Synthesis and Fluorescent Property of Biodegradable

Article pubs.acs.org/Macromolecules

Synthesis and Fluorescent Property of Biodegradable Polyphosphazene Targeting Long-Term in Vivo Tracking Zhaohui Huang,† Lika Yang,† Xuehui Zhang,‡ Bingyuan Ruan,† Xiaoqing Hu,§ Xuliang Deng,‡ Qing Cai,*,† and Xiaoping Yang† †

State Key Laboratory of Organic-Inorganic Composites, Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029, P. R. China ‡ Department of Geriatric Dentistry, Peking University School and Hospital of Stomatology, Beijing 100081, P. R. China § Institute of Sports Medicine, Beijing Key Laboratory of Sports Injury, Peking University Third Hospital, Beijing 100191, P. R. China S Supporting Information *

ABSTRACT: The importance of developing photoluminescent biodegradable scaffolding materials for tissue engineering is obvious, but it meets challenges with conventional biodegradable polymers such as aliphatic polyesters. In this study, photoluminescent biodegradable polyphosphazenes (PTA) were suggested as alternatives to target for long-term in vivo tracking applications. The PTA polymers were synthesized via nucleophilic cosubstitution of linear poly(dichlorophosphazene) with a fluorescent compound (TPCA) and alanine ethyl ester. The TPCA, with high fluorescent intensity and high quantum yield (∼0.5), was synthesized from citric acid and 2-aminoethanethiol. The resulted PTA polymers demonstrated adjustable degradation rates and fluorescent intensities in relating to their chemical compositions. In comparison with TPCA, the photostability of PTA polymers has been significantly improved, which made the long-term in vivo tracking feasible. PTA polymers were proven biocompatible and noncytotoxic for biomedical applications via both in vitro cell culture and in vivo implantation evaluations. During the 24-week subcutaneous implantation in mouse, the location and the degradation of PTA polymer were clearly visualized with the aid of fluorescent excitation and emission. In summary, PTA polymers were envisioned as good choices for tissue regeneration as scaffolding materials with in situ bioimaging potentials. have high fluorescent intensities and, moreover, highly tunable excitation and emission spectra.20,21 The potential toxicity originating from the heavy metal core of Qdots, however, prohibits their applications for long-term tracking or diagnosis in human bodies.18 Qdots labeling had been reported able to downregulate gene expressions in relating to osteogenic differentiation of human mesenchymal stromal cells (MSCs).22 Small organic fluorophores are preferred over Qdots for their lower toxicity and structural versatility, which enables easy conjugation with polymeric materials and facile tuning in optical properties.23 In order to monitor the mass loss of materials to model their hydrolytical degradation in vivo and in vitro, for example, Artzi et al. synthesized fluorescein-labeled poly(ethylene glycol) (PEG) and Texas Red-labeled collagen3 and Owens et al. synthesized a series of meso-brominated nearinfrared (NIR) fluorophores and conjugated them to biodegradable gelatin scaffold for real-time trafficking.24 Nevertheless, the main disadvantage of small molecular organic dyes is their short lifetime due to the photobleaching effect, i.e.,

1. INTRODUCTION In tissue engineering, biodegradable biomaterials are irreplaceable by serving as scaffolds for cell migration, proliferation, and differentiation as well as for defect filling and structural stabilization.1 The materials will degrade gradually along with the tissue regeneration. Although intensive studies have been conducted on in vitro degradation properties of various biomaterials,2 the in vitro results cannot fully reflect the in vivo behaviors of corresponding materials because of the complex in vivo environment.3 Thus, great interests have been evoked in tracking the in vivo degradation of implanted scaffolding materials via noninvasive techniques such as ultrasound elasticity imaging,4−6 X-rays,7−9 magnetic resonance imaging (MRI), 10−12 or photoluminescence excitation (PLE). 13−16 Among them, fluorescence techniques are suggested able to readily achieve the goal due to their fast, sensitive, reliable, and reproducible detection properties.17 However, the majority of biodegradable scaffolding materials are polymers like polyesters and polyanhydrides, etc., which are inherently nonfluorescent. To endow them photoluminescent properties, modifications are necessary. Quantum dots (Qdots) and organic dyes are the two kinds of popular labeling markers currently used in modifying biodegradable polymers.18,19 Qdots are chosen because they © XXXX American Chemical Society

Received: September 8, 2016 Revised: October 23, 2016

A

DOI: 10.1021/acs.macromol.6b01976 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules the fluorescence intensity decaying quickly with continuous exposure to light.18 Some studies showed that the photostability of organic dyes and Qdots could be significantly improved after they were integrated into polymeric structures.23,25 In comparison with commercial Qdots, the photostability of di(thiophene-2-yl)diketopyrrolopyrrole was significantly enhanced when it was covalently linked into the backbone of bioresorbable poly(εcaprolactone) (PCL).23 It was proposed that the integrated fluorescent molecules were isolated from each other by those covalent bonds on the polymeric backbone, and thus π−π aggregation was suppressed to avoid significant photobleaching. The result suggested a potential way to use fluorescence for long-time tracking. From the viewpoint of biocompatibility, however, a kind of fluorescent biodegradable aliphatic polyesters based on citric acid, dihydroxylalkane, and amino acids was more welcomed by tissue engineers in comparison with organic dyes and quantum dots.13 The fluorophores in these polyesters were assumed the reaction products of citric acid and amino acids.26 Among various amino acids, especially, the molecules derived from citric acid and thiol-containing cysteine displayed high fluorescent intensity and high quantum yield. These studies inspired our keen interests to develop other kinds of fluorescent biodegradable polymers for tissue engineering applications. Biodegradable polyphosphazenes can be a unique alternative for the purpose. Biodegradable polyphosphazenes are hybrid inorganic−organic polymers composed of a phosphorus− nitrogen backbone with two degradable side groups linking to each phosphorus atom.27 The degradability and biological performances of polyphosphazenes are highly tunable by selecting different side groups and by regulating their ratios.28−30 Amino acid ester-substituted polyphosphazenes are well-known for their sensitivity to hydrolysis and their nontoxic degradation products and have been widely studied as scaffolding materials.28,31 Because of their unique feature of containing phosphorus element, in particular, amino acid estersubstituted polyphosphazenes are identified bioactive in enhancing osteogenic differentiation of bone-related cells.32−35 Moreover, polyphosphazenes are superior to polyesters in their pH-buffering degradation products, which is helpful in alleviating the uncertainty caused by the accumulation of acidic degradation products in the case of polyesters.36,37 The synthetic chemistry of polyphosphazenes provides a feasible way to attach organic fluorophores onto their backbones by nucleophilic substitution in using linear poly(dichlorophosphazene) (PDCP) as the starting polymer.38 Therefore, in this study, photoluminescent biodegradable polyphosphazenes were designed and synthesized via substituting PDCP in sequence with a fluorescent derivative (TPCA) of citric acid, 2-aminoethanethiol, and amino acid esters. The photoluminescent properties of both the TPCA and the TPCAcontaining polyphosphazenes (PTAs) were investigated. Especially, the photostability of PTAs was monitored with their in vitro hydrolysis degradation. Cytotoxicity and biocompatibility of PTAs were evaluated by both in vitro culture of bone MSCs (BMSCs) and in vivo implantation. At the same time, the subcutaneous implantation in mouse was continued for as long as 24 weeks to verify the possibility in long-term tracking the in vivo degradation of PTAs via the photoluminescent feature.

2. EXPERIMENTAL SECTION 2.1. Materials. Hexachlorocyclotriphosphazene (HCCP) monomer was purchased from Aladdin and used after recrystallization and sublimation (50 °C, 10 mbar). Alanine ethyl ester hydrochloride and glycine ethyl ester hydrochloride were purchased from Alfa Aesar (USA) and used after vacuum dehydration (35 °C, 10 mbar). 2Aminoethanethiol was purchased from Tokyo Chemical Industry (Japan) and used directly. All other reagents and solvents used in the study were of analytical grade and supplied by Beijing Chemical Reagent Co., Ltd. (China). 2.2. Synthesis of 5-Oxo-2,3-dihydro-5H-thiazolo[3,2-α]pyridine-7-carboxylic Acid (TPCA). Referring to an early publication,26 TPCA was synthesized readily in one step. Briefly, 1.21 g of citric acid (6.3 mmol) was mixed with 0.48 g of 2aminoethanethiol (6.3 mmol) with the addition of DI water (1 mL) to dissolve the ingredients. Then vacuum (10 mbar) was applied to remove the water in order to get the homogeneous mixture of citric acid and 2-aminoethanethiol. Afterward, the mixture was heated up to 140 °C and kept for 5 h to complete the reaction as shown in Figure 1a. The raw product was purified by LC-20AP preparative high

Figure 1. Synthesis and chemical structures of (a) TPCA and (b) PTA polymers. pressure liquid chromatography (Shimadzu, Japan) at RT on a C-18 column and eluted by 1‰ (v/v) trifluoroacetic acid in water/ acetonitrile (v/v = 90/10) at a flow rate 10 mL/min (Figure S1). Liquid chromatography electrospray ionization tandem mass spectrometry (ESI-MS) measurement was performed on a UHD AccurateMass Q-TOF liquid chromatography liquid/mass spectrometer (Agilent Technologies, USA) at RT on a C-18 column and eluted by water at a flow rate 1 mL/min. The fraction with the molecule weight of 198 was collected and freeze-dried (Figure S2). 2.3. Synthesis of Poly[(TPCA)x(ethylalanato)yphosphazene] (PTA). The synthesis of PTA was carried out as schemed in Figure 1b. At first, linear PDCP was obtained from the bulk polymerization of HCCP under vacuum at 250 °C for 24 h. After being purified by removing unreacted HCCP, the linear PDCP, containing 0.038 mol of the −PNCl2− unit, was dissolved in 200 mL of anhydrous tetrahydrofuran (THF), followed by the addition of alanine ethyl ester solution in THF. This alanine ethyl ester solution was preprepared by refluxing the mixture of alanine ethyl ester hydrochloride (0.076 mol) and triethylamine (TEA, 0.174 mol) in THF (200 mL) for 6 h and filtrated for further use. The system containing both PDCP and alanine ethyl ester was allowed to react at 35 °C for 24 h under continuous agitation. Subsequently, TPCA (0.015 or 0.03 mol) solution in THF (200 mL) was added into the system and reacted at 35 °C for another 36 h. Finally, a second portion of alanine ethyl ester solution (0.038 mol) was added, and the system was further kept at 35 °C for 24 h. Then the reaction was stopped, followed by filtration to obtain a kind of yellowish viscous solution. The solution was concentrated by vacuum rotary evaporation to remove some solvent and precipitated into DI water to get the PTA polymer. The resulted PTA polymer was further purified with petroleum ether (30− 60 °C) and DI water extractions to ensure the complete removal of unreacted TPCA, alanine ethyl ester, and residual TEA or TEA B


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Macromolecules Table 1. Identification and Properties of the Synthesized TPCA and Polyphosphazenes intrinsic viscosity (dL/g)

31 a

sample

P (ppm)

TPCA PAGP

−1.92

PTAl

−0.77

PTA2

−1.72

1

a

H (ppm)

3.52 (2H, AE), 4.36 (2H, AE), 6.54 (1H, CA), 6.58 (1H, CA) 1.27 (3H, Ala), 1.44 (6H, Gly, Ala), 3.69 (1H, Ala), 3.95 (2H, Gly), 4.12 (2H, Gly), 4.20 (2H, Ala) 1.27 (3H, Ala), 1.43 (3H, Ala), 3.45 (2H, AE), 3.59 (1H, Ala), 4.18 (2H, Ala), 4.51 (2H, AE), 6.68 (1H CA), 6.88 (1H, CA) simlar to PTAl

yield (%)

atom ratio (S:P)c

mol % TPCA

quantum yield (%) 49.9 23.8

87

53.7

53.1

46.3

0.23:1

11.5

25.8

32.7

42.7

0.37:1

18.5

27.9

a

NMR spectra were obtained by dissolving TPCA in DMSO and polyphosphazenes in CDC13. bCA refers to citric acid, AE refers to 2aminoethanethiol, Gly refers to glycine ethyl ester, and Ala refers to alanine ethyl ester. cAtom ratio was determined by XPS. culture plates (Corning, USA) or 24-well Transwell (0.4 μm, Corning, USA). In the former case, the cells were seeded onto the materials directly (i.e., contact manner), while in the latter case, the cells did not touch the materials (i.e., noncontact manner). In both cases, BMSCs cultured on tissue culture polystyrene (TCPS) were taken as the control. Before cell seeding, the films were exposed to ultraviolet (UV) light for 2 h, followed by 3 times PBS washing and being immersed in DMEM overnight. Then, 500 BMSCs were added into each well and cultured for 1−7 days to evaluate cell proliferation. One day after cell seeding, the culture medium was taken away, and the wells were rinsed one time with PBS. The Alamar blue stock solution (Yeasen, USA) was diluted with DMEM (1:10), and a volume of 100 μL was added into each well. After 2 h incubation at 37 °C, fluorescence of the liquid was quantified at the excitation and emission wavelength of 530 and 590 nm, respectively, using a Luciferase tester (Polarstar, Australia). Because of the nontoxicity of Alamar blue, DMEM was supplied into the culture plates, and the cells were cultured continuously. At day 3, 5, and 7 after cell seeding, Alamar blue analysis was applied similarly to determine cell proliferation. For each sample, three independent experiments were conducted for averaging. For cell attachment evaluation, patches of samples (ϕ = 6 mm) were placed into 96-well culture plates, and 5000 cells were seeded onto the materials directly and cultured for 4, 8, 12, and 24 h. At each time point, the media were replaced by fresh media with 10% CCK-8 (Beyotime, China), and the OD values were measured at the wavelength of 490 nm with a microplate reader (Bio-Rad 680, USA). For each sample, three independent experiments were conducted for averaging. For cell morphology observation, the cell/material complexes were retrieved after 7 days culture and then gently washed with PBS, followed by being soaked in 3% glutaraldehyde 4 °C for 3 days. Subsequently, the complexes were allowed to air-dry overnight at room temperature and submitted to scanning electron microscope (SEM) observation. Before observation, the samples were sputtercoated with gold (30 mA, 20 s) using an auto-sputter-coater (Cressington 108, England) and then examined under a Hitachi S4700 SEM at an accelerating voltage of 20 kV. 2.7. In Vivo Implantation. Fifteen 4-week-old mice, weighing 30− 35 g, were used in this study. All animal experimental procedures followed the guidelines of Peking University School and Hospital of Stomatology (China). PTA films for implantation were cut into circular shape (ϕ = 1 cm) and sterilized by being exposed to UV light for 2 h before the implantation. The mice were anesthetized by intraperitoneal injection, and the prepared films were implanted subcutaneously. At each predetermined time points (2, 4, 8, 16, and 24 weeks), two mice were sacrificed to harvest the implants, kidneys, and livers for histological and fluorescent analysis. For histological analysis, the samples were fixed in 4% paraformaldehyde, dehydrated by ethanol solutions of series concentrations, embedded by paraffin, and sliced.40 Then the sections were stained with hemothoxylin and eosin (H&E) and observed under an optical microscope (CKX41, Olympus, Japan) to determine the biocompatibility of the PTA material. For fluorescent analysis, the samples containing implants were frozen and sectioned, followed by observation using a laser confocal scanning microscope (LCSM, Leica TCS SP8, Leica) to trace the remaining implants.

hydrochloride. The purified PTA polymer was then freeze-dried for characterization and further use. With the different feeding doses (0.015 or 0.03 mol) of TCPA in synthesizing PTA, two PTA polymers were obtained and termed as PTA1 and PTA2, respectively. For comparison, alanine ethyl ester and glycine ethyl ester cosubstituted polyphosphazene (PAGP) was synthesized in referring our previous study.39 2.4. Characterizations. 1H, 13C, and 31P nuclear magnetic resonance (NMR) spectra were obtained using a Bruker AV600 instrument operated at 400 MHz, in which the 31P shifts are relative to an 85% H3PO4 at 0 ppm as reference. Intrinsic viscosity was measured by a capillary viscosimeter in a water bath thermoset at 30 °C using THF as solvent. X-ray photoelectron spectroscopy (XPS) experiments were performed on an ESCALAB 250Xi electron spectrometer from VG Scientific using monochromatic Al Kα radiation (1486.7 eV) as the excitation source. All spectra were acquired at pass energy of 80 eV with the anode operated at 300 W. The base pressure was about 3 × 10−9 mbar. The binding energies were referenced to the C1s line at 284.8 eV from adventitious carbon. Atomic percentages were calculated by normalizing the area of each peak with the total peak area of all atomic elements. For fluorescence studies, TPCA was dissolved in DI water or THF at different concentrations, and PTA polymers were dissolved in THF at different concentrations. Then the solutions were submitted to fluorescence measurement on an F-7000 FL spectrophotometer (Hitachi, Japan). The slit width for excitation and emission was 5 nm, the voltage was 400 V, and the scan speed was 240 nm/min. The fluorescence quantum yields were measured by using a FLS980 lifetime and steady state spectrometer with a calibrated integrating sphere (Edinburgh). 2.5. Hydrolysis Studies. Hydrolysis of PTA polymers was performed by soaking PTA films in phosphate buffered saline (PBS, pH 7.4) at 37 °C for 24 weeks. The PTA films were prepared by dissolving PTA polymers in THF and solution-casting, followed by solvent evaporation and vacuum-drying to constant weight. The films, ∼100 μm in thickness, were cut into square patches (1 cm × 1 cm), weighed (W0), and immersed into the PBS, followed by continuous rotation (60 rpm) at 37 °C. The PBS was refreshed every week. At each predetermined time point, three patches were retrieved, freezedried, and weighted again (Wt). Weight loss was then calculated as 100 × (W0 − Wt)/W0. The fluorescent intensities of the residual polymers were determined by dissolving a 50 mg sample in 2 mL of THF and measured as aforementioned. 2.6. In Vitro Cell Culture. Sprague Dawley rat BMSCs were purchased from Cyagen Biosciences (Guangzhou, China). Cells were maintained in a complete Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 IU/mL penicillin (Sigma, USA), and 100 mg/mL streptomycin (Sigma, USA). BMSCs were cultured in an incubator (Sanyo, Japan) with 5% CO2 supply and saturated humidity at 37 °C. Once reaching 80% confluence, the cells were digested by 0.25% trypsin (Sigma) and 0.02% ethylenediaminetetraacetic acid (EDTA) for further use. The culture medium was changed every 2 days. The fourth generation of BMSCs was used for the cell viability assay. Cell viability was evaluated with Alamar blue assay in two culture manners. Patches of PTA films (ϕ = 6 mm) were placed into 96-well C


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Figure 2. (a) Excitation and emission spectra of TPCA in THF and H2O (0.03 g/L), together with emission spectra of TPCA in (b) H2O and (c) THF along with the storing time in dark conditions.

Figure 3. Photostability of TPCA evaluated by changing (a−c) its solution concentrations and (d−f) exposure times to 365 nm light (3 W): (a, d) emission spectra of TPCA in H2O; (b, d) emission spectra of TPCA in THF; (c, f) dependence of emission intensity of TPCA on concentrations (c) and irradiation times (f). The solution concentration for exposure time evaluation was 0.03 g/L. 2.8. Statistical Analysis. All quantitative data were expressed as mean ± standard deviation (SD) for n = 3. Statistical analysis was performed using the SPSS 22.0 software. Statistical difference was determined using ANOVA for independent samples. Differences between groups of *p < 0.05 were considered statistically significant.

Concentration quenching and photobleaching are two common phenomena associating with small organic photoluminescent molecules. In this study, both significant concentration quenching and photobleaching were also identified for TPCA. As shown in Figure 3a−c, a significant concentration quenching effect was identified for TPCA solutions along with increasing concentrations. A linear relationship between the fluorescent intensity and the TPCA concentration could be obtained only when the concentration of TPCA was below 0.1 g/L. When the TPCA solutions (0.03 g/L in water or in THF) were exposed to 365 nm light (3 W) continuously, it was found the emission intensity of TPCA could only remain ∼32% and ∼2% of their initial values, respectively, after 60 min exposure (Figure 3d−f), indicating the occurrence of significant photobleaching. The fact that the photostability of TPCA being higher in water than in THF, was suggested being ascribed to the stronger interaction between TPCA and water molecules than it with THF molecules. 3.2. Characterizations of PTA Polymers. PTA polymers were synthesized by nucleophilic substitution of linear PDCP with both alanine ethyl ester and TPCA in sequence. To ensure the random distribution of TPCA along the polyphosphazene backbone, the substitution of alanine ethyl ester was completed in two steps, i.e., the feeding order of nucleophilic reagents being alanine ethyl ester, followed by TPCA and alanine ethyl ester again. Two feeding doses of TPCA (MTPCA:Mchlorine = 0.2:1 or 0.4:1) were applied to prepare PTA polymers with different fluorescent intensities and degradation rates. The

3. RESULTS 3.1. Characterizations of TPCA. The synthesis of TPCA was based on the condensation reaction of citric acid and 2aminoethanethiol (Figure 1a). Its chemical structure was confirmed by mass spectrometry (MS) as well as 1H and 13C NMR spectra as presented in Table 1 and Figures S2−S4. Afterward, the fluorescent property of TPCA was examined. By dissolving TPCA in water or THF (0.03 g/L), the excitation and emission spectra of TPCA solutions were obtained and are shown in Figure 2a. In water, the excitation peak and the emission peak were detected at λexc = 341 nm and λemm = 416 nm, respectively, showing a Stokes shift Δλ = 75 nm. In THF, the λexc and λemm were identified moving to 362 and 427 nm, respectively, showing a Stokes shift Δλ = 65 nm. The different polarity of water and THF, as well as their different abilities in interacting with TPCA, had contributed to the difference in the excitation and emission spectra of TPCA. TPCA was then dissolved in water or THF and stored in dark conditions for 4 weeks to check the stability of the fluorophore. As expected, the fluorescent intensity did not change apparently (Figure 2b,c), confirming the fluorophore able to kept stable in these solvents. The absolute quantum yield of TPCA was calculated ∼49.9% in THF, and the data are shown in Table 1. D


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Figure 4. (a, b) Excitation and emission spectra of (a) PAGP, (b) PTA1 and PTA2 in THF (4 g/L). (c, d) Appearances of PTA1 films under (c) natural light and (d) 365 nm light.

Figure 5. Photostability of PTA polymers evaluated by changing (a−c) their concentrations in THF, (d−f) exposure times to 365 nm light (3 W), and (g−i) the storing conditions: (a, d) emission spectra of PTA1; (b, e) emission spectra of PTA2; (c, f) dependence of emission intensities of PTA polymers on concentrations (c) and irradiation times (f); (g, h) emission spectra of PTA1 being stored under normal (g) or dark conditions (h). The solution concentration for exposure time and storage evaluations was 4 g/L.

the total amounts of the two side groups (Table 1). It could be noticed that the intrinsic viscosity of PTA2, which had a higher content of TPCA side group, displayed a lower value than that of PTA1. Because of the presence of secondary and tertiary amine in amino acid ester-substituted polyphosphazene, PAGP was suggested to have intrinsic photoluminescent feature. By dissolving PAGP in THF, the excitation and emission spectra were measured as shown in Figure 4a. However, both the excitation and emission wavelengths were wide and weak, which was not strong enough to meet the requirements of in vivo bioimaging. After the incorporation of photoluminescent TPCA, as shown in Figure 4b, the excitation and emission patterns of PTA polymers resembled those of TPCA, and the fluorescent spectrum showed a red-shift of about 3 nm. The fluorescent intensities of PTA polymers displayed strong dependence on the contents of TPCA being introduced that PTA2 had obviously stronger fluorescent intensity than PTA1.

yields of PTA polymers were around 40−45% after the series of reactions. The chemical structures of the obtained PTA1 and PTA2 were characterized by 31P and 1H NMR spectra as well as intrinsic viscosity measurement. As presented in Table 1 and Figure S5, the appearance of chemical shifts at 3.45, 4.51, 6.68, and 6.88 on the 1H spectrum confirmed the successful incorporation of TPCA into the polymer, and other signals indicated the presence of alanine ethyl ester. The characteristic signal relating to phosphorus atom in PTA backbone was identified by the 31P NMR spectrum (Figure S6). For comparison, 1H and 31P spectra of PAGP were also provided as Figures S7 and S8. Since TPCA has the sulfur element and the polyphosphazene backbone contains the phosphorus element, XPS analysis was performed to judge the graft ratio of TPCA molecule onto the polyphosphazene backbone. Therefore, in PTA1 and PTA2, the graft ratios of TPCA were calculated 11.5 and 18.5 mol %, respectively, in relating to E


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Figure 6. Luminescence efficiency of PTA polymers at different solution concentrations: (a) emission spectra of PTA1; (b) emission spectra of PTA2; (c) dependence of emission intensities of PTA polymers on solution concentrations.

Figure 7. Hydrolysis of PTA polymers and the change in their fluorescent intensities along with weight loss for a period of 24 weeks: (a) weight loss; (b) normalized fluorescent intensity in relating to sample weight; (c, d) emission and excitation spectra of PTA1 (c) and PTA2 (d) vs degradation time in THF (4 g/L).

As shown in Figure 5a−c, the fluorescent intensities of PTA polymers displayed a good linear relationship to the concentrations with no obvious concentration quenching phenomenon being detected. Next, the luminescence efficiency of PTA polymers was tested at even higher concentrations (10−100 g/L). As shown in Figure 6, the concentration quenching effect was detected only when the concentrations of PTA1 and PTA2 had been above 60 and 40 g/L, respectively. Because PTA2 contained higher content of TPCA component than PTA1, the concentration quenching effect of PTA2 would occur at a lower solution than that of PTA1. It could be seen these critical values were much higher than that of TPCA (∼0.1 g/L). As for the photobleaching effect along with irradiation time, the PTA1 and PTA2 were also able to remain about 38% and 21% of their initial intensities after 60 min exposure to 365 nm light, respectively, which exhibited superior resistance to photobleaching in comparison with TPCA (Figure 5d−f). Taking PTA1 as an example, the long-term photostability of PTA polymer was further investigated by storing the polymer under normal conditions or keeping it in dark conditions for 24

The quantum yields of PTA polymers were calculated relatively high, 25.8% for PTA1 and 27.9% for PTA2 as presented in Table 1. The photographs of PTA1 membrane under natural light and 365 nm light are shown in Figures 4c and 4d, respectively, and the photoluminescent feature of PTA polymer could be vividly seen. The photostability of PTA polymers was then investigated. Different from the case of TPCA (Figure 3), the change in emission spectra of PTA polymers along with concentration did not show a descending trend even at relative high concentrations (0.25−8 g/L) (Figure 5a−c). To make the results reliable and comparable, PTA1 was dissolved in THF to get approximately the same concentration of TPCA unit to those in Figure 3, calculating from the graft ratio of TPCA, while the concentrations of PTA2 solutions were kept constant with those values of PTA1 solutions for the consideration of avoiding the effect of polymer concentration on the concentrationquenching effect. Thus, the PTA2 solutions actually had higher contents of TPCA units than PTA1 solutions when they were at the same solution concentration. F


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Figure 8. (a−c) Proliferation and (d) attachment of BMSCs on polyphosphazenes (PTA 1, PTA2, PAGP) in comparison with TCPS (control) in (a) noncontact and (b−d) contact culture manners: (a) being cultured in transwell chamber; (b) being cultured on polyphosphazene films; (c) normalized cell proliferation basing on data from (b); (d) cell attachment. *p < 0.05, significant.

weeks. Although a decrease in fluorescent intensity was also detected after 24 weeks of exposure in normal condition, its fluorescent intensity was still able to retain as half as the initial intensity (Figure 5g). This finding revealed that the long-term photostability of PTA polymer was quite impressive. If PTA1 was kept in a dark place, undoubtedly, its fluorescent intensity would remain constant all the time as shown in Figure 5h. 3.3. In Vitro Degradation. Hydrolysis of PTA1 and PTA2 was conducted in pH 7.4 PBS at 37 °C as long as 24 weeks by applying a dark environment. At each time point, the samples were retrieved and freeze-dried to constant weight. The weight remaining was plotted as a function of degradation time and is shown in Figure 7a. Noticeably, except the sharp initial weight loss was observed for PTA2, both PTA1 and PTA2 displayed gradual weight loss along with longer soaking time. Up to the 24 weeks of hydrolysis, the weight losses of PTA1 and PTA2 reached ∼35 and ∼50 wt %, respectively, indicating the faster degradation rate of PTA2 than that of PTA1. The degradation residuals were then dissolved in THF at 4 g/L and submitted to fluorescent measurements. As shown in Figure 7c,d, the excitation and emission spectra of PTA1 were quite similar for all the hydrolyzed samples, showing independence on degradation time. By normalizing the fluorescent intensity to the polymer amount, the photoluminescent feature of hydrolyzed PTA1 almost remained unchanged along with its continuous weight loss. The results strongly suggested that the degradation of PTA1 occurred randomly along the polyphosphazene backbone, and the loss of photoluminescent TPCA side group was associated with the random breakage of the polyphosphazene backbone. From Figure 7b, in general, the fluorescent intensity of residual PTA2 also remained constant after being hydrolyzed for different times, displaying a similar trend to that of PTA1.

Different from the case of PTA1, however, a rapid initial decrease in the fluorescent intensity was detected for PTA2 within the 1−4 weeks of hydrolysis. This phenomenon was thought in accordance with the initial fast weight loss of PTA2 in hydrolysis, which was thought closely relating to its molecular weight. From Table 1, it was known that the intrinsic viscosity of PTA2 was significantly smaller than that of PTA1, which meant PTA2 having more fractions of oligomers than PTA1. Therefore, the fast loss of the oligomers would result in rapid weight loss at the initial stage of hydrolysis and, accordingly, which had possibly caused the significant initial decrease in fluorescent intensity in the case of PTA2. 3.4. Cytotoxicity of PTA Polymers. To evaluate the cytotoxicity of the synthesized PTA polymers, in vitro cell culture using BMSCs was performed in two culture manners, i.e., the noncontact and the contact manner. In the noncontact culture manner, cells were cultured in the presence of materials but not being seeded onto the materials by using the transwell culture chambers. Thus, only soluble ingredients from the materials could reach the cells and influence the cell proliferation. If there was any cytotoxic ingredient coming out of the materials, it could be easily detected. For comparison, BMSCs were also cultured in the presence of PAGP or without material (control). As shown in Figure 8a, continuous cell growth was identified in all the groups, indicating the viability of BMSCs. In the presence of the two PTA polymers, the cell proliferation rates displayed no significant difference from those in the presence of PAGP and the control group. These results revealed that no cytotoxic ingredient was found for the three degradable polyphosphazenes, indicating the introduction of TCPA not bringing in extra cytotoxicity. G


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Figure 9. Morphology of BMSCs being cultured on (a) PTA1, (b) PTA2, and (c) PAGP films for 7 days.

Figure 10. (a) Surgical procedure of the implantation and (b−f) fluorescent imaging of subcutaneously implanted PTA1 films at (b) 2, (c) 4, (d) 8, (e) 16, and (f) 24 W postimplantation.

3.5. In Vivo Bioimaging. In vivo implantation of PTA1 was carried out to verify the hypothesis that the location and the degradation of photoluminescent polyphosphazene could be monitored via the fluorescent imaging. PTA1 films were subcutaneously implanted in mice for as long as 24 weeks. From Figure 7a, it had been known that significant degradation had taken place for PTA1 with the weight loss of ∼35 wt % at the end of 24 weeks. During the implantation, therefore, PTA1 films should also degrade, turn cracked, and lose integrity, and these changes should be reflected by fluorescent detection. As shown in Figure 10, excitedly, the morphological changes of the implanted PTA1 films were visualized clearly with the aid of LCSM, and the local fluorescent intensity did not fade with the implantation time obviously. Besides, no accumulation of fluorescent degradation products was found in surrounding tissues. Histological evaluations on the sections of harvested implantations, livers, and kidneys were carried out at all the implantation time points. As shown in Figure 11, no inflammation was detected in all the cases. The tissues of both the liver (Figure 11f−j) and the kidney (Figure 11k−o) displayed normal histological morphology with no hint of accumulation of foreign matters. Fluorescent detection on these liver and kidney sections revealed no fluorescent stain (dark pictures, data not shown). As for the harvested skin tissues containing PTA1 films, the skin tissues also presented normal histological morphology (Figure 11a−e), indicating no

By culturing BMSCs directly on the materials (i.e., contact manner), as shown in Figure 8b, it was unexpected to find that the cell proliferation rates on the three polyphosphazenes were well slower than that on TCPS, while no significant difference was identified among the polyphosphazenes. By normalizing the fluorescent intensities to the first day data of each corresponding case, however, the relative cell growth rates on polyphosphazenes were found comparable with that on TCPS (Figure 8c). These two phenomena seemed being contradictory. Therefore, cell attachments on these materials within 24 h were conducted and are presented as Figure 8d. Apparently, the attachments of BMSCs onto polyphosphazenes were inferior to the TCPS case, which was suggested closely relating to surface features of substrates. For those attached BMSCs on polyphosphazenes, whether on the PTA polymers or on the PAGP, could proliferate gradually. As revealed by Figure 8c, moreover, the polyphosphazenes actually demonstrated enhancement in cell growth in comparison with the control. In Figure 9, morphology of BMSCs being cultured on the three polyphosphazenes was shown via SEM observation. In all the cases, it could be seen the cells attached firmly on the substrates and spread widely. The cells were in normal spindlelike shape with abundant extracellular matrix (ECM) being secreted. All these results confirmed that the synthesized PTA polymers were cell affinitive and supporting the proliferation of BMSCs, although the cell attaching efficiency on polyphosphazenes was inferior to TCPS. H


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Figure 11. Histological evaluations on skin sections where the implanted PTA1 films are located as well as liver and kidney sections harvested at 2, 4, 8, 16, and 24 weeks postimplantation. The letter P represents the site where the implanted PTA1 film is located (scale bar = 100 μm).

groups, photoluminescent polyphosphazenes were readily synthesized to target for in vivo applications.42 Although PAGP is able to be detected showing weak photoluminescent property, it is not strong enough for in vivo imaging (Figure 4a). To endow polyphosphazene with strong fluorescent intensity and high quantum yield, in this study, TPCA was coattached onto alanine ethyl ester substituted polyphosphazene at two graft ratios. TPCA is a small organic fluorophore deriving from citric acid and 2-aminoethanethiol, which suffers concentration quenching and photobleaching problems like many other small organic fluorophores (Figure 3).43,44 In its molecular structure, however, TPCA had a free carboxyl group to react with the phosphorus chloride group in PDCP. Thus, TPCA was able to be grafted onto polyphosphazene backbone via nucleophilic substitution, and its graft ratio was regulated by controlling the feeding dose. To ensure the random substitution of TPCA, a part of alanine ethyl ester was grafted onto PDCP at first, followed by the TPCA reaction. Finally, another part of alanine

inflammatory reactions presenting. The sites where the materials located were left blank because PTA1 had dissolved in the preparation of histological sections with the use of paraffin and solvent. After 16 weeks postimplantation, fibrous tissues had grown into the implantation area with the degradation and cracking of the implanted films. All these observations suggested that PTA1 possessed good biocompatibility and would not cause adverse effects on the mouse.

4. DISCUSSION It is important and quite attractive if the location and the degradation of scaffolding materials for tissue engineering can be visualized in vivo.41 Different from many conventional biodegradable polymers, which are hard or complex to be modified to have intrinsic photoluminescent features, biodegradable polyphosphazenes stand out due to their strong flexibility in designing chemical structures.38 By introducing fluorescent side groups onto the polyphosphazene backbone, together with other biocompatible and cell affinitive side I


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into the material area along with the degradation of PTA1. Especially, no hint of accumulation of fluorescent degradation products was found in both the liver and kidney tissues. The degradation products of PTA1 did not trigger the formation of fibrous capsule around the implantation. This might be an advantage over biodegradable aliphatic polyesters, in which their acidic degradation products might stimulate the growth of fibrous capsule.33 The degradation products of amino acid ester-substituted polyphosphazenes were reported mainly pHbuffered phosphate, amine, and corresponding side groups.29,37 Therefore, PTA polymers were envisioned as good scaffolding materials with adjustable degradation rate, acceptable biocompatibility, and in vivo bioimaging capacity.

ethyl ester was added to substitute the residual phosphorus chloride groups to avoid unwanted cross-linking during the storage and the use of PTA polymers. Owing to the hydrolysis of amino acid ester side group, the resulted PTA polymers were degradable in aqueous environment, but showing dependence on the graft ratio of TPCA side group (Figure 7a). The fluorescent intensities of PTA polymers also displayed a dependence on the graft ratio of TPCA side group, that higher graft ratio of TPCA resulted in stronger fluorescent intensity (Figure 4b). The quantum yields of PTA polymers were about 25−30%. Although the values were lower than that of TPCA (49.9%), they were comparable to some fluorophores applied in publications relating to cell or in vivo imaging.23,40 This inferred the feasibility of using photoluminescent PTA polymers for in vivo bioimaging. In comparison with TPCA, the photostability of PTA polymers was remarkably enhanced in both aspects of concentration quenching and photobleaching (Figures 5 and 6). The explanation was supposed due to the backbone structure of polyphosphazene. As we have known, TPCA was randomly grafted onto the polyphosphazene backbone, and the polyphosphazene backbone was composed of nonconjugated alternating single and double phosphorus−nitrogen bonds.45 Therefore, the polyphosphazene segments could serve as spacers to isolate TPCA side groups, which was able to efficiently suppress the collision and prevent the transfer of excited electrons between TPCA moieties. Thereby, photobleaching and concentration quenching effects were alleviated significantly.23,25 Even so, a dark environment was still preferred for the storage of photoluminescent PTA polymers (Figure 5g−i). In performing the hydrolysis of the PTA polymers, a major concern was the worry about the premature loss of TCPA moieties. If so, it would not achieve the goal to visualize the gradual in vivo degradation of polyphosphazenes. Promisingly, from Figure 7b−d, the fluorescent intensity per unit mass of both PTA1 and PTA2 remained constant even remarkable weight loss had occurred, except a fast initial decrease in fluorescent intensity was detected for PTA2 in relating to its rapid initial weight loss. The hydrolysis of amino acid estersubstituted polyphosphazenes was identified starting from the side group, followed by the breakage of polyphosphazene backbone.31 Since the constant fluorescent intensities were detected for hydrolyzed PTA polymers, it was inferred that the alanine ethyl ester was more sensitive to hydrolysis than the TPCA. Thus, the loss of TPCA was not initiated from itself degradation but associated with the breakage of polyphosphazene backbone that was triggered by the hydrolysis of an alanine ethyl ester side group. Reasonably, photoluminescent PTA polymers could be used as implantable materials, which could be monitored with the aid of fluorescent excitation and emission. As expected, the in vivo implantation of PTA1 films gave out satisfactory fluorescent images to show the contour and the degradation of the materials (Figure 10). After 24 weeks subcutaneous implantation, the remaining fluorescent intensities in local areas were still very strong although the total fluorescent intensity had decreased due to the degradation. PTA polymers were found biocompatible, noncytotoxic, and supporting cell growth from both the in vitro culture of BMSCs (Figures 8 and 9) and the histological evaluations of tissue sections (Figure 11). No inflammation was identified around the implanted PTA1 film, and fibrous tissue was able to grow

5. CONCLUSIONS The chemistry of polyphosphazene provided a feasible way to design and synthesize photoluminescent scaffolding materials with controllable fluorescent properties and degradation rates. By introducing photoluminescent side group as TPCA and hydrolyzable side group as amino acid ester, photoluminescent biodegradable polyphosphazenes as PTA polymers were readily prepared. Their degradation rates and fluorescent intensities were closely related to the graft ratios of the two kinds of side groups. More importantly, the photostability of PTA polymers was significantly improved in comparison with TPCA small molecule by alleviating both concentration quenching and photobleaching effects. With good cell affinity and biocompatibility, promisingly, photoluminescent biodegradable polyphosphazenes were able to serve as scaffolding materials for tissue engineering applications benefiting from their long-term in vivo tracking feature.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b01976. Chromatograms of reaction product from citric and 2aminoethanethiol containing TPCA, characterization spectra including MS, 1H NMR, and 13C NMR for TPCA, and characterization spectra including 1H and 31P NMR for both PTA and PAGP polymers (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone and Fax +86-10-64412084; e-mail [email protected]. edu.cn (Q.C.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support from National Natural Science Foundation of China (No. 51473016, 51373016), and Beijing Municipal Commission of Education (ZDZH20141001001).

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REFERENCES

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Synthesis and Fluorescent Property of Biodegradable

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