Fluorescent Poly(glycerol-co-sebacate) Acrylate Nanoparticles for

Mar 1, 2017 - ... National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Na...
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Fluorescent Poly(glycerol-co-sebacate) Acrylate Nanoparticles for Stem Cell Labeling and Longitudinal Tracking Lifeng Wang,†,‡ Keming Xu,†,‡ Xiaochun Hou,‡,§ Yiyuan Han,‡ Shiying Liu,‡ Christian Wiraja,‡ Cangjie Yang,‡ Jun Yang,† Mingfeng Wang,‡ Xiaochen Dong,*,† Wei Huang,*,† and Chenjie Xu*,‡,∥ †

Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing 211816, P. R. China ‡ School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, Singapore 637457, Singapore § Key Laboratory for Organic Electronics and Information Displays, Nanjing University of Posts and Telecommunications, Nanjing 210046, P. R. China ∥ NTU-Northwestern Institute for Nanomedicine, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore S Supporting Information *

ABSTRACT: The stable presence of fluorophores within the biocompatible and biodegradable elastomer poly(glycerol-cosebacate) acrylate (PGSA) is critical for monitoring the transplantation, performance, and degradation of the polymers in vivo. However, current methods such as physically entrapping the fluorophores in the polymer matrix or providing a fluorescent coating suffer from rapid leakage of fluorophores. Covalent conjugation of fluorophores with the polymers and the subsequent core-cross-linking are proposed here to address this challenge. Taking rhodamine as the model dye and PGSA nanoparticles (NPs) as the model platform, we successfully showed that the synthesized rhodamine-conjugated PGSA (PGSAR) NPs only released less than 30% rhodamine at day 28, whereas complete release of dye occurred for rhodamine-encapsulated PGSA (PGSA-p-R) NPs at day 7 and 57.49% rhodamine was released out for the un-cross-linked PGSAR NPs at day 28. More excitingly, PGSAR NPs showed a strong quantum yield enhancement (26.24-fold) of the fluorophores, which was due to the hydrophobic environment within PGSAR NPs and the restricted rotation of (6-diethylamino-3H-xanthen-3-ylidene) diethyl group in rhodamine after the conjugation and core-cross-linking. The stable presence of dye in the NPs and enhanced fluorescence allowed a longitudinal tracking of stem cells both in vitro and in vivo for at least 28 days. KEYWORDS: poly(glycerol-co-sebacate) acrylate, rhodamine, nanoparticles, stem cell labeling, longitudinal tracking

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entrapment of the fluorophores in the polymer matrix or the use of a fluorescent coating.10−12 Yet, the physically trapped fluorophores easily leak out, and the fluorescent coating might detach from PGSA in the biological environment, both of which would decrease the visibility of PGSA and generate false signal from bystander cells/tissues.13 Another strategy is the utilization of fluorescent contrast agents, such as quantum dots,14−16 upconversion nanoparticles,17−19 or fluorescent polymers.20−23 However, the potential system toxicity of inorganic nanoparticles (e.g., the quantum dots and upconversion nanoparticles) has hindered further bioimaging applications in vivo.24−29 The fluorescent polymers, on the other hand, often lack sufficient stability and possess a relatively low quantum yield in aqueous solution.30−33 Therefore, there is an

INTRODUCTION Poly(glycerol-co-sebacate) acrylate (PGSA) is an acrylated derivative of the biocompatible and biodegradable elastomer poly(glycerol-co-sebacate) (PGS).1−4 The cross-linking of vinyl bonds in PGSA can be achieved through either redox or photoinitiated free radical polymerization within minutes at ambient temperature.5 This strategy drastically reduces the curing time to few minutes from the 48 h typically required in PGS synthesis. By controlling the percentage of acrylate moieties in the PGSA, it is also possible to control the mechanical and degradation properties of PGSA to meet the specific requirements of various applications.6 These unique properties have attracted great interest to explore the roles of PGSA in the form of solution, patterned film, or nanoparticles (NPs) for drug delivery and tissue engineering.7−9 To visualize the transplantation, presence, and degradation of polymers and cells both in vitro and in vivo, it is often necessary to label them with fluorophores. One strategy is the physical © 2017 American Chemical Society

Received: January 24, 2017 Accepted: March 1, 2017 Published: March 1, 2017 9528

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ACS Applied Materials & Interfaces urgent need to develop biocompatible fluorescent contrast agents that hold excellent stability and enhanced fluorescence quantum yield for bioimaging applications. Core-cross-linking has been demonstrated to be an effective method to improve the in vivo stability of micelles.34−37 Corecross-link is a bond that makes polymer chains in the core of micelles link each other,38−43 and it can be realized through methods like photo-cross-linking.44,45 Inspired by this observation in micelles, we propose to conjugate the fluorophores onto PGSA chain through covalent bonds and subsequently induce the core-cross-linking within the particles through photoinitiated free radical polymerization. We hypothesize that this would significantly improve the stability of fluorophores in the PGSA NPs. Taking rhodamine as the model fluorophore, we showed that the chemical conjugation onto PGSA and photocore-cross-linking minimized the detachment of rhodamines from PGSA NPs and improve their stability in the physiological conditions. More interestingly, the conjugation and core-crosslinking significantly improved the quantum yield of the fluorophore (i.e., 26.24-fold of that of free rhodamine). Excited by these observations, we explored the usage of rhodaminelabeled PGSA NPs for labeling and tracking mesenchymal stem cells in vitro and in vivo.

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Synthesis and Characterization of Poly(glycerol-co-sebacate) (PGS) Prepolymer. PGS prepolymer was synthesized through the condensation reaction as reported.1 Briefly, 0.03 mol of sebacic acid and 0.03 mol of glycerol were mixed in a 250 mL Schlenk flask and heated to 120 °C under argon gas for 24 h. Then the pressure was reduced to 40 mTorr over 5 h, and the reaction was continued for another 12 h. The PGS prepolymer was dissolved in anhydrous ethanol and dialyzed against anhydrous ethanol using the dialysis membrane with a molecular weight cutoff of 3500 Da (Spectrum Laboratories, USA) for 5 days to purify. Then the PGS prepolymer was obtained (yield: 90%), and its molecular weight distribution and structure were analyzed and characterized through gel permeation chromatography (GPC), nuclear magnetic resonance (1H NMR), and Fourier transform infrared spectroscopy (FTIR). Synthesis and Characterization of Rhodamine-Conjugated Poly(glycerol-co-sebacate) (PGSR). PGS prepolymer (0.5 g) was dissolved in 2 mL of anhydrous DMSO under a nitrogen atmosphere for 15 min at room temperature. Then 0.02 mL of pyridine, 0.1 g of RITC, and 0.02 mL of DBTDL were added, and the mixture was stirred for 4 h at 65 °C The final solution was added dropwise into a mixture of diethyl ether and ethanol (9:1, v:v) to precipitate the PGSR and remove the excessive pyridine, RITC, and DBTDL. The isolated viscous oily PGSR was dissolved in anhydrous ethanol and dialyzed against anhydrous ethanol with the same dialysis membrane as above. After 1 week dialysis, the final solution was concentrated with rotary evaporator to obtain an oily product, and then the product was subjected to high vacuum oven at 50 °C for 12 h to remove water and obtain the pure PGSR (yield: 80%). Then the molecular weight distribution and structure of PGSR were analyzed and characterized through GPC, 1H NMR, and FTIR. Synthesis and Characterization of Rhodamine-Conjugated Poly(glycerol-co-sebacate) Acrylate (PGSAR). PGSR was further acrylated with acryloyl chloride. Briefly, 0.5 g of PGSR and 0.5 mg of 4-(dimethylamino)pyridine (DMAP) were dissolved together in 20 mL of anhydrous dichloromethane. Then the reaction flask was cooled to 0 °C with an ice bath under a positive pressure of argon gas. Next, acryloyl chloride (0.5 mol/mol glycerol on PGS) and equimolar triethylamine were added dropwise with vigorous stirring in an ice bath. After addition, the reaction was allowed to reach room temperature and kept at this temperature for an additional 24 h with vigorous stirring. Then the resulting mixture was dissolved in ethyl acetate, filtered, and dried in a vacuum oven at 40 °C for 6 h (yield: 94%). Finally, the molecular weight distribution and structure of PGSAR were analyzed and characterized through GPC, 1H NMR, and FTIR. Synthesis and Characterization of PGSAR NPs. PGSAR NPs were prepared using the single emulsion method as reported.9 Briefly, a 10 mL solution of 10 mg/mL PGSAR in acetone supplemented with 12 mg of photoinitiator (Irgacure 2959) was added into 20 mL of 0.1% w/v sodium alginate (low viscosity) solution in water (pH = 7.0) dropwise under magnetic stirring (speed: 14 000 rpm) with a rate of 0.7 mL/min. The mixture was placed in dark fume hood for overnight to evaporate acetone. Then 500 μL of uncured PGSAR NPs solution was poured into a Petri dish to form an aqueous layer (with thickness of 1 mm) and exposed to the UV light (light intensity: 0.3 W/cm2) for 20 s to cure the NPs. Finally, the PGSAR NPs were purified through centrifugation and washed with PBS to remove the free surfactants. A similar procedure was applied to form PGSA-p-R NPs in water. Measurement of Fluorescence Quantum Yield. PGS, PGSA, RITC, PGSA-p-R NPs, uncured PGSAR NPs, and cured PGSAR NPs with the same size were fabricated, and a rhodamine solution (10 μg/ mL) was prepared as a control. Next, UV−vis spectra of all samples were tested, and the absorbance was collected at 550 nm. Then fluorescence of all samples was examined with an excitation wavelength at 550 nm and emission collection window from 560 to 700 nm. Subsequently, the fluorescence spectra and absorption spectra were analyzed, and the baselines of absorption curves of all samples were adjusted to zero through a peak analyzer. Finally, the fluorescence quantum yields (Φf) of different samples in aqueous solution were calculated using the equation46

MATERIALS AND METHODS

Glycerol (analytical grade, 99% pure), sebacic acid (analytical grade, 99% pure), rhodamine B isothiocyanate (RITC), di-n-butyltin(IV) dilaurate (DBTDL), pyridine (anhydrous, 99.8% pure), dimethyl sulfoxide (DMSO, anhydrous, ≥99.9%), ethanol (anhydrous, ≥99.8%) dichloromethane (DCM, anhydrous, ≥99.8%), diethyl ether (anhydrous, ACS reagent, ≥99%), triethylamine (≥99%), 4(dimethylamino)pyridine (DMAP) (≥99%), ethyl acetate (EA, analytical grade, ≥99%), 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), sodium alginate (low viscosity), bovine serum albumin (BSA), Triton X-100, formalin solution (10%), 4′,6-diamidino-2-phenylindole (DAPI), and matrigel were all purchased from Sigma-Aldrich and used without further purification. Acryloyl chloride (96%) was purchased from Aladdin (China). Dulbecco’s modified eagle medium (DMEM) with high glucose (4.5 g/L) and L-glutamine, fetal bovine serum (FBS), penicillin− streptomycin (10 000 U/mL), Alexa Fluor 633 phalloidin, and trypsin-EDTA (0.5%, 10×) were obtained from Life Technologies (USA). AlamarBlue cell viability reagent was purchased from Thermo Fisher Scientific (USA). The methyl cellulose (Metolose 90SH100000SR) was acquired from Shin-Etsu Chemical Co., Ltd. Equipment. 1H NMR spectra were tested on a Bruker Avance II 300 MHz spectrometer. FTIR spectra were obtained from a Spectrum One FTIR spectrometer. UV−vis absorption measurement was performed on a Shimadzu UV 2450 ultraviolet−visible spectrometer. Fluorescence measurement was performed on PerkinElmer LS-55 fluorometer. The number-average molecular weight (Mn) and polydispersity (Mw/Mn) of polymers were measured on an Agilent Infinity-1260 GPC at room temperature, using polystyrene as standard and THF as eluent at an elution rate of 1 mL/min. Zeta potential and dynamic light scattering (DLS) measurements were performed on a Zetasizer Nano ZS (Malvern Instruments, UK). Fluorescence imaging was performed on a confocal laser scanning microscopy (LSM 710, Carl Zeiss Pte Ltd.). The fluorescence intensity of individual cells in fluorescence images was obtained by using ImageJ. Transmission electron microscopy (TEM) images were acquired by JEM-2100 highresolution transmission electron microscope (JEOL, Japan). Flow cytometry data were collected on an LSR II cytometer (BD Biosciences, USA) and analyzed with FlowJo (Tree Star). At least 10 000 gated events were collected per sample. The in vivo experiments were performed on IVIS SpectrumCT in vivo imaging system (PerkinElmer). 9529

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ACS Applied Materials & Interfaces Φf = Φst

FA st FstA

set at least 10 000, and analysis was performed on a LSR II flow cytometer (BD Biosciences, USA). The PGSAR NPs-labeled cells were detected by using PE (excitation: 561 nm; emission: 567−597 nm) channel. In Vivo Imaging of PGSAR NPs. Freshly prepared fluorescent PGSAR NPs and PGSA-p-R NPs were encapsulated into methyl cellulose hydrogel (100K) with the concentration of 0.5 mg/mL, and then 200 μL aliquots of PGSAR NPs-entrapped or PGSA-p-R NPsentrapped gels were subcutaneously injected into one side of each healthy mouse’s back (female NCr nude mice, 8 weeks old, InVivos Pte Ltd.). The time-dependent in vivo imaging of the PGSAR NPs and PGSA-p-R NPS in mice was performed at day 0, 3, 7, 14, 21, and 28 by using an IVIS Spectrum CT in vivo imaging system (PerkinElmer). The light with a central wavelength at 535 nm was selected as the excitation source. In vivo spectral imaging from 580 to 640 nm (20 nm step) was carried out with an exposure time of 1 s for each image frame. The autofluorescence from mice skin was removed using spectral unmixing function in the software of IVIS system. The care and use of laboratory animals were performed according to the approved protocols of the Institutional Animal Care and Use Committee (IACUC) at Nanyang Technological University, Singapore. Tracking of PGSAR NP-Labeled MSCs in Vivo. The hMSCs (passage 7) (1.3 × 106) were seeded in each T175 flask and cultured at 37 °C. Then the original medium was replaced with culture medium (2% FBS, 1% penicillin−streptomycin), containing PGSAR NPs or PGSA-p-R NPs with rhodamine concentration of 0.8 μg/mL, respectively. After 6 h, the labeling medium was removed, and the labeled hMSCs were detached with trypsin. Then the PGSAR NPs or PGSA-p-R NPs labeled cells (5.3 × 106) were encapsulated in 800 μL of Matrigel. Next, 150 μL of hMSCs encapsulated Matrigel was subcutaneously injected into the back of each mouse. The timedependent in vivo imaging of the transplanted MSCs was performed at day 0, 3, 7, 14, 21, and 28 with the IVIS imaging system described above. In Vivo Biocompatibility. On day 28 postadministration, the skin of mice treated with PGSAR NPs was dissected based on fluorescence using the IVIS imaging system. Skin samples were fixed in 10% formalin for 24 h for histological analyses. Briefly, samples were dehydrated with following gradients: 30%, 50%, and 60% ethanol for 5 min, followed by 70%, 80%, 90%, 95%, and 100% ethanol for 10 min. Afterward, tissues were treated with 1 mL of xylene for 30 min, embedded into paraffin, and sectioned at a thickness of 5 μm. Then skin sections on slides were placed in oven at 50 °C for 3 h and treated with xylene for 12 h. Subsequently, they were washed with xylene and rinsed in 100% ethanol for 10 min two times, 90% ethanol for 5 min, 70% ethanol for 1 min, 50% ethanol for 1 min, 30% ethanol for 1 min, and PBS for 5 min, followed by H&E staining (Sigma). Finally, the slides were mounted and imaged with optical microscopy. Statistical Analysis. All data were expressed as mean ± standard deviation. Statistical difference between two sets of data was determined by Student’s t test, and P < 0.05 was considered statistically significantly.

(1)

where Φst is the fluorescence quantum yield of rhodamie B. F and Fst are the integrated values of fluorescence spectra for the sample and standard material, respectively. A and Ast are the absorbance of the sample and standard material at excitation wavelength (550 nm), respectively. Dye Release. PGSA-p-R NPs and uncured or cured PGSAR NPs stock solution (0.5 mL, 5 mg/mL) was injected into the dialysis cassette with a molecular weight cutoff (MWCO) of 10 kDa (Thermo Scientific, USA) and then dialyzed against 10 mL of phosphate buffer solution (pH = 7.4) at 37 °C. Pure rhodamine solution was used as a control. For sample collections, 0.5 mL of buffer was withdrawn, and an equal volume of buffer was refilled at the predetermined time points. The cumulative release of dye was measured by fluorescence and normalized to the percentage of the total rhodamine in the 0.5 mL stock solution. A similar procedure was also performed to examine the cumulative release of pure rhodamine and PGSAR NPs in acetate buffer solution (pH = 5.0). Cytotoxicity Assay of PGSAR NPs. The hMSCs (Lonza) (passage 5) were cultured in DMEM supplemented with 10% FBS and 1% penicillin−streptomycin in a humidified cell incubator with 5% CO2. Cytotoxicity of PGSAR NPs was analyzed by using Alamar Blue assay. Briefly, hMSCs were seeded in 96-well plates at a density of 8000 per cm2 12 h before the experiment. Then cells were incubated with 0, 6.25, 12.5, 25, 50, and 100 μg/mL PGSA-p-R NPs, uncured or cured PGSAR NPs in culture medium supplemented with 2% FBS and 1% penicillin−streptomycin for 6 h, respectively. Later the labeling solution was removed, and 0.1 mL of medium containing 10% Alamar Blue reagent was added into each well. After 4 h incubation, the fluorescence emission was recorded at 590 nm with an excitation wavelength of 545 nm through a spectromax M5 plate reader (Molecular Devices, USA). For long-term cytotoxicity experiment, PGSAR NPs were incubated with hMSCs at various concentrations (18−30 μg/mL) for 7 days before Alamar Blue assay. All samples were examined in at least triplicates. hMSCs Labeling with PGSAR NPs. The hMSCs (passage 7) were seeded at a density of 10 000 per cm2 into a 6-well plate 12 h before experiments. Then the original medium was replaced with culture medium (2% FBS, 1% penicillin−streptomycin), uncured or photocured PGSAR NPs or PGSA-p-R NPs (with a final rhodamine concentration of 0.125 μg/mL). After 6 h, the labeling medium was replaced with complete culture medium for any further experiment. Confocal Laser Scanning Microscope Imaging. The hMSCs (passage 7) were labeled as stated above and then incubated with full growth medium and refilled with fresh medium every 3−4 days. After 7, 14, 21, and 28 days, the full growth medium was removed and washed with PBS for 3 times; then the cells were fixed with formalin (10%) and stained with DAPI. Subsequently, each well was refilled with 2 mL of PBS. Finally, the cells labeled with different samples were observed with confocal laser scanning microscopy (LSM 710, Carl Zeiss Pte Ltd.) for DAPI (excitation: 405 nm; emission: 410−480 nm) and rhodamine (excitation: 561 nm; emission: 566−685 nm) in different samples. To study the subcellular localization of PGSAR NPs, live hMSCs were labeled by PGSAR NPs for 6 h and then stained with lysotrack green (50 nM) (excitation: 488 nm; emission: 495−547 nm) and DAPI (1 μg/mL) (excitation: 405 nm; emission: 421−481 nm) for 10 min, followed by confocal microscopy. To study the endocytic pathways for the internalization of PGSAR NPs, hMSCs were preincubated at 4 or 37 °C with chlorpromazine (30 μM), Nystatin (100 μM), Wortmannin (80 μM), or Cytochalsin D (20 μM) for 1 h followed by labeling with PGSAR NPs for 2 h before observation under fluorescence microscope. Flow Cytometry. The hMSCs (passage 7) were labeled as stated above and then incubated with full growth medium and refilled with fresh medium every 3−4 days. After 7, 14, 21, and 28 days, the full growth medium was removed and washed with PBS for 3 times and then collected through trypsinization. Subsequently, the cells were resuspended in PBS for flow cytometry analysis. The gated events were

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RESULTS AND DISCUSSION Rhodamine-conjugated poly(glycerol-co-sebacate) acrylate (PGSAR) was synthesized through a three-step reaction (Scheme 1). First, PGS prepolymer was prepared through the condensation of sebacic acid and glycerol.1 Gel permeation chromatography (GPC) analysis (Figure 1a) revealed that prepolymer had a number-average molecular weight (Mn) of 2200 with a polydispersity index (PDI) of 1.96 (Table 1). The chemical structure of this prepolymer was examined through 1 H-NM Rand FTIR spectroscopy. The peaks at 1.30, 1.62, and 2.34 ppm in the 1H NMR spectrum (Figure S1,Supporting Information) belong to methylene protons of sebacic acid while peaks at 4.0−4.5 and 4.8−5.3 ppm are from protons of secondary carbon and tertiary carbon of glycerol, respectively. 9530

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ACS Applied Materials & Interfaces Scheme 1. Synthetic Routes of Rhodamine-Conjugated Poly(glycerol-co-Sebacate) Acrylate (PGSAR)

Table 1. Characteristic Information of PGS, PGSR, and PGSAR Polymersa sample

Mn

Mw

PDI

[R] (%)

[A] (%)

PGS PGSR PGSAR

2200 2400 2800

4400 6600 7900

1.96 2.69 2.78

0.4 0.4

40

a

Mn is the number-average molecular weight, Mw is the weight-average molecular weight, and PDI is the polydispersity index; [R] is the value of mole percentage of conjugated rhodamine in the PGSR polymer; [A] is the value of mole percentage of conjugated acrylate groups in the PGSAR polymer.

Next, rhodamine was conjugated to PGS through the reaction between the hydroxyl groups of PGS and isothiocyanate groups of rhodamine B isothiocyanate (RITC). The successful conjugation between rhodamine and PGS was confirmed through GPC and 1H NMR. In the GPC chromatogram, the elution time showed a slight decrease after rhodamine conjugation (Figure 1a). Respectively, the Mn of the polymer changed from 2200 to 2400 (Table 1). In the 1 H NMR spectrum (Figure S2), the presence of rhodamine brought in additional peaks at 6.67, 7.22, 8.24, and 8.27 ppm corresponding to aromatic protons. The molar ratio between rhodamine and the glycerol of PGS was estimated to be 4:1000 according to the ratio of the integral of the peak at 8.27 ppm (position “g”, rhodamine segment) and one-fourth integral of the peak at 1.5 ppm (position “d”, methylene groups on sebacic acid segment) in the 1H NMR spectrum. The final step of the synthesis was to modify rhodamineconjugated PGS (PGSR) with acryloyl chloride that further decreased the elution time of polymers (Figure 1a) and increased the Mn to 2800 g/mol (Table 1). The appearance of new peaks at 5.89, 6.13, and 6.39 ppm in 1H NMR associated with the protons of the double bond in acryloyl groups confirmed the conjugation (Figure S3). The degree of acrylation was approximately 0.4, calculated by comparing the integrals of peaks at 2.31 ppm (position “c”, methylene groups in sebacic acid segment) and 6.13 ppm (position“o”, vinyl groups in acryloyl segment). The absorbance at 1637 cm−1 in the FTIR spectrum was caused by the CC stretching of the acrylate groups as well (Figure 1b). To prepare PGSAR NPs, uncured PGSAR NPs were formed through a single-emulsion method as our previous report (Figure 2a).9 These uncured PGSAR NPs with a hydrophobic

The molar ratio of glycerol and sebacic acid in the PGS prepolymer is approximately 1:1 according to the ratio of the integrals of the peaks at 1.62 ppm (position “d”, methylene groups on sebacic acid segment) and 4.16 ppm (position “a”, methylene groups on glycerol segment) in the 1H NMR spectrum. 47 In the FTIR spectrum (Figure 1b), PGS prepolymer displayed a strong absorption band at 1742 cm−1 corresponding to carbonyl groups (CO stretching) from the ester groups and a broad absorption band at 3457 cm−1 corresponding to free hydroxyl groups (−OH stretch) that were later used for the further conjugation with rhodamine or acrylate.

Figure 1. Characterization of rhodamine-modified poly(glycerol-co-sebacate) acrylate (PGSAR). (a) Normalized GPC spectra of PGS, PGSR, and PGSAR. (b) FTIR spectra of PGS, PGSR, and PGSAR. 9531

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Figure 2. Synthesis and characterization of PGSAR NPs. (a) Schematic of the PGSAR NP synthesis. (b) TEM image of PGSAR NPs. (c) Hydrodynamic diameter of PGSAR NPs; inset: photo of the PGSAR NP aqueous solution.

Figure 3. Characterization of optical properties of PGS, PGSA, RITC, PGSA-p-R NPs, uncured PGSAR NPs, and PGSAR NPs: (a) UV−vis absorption spectra; (b) fluorescence spectra. All samples contained 2 μg/mL rhodamine.

by TEM and that from dynamic light scattering (DLS) is most likely due to the shrinkage of alginate coating during TEM preparation. The optical properties of PGS, PGSA, RITC, PGSA-p-R NPs, uncured PGSAR NPs, and PGSAR NPs were examined in deionized water (Figure 3). First, PGS or PGSA did not show any absorption peak or fluorescence signals in regions we tested. For RITC, PGSA-p-R NPs, uncured PGSAR NPs, and PGSAR NPs, the maximum absorption wavelengths were 550, 560, 554.5, and 555 nm, respectively (Figure 3a). Comparing to the absorption peak of RITC, the absorption peaks of PGSA-p-

PGSAR core and a hydrophilic coating of sodium alginate were solidified or cured through UV irradiation to form PGSAR NPs. The curing was confirmed through observing the weakening of peaks related with CC stretching of acrylate groups in the FTIR spectrum (Figure S4). TEM examination showed that PGSAR NPs displayed a spherical shape, with an average diameter of 118.25 ± 5.08 nm (Figure 2b). They were negatively charged with the zeta potential at −60.5 ± 3.11 mV (Figure S5a) and had an average hydrodynamic diameter of 190.80 ± 2.11 nm (Figure 2c). The solution presented a pink milky color (Figure 2c). The discrepancy of the sizes measured 9532

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Figure 4. (a) Release profiles of pure rhodamine solution, PGSA-p-R NPs, uncured PGSAR NPs, and PGSAR NPs in PBS (pH 7.4) at 37 °C. (b) The zoom-in release profile of (a) in the first 50 h.

Besides the fluorescence enhancement, the release profile of PGSAR NPs was also investigated. A dialysis cassette with a molecular weight cutoff (MWCO) of 10 kDa (larger than the molecular weight of PGSAR ∼ 3 kDa) was used to examine the stability of PGSAR nanoparticles before and after photo-crosslinking. As a control, rhodamine was also physically trapped within PGSA NPs that were prepared through a similar procedure. As shown in Figure 4, the free rhodamine had the fastest release from the dialysis cassette and ∼80% of rhodamine was released out at 8 h. PGSA NPs with physically entrapped rhodamine (PGSA-p-R NPs) had a slower release rate, and the cumulative releases at 8 h and 7 days were 40% and 100%, respectively. In contrast, the rhodamine-conjugated PGSAR NPs without core-cross-linking (uncured PGSAR NPs) showed the much lower dye release rate, and at 8 h and 28 days, the cumulative release was 28.19% and 57.49%, respectively. Most importantly, PGSAR NPs displayed the slowest release rate of the fluorophore. After 8 h, there was only 10.60% release. Since rhodamine was covalently linked to PGSA polymers, the initial release that we observed in uncured PGSAR NPs and PGSAR NPs in the first several hours could be attributed to the signals from un-cross-linked and insufficiently cross-linked PGSAR molecules (molecular weight 90% PGSAR NP-labeled cells were still fluorescent. However, only 28.1% and 58.7% PGSA-pR NP-labeled and uncured PGSAR NPs-labeled cells were fluorescent. At week 3 and 4, the percentage of fluorescent cells labeled with PGSAR NPs decreased to 86.9% and 51.5%, respectively. For cells labeled with PGSA-p-R NPs and uncured PGSAR NPs, the number decreased to 24.8% and 11% and 44.9% and 19.3%, respectively, which were roughly one-fifth and one-third of those labeled with PGSAR NPs, respectively (Figure 5b). And the difference can also be observed from the fluorescence intensity normalized against that of unstained cells (Figure 5d). For cells labeled with PGSAR NPs, the number changed from the initial value of 54.08 (day 0) to 31.23 (week 2) to 21.95 (week 3) and 11.62 (week 4). And for cells labeled with PGSA-p-R NPs and uncured PGSAR NPs, the values at day 0, week 2, week 3, and week 4 were 25.79 and 30.51, 5.93 and 7.56, 4.88 and 5.16, and 3.45 and 3.72, respectively. At day 0, the normalized fluorescence intensity of PGSAR NP-labeled hMSCs was only 2.10 and 1.77 times of PGSA-p-R NP-labeled and uncured PGSAR NPs-labeled cells. However, at week 2, 3, and 4, this difference increased to 5.27 and 4.13, 4.50 and 4.25, and 3.37 and 3.12, respectively. These findings in flow cytometry were consistent with the fluorescence images we observed in confocal microscopy and 9535

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injected was observed on the back immediately after injection (day 0). As the time went on, the fluorescence signal gradually decreased (2 and 3 weeks later, the fluorescence signal became 24.11% and 15.7% of the original value), but it was still observable (15.66% of the original value) after 28 days (Figure 6d). In comparison, the fluorescence signal where PGSA-p-R NPs-labeled hMSCs were injected was almost undetectable even at the beginning, which may be due to the rapid cellular release of rhodamine from the PGSA-p-R NPs. And the fluorescence signal of injected PGSA-p-R NPs-labeled hMSCs also decreased much faster and was only 6.04%, 3.03%, and 2.25% of the original value for the day 14, 21, and 28 (Figure 6d). This result was consistent with the in vitro experiments and confirmed the stability of the fluorescence of PGSAR NPs for cell tracking both in vitro and in vivo. Finally, we investigated the biocompatibility of PGSAR NPs through subcutaneous injection in mice. After 28 days of NP administration, we collected the skin tissues that showed fluorescence (using IVIS imaging system stated in Figure 6a) for histological analyses. As shown in Figure 7, we did not

Research Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b01203. 1 H NMR spectra of PGS prepolymer in CDCl3, PGSR polymer in DMSO-d6, and PGSAR polymer in CDCl3; FTIR spectra of uncured and cured PGSAR NPs; zeta potential of uncured and cured PGSAR NPs; fluorescence quantum yield of free rhodamine, PGSA-p-R NPs, uncured PGSAR NPs, and PGSAR NPs; cumulative release of pure rhodamine solution and PGSAR NPs in acetate buffer (pH = 5) at 37 °C; contents of released PGSAR and residual PGSAR after 14 days release; cell viabilities of hMSCs after incubating with various concentrations of PGSA-p-R NPs, uncured PGSAR NPs, and PGSAR NPs for 6 h; cell viabilities of MSCs after incubating with various concentrations of PGSAR NPs for 7 days; fluorescence images of hMSCs labeled with RITC at the same fluorophore concentration as PGSAR over a period of 4 weeks; flow cytometry analysis of PGSA-p-R NPs-labeled, uncured PGSAR NPs-labeled, and PGSAR NPs-labeled hMSCs at day 0; internalization of PGSAR NPs in hMSCs cells (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (C.X.). *E-mail: [email protected] (W.H.). *E-mail: [email protected] (X.D.).

Figure 7. Photomicrographs of H&E analyses of the skins without (a) or with (b) the injection of PGSAR NPs after 28-day administration. Arrows indicate the boundary of skin’s muscle layer. Scale bar = 100 μm.

ORCID

Wei Huang: 0000-0001-7004-6408 Chenjie Xu: 0000-0002-8278-3912 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Specifically, L.W., K.X., X.D., W.H., and C.J.X. conceived the project and wrote the manuscript. L.W. and K.X. performed the experiments and analyzed the data. X.H., Y.H., S.L., C.W, J.Y., and M.W. helped plan the experiments and provided valuable input during the manuscript preparation.

observe the presence of NP aggregates, suggesting the total uptake of NPs by surrounding cells. Comparing to the healthy skin (control, without PGSAR NPs), only mild tissue reactions were observed as indicated by the presence of lymphocytes and fibroblasts in elevated numbers. Yet, no fibrous capsule was observed around the muscle layer of the skin where the PGSAR NPs were injected. The formation of fibrous capsule usually occurs after the implantation of foreign objects as a result of acute or chronic inflammation,58−60 and the thickness of fibrous capsule has been widely reported as an indicator for the severity of inflammation.61,62 The absence of fibrous capsule in these fluorescent skin tissues further confirmed the excellent biocompatibility of PGSAR NPs.

Author Contributions

L.W. and K.X. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by Ministry of Education Tier 1 Academic Research Fund (RG131/15), NTU-Northwestern Institute for Nanomedicine (M4081502.F40), and Primary Research & Development Plan of Jiangsu Province (BE2016770).

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CONCLUSIONS To summarize, the chemical conjugation and core-cross-linking approaches were utilized to improve the stable presence of fluorophores within PGSAR NPs. Excitingly, this strategy not only significantly improved the stability of fluorophores in the PGSA NPs but also increased the quantum yield of the fluorophores. This observation allowed us to explore the utilization of the derived PGSAR NPs for labeling and longitudinal tracking stem cells both in vitro and in vivo. In the future, this strategy (i.e., chemical conjugation and corecross-linking) can be further utilized to stabilize other small molecules such as drugs to minimize the burst release and provide a sustainable release.

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