were placed in an incubator in a humidified 5% CO2 atmosphere at 37  C for 24 h. Then the cells were washed carefully with phosphate-buffered saline (PBS), and 2 mL of fresh medium was replaced into each well. Certain concentration of PGL NPs or TPD NPs solution (as determined by absorption measurements) was added to each plate and carefully mixed to obtain the final concentration of 5 mM on each well. The treated cells were returned to the incubator (37  C, 5% CO2). After 4 h of incubation, the plates were washed thoroughly with sterile PBS and fresh medium was added, followed by fixation and counterstaining with DAPI. Finally, the cells were placed under a confocal laser scanning microscope (Zeiss, LSM 510) equipped with a 63 oil immersion objective for observation and imaging. The images were firstly recorded separately in each fluorescence channel and merged afterwards. 2.7.2. In vitro PDT efficacy assay The cells were seeded on 96-well plates at a density of 2  104 cells/well. After growing overnight, the cells were used for the experiments. Predetermined concentrations of PGL NPs or TPD NPs, as evaluated by UVeVis absorption measurements, were added to the designated wells, the final sample concentration of each well was ranging from 0.01 to 30 mM. After incubation for 4 h in the dark at 37  C, the wells were carefully rinsed three times with sterile PBS. Next, a 0.2 mL of fresh medium was added, and each well was immediately irradiated for 20 min with broadband visible light using xenon lamp (150 W) equipped with a filter passing light of 400e700 nm (The power at the cell level was 180 J/cm2) [45]. Finally, the plates were incubated at 37  C in the dark overnight. Cell viability was estimated by MTT assay. 2.7.3. MTT assay The number of live cells is directly proportional to the absorbance of formazan at 570 nm (produced in living cells by the cleavage of MTT by dehydrogenases). Briefly, solution of MTT in sterile PBS were prepared with a concentration of 5 mg/mL, and 20 mL was added to each well and incubated for another 4 h at 37  C with 5% CO2. Next, the medium was carefully removed and 150 mL of DMSO was added into the well. Then the absorbance at 570 nm was measured using a microplate reader (Multidkan MK3, Thermo). Cells incubated with serum-supplemented medium represent 100% cell survival. Each concentration and light dose were run in five replicate wells, and each experiment was repeated for three times. 2.8. Inductively coupled plasma optical emission spectrometry (ICP-OES) Manganese quantification of TPD NPs was performed on an Optima 5300 DV (PerkinElmer, Waltham, USA) ICP-OES instrument. Briefly, 5 mg of TPD NPs was first hydrolyzed by nitric acid (65%; 3 mL), then the solvents were completely removed and the residues were dissolved in 3 mL of perchloric acid and dried again. Later the residue was redispersed in 5 ml of water for detection. 2.9. In vitro and in vivo MRI measurement In vitro MR imaging of TPD NPs with different Mn-porphyrin/PGL molar ratios including 0%, 9.9%, 19.8%, 40.1% and 51.6% were carried out on a 0.5 T Shanghai Niumag Corporation ration NM120-Analyst at 25  C. For T1-weighted MR imaging, the following parameters were adopted: field of view (FOV) ¼ 40 mm  40 mm, matrix size ¼ 128  256, section thickness ¼ 2 mm, echo time (TE) ¼ 19 ms, repetition time (TR) ¼ 520 ms, number of averages ¼ 3. The MR imaging of aqueous TPD NPs with Mn-porphyrin/PGL molar ratio of 40.1% was acquired on a 3 T MRI scanner of PHILIPS-14B464A, and the following parameters were adopted: TR ¼ 2300 ms, TE ¼ 15.5 ms. For animal imaging, nude mice bearing HT-29 tumor were first anesthestized and then intravenously injected with aqueous TPD NPs with Mn-porphyrin/PGL molar ratio of 40.1% (or PGL NPs) (dose: 200 mL, 3.8 mg mL
1), later imaged with the Pharmascan 70/16 US In-vivo MRI system (Bruker, 7.0 T) with the parameters TR ¼ 1000 ms; TE ¼ 9 ms. MRI data sets were first acquired to serve as baseline controls, and then animals were rescanned 30 min, 24 h post-injection. 2.10. Therapeutic efficacy of TPD NPs in HT-29 tumor-bearing mice The protocol experiments were approved by the Institutional Animal Care and Use Committee at the Institute of Biophysics of Peking University. Female BALB/c athymic nude mice (5e6 weeks old) were implanted with HT-29 tumors by a subcutaneous injection of 106 cells in PBS. Treatments were started when the tumors volume reached 110  5 mm3, which was designated as day 0. Mice were divided into four groups for the treatments (saline, light alone, drug alone and both drug and light), consisting of seven mice in each group. TPD NPs with Mn-porphyrin/PGL molar ratio of 40.1% (200 mL, 3.8 mg mL
1 suspension in PBS) were injected into

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the mice via tail vein at double doses on days 1 and 5. At 24 h post-injection, mice were irradiated for 30 min at the tumor site with a laser (650  5 nm, 200 mW). Tumor sizes were measured every 3e4 days for the duration of the experiment. Tumor volume was calculated as V ¼ (L  W2)p/6, where L and W are the longer and shorter diameter of the tumor. In addition, after treatment for 32 days, animals were sacrificed, liver, spleen, kidney, skin, heart, lung, tumor tissues or tissues at the original site of tumor were collected from the four group mice and fixed in formalin. Fixed tissue specimens were embedded in paraffin, stained with hematoxylin and eosin (H&E), and subsequently examined by light-field microscopy to evaluate the dark toxicity and photocytotoxicity of TPD NPs.

3. Results and discussion 3.1. Preparation and characterization of TPD NPs The porphyrin grafted lipid was synthesized by coupling 5-(4aminophenyl)-10,15, 20-triphenylporphyrin and dihexadecylamine to multivalent pentaerythritol. Then, the obtained PGL was used to assemble into nanoparticles by hydrating its dry thin film upon ultrasonication with a probe-type sonicator. The covalent linkage of metal free porphyrin to the PGL NPs resulted in a photosensitizer loading content of 38.45%, significantly higher than the liposomal photosensitizer (usually 9.9% > 19.8% > 40.1% > 51.6%. These results were in good accordance with the singlet oxygen generation data (Supplementary Fig. S6c). MTT results were further confirmed by the bright field images and fluorescence images of Hela cells before and after photoirradiation (Fig. 4B). After treatment with 1 mM TPD NPs with a Mnporphyrin/PGL molar ratio of 40.1% followed by 20 min irradiation and 24 h incubation in the dark. Cell survival was then determined with Calcein AM, a fluorescence dye that stains the cell green once the cell is still living. The impregnated Hela cells displayed shrinkage and deformation, showing significant damage (Fig. 4Bb). We could clearly observe that the number of living cells decreased greatly (Fig. 4Bd). In contrast, the control group without light irradiation showed that such TPD NPs alone did not cause cell death (Fig. 4Bc). These results further demonstrated the low cytotoxicity and significant phototoxicity of TPD NPs. 3.4. MR contrast properties of TPD NPs The effect of the Mn-porphyrin/PGL molar ratios on MR imaging capability of TPD NPs were investigated on a Shanghai Niumag Corporation ration NM120-Analyst at 25  C (0.5 T) [52]. The concentration of TPD NPs was set at 100 mM (as determined by the UV/ Vis spectra) while the Mn-porphyrin/PGL molar ratio was varied from 0 to 51.6%. T1-weighted MR images of phantoms containing TPD NPs showed that MR signal intensities increased with the increasing the Mn-porphyrin/PGL molar ratios. The longitudinal relaxation times (T1), represented as a bar at the right side of the adjacent corresponding samples in Supplementary Fig. S9, were

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Fig. 4. (A) Hela cell viability after incubation for 4 h at different concentration of TPD NPs with irradiation for 20 min with reference to the irradiated but untreated cells as having 100% survival. Cell viability was assayed by the MTT method (values: mean  standard deviation). (B) Fluorescence microscopic observations of Hela cells treated with 1 mM TPD NPs with a Mn-porphyrin/PGL molar ratio of 40.1% and stained with calcein AM. a,b) Bright-field images. c,d) Fluorescence images. a,c) Before irradiation. b,d) After irradiation with light of 400e700 nm for 20 min followed by incubation for 24 h at 37  C in the dark (Scale bar is 20 mm).

found to decrease gradually with the increasing the Mn-porphyrin/ PGL molar ratios and nearly reach a plateau at the 40.1% molar ratio, which was similar to that of 51.6%. It was well in accordance with the result shown in the corresponding T1-weighted images. Taking account of the low cytotoxicity and significant phototoxicity of the TPD NPs with the molar ratio of 40.1%, this structural characteristics of the porphyrin dyad is highly beneficial for the use as a theranostic agent where the inner metal free porphyrin functions as photosensitizers for PDT and the outer Mn-porphyrin serves as MRI contrast agent. The MR contrast properties of the TPD NPs with Mn-porphyrin/ PGL molar ratio of 40.1% were further investigated at different Mn concentrations of 18.75, 37.5, 75, 112.5, 150 and 300 mM (Fig. 5), in comparison with Mn-porphyrin molecules. The relaxivities were determined to be r1 ¼ 20.58 s
1 mM
1 for the nanoparticles, which was approximately four times as much as that of Mn-porphyrin (5.16 s
1 mM
1) (Fig. 5b). To further verify the capability to be a positive T1 contrast agent, T1-weighted MR images of the nanoparticles were acquired at 25  C on a 3 T MRI scanner of PHILIPS14B464A. As seen in Fig. 5c, MRI signals were significantly enhanced compared to the control Mn-porphyrin phantom. The immobilization of Mn-porphyrin complexes on the surface of TPD NPs results in an enhanced relaxivity of the central Mn(III) ions caused by an increase in rotational correlation time (tR) [53,54]. It is consistent with the SolomoneBloembergeneMorgan theory [55]. In addition, it greatly enhanced water accessibility to the manganese paramagnetic centers and therefore resulting in highly improved MRI efficacy [56,57]. After intravenously injecting TPD NPs with the Mn-porphyrin/ PGL molar ratio of 40.1% to the nude mice bearing HT-29 tumor, the T1-weighted MR images of the tumor were obtained with the Pharmascan 70/16 US In-vivo MRI system (Bruker, 7.0 T). It was seen that the MR signals increased gradually with the time prolonging (Fig. 6aec). The tumor area turned into obviously bright after 0.5 h post-injection of nanoparticles, indicating a homogeneous distribution of the contrast agent within the tumor. Moreover, after 24 h injection, the whole tumor area was becoming much brighter, suggesting large amount of TPD NPs were accumulating in the tumor area. The tumor sites exhibited obvious increase in T1-weighted MR intensity compared with that before injection, indicating that the TPD NPs remain in the tumor sites as long as 24 h to provide adequate time for the following treatment. The MRI signals of the tumor tissue were much stronger than those

of normal tissue, showing that TPD NPs could target the tumor passively. This effect was very important for the subsequent PDT, because the obvious MR images allowed the laser light to be precisely delivered to the desired tumor site for photodynamic ablation, thus improving the therapeutic efficiencies without causing damages to the healthy tissues. On the contrary, the administration of PGL NPs caused no signal enhancement within the studied time course (Fig. 6def), indicating that MRI signal enhancement of tumor was mainly due to the existence of Mn-porphyrin on the surface of TPD NPs. 3.5. In vivo PDT effect of TPD NPs The in vivo anti-tumor efficiency of TPD NPs with the Mnporphyrin/PGL molar ratio of 40.1% were validated in mice bearing HT-29 human colon cancer, as shown in Fig. 7. The tumorbearing nude mice were divided into 4 groups with 7 mice in each group: PBS only (no drug and no light), drug-only, light-only and drug þ light. The nanoparticles (200 mL, 3.8 mg mL
1 suspension in PBS) were injected into the mice drug-only and drug þ light groups via the tail vein, while PBS (200 mL) in control and light-only groups, respectively. Then, the tumor-bearing mice of light-only and drug þ light groups with continuous anesthesia were irradiated at the tumor site (two times; 30 min each time; 4-day intervals) with a 650  5 nm laser (200 mW) at 24 h post-injection. The therapeutic effectiveness was estimated by the tumor volume measurement (Fig. 7a). Inoculated tumors in PBS only, drug-only, light-only groups grew rapidly, with no significant difference in final tumor sizes. On the day 32, the mean tumor volume reached to about 16 times as big as the initial volume. The result indicated that the tumor growth could not be affected by either TPD NPs or laser irradiation alone. In contrast, unlike the other 3 groups, tumors in drug þ light group deceased from 110  5 mm3e0 mm3 on the 32nd day after treatment. There was no palpable tumor at the original tumor site on days 7e32 in 100% of the mice. Compared with control group (1735  231 mm3 at day 32), the tumor growth was inhibited by 100%, suggesting that sufficient accumulation of TPD NPs could trigger the great significant PDT effects locally for effective tumor ablation. That is to say, although metal free porphyrin units were covalently encapsulated inside the nanoparticles, the formation of singlet oxygen through light activating could diffuse out to result in damage to the tumor tissue.

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Fig. 5. The aqueous dispersions of TPD NPs with Mn-porphyrin/PGL molar ratio of 40.1% at different Mn concentrations: 18.75, 37.5, 75, 112.5, 150, 300 mM: (a) Photographs; (b) Spinlattice 1/T1 relaxation rates compared to Mn-prophyrin, and relaxivity rates (r1) were obtained by comparing the measured (symbols) and theoretical (lines) values; (c) T1-weighted MR images of phantoms containing TPD NPs compared to Mn-porphyrin.

The above results were confirmed by the photographs of the representative mice from the 4 different groups recorded during treatments (Fig. 7b, c and Supplementary Fig. S10). At 6 days postinjection, the mice treated with both TPD NPs and light demonstrated a severe tumor necrosis at the site of the laser irradiation, showing significant therapeutic effects (Fig. 7b). 15 days after PDT, it was apparent that the necrotic scar tissue was healing and that normal tissue had begun to regenerate, and 25 days later, complete tumor regression could be observed without any signs of tumor regrowth. In contrast, the animals treated with PBS (Fig. 7c), TPD NPs alone (Supplementary Fig. S10A) or light irradiation alone

(Supplementary Fig. S10B) continued to increase in tumor size, indicating that only combination of both light and TPD NPs can perform photodynamic therapy of tumor. These results further demonstrated the accumulation of TPD NPs with the Mnporphyrin/PGL molar ratio of 40.1% into tumor and their excellent photodynamic effect to destroy tumor tissue. The potential in vivo toxicity induced by different treatments was evaluated by the body weight of animals after treatment (Supplementary Fig. S11). There was no significant body weight loss after treatments up to 32 days in all groups, demonstrating that all treatments including intravenous injection of the agent and

Fig. 6. In vivo T1-weighted MRI images of nude mice bearing HT-29 tumor before and after administration of TPD NPs with the Mn-porphyrin/PGL molar ratio of 40.1% (aec) and PGL NPs (def) acquired at different time intervals: before (a, d) and after injection for 30 min (b, e) and 24 h (c, f). (Red circles indicate the tumor). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 7. (a) Change of tumor volumes as a function of time for the different treatment groups. Photographs showing therapeutic response of the tumor bearing nude mice after treatment with (b) TPD NPs with the Mn-porphyrin/PGL molar ratio of 40.1% or (c) PBS followed by irradiation with light of 650  5 nm.

photodynamic effect were reasonably well-tolerated by the tumorbearing animals and the photodynamic therapy showed no unacceptable toxicity during the entire observation time. After treatments for 32 days, the mice were sacrificed, and the tissue slice such as tumor, heart, kidney, liver, lung and spleen were collected and stained with hematoxylin and eosin (H&E). As seen in Fig. 8, tumor cells in the original site of tumor tissue were completely disappeared in the drug þ light group (Fig. 8B), indicating severe death of tumor cells and the successful results of PDT. In contrast, in the other three control groups (Fig. 8A and Supplementary Fig. S12), there were no observed apoptosis and necrosis in the tumor tissue, further confirming that only combination of TPD NPs and laser irradiation can generate singlet oxygen to induce tumor cell death. In addition, H&E results demonstrated that there were no observation of toxicological lesions in normal tissues from the TPD NPs treated mice (Irradiation or not), all of them were in good condition and wellpreserved. TPD NPs exert cytotoxicity only upon laser irradiation, showing an enormous advantage over traditional chemotherapeutic drugs.

4. Conclusions A versatile theranostic porphyrin dyad nanoparticles have been successfully fabricated through a self-assembly of a metal free porphyrin grafted lipid, followed by covalent attachment of Mnporphyrin onto the surface of nanoparticles for MRI guided PDT of cancer. Both PDT and MRI capabilities of TPD NPs can be conveniently modulated by optimizing the molar ratio of metal free porphyrin/Mn-porphyrin. At the molar ratio of 40.1%, the total drug loading content of TPD NPs is up to 49.8%, much more than liposomes (less than 10%). It substantially minimize use of inactive materials. In addition, the premature release is avoided during systemic circulation. The in vivo experiments showed that sufficient accumulation of TPD NPs triggered the great significant PDT effects locally for tumor ablation. The immobilization of Mn-porphyrin on the surface of TPD NPs results in significantly enhanced MRI signal intensity. The obvious MR images allowed the laser light to be precisely delivered to the desired tumor site to improve the therapeutic efficiencies, causing no damage to the surrounding healthy tissues. Moreover, the unique nanostructure of TPD NPs may be

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Fig. 8. Representative hematoxylin and eosin stained sections of the indicated organs from mice after treatments up to 32 days: (A) PBS only; (B) TPD NPs with the Mn-porphyrin/ PGL molar ratio of 40.1% followed by laser irradiation.

used as a carrier for different chemotherapeutic drugs and probes, facilitating fabrication of platform for multimodal imaging guided combination of photodynamic- and chemo-therapy.

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Acknowledgments

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This work was financially supported by National Natural Science Foundation for Distinguished Young Scholars (No. 81225011), State Key Program of National Natural Science of China (Grant No. 81230036), National Natural Science Foundation of China (No. 21273014 and No. 81201186) and Xiaolong Liang was supported by the China Postdoctoral Science Foundation (No. 2013M530014).

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Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2014.04.094.

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