Hyaluronic Acid Nanoparticles Based on a Conjugated Oligomer

Article Cite This: ACS Appl. Bio Mater. 2019, 2, 2421−2434

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Hyaluronic Acid Nanoparticles Based on a Conjugated Oligomer Photosensitizer: Target-Specific Two-Photon Imaging, RedoxSensitive Drug Delivery, and Synergistic Chemo-Photodynamic Therapy Yan-Qin Huang,*,† Li-Jie Sun,† Rui Zhang,*,∥ Jian Hu,† Xing-Fen Liu,† Rong-Cui Jiang,† Qu-Li Fan,† Lian-Hui Wang,† and Wei Huang*,†,‡,§ Downloaded via BUFFALO STATE on July 29, 2019 at 12:35:01 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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Key Laboratory for Organic Electronics & Information Displays (KLOEID) and Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, Nanjing 210023, China ‡ Shaanxi Institute of Flexible Electronics (SIFE), Northwestern Polytechnical University (NPU), 127 West Youyi Road, Xi’an 710072, China § Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), Nanjing 211816, China ∥ Department of Ophthalmology, Zhongda Hospital, Southeast University, Nanjing 210009, China S Supporting Information *

ABSTRACT: Self-assembled hyaluronic acid (HA) nanoparticles have been extensively investigated as anticancer therapeutic agents due to the biocompatibility, biodegradability, and active targeting characteristics of HA. However, many HA nanoparticles are restricted to the applications in drug delivery for chemotherapy or lack effective imaging agents. Hence, we developed the camptothecin (CPT)-loaded HA-SS-BFVPBT nanoparticles (HSBNPs) as a multifunctional platform for two-photon imaging and synergistic chemo-photodynamic therapy at the same time. A novel conjugated oligomer photosensitizer, BFVPBT, which was conjugated onto HA through the redox-responsive disulfide linkage (SS), could not only provide a hydrophobic domain for the formation of nanoparticles and drug entrapment but also act as a two-photon photosensitizer that can be directly excited and simultaneously used in two-photon imaging and photodynamic therapy (PDT). HeLa cells overexpressing the HA receptor (CD44) were used for in vitro studies, which proved the specific cellular uptake of CPT-loaded HSBNPs and excellent two-photon PDT/chemotherapy synergistic effect. The nanoparticles have also been shown to realize tumor-targeting in vivo imaging in HeLa-tumor-bearing mice. Moreover, the fluorescence of CPT-loaded HSBNPs could be activated due to the degradation by the reductive glutathione (GSH) and overexpressed hyaluronidases (Hyal-1) in cancer cells, and the intracellular drug release rate was quickened, thus improving the probability of precise cancer diagnosis and therapy. Accordingly, this HSBNPs system is also anticipated to be a precise nanocarrier for other imaging and therapeutic agents besides CPT, offering a promising new avenue for imaging-guided efficient cancer therapy. KEYWORDS: hyaluronic acid nanoparticles, conjugated oligomer photosensitizer, two-photon imaging, chemo-photodynamic therapy, redox-responsive

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attention recently.6−12 HA is an anionic disaccharide polymer that naturally exists in extracellular matrices, connective tissue, epithelial tissue, and neural tissues.13−15 Importantly, HA possesses strong specificity and affinity for CD44a receptor overexpressed in different cancer cells. It enters into cancer cells through CD44-mediated endocytosis and can be

INTRODUCTION Nanomaterials with multiple functions, which provide both diagnostic and therapeutic features in one platform, have recently sparked tremendous interest.1−6 Generally, an ideal multifunctional nanoplatform for treatment needs good biocompatibility, effective drug delivery and imaging, and a targeting ability. Various kinds of nanoparticles were designed to optimize treatment strategies. Particularly, the studies and applications of self-assembled hyaluronic acid (HA) nanoparticles as anticancer therapeutic agents have attracted much © 2019 American Chemical Society

Received: February 15, 2019 Accepted: May 7, 2019 Published: May 7, 2019 2421

DOI: 10.1021/acsabm.9b00130 ACS Appl. Bio Mater. 2019, 2, 2421−2434

Article

ACS Applied Bio Materials Scheme 1. Schematic Model of Effective Intracellular CPT Deliverya by HA-SS-BFVPBT with Redox Response and Biodegradability

a

CPT delivery involves CD44-mediated endocytosis by cancer cells and CPT release in cytoplasm initiated by GSH and Hyal-1.

imaging-guided PDT.30 Organic conjugated materials with expanded π-conjugated systems have been employed as effective two-photon imaging and therapeutic agents.29−33 In contrast with quantum dots or organic fluorescent dyes, they have been proven to be long-term probes for intracellular twophoton imaging because of their low cytotoxicity and excellent photostability.30,34 Among them, conjugated oligomers with two-photon absorption, which displayed fascinating merits including tiny size, small interference to biological systems, and easy synthesis, are especially conducive to the practical application of two-photon imaging and PDT.31,35−38 However, many conjugated oligomers always suffer from poor water solubility or the lack of an active targeting function, tremendously restricting their biological applications. To overcome the above obstacles, it would be meaningful to develop HA nanoparticles based on a conjugated oligomer photosensitizer for simultaneous two-photon imaging and synergistic chemo-photodynamic therapy. Herein, we developed a novel HA derivative, HA-cystamine-BFVPBT (HA-SSBFVPBT), by conjugating a hydrophobic conjugated oligomer, BFVPBT, onto HA by using cystamine, a disulfide-based bifunctional primary amine, as the linker molecule (Scheme 1). The amphiphilic HA-SS-BFVPBT was easy to self-assemble into nanoparticles (HSBNPs) in aqueous solution and could encapsulate the hydrophobic model anticancer drug camptothecin (CPT). BFVPBT can not only provide a hydrophobic domain for the formation of nanoparticles and drug entrapment but also serve as a photosensitizer that can be directly excited and simultaneously used in two-photon imaging and PDT. Thus, a multifunctional nanoplatform, CPT-loaded HSBNPs for simultaneous target-specific twophoton imaging and synergistic chemo-photodynamic therapy, was constructed. Furthermore, the redox-responsive disulfide bond linkages and biodegradable HA in the nanoparticles enabled this system to release anticancer drugs rapidly in cancer cells, where the concentrations of GSH and Hyal-1 are much higher than those in normal cells.10,11 Because of the aggregation of BFVPBT and CPT in the inner core, CPTloaded HSBNPs exhibited almost quenched fluorescence, and the fluorescence could be activated by the disassembly of the

degraded by hyaluronidases (mainly Hyal-1), whose levels were reported to rise in numerous malignant tumors.13−15 The biocompatibility, biodegradability, active targeting characteristics, and easily modified chemical structure of HA make it widely employed in various cancer treatment methods.6−15 HA can self-assemble into nanoparticles when it is chemically bound to hydrophobic moieties, including 5βcholanic acid, 1-pyrenebutanamide, and octadecylamine.6−9,15 However, many HA nanoparticles are restricted to the applications in drug delivery for chemotherapy,7−11 which may show some deficiencies like endotoxicity or drug resistance and lead to side effects.5 Moreover, when it comes to diagnosing diseases and monitoring the treatment through imaging, HA always needs to be further modified with quantum dots16,17 or organic fluorescent dyes like Cy 5.5,6,7 fluorescein,9 rhodamine,10 and coumarin.11 Organic fluorescent dyes are inappropriate to long-time imaging because of photobleaching,18 and quantum dots are often inherently toxic,19 which greatly restrict their clinical use. Therefore, to enhance the efficacy of HA nanoparticles, there is a great demand for the incorporation of additional diagnostic and therapeutic modalities in this single platform. Currently, synergistic chemo-phototherapy has led to wide interest in clinical treatment research.20−22 Photodynamic therapy (PDT) is a noninvasive cancer treatment that has great prospects in the future. Particularly, two-photon PDT exhibits many advantages, such as deep penetration, inherent threedimensional space selectivity, less light damage, and less photobleaching. It is superior to traditional single-photon PDT in precise cancer treatment.23,24 Moreover, because of the advantages of large penetration depth, high spatial resolution, and minimized autofluorescence,25−27 two-photon imaging was integrated with PDT to diagnose diseases, to guide and monitor the treatment, and to assess the success of therapy. Two-photon imaging-guided therapy was successfully employed in preclinical studies and has shown many unique advantages.28−31 Excellent photosensitizers, which can produce both fluorescence and singlet oxygen effectively under two-photon laser irradiation, are a very important premise for two-photon 2422


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ACS Applied Bio Materials

NHS, and 2-mercaptoethanol (MW cut off 3500). After freeze-drying, an orange powder (Figure S3) is obtained as a product, which is stored in darkness at −20 °C. The lyophilized HA-SS-BFVPBT is measured by 1H NMR (400 MHz, DMSO/D2O: 2v/1v). It is also redistributed in DMF to measure the degree of substitution (DS, the molar content of BFVPBT in HA-SS-BFVPBT) on HA-SS-BFVPBT using UV−vis absorption spectra. Briefly, the UV−vis absorption spectra of DMF solutions of BFVPBT with different concentrations (2−10 μM) are measured. The standard curve at 425 nm is thus obtained, and the DS is determined according to the standard curve. The critical micelle concentration (CMC) is measured by a fluorescence technique using pyrene as the hydrophobic molecule. CMC is calculated from the ratio of fluorescent intensities at 373 and 384 nm (I373/I384) and the logarithm of HA-SS-BFVPBT concentrations (1 × 10−4 to 0.5 mg/ mL). Preparation and Characterization of HA-SS-BFVPBT NPs (HSBNPs) and CPT-Loaded HSBNPs. HSBNPs are prepared by a simple method including the steps of sonication, dialysis, and lyophilization. A 10 mg portion of HA-SS-BFVPBT is dissolved in a mixture of 5 mL of DMSO and 5 mL of deionized water. Next, in an ice bath, the solution is sonicated at 50% amplitude with a probe sonicator (ultrasonic processor 130 W model, Sonics, Newtown, CT) for 10 min, in which the pulse is closed for 2 s, and the interval is 10 s. Then, the solution is stirred for 2 h and dialyzed against deionized water (1 L) for 24 h (MW cut off 3500) to eliminate DMSO and promote the self-assembly of HSBNPs. Finally, the solution is filtered by a 0.45 μm membrane and lyophilized. CPT-loaded HSBNPs are prepared and purified by the same method as HSBNPs. A 2 mg portion of CPT is dissolved in 5 mL of DMSO containing 10 mg of HA-SS-BFVPBT, and then, the solution is mixed with 5 mL of deionized water. The follow-up steps are consistent with the selfassembly process of HSBNPs. After filtration using a 0.45 μm syringe filter to eliminate unloaded CPT, the solution is lyophilized to obtain CPT-loaded HSBNPs. For the measurement of CPT loading content (LC) and loading efficiency (LE), CPT-loaded HSBNPs are disassembled with DMF, and the drug content is analyzed by UV− vis absorption spectra. The CPT UV−vis absorption spectrum is given in Figure S4, and the standard curve at 366 nm is obtained from the DMF solutions of CPT with different concentrations. The content of CPT in HSBNPs is obtained on the basis of the standard curve. LC and LE are calculated according to the following formula.8

nanoparticles in the typical microenvironment of cancer cells. This type of activatable nanoparticles is anticipated to provide a precise cancer diagnosis and an optimized therapeutic strategy.

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EXPERIMENTAL SECTION

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METHODS

Materials and Instruments. HA (Mw = 210 kDa) was bought from Freda Biopharm Co. Ltd. (Shandong, China). β-Mercaptoethanol, camptothecin (CPT), and dithiothreitol (DTT) were purchased from Aladdin (Shanghai, China). 2,2′-(Anthracene-9,10-diylbis(methylene) dimalonic acid (ABDA), propidium iodide (PI), and 2′,7′-dichlorodihydro-fluorescein diacetate (DCFH-DA) were bought from Sigma-Aldrich Chemical Co. We purchased other chemicals from Energy Chemical Co. (Shanghai, China). Milli-Q ultrapure water (18.2 MΩ cm) was used to prepare the buffers. Unless otherwise noted, all the chemicals are analytical and utilized without purification. The synthesis and characterization of BFVPBT are shown in the Supporting Information (Scheme S1 and Figures S1 and S2). NMR spectra were obtained with a Bruker Ultra Shield Plus 400 MHz NMR instrument. Elemental microanalyses were carried out on a Vario EL III CHNOS elemental analyzer. UV−vis absorption spectra were measured by a PerkinElmer Lambda 650 UV−vis−NIR spectrophotometer. Photoluminescence (PL) spectra were recorded on a PerkinElmer LS 55 luminescence spectrophotometer. ζ potentials were recorded on a ζ potential analyzer (Zeta PALS, Brookhaven Instruments Corp.). The hydrodynamic sizes of nanoparticles were obtained with dynamic light scattering (DLS) on a 90 Plus particle size analyzer (Brookhaven Instruments). TEM images were obtained using a Hitachi HT7700 transmission electron microscope at an acceleration voltage of 100 kV. Photoluminescence quantum yields were measured by an Edinburgh FLSP920 fluorescence spectrophotometer equipped with an integrating sphere and a xenon lamp. Using a 720 nm Ti-Sapphire oscillator as the twophoton excitation source, 100 fs pulses were generated at an 80 MHz repetition frequency. The fluorescence images were recorded on a confocal laser scanning microscope (CLSM, Olympus, FV1000MPE). The (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed using a microplate reader (BioTek, PowerWave XS/XS2). The in vivo fluorescence imaging test was performed using an IVIS Lumina K small-animal imaging system (PerkinElmer).

LC = (weight of CPT in CPT‐loaded HSBNPs /weight of CPT‐loaded HSBNPs) × 100

Synthesis and Characterization of Cystamine-Conjugated Hyaluronic Acid (HA−Cystamine). Sodium hyaluronate (200 mg, 0.5 mmol) is dissolved in 40 mL of PBS (0.02 M, pH 6.8) and stirred to be completely dissolved. 1-Ethyl-3-[3-(dimethylamino)-propyl] carbodiimide (EDC, 14.4 mg, 0.075 mmol) and N-hydroxysuccinimide (NHS, 8.6 mg, 0.075 mmol) dissolved in PBS are added in and stirred for 2 h. Next, cystamine dihydrochloride (84.4 mg, 0.375 mmol) dissolved in PBS is added dropwise, and the solution is stirred for 24 h at room temperature. Then, the solution is dialyzed against deionized water for 24 h to eliminate the excessive cystamine, EDC, and NHS (MW cut off 3500). Finally, the product HA−cystamine is lyophilized for 3 days and preserved at room temperature for use. The degree of cystamine substitution on HA is obtained by 1H NMR analysis (400 MHz, D2O). Synthesis and Characterization of HA-Cystamine-BFVPBT (HA-SS-BFVPBT). BFVPBT (9.57 mg, 10 μmol) is dissolved in DMSO (10 mL). EDC (2.3 mg, 12 μmol) and NHS (1.38 mg, 12 μmol) are dissolved in 5 mL of PBS (0.1 M, pH 8.0) and then added to the solution of BFVPBT. The mixture is stirred for 2 h, and then, 2mercaptoethanol (12 μmol, 1.4 μL) is added to terminate this reaction. After 5 min, HA−cystamine (C = 5 mg/mL) in PBS solution (0.1 M, pH 8.0) is added to the activated BFVPBT solution, which is stirred overnight at room temperature. Then, the solution is dialyzed against methanol/deionized water (2v/1v−1v/1v) for 12 h and deionized water for 24 h to eliminate the excessive BFVPBT, EDC,

LE = (weight of CPT in CPT‐loaded HSBNPs /weight of the feeding CPT) × 100 Reduction-Triggered Disassembly of HSBNPs. The reductive extracellular and intracellular conditions are simulated by using different concentrations of DTT solution. The redox sensitivity of HSBNPs to 10 μM and 10 mM DTT in PBS (pH 7.4, 0.02 M) is studied by DLS and TEM. First, HSBNPs are dissolved in 3 mL of PBS (c = 0.08 mg/mL), and O2 in the solution is eliminated under a mild nitrogen (N2) gas flow for 20 min at room temperature. Next, after the addition of DTT, the solutions are incubated at 37 °C; then, the morphology is monitored by DLS at predetermined time intervals and finally observed by TEM. In Vitro Drug Release. The concentrations of released drug from CPT-loaded HSBNPs are measured by a UV spectrophotometer. In brief, 10 mg of CPT-loaded HSBNPs is dispersed in 2.5 mL of PBS (pH 7.4, 0.02 M) and then shifted into a dialysis bag (MW cut off 3500), which is then dipped in 150 mL of PBS (pH 7.4 or pH 5.0) containing 0.1% (w/v) Tween 80. After the addition of DTT to PBS (0 and 10 mM), the experiment is carried out in an incubated shaker at 37 °C with vibrating speed of 100 rpm. A 3 mL portion of the sample solution is removed within the selected time intervals, and the solution is supplemented with the same volume of corresponding 2423


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ACS Applied Bio Materials Scheme 2. Synthetic Routes for HA-SS-BFVPBT

concentration: 0, 0.2, 1, 2, 3, 4 μg/mL) are added into the wells, separately. After incubation for 48 h, the cells are gently washed with PBS two times. Then, 100 μL of MTT in fresh medium (0.6 mg/mL) is added, and the cells continue to be incubated for another 3 h. The culture medium is then discarded, and DMSO is added at 100 μL/ well. The absorbance is obtained by a microplate reader at 490 nm. The MTT assay is also performed for an untreated cell population as a control to set up 100% cell viability. On the other hand, for the phototoxicity test, HeLa cells are treated with the medium containing HSBNPs or CPT-loaded HSBNPs (equivalent BFVPBT concentration, 0.26 μg/mL; equivalent CPT concentration, 0.2 μg/mL). They are then incubated for 24 h before exposure to a two-photon laser (λ = 720 nm, power = 2 W/cm2) for 5, 10, or 15 min. The solutions are discarded, followed by the addition of fresh medium. The treated cells continue to culture for another 24 h. The following experimental procedures are similar to those in the dark cytotoxicity test after incubation. The phototoxicity of HSBNPs and CPT-loaded HSBNPs is also assessed by PI staining. HeLa cells (1 × 106) are cultured in CLSM dishes overnight. Cells are, separately, incubated with these two kinds of nanoparticles at an equivalent BFVPBT concentration of 5 × 10−5 M for 6 h and then exposed to a two-photon confocal laser (λ = 720 nm, power = 2 W/cm2) for 20 min. Finally, the dead cells are stained by PI (5 μM) according to the suggested procedures. The red fluorescent signals of PI at 615 nm are captured under the excitation at 535 nm by using CLSM. In Vivo Fluorescence Imaging. Female nude mice aged 5−6 weeks are bought from Jiangsu KeyGEN Bio TECH Corp., Ltd. HeLa cells in 50 μL of PBS (4 × 106) are subcutaneously injected into the right forelimb armpit of every mouse to establish the HeLa tumor. When the tumors become about 120−150 mm3 in size, CPT-loaded HSBNPs or HA-SS-BFVPBT (150 μL, equivalent BFVPBT concentration: 400 μM) are intravenously injected into HeLatumor-bearing mice. Fluorescent images are obtained at different time points postinjection (0, 0.5, 1, 3, 5.5, 7.5, 10, 12, and 24 h) with excitation at 420 nm and emission at 620 nm, using an IVIS Lumina K small-animal imaging system (PerkinElmer).

fresh PBS. UV−vis absorption at 366 nm is used to determine the cumulative CPT release. Cellular Uptake of CPT-Loaded HSBNPs and Cell Imaging. CPT-loaded HSBNPs are tested in HeLa cells and NIH-3T3 cells to study the cellular uptake efficiency and specificity. First, the cells are cultured in culture medium for 24 h (density, 5 × 105/mL, 37 °C) in CLSM dishes. Next, the medium is substituted by fetal bovine serum (FBS)-free and CPT-loaded HSBNPs-containing medium (0.05 mg/ mL), and the cells continue to be incubated for 4 h at 37 °C. Then, they are rinsed with PBS (0.01 M, pH 7.4) two times. Additionally, a high dose of free-HA (3 mg/mL) is used to incubate another dish of HeLa cells for 2 h at 37 °C, which are then treated with CPT-loaded HSBNPs.8,9 The cell imaging test is then performed on CLSM with two-photon excitation at 720 nm. The emissions in the range 500− 700 nm are collected. Detection of Singlet Oxygen (1O2). The photodegradation of an 1 O2 trapping agent ABDA is employed to detect the generation of 1O2 under one-photon excitation. Briefly, the aqueous solution of HSBNPs (equivalent BFVPBT concentration of 1 × 10−4 M) containing 3.6 μM ABDA is exposed to xenon lamp irradiation (λ = 400 nm, power = 0.2 W/cm2) for different periods of time. Then, the photodegradation rate of ABDA is obtained from the decrease of the absorption at 399 nm with the excitation time. Taking tris(2,2′bipyridine)dichlororuthenium(II) (Ru(II)(bpy)3Cl2) as the standard reference, the 1O2 quantum yield of HSBNPs can be determined. The intracellular 1O2 generation is characterized using the DCFHDA fluorescent probe. HSBNPs are added to the HeLa cells cultured in CLSM dishes at the BFVPBT concentration of 1 × 10−5 M. After the cells are incubated for another 3 h, the culture medium is substituted by 10 μM DCFH-DA. After 30 min, the cells are irradiated by a two-photon confocal laser (λ = 720 nm, power = 2 W/ cm2) for 10 min to produce singlet oxygen. The green fluorescent signals of DCF at 505−555 nm are captured under the irradiation at 488 nm by using CLSM. In Vitro Cytotoxicity Evaluation. The standard MTT assay is employed to study the dark cytotoxicity and phototoxicity of HSBNPs and CPT-loaded HSBNPs in HeLa cells. HeLa cells are cultured for 24 h at 37 °C in a 96-well plate at 1 × 104/well before the experiments. Next, for the dark cytotoxicity test, a series of doses of free-CPT or CPT-loaded HSBNPs solutions (equivalent CPT 2424


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Figure 1. 1H NMR spectra of (a) HA−cystamine and (b) HA-SS-BFVPBT.

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RESULTS AND DISCUSSION

to hyaluronic acid. According to the literature, when the carboxyl group on the main chain of the HA is oversubstituted, the targeting ability of HA to the receptor will be weakened, but it can maintain enough targeting ability when the degree of substitution is below 35%.16 Hence, in the next reaction, the conjugation of BFVPBT to HA−cystamine was performed using HA−cystamine with 18% and 31% degree of substitution, respectively. Then, the primary amine groups on HA−cystamine were conjugated to the carboxylic acid groups of BFVPBT by using EDC/NHS. By controlling the ratio of feeding, only one-fourth of the carboxyl groups in BFVPBT were activated, and the excessive EDC was inactivated by 2-mercaptoethanol, which facilitated the one-pot reaction.16,39 The successful conjugation

Synthesis and Characterization of HA-SS-BFVPBT. Scheme 2 showed the synthetic route for HA-SS-BFVPBT. First, the cystamine was conjugated to the carboxylic acid groups of HA by using EDC/NHS coupling agents.39,40 The degree of cystamine substitution on HA was estimated by 1H NMR analysis. As shown in Figure 1a, from the ratio of integrated peaks at ∼3.0 ppm for protons of cystamine (b, 4H, −CH2SSCH2−) to that at ∼2.0 ppm for the N-acetyl group of HA (c, 3H, −NCOCH3), the degree of cystamine substitution was determined to be 18% on the molar basis. HA−cystamine with various degrees of substitution can be prepared by changing the molar ratio of the coupling agents and cystamine 2425


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ACS Applied Bio Materials Table 1. Characterization of HSBNPs with Different DS Values of BFVPBT sample

DS (%)

CMC (mg/L)

DLS (nm)

PDIa

TEM (nm)

ζ potential (mV)

1 2

24.9 9.3

30 48

79.8 ± 1.4 139.3 ± 2.0

0.16 0.18

53 107

−43.57 ± 0.12 −44.05 ± 0.17

a

PDI: polydispersity index.

Figure 2. (a, b) TEM images of HSBNPs with 24.9% DS. (c, d) TEM images of HSBNPs with 9.3% DS. Inset of part b: nanoparticle sizes of HSBNPs with 24.9% DS determined by DLS. Inset of part d: nanoparticle sizes of HSBNPs with 9.3% DS determined by DLS.

S7).10,41,42 As the DS of BFVPBT increased from 9.3% to 24.9% (Table 1), the CMC of HA-SS-BFVPBT declined from 48 to 30 mg/L, indicating the formation of a more compact core because of the enhancement of hydrophobic interactions between conjugated segments in BFVPBT groups.10 Such low CMCs will ensure that the nanoparticles keep their original shape in vivo in highly diluted environments before reaching the target position.11 Table 1 presents all the data on the properties of HSBNPs nanoparticles. As measured by DLS, the hydrodynamic size of HSBNPs decreased from 139.3 ± 2.0 to 79.8 ± 1.4 nm when the DS of BFVPBT increased from 9.3% to 24.9%. The ζ potential of each kind of HSBNPs was around −44 mV, implying that HA was located on the surface of the nanoparticles.10 The TEM observation is shown in Figure 2. The 9.3% DS sample was spherical with good dispersion, and the inner core was dark, while the 24.9% DS sample exhibited near spherical shapes. The sizes of HSBNPs from TEM were smaller than that from DLS probably because the micelles shrank at the drying steps of TEM samples.8,10 Moreover, the particle size of the 9.3% DS sample was also about 50 nm larger than that of the 24.9% DS sample. Therefore, with the change of DS on HA, the DLS and TEM data of HSBNPs

was observed in the 1H NMR spectrum (Figure 1b). From the ratio of integrated peaks at ∼1.2 ppm for protons on the side chains of BFVPBT (a, 8H, −CH2CH2CO2Na−) to that at ∼1.8 ppm for the N-acetyl group of HA (c, 3H, −NCOCH3), the DS of BFVPBT on HA was estimated to be 9.8% on the molar basis. DS was also calculated according to the standard curve obtained from the BFVPBT UV−vis absorption spectra (Figure S5). The calculated result was 9.3%, which was very close to the above value 9.8%. Similarly, using this standard curve, the DS in the other product was calculated to be 24.9%. In addition, the FT-IR spectra of HA, HA−cystamine, BFVPBT, and HA-SS-BFVPBT are shown in Figure S6, which also demonstrated the successful conjugation of BFVPBT to HA−cystamine. Preparation and Characterization of HA-SS-BFVPBT NPs (HSBNPs) and CPT-Loaded HSBNPs. The above synthesis produced amphiphilic HA-SS-BFVPBT with varying DS values of hydrophobic BFVPBT. As shown in Scheme 1, such an amphiphilic structure was necessary to promote selfassembly and the encapsulation of CPT. HA-SS-BFVPBT selfassembled into nanoparticles at a low critical micelle concentration (CMC), which was measured by a fluorescence technique using pyrene as a hydrophobic molecule (Figure 2426


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Figure 3. (a) UV−vis absorption spectra of BFVPBT, HA-SS-BFVPBT, HSBNP, and CPT-loaded HSBNP in H2O (solid lines) and DMF/H2O (9v/1v, dashed lines). (b) PL spectra of BFVPBT, HA-SS-BFVPBT, HSBNP, and CPT-loaded HSBNP in H2O with excitation at 425 nm. (c) PL spectra of HA-SS-BFVPBT, HSBNP, and CPT-loaded HSBNP in DMF/H2O (9v/1v) with excitation at 425 nm.

evident changes in size were observed for CPT-loaded HSBNPs after 7 days of storage in PBS (pH = 7.4), FBS, or Dulbecco’s modified Eagle’s medium (DMEM), indicating the excellent stability of the particles under physiological conditions. Optical Properties. Optical properties of the four products BFVPBT, HA-SS-BFVPBT, HSBNPs, and CPT-loaded HSBNPs were studied by UV−vis and PL spectrophotometer in aqueous solutions and in DMF/H2O (9v/1v), in which the concentrations of BFVPBT were all 5.5 × 10−6 mol/L. The absorption spectra of these products in DMF/H2O (9v/1v) all featured a short wavelength band with a peak at 356 nm and a long wavelength band with a peak at 425 nm except CPTloaded HSBNPs, which exhibited a much stronger band at 366 nm due to the absorption of CPT (Figure 3a). By contrast, the absorption value in aqueous solution was lower than that in DMF/H2O (9v/1v); moreover, the maximum absorption peak in the short wavelength band remained at 356 nm while the peak in the long wavelength band red-shifted to 445 nm, likely due to aggregation effects of BFVPBT in the core of HSBNPs. As the four products in aqueous solutions showed similar absorbance at 425 nm, their PL spectra were compared with each other with excitation at 425 nm (Figure 3b). All the PL spectra ranged from 500 to 750 nm with the emission maxima at around 590 nm. The PL intensity of HA-SS-BFVPBT was obviously higher than that of BFVPBT, probably because of the increased hydrophilicity of HA-SS-BFVPBT. By contrast, the PL intensity of HSBNPs was about half of that of HA-SSBFVPBT, suggesting that BFVPBT was aggregated within HSBNPs.15 Furthermore, CPT-loaded HSBNPs only exhibited about one-fourth of the PL intensity of HSBNPs, most likely

exhibited the trend of changes consistent with that of the CMC value. The sizes of both kinds of HSBNPs were within the size range 70−200 nm, suitable for the circulation in the body.43 However, the 9.3% DS sample showed better water solubility than the 24.9% DS sample. Hence, to maintain a good water solubility after the entrapment of hydrophobic CPT in the nanoparticles, HA-SS-BFVPBT with 9.3% DS of BFVPBT was employed in the following CPT loading experiment. Because of the hydrophobicity of conjugated segments in BFVPBT groups, CPT was entrapped in the inner core, which can dissolve the free-CPT more effectively.10 The loading content (LC) and loading efficiency (LE) of CPT-loaded HSBNPs were analyzed using the standard curve obtained from UV−vis absorption spectra. At 10% CPT feed ratio, LC and LE were measured to be 7.4% and 74.2%, respectively. At 20% feed ratio, LC and LE were 12.9% and 65.9%, respectively. When the feed ratio exceeded 30%, LE was less than 50%. Therefore, CPT-loaded HSBNPs of 20% CPT feed ratio were employed for further research. They displayed a similar ζ potential and particle size to their corresponding empty nanoparticles, suggesting that the encapsulation of CPT had little influence on the surface properties of nanoparticles (Figure S8). Furthermore, the water solubilities of HA-SSBFVPBT, CPT, and CPT-loaded HSBNPs are compared in Figure S9. The aqueous solutions of HA-SS-BFVPBT (2 mg/ mL) and CPT-loaded HSBNPs (1 mg/mL) presented an optically transparent yellow color, while free-CPT cannot be completely dissolved in the aqueous solution (0.1 mg/mL), confirming that the formation of CPT-loaded HSBNPs increased the water solubility of CPT. As shown in Figure S10, on the basis of DLS measurements, no precipitation and 2427


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Figure 4. (a) Particle size changes of HSBNPs during 24 h of 0 or 10 μM or 10 mM DTT treatment in PBS (0.02 M, pH = 7.4) at 37 °C measured by DLS. Data were expressed as mean ± SD (n = 3). (b) TEM image of HSBNPs after 24 h of 10 mM DTT treatment. Inset: corresponding nanoparticle sizes of HSBNPs as measured by DLS.

because the hydrophobic CPT was aggregated with BFVPBT in the core of the nanoparticles. Figure 3c indicated that the disassembly of HSBNPs and CPT-loaded HSBNPs was induced by the addition of DMF. The PL intensity of HSBNPs in DMF/H2O (9v/1v) was close to that of HA-SSBFVPBT and was about 6 times that of HSBNPs in aqueous solution. Moreover, the PL intensity of CPT-loaded HSBNPs in DMF/H2O (9v/1v) was about 15 times that of CPT-loaded HSBNPs in aqueous solution, indicating an even stronger fluorescence activation because of the release of CPT.15 Reduction-Triggered Disassembly and Drug Release of Nanoparticles. The morphological changes of HSBNPs were observed by DLS and TEM during 24 h of 10 μM or 10 mM DTT treatment to study reduction-triggered disassembly.10,11 In Figure 4a, the particle size of HSBNPs displayed an obvious enhancement in the case of 10 mM DTT (simulating the reductive microenvironment of cancer cells);44 moreover, the nanoparticles were directly observed to be disassembled from TEM image after reacting with 10 mM DTT for 24 h, which was consistent with the result of DLS (Figure 4b). However, the particle size of HSBNPs did not change much even after 24 h of culture in 10 μM DTT (simulating the microenvironment in human plasma). Similarly, particle sizes of HSBNPs were essentially unchanged without DTT. These results indicated that HSBNPs exhibited excellent stability in the blood but could disassemble quickly in the reductive microenvironment of cancer cells.10 This is because the disulfide linkages in nanoparticles were cleaved by the high concentration of DTT.10,11,42 Thus, it was reasonable to predict that CPT-loaded HSBNPs, because of the response to the reductive microenvironment, could disassemble and release the drug quickly once they entered cancer cells. As shown in Figure 5, upon treatment with 10 mM DTT at pH 7.4, corresponding to the level of intracellular GSH in cancer cells,11,45 CPT release was quickened, with approximately 90% of CPT released in 10 h from CPT-loaded HSBNPs. Tumors usually exhibit acidic conditions and can be employed to promote drug delivery. In this case, the amount of CPT released upon treatment with 10 mM DTT at pH 5.0 was also observed to be slightly higher than that of CPT at pH 7.4. In a physiological solution (i.e., pH 7.4), the stiff helical configuration that HA tends to adopt displays a coil structure

Figure 5. In vitro CPT release from CPT-loaded HSBNP during 25 h of 0 or 10 mM DTT treatment in PBS (0.02 M, pH = 7.4 or 5.0) at 37 °C. Data were expressed as mean ± SD (n = 3).

of large hydrodynamic volume, which has been reported to result in obstacles to fast drug release.11 Probably for this reason, CPT-loaded HSBNPs exhibited more rapid release upon treatment with DTT at pH 5.0. Nevertheless, in the medium without DTT at pH 7.4 or 5.0, only about 20% of CPT was released after 25 h. All the results suggest that the disulfide bonds in the redox-responsive CPT-loaded HSBNPs were responsible for greatly accelerating the drug release rate in cancer cells, and the anticancer effect can thus be improved. Cell Imaging and Drug Delivery. To investigate the cell imaging and drug delivery capability, the target-specific cellular uptake of CPT-loaded HSBNPs by HeLa cells was studied using CLSM. HeLa cells have been previously reported to overexpress the HA receptorCD44.46 Bright orange fluorescence was obviously observed in HeLa cells with twophoton excitation at 720 nm, suggesting that CPT-loaded HSBNPs were readily taken up by HeLa cells due to the strong affinity between HA and CD44 (Figure 6a).8,9,12 The fluorescence of the nanoparticles was activated, and CPT was released due to the degradation by overexpressed GSH and Hyal-1 in cancer cells. By comparison, if a high 2428


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Figure 6. (1) Two-photon excitation images, (2) bright-field images, and (3) overlap images of (a) HeLa cells, (b) HeLa cells pretreated with HA, and (c) NIH 3T3 cells after 4 h of treatment with CPT-loaded HSBNPs (two-photon excitation at 720 nm). Scale bar: 20 μm.

performed for ABDA alone or in the presence of Ru(II)(bpy)3Cl2 under the same experimental conditions (Figure S12). The 1O2 quantum yield of HSBNPs was thus determined to be 0.35 by using Ru(II)(bpy)3Cl2 (ΦΔ = 0.41 in water) as a reference.51 The intracellular 1O2 generation of HSBNPs was also confirmed under two-photon excitation by using DCFH-DA as a fluorescent probe. Intracellular esterases in living cells would hydrolyze nonfluorescent DCFH-DA to DCFH, which would then be oxidized to green emitting 2,7-dichlorofluorescein (DCF) by 1O2.51,52 As shown in Figure 7c, the green fluorescence of DCF could be hardly observed in cells treated with PBS and two-photon laser irradiation, while in the HSBNPs with two-photon laser irradiation-treated cells, the green fluorescence was remarkably enhanced, suggesting the intracellular 1O2 generation capability of HSBNPs. In Vitro Cytotoxicity Evaluation. Cytotoxicity is critical for imaging agents and drug carriers. First, the MTT method was employed to determine the cytotoxicity of HSBNPs.53 Figure S13 shows the quantitative effect of HSBNPs on cell viability. After culturing HeLa cells with different concentrations of BFVPBT (0−90 μg/mL) for 48 h, the cell viability decreased slightly, which was still more than 90% even at a concentration of 90 μg/mL. The low toxicity of HSBNPs ensured its viability for cellular imaging and further biological applications. Then, HeLa cells were treated with different doses of free-CPT or CPT-loaded HSBNPs without laser irradiation (equivalent CPT concentration: 0−4 μg/mL), and the relative viability shown in Figure 8a declined remarkably with the enhancement of CPT concentration. Furthermore, CPT-loaded HSBNPs exhibited evidently higher cytotoxicity as compared to free-CPT because of their better water

concentration of free-HA was employed to pretreat HeLa cells before the treatment with nanoparticles, only very weak signals were detected. This result suggested that it was difficult for HeLa cells to take up the nanoparticles through CD44mediated endocytosis because CD44 was preblocked by freeHA (Figure 6b).8,9,12 Moreover, when CPT-loaded HSBNPs were incubated with NIH-3T3 cells, the normal cells that display low CD44 expression, fluorescence could hardly be detected in cells (Figure 6c), further demonstrating the targetspecific drug delivery capability of CPT-loaded HSBNPs related with HA-CD44 binding. Good photostability is essential for fluorescent probes used in bioimaging. Hence, We further applied HSBNPs to longtime cell imaging to detect its photostability. After 720 nm (0.6 w/cm2) two-photon laser irradiation of HeLa cells for 70 min, strong fluorescence could still be detected in the cells, and the outlines of the cells were still very clear (Figure S11). This confirmed the excellent photostability of HSBNPs under 720 nm two-photon excitation. All of the above results demonstrated that CPT-loaded HSBNPs can realize effective targetspecific two-photon imaging and redox-sensitive CPT delivery at the same time. Detection of Singlet Oxygen (1O2). Cancer cells are killed by singlet oxygen in PDT.47 Using ABDA as an 1O2 indicator, the production of 1O2 by HSBNPs in water was measured under one-photon excitation. 1O2 can cause the photodegradation of ABDA, thereby leading to the decrease of UV−vis absorption of ABDA.48−51 Thus, the 1O2 quantum yield (ΦΔ) can be evaluated indirectly by monitoring the absorption spectra of ABDA (Figure 7a). The absorbance of ABDA at 399 nm displayed a linear relationship with the light irradiation time (Figure 7b). The control experiments were 2429


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Figure 8. (a) Relative viability of HeLa cells treated with different doses of free-CPT or CPT-loaded HSBNPs without laser irradiation. (b) Influence of two-photon irradiation time on the viability of HeLa cells treated with HSBNPs or CPT-loaded HSBNPs.

HeLa cells incubated with HSBNPs declined remarkably with the extension of irradiation time, and the cell viability declined to 52% as the irradiation time was extended to 15 min, verifying a good potential of HSBNPs in highly efficient twophoton PDT. Moreover, the viability of HeLa cells treated with CPT-loaded HSBNPs decreased from 72% to 38% after irradiation for 15 min, demonstrating the efficient synergistic effect of chemo-photodynamic therapy. Furthermore, the therapeutic efficiency of HSBNPs and CPT-loaded HSBNPs on cancer cells was studied with the fluorescence probe PI, which can stain dead cells instead of living ones.5,31,51 In Figure 9a, HeLa cells were incubated with PBS with two-photon irradiation for 20 min, and the red fluorescence from PI was not observed, suggesting the safety of irradiation under such conditions. As shown in Figure 9b, the cell death could also be hardly observed, implying that HSBNPs alone had little effect on cell viability. This result was consistent with that obtained from the MTT assay. In contrast, HeLa cells treated with CPT-loaded HSBNPs without twophoton laser irradiation exhibited evident red fluorescence from PI, indicating the chemotherapy effect of CPT-loaded HSBNPs (Figure 9c).31 In Figure 9b,c, PI both showed remarkably increased red fluorescence after exposure to the two-photon laser for 20 min, as compared with the case

Figure 7. (a) UV−vis absorption spectra of ABDA in the aqueous solution containing HSBNPs at different irradiation times. (b) Irradiation time dependent absorbance of ABDA at 399 nm in the aqueous solution containing HSBNPs (black line) or Ru(bpy)3Cl2 (red line). (c) 1O2 detection of HeLa cells by using DCFH-DA as a fluorescent probe after incubation with HSBNPs or PBS.

solubility and efficient targeting to cancer cells.6,54,55 Then, we chose CPT-loaded HSBNPs with a low CPT concentration (0.2 μg/mL) to investigate the synergistic effect of chemophotodynamic therapy. The viability of HeLa cells treated with HSBNPs or CPT-loaded HSBNPs (with the same equivalent concentration of BFVPBT, 0.26 μg/mL) was obviously influenced by two-photon irradiation time (Figure 8b). No evident decrease in cellular viability was observed for twophoton excitation alone, suggesting that the laser irradiation had little effect on cell activity. By comparison, the viability of 2430


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Figure 9. CLSM images of PI staining HeLa cells treated with (a) PBS, (b) HSBNPs, and (c) CPT-loaded HSBNPs without and with two-photon confocal laser irradiation. Scale bar: 50 μm.

Figure 10. (a) In vivo fluorescent images of HeLa-tumor-bearing mice obtained at different time points within 24 h after injection with excitation at 420 nm and emission at 620 nm. The mice were intravenously administered with CPT-loaded HSBNPs (top) or HA-SS-BFVPBT (bottom). Red and green circles indicated the location of tumors in two mice, separately. (b) Tumor/muscle tissue (T/M) ratios of fluorescence intensity at different time points after injection for the mice treated with CPT-loaded HSBNPs (n = 3).

without two-photon irradiation. Moreover, it was obviously revealed that CPT-loaded HSBNPs with two-photon laser irradiation caused the most serious cell apoptosis due to a laser-activated PDT/chemotherapy synergistic effect. In Vivo Fluorescence Imaging. In vitro studies demonstrated that CPT-loaded HSBNPs could target CD44-positive HeLa cells; hence, we were motivated to study in vivo tumor selectivity of CPT-loaded HSBNPs. CPT-loaded HSBNPs

were injected into the tail vein of HeLa-tumor-bearing nude mice. As a control, we intended to simultaneously study in vivo fluorescence imaging of BFVPBT but failed because its water solubility was unsatisfactory. Therefore, we used the macromolecule HA-SS-BFVPBT as a control instead. The fluorescent images for two groups of mice were obtained at different time points within 24 h after injection. In Figure 10a, just 1 h after injection, obvious fluorescence was observed in the tumor of 2431



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the mouse injected with CPT-loaded HSBNPs, and the fluorescence intensity in the tumor increased gradually in the following few hours. Most of the fluorescent signals accumulated in the tumor 5.5 h after injection, suggesting good tumor targeting. This may be the combined effect of CD44-mediated endocytosis and the enhanced permeation and retention effect.6,8,10 Moreover, tumor/muscle tissue (T/M) ratios of fluorescence intensity are recorded in Figure 10b, and the tumor fluorescence intensity was found to reach its maximum at 12 h after injection and maintain a high value at 24 h after injection. Although CPT-loaded HSBNPs rapidly reached the tumor area, their fluorescence was only visible over time after the degradation by the GSH and Hyal-1 in cancer cells. This phenomenon was similar to what has been reported for HA nanoparticles in some previous literature.6,56 By comparison, fluorescence was hardly observed in the tumor for the mouse injected with HA-SS-BFVPBT, probably because it is a macromolecule that cannot exhibit the EPR effect of the nanoparticle when circulating in vivo.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone/Fax: +86 25 8586 6396/8586 6396. (Y.-Q.H.) *E-mail: [email protected]. (R.Z.) *E-mail: [email protected]. Phone/Fax: +86 25 8586 6396/8586 6396. (W.H.) ORCID

Qu-Li Fan: 0000-0002-9387-0165 Lian-Hui Wang: 0000-0001-9030-9172 Wei Huang: 0000-0001-7004-6408 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

The authors are grateful for the financial support provided by the National Natural Science Foundation of China (51073078, 21674048, 51503103, 61378081), the Natural Science Foundation of Jiangsu Province, China (BK20161515), the State Key Laboratory of Bioelectronics-Zhongda Hospital Cross Innovation Cooperation Research Fund (2018yyjccx012), the Ministry of Education of China (IRT1148), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM).

CONCLUSION

In summary, CPT-loaded HSBNPs, the hyaluronic acid nanoparticles based on conjugated oligomer photosensitizer BFVPBT, were successfully developed as a multifunctional nanoplatform for target-specific two-photon imaging and synergistic chemo-photodynamic therapy. The BFVPBT moieties in HA-SS-BFVPBT can not only provide a hydrophobic domain for the formation of nanoparticles and drug entrapment, but also act as excellent two-photon imaging and PDT agents. CPT-loaded HSBNPs can be efficiently degraded by the reductive GSH and overexpressed Hyal-1 in cancer cells and consequently release drugs rapidly and boost strong twophoton fluorescent signals to indicate tumor presence. In vitro two-photon and in vivo one-photon fluorescence imaging demonstrated the target-specific drug delivery capability of CPT-loaded HSBNPs. Moreover, in vitro cytotoxicity experiments indicated the excellent two-photon laser-activated PDT/ chemotherapy synergistic effect of CPT-loaded HSBNPs. Accordingly, this HSBNPs system is also anticipated to be a precise nanocarrier for other imaging and therapeutic agents besides CPT and to support cancer diagnosis and therapy at the same time. Our studies may greatly promote the HA-based nanoplatform to be employed in imaging-guided synergistic cancer therapy in future clinical applications.

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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/acsabm.9b00130. Synthesis and characterization of BFVPBT, photograph of HA-SS-BFVPBT, standard curve obtained from the CPT and BFVPBT UV−vis absorption spectra, FT-IR spectra, CMC determination, TEM image, photograph of aqueous solutions, stability of CPT-loaded HSBNPs under physiological conditions, photostability of HSBNPs in two-photon fluorescence cell imaging, control experiments, and the cytotoxicity of HSBNPs against HeLa cells (PDF) 2432


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