- Email: [email protected]
NIR and UV-responsive degradable hyaluronic acid nanogels for CD44-targeted and remotely triggered intracellular doxorubicin delivery
Colloids and Surfaces B: Biointerfaces 158 (2017) 547–555
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
NIR and UV-responsive degradable hyaluronic acid nanogels for CD44-targeted and remotely triggered intracellular doxorubicin delivery Chunfeng Hang 1 , Yan Zou 1 , Yinan Zhong, Zhiyuan Zhong ∗ , Fenghua Meng ∗ Biomedical Polymers Laboratory, and Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, PR China
a r t i c l e
i n f o
Article history: Received 6 May 2017 Received in revised form 11 July 2017 Accepted 16 July 2017 Available online 18 July 2017 Keywords: Nanogels Light-sensitive Targeted delivery Anticancer drug Cancer chemotherapy
a b s t r a c t Hyaluronic acid (HA) is an endogenous polysaccharide that shows intrinsic targetability to CD44+ cancer cells. Here, we developed NIR and UV-responsive degradable nanogels from hyaluronic acidg-7-N,N-diethylamino-4-hydroxymethylcoumarin (HA-CM) for CD44 targeted and remotely controlled intracellular doxorubicin (DOX) delivery. Nanometer-sized HA-CM nanogels could readily load DOX, and both NIR and UV irradiation could significantly enhance DOX release from the nanogels, resulting from light-triggered cleavage of urethane bonds that connect CM to HA. MTT assays showed that DOX-loaded HA-CM nanogels combined with NIR irradiation induced much higher antitumor activity to MCF-7 cells (CD44+) than to U-87MG cells (CD44-) and free HA pretreated MCF-7 cells. CLSM observations confirmed that DOX-loaded HA-CM nanogels were internalized by CD44+ cells via receptor mediated endocytosis mechanism, and intracellular DOX release was triggered by NIR. These HA-CM nanogels with easy preparation, CD44 targetability and photo-controlled intracellular drug release are interesting for cancer chemotherapy. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Stimuli-responsive nanosystems can achieve better control over drug release leading to not only better therapeutic effects but also reduced adverse effects [1,2]. In the past years, nanosystems responding to varying internal stimuli (e.g. redox potential, pH, enzymes) and external stimuli (e.g. light, temperature, magnetic field) have been designed and explored for drug delivery [3,4]. Fast intracellular drug release of stimuli-responsive nanosystems has been reported to effectively overcome multidrug resistance (MDR) [5–8]. The internal stimulus-responsive nanosystems would achieve accelerated drug release only when arriving at the right compartments [9]. For example, to take effect, reduction-sensitive nanoparticles need to be delivered to the cytosols of cancer cells. Reduction-sensitivity can hardly work if the nanotherapeutics can’t escape from endo/lysosomal compartments. The other issue with
∗ Corresponding authors. E-mail addresses: [email protected] (Z. Zhong), [email protected] (F. Meng). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.colsurfb.2017.07.041 0927-7765/© 2017 Elsevier B.V. All rights reserved.
internal stimulus-responsive nanosystems is that there is limited control over rate of drug release. In contrast, external stimulus-responsive nanosystems have the advantages of superior spatiotemporal control over drug release. These nanosystems enabling repeated on-demand fast dosing allow multiple dosages from a single administration and can be compliant to the patients’ schedule [10]. Light is an attractive stimulus for constructing responsive nanosystems [11,12]. Various light responsive nanosystems including liposomes [13], nanoparticles [14–16], micelles [17–19] and polymersomes [20–22] have been reported for controlled release of different drugs. Light responsive nanosystems are usually prepared by using inorganic nanoparticles such as gold nanorods or nanoshells [23–25], graphene oxides [26,27] and up-conversion nanoparticles [28], or by incorporating photochromic groups such as o-nitrobenzyl (ONB) [29–32], spiropyran [33], 2-diazo-1,2-naphthoquinone [19] and coumarin [34,35] into polymer backbone or side chains. Coumarin-based nanosystems are particularly interesting in that coumarin has an adequately high two-photon absorption cross section thus suitable for NIR or UV triggered drug release [34–37]. Various coumarin derivatives were able to undergo photo-triggered cleavage [38–40], which has been employed for controlled activation or release of
548
C. Hang et al. / Colloids and Surfaces B: Biointerfaces 158 (2017) 547–555
Scheme 1. Light-responsive HA-CM nanogels for CD44 targeted and remotely controlled DOX delivery. (i) Receptor-mediated endocytosis, and (ii) nanogel swelling and drug release upon light irradiation.
biomacromolecules like peptides [41], proteins [42] and cells [43]. However, most reported systems involve either complicated synthesis or non-degradable materials, which restricts their potential biomedical applications. Hyaluronic acid (HA) is an endogenous polysaccharide that is highly water soluble, biocompatible and biodegradable and moreover shows intrinsic targetability to cancer cells over-expressing CD44 [44,45]. For example, we have reported that reversibly crosslinked HA nanoparticles actively targeted and delivered doxorubicin to CD44 over-expressing, drug resistant human breast tumor xenografts in mice [46]. Mansoor et al. reported that HA-based nanosystem encapsulated SSB/PLK1 siRNA exhibited selective gene knockdown in solid as well as metastatic tumor models [47]. Here, we designed and developed NIR and UV-responsive degradable HA nanogels from HA-(7-N,N-diethylamino-4hydroxymethylcoumarin) (HA-CM) conjugates for CD44 targeted and triggered intracellular doxorubicin (DOX) delivery (Scheme 1). Notably, HA-CM4 with only four CM molecules per HA chain could form small-sized, negatively charged and stable nanogels. Moreover, HA-CM is easy to synthesize. The in vitro studies demonstrate that DOX-loaded HA-CM4 nanogels possess active targeting ability to CD44 positive MCF-7 cells and intracellular DOX release can be triggered by NIR irradiation. 2. Experimental section 2.1. Synthesis of 7-N,N-diethylamino-4-hydroxymethylcoumarin (CM-OH) CM-OH was synthesized according to a reported procedure [35]. Briefly, to 7-N,N-diethylamino-4-methylcoumarin (20 g, 0.864 mmol) dissolved in hot xylene (150 mL) was added selenium dioxide (13.4 g, 0.12 mol). After refluxing the mixture for 16 h, the insoluble selenium dioxide was filtrated off. The filtrate was
concentrated and dried by rotary evaporation. The solid residue was dissolved in 150 mL of dry ethanol/THF (1/1, v/v) mixture. NaBH4 (3.86 g, 8.64 mmol) was added slowly under stirring. After 4 h, remaining NaBH4 was hydrolyzed with 1 M HCl (20 mL). The residues following evaporating organic solvent were dissolved in dichloromethane (DCM) and washed with K2 CO3 aqueous solution (×3). The organic phase was desiccated over magnesium sulfate, filtered and dried by evaporation under vacuum. CM-OH was recovered by chromatography on silica gel (CH2 Cl2 /acetone 5:1). Yield: 22%. 1 H NMR (400 MHz, CDCl3 , ppm): ı 7.30 (-CH-CH-C-), 6.57 (-NC-CH-CH-), 6.52 (-C-CH-CO-), 6.25 (-N-C-CH-C-), 4.83 (-C-CH2 -OH), 3.40 (N-CH2 -CH3 ), and 1.20 (-CH2 -CH3 ). 2.2. Synthesis of [7-(diethylamino)coumarin-4-yl]methyl 4-nitrophenyl carbonate (CM-NPC) Under a nitrogen flow, to a stirred solution of CM-OH (0.5 g, 2 mmol) in anhydrous DCM (30 mL) were added triethylamine (0.85 mL, 6 mmol), pyridine (0.16 g, 2 mmol) and NPC (0.49 g, 2.4 mmol). The mixture was constantly stirred for 16 h at r.t. CMNPC was recovered by adding 15-fold ice cold n-hexane, filtration, washing with anhydrous diethyl ether (×3) and vacuum drying. Yield: 90%. 1 H NMR (400 MHz, CDCl3 , ppm): ı 8.35 (-CH-CH-CNO2 ), 7.40 (-O-C-CH-CH-), 7.30 (-CH-CH-C-), 6.57 (-N-C-CH-CH-), 6.52 (-C-CH-CO-), 6.25 (-N-C-CH-C-), 5.44 (-C-CH2 -O-CO-), 3.40 (N-CH2 -CH3 ), and 1.20 (-CH2 -CH3 ). 2.3. Synthesis of HA-Lys HA-Lys was synthesized according to a previously reported procedure [46] with slight modifications. Into 10 mL methanol solution of H-lys(Boc)-OMe·HCl (0.40 g, 1.35 mmol) was added triethylamine (0.14 g, 1.35 mmol). The mixture was stirred for 30 min at r.t. Then an aqueous solution (pH 8.5) containing HA (molecular weight: 8.9 kDa, 0.50 g, 1.32 mmol carboxyl groups), EDC (0.77 g,
C. Hang et al. / Colloids and Surfaces B: Biointerfaces 158 (2017) 547–555
4.03 mmol) and NHS (0.23 g, 2.02 mmol) were added at r.t. The reaction proceeded for 48 h. The resulting solution was extensively dialyzed (MWCO 3500) against water followed by lyophilization. The obtained HA-Lys(Boc) was deprotected by adding diluted hydrochloric acid (10 mL, 1 M) and TFA (1/1 v/v). After stirring for 6 h, HA-Lys was obtained after dialysis (MWCO 3500) against water (pH 7.0) followed by lyophilization. Yield: 91%. 1 H NMR (400 MHz, D2 O, ppm): ı 4.41 (-CH-CH-CO-NH-), 3.84–3.28 (HA main chain), 3.12 (-CH-CH2 -CH2 -), 2.84 (-CH-CH2 -NH2 ), 1.96 (-NH-CO-CH3 ), and 1.14 (-CH-CH2 -CH2 -NH2 ). At Lys/COOH molar ratio of 1:2 and 1:1, HA-Lys conjugates with 5 and 12 Lys residues per HA chain were obtained, denoted as HA-Lys5 and HA-Lys12, respectively. 2.4. Synthesis of HA-CM Under nitrogen atmosphere, into 10 mL dry methanamide solution of HA-Lys5 (0.15 g, 0.08 mmol amines) was added 2 mL DMF solution of CM-NPC (65.5 mg, 0.16 mmol) and DMAP (19.3 mg, 0.16 mmol). The mixture was stirred for 24 h. HA-CM was recovered by extensive dialysis (MWCO 3500) against methanamide/DMF (1/1, v/v) for 4 times and then against deionized water for 3 times followed by lyophilization. Yield: 85%. UV spectroscopy showed that thus-obtained HA-CM had 4 CM molecules per HA chain (denoted as HA-CM4). In a similar way, HA-CM9 was obtained from HA-Lys12.
549
range of 315–400 nm (100 mW/cm2 ), or a near-infrared semiconductor laser (Changchun Femtosecond Technology Co., Ltd.) operated at 785 nm (2 W/cm2 ). After irradiation for predetermined time, the dispersions were stirred and incubated at 37 ◦ C (200 rpm). The changes in size, size distribution or morphology of nanogels with time were evaluated using DLS and TEM, respectively. 2.7. Loading and light-triggered release of DOX of HA-CM nanogels DOX was loaded into the nanogels by dropwise addition of 1.8 mL PB (10 mM, pH 7.4) to a mixture of 0.2 mL of HA-CM in methanamide/DMF (3 mg/mL) and 12 L DOX in DMSO (5 mg/mL) under stirring at 25 ◦ C, followed by dialysis (MWCO 3500) for 24 h with five times change of dialysis medium. The whole procedure was performed in the dark. To determine drug loading content (DLC), the obtained dispersions were diluted with 5- fold DMSO and analyzed using fluorometry (ex. 480 nm and em. 560 nm) based on a calibration curve obtained with solutions of known DOX concentrations. DLC and DLE (drug loading efficiency) were calculated according to the following formula: DLC(wt.%) = (weight of loaded DOX/weight of polymer and DOX) × 100
2.5. Formation and characterization of nanogels HA-CM nanogels were prepared by dropwise addition of 1.8 mL phosphate buffer (PB, 10 mM, pH 7.4, Na2 HPO4 ·12H2 O: 2.9 g/L, NaH2 PO4 ·2H2 O: 0.3 g/L) to 0.2 mL of HA-CM solution (3 mg/mL) in methanamide/DMF (1/1, v/v) under stirring at 25 ◦ C followed by extensive dialysis (MWCO 3500) against PB for 24 h. The size and size distribution of the nanogels were determined by DLS, and CAC was determined by fluorometry [24,48]. 2.6. Light-triggered destabilization of HA-CM nanogels 0.5 mL of HA-CM nanogels in PB (10 mM, pH 7.4) were irradiated by a UVITRON INTELLI-RAY-400 system operated at a wavelength
DLE(%) = (weight of loaded DOX/weight of DOX in feed) × 100 The release profiles of DOX from HA-CM nanogels in PB were studied using a dialysis tube (MWCO 12000) at 37 ◦ C either under dark or irradiated by UV (100 mW/cm2 ) or NIR (785 nm, 2 W/cm2 ). In order to acquire sink conditions, the study was conducted at low DLCs (ca. 1.0 wt.%) with 0.7 mL of nanogels dialyzed in 25 mL PB. At predetermined time points, 6 mL release medium was withdrawn and 6 mL fresh medium was added. The DOX and coumarin released in the medium were quantified using a fluorometry (DOX: ex. 480 nm and em. 560 nm; coumarin: ex. 380 nm, em. 430 nm). The release experiments were conducted in triplicate, and the results presented are the average ± standard deviations (SD).
Scheme 2. Synthesis of HA-CM. Conditions: (i) H-lys(Boc)-OMe·HCl, Et3 N, methanol, r.t., 0.5 h; EDC, NHS, water, r.t., 2 d; (ii) hydrochloric acid (1 M), TFA, r.t., 6 h; (iii) CM-NPC, DMAP, DMF, r.t., 24 h.
550
C. Hang et al. / Colloids and Surfaces B: Biointerfaces 158 (2017) 547–555
Fig. 1. The size distribution profiles of HA-CM nanogels (0.3 mg/mL) in PB (10 mM, pH 7.4) determined by DLS (A) and TEM (B). The changes in size of HA-CM4 nanogels after (C) UV irradiation (100 mW/cm2 ) and (D) NIR irradiation (785 nm, 2 W/cm2 ) followed by 48 h incubation. Data are presented as the average ± standard deviation (n = 3).
2.8. Cell viability assays MCF-7 cells (CD44 positive) were plated in a 384-well plate (2 × 103 cells/well) using DMEM medium supplemented with FBS (10%), glutamate (1%), antibiotic penicillin (100 IU/mL) and streptomycin (100 g/mL) for 24 h. The medium was removed and fresh medium was added followed by addition of 5 L DOX loaded HACM4 nanogels (25 g/mL) or PBS. After incubation for 4 h, medium was replaced with fresh medium. MCF-7 cells were subjected to NIR light irradiation (785 nm, 2 W/cm2 ) for 60 min and further incubated for 72 h. The medium was aspirated and cells were subjected to MTT assays as described previously [46]. MCF-7 cells incubated with DOX loaded HA-CM4 without NIR irradiation or free DOX were used as controls. Low CD44 expressing U-87MG cells were used as negative controls. To study competition binding to CD44, free HA (5 mg/mL, 5 L) were applied to pretreat MCF-7 and U-87MG cells for 4 h before addition of DOX loaded HA-CM4 and NIR irradiation. The cytotoxicity of empty HA-CM nanogels and the prepolymer HA-Lys12 with or without 32.5 M coumarin (equivalent amount of coumarin from full degradation of 1.0 mg/mL HA-CM9 nanogels) was investigated using MCF-7 and U87MG cells following 24 h incubation.
2.9. Cellular uptake and intracellular DOX release using CLSM MCF-7 cells were seeded in 8-well glass-bottomed Lab-TekTM Chamber Slide System (∼5 × 104 cells/well) for 24 h. 20 L of DOXloaded HA-CM4 nanogels or free DOX in PB (10 g DOX/mL) were added. After 4 h incubation, culture medium was replaced with
fresh medium, and the cells were illuminated by NIR (785 nm, 2 W/cm2 ) for 60 min followed by 8 h incubation. DOX loaded HACM4 nanogels without NIR irradiation, and DOX loaded HA-CM4 incubated with free HA (5 mg/mL, 5 L, 4 h) pre-treated MCF-7 cells with NIR irradiation were used as controls. The cells were fixed using formaldehyde, stained with 4 ,6-diamidino-2-phenylindole (DAPI) and washed with PBS, before subject to CLSM observation (TCS SP5). 2.10. Statistical analyses Data were expressed as mean ± SD. Differences between groups were assessed using the paired, two-sided Student’s t-test. *p < 0.05 was considered significant, and **p < 0.01, ***p < 0.001 were considered highly significant. 3. Results and discussion 3.1. Synthesis of HA-CM conjugates HA-CM conjugates were readily synthesized by reacting 4-nitrophenyl carbonate (NPC) activated coumarin (CM-NPC) with HA-lysine (HA-Lys) conjugate (Scheme 2). Here CMNPC was obtained in two steps (Scheme S1). Firstly, 7diethylamino-4-methylcoumarin (CM) was oxidized by SeO2 and then reduced using NaBH4 to yield 7-N,N-diethylamino4-hydroxymethylcoumarin (CM-OH) [35]. 1 H NMR spectrum of CM-OH displayed appearance of a peak assignable to methylene protons neighboring hydroxyl group (ı 4.83) (Fig. S1A). The sig-
C. Hang et al. / Colloids and Surfaces B: Biointerfaces 158 (2017) 547–555
551
Table 1 Characteristics of HA-CM nanogels. Entry
Polymer
Sizea (nm)
PDIa
Zeta potentialb (mV)
CACc (mg/L)
1 2
HA-CM4 HA-CM9
147.2 ± 5.8 165.4 ± 7.2
0.17 ± 0.03 0.18 ± 0.04
−16.8 ± 1.2 −9.5 ± 0.8
9.1 15.8
a b c
Determined by DLS. Determined in PB (10 mM, pH 7.4); data are presented as the average ± standard deviation (n = 3). Using pyrene as a fluorescent probe.
nals at ı 4.83, 3.40 and 1.20 had an integral ratio close to 1:2:3, confirming that CM-OH has been successfully synthesized [35]. Then, the hydroxyl group of CM-OH was activated using NPC to get CM-NPC. 1 H NMR spectrum showed complete shift of methylene protons from ␦ 4.83–5.44 while all other peaks of CM moieties remained unchanged (Fig. S1B). The peaks assignable to the NPC were discerned at ı 8.29 and 7.39. The signals at ı 8.29, 5.44 and 3.40 demonstrated an integral ratio close to 1:1:2, confirming the successful synthesis of CM-NPC. HA-Lys was prepared according to our previous report [46]. 1 H NMR spectrum showed characteristic peaks of HA at ı 4.41 (anomeric methine proton) and 1.98 (methyl proton) and lysine at ı 3.12 (methine proton) and 2.84 (methylene proton) (Fig. S2). By varying Lys(Boc)-OMe·HCl/carboxyl group of HA ratios from 1:2 to 1:1, we have obtained HA-Lys conjugates with 5 and 12 Lys per HA chain (denoted as HA-Lys5 and HA-Lys12, respectively), as determined by comparing the integral ratio of signals at ␦ 3.12–4.41. The treatment of HA-Lys5 and HA-Lys12 with CM-NPC gave HA-CM conjugates. The number of CM per HA chain was quantified using
UV spectroscopy at 380 nm using the formula A = 22.452 × C + 0.14 (C: in mg/mL). The results showed that HA-CM conjugates with 4 and 9 CM per HA chain (i.e. HA-CM4 and HA-CM9) were obtained from HA-Lys5 and HA-Lys12, respectively. It should be noted that CM was linked to HA-Lys via a urethane bond, which can be cleaved upon photo irradiation [38–40]. 3.2. Preparation and light-responsiveness of HA-CM nanogels HA-CM conjugates readily formed nanogels in aqueous solutions. DLS showed that HA-CM4 and HA-CM9 nanogels had hydrodynamic sizes of 147.2 and 165.4 nm, respectively (Fig. 1A). TEM micrograph displayed an average size of 40–80 nm for HA-CM4 nanogels (Fig. 1B). The much smaller size than the hydrodynamic size measured using DLS was mainly owing to nanogel dehydration and shrinkage upon drying. HA-CM4 and HA-CM9 nanogels revealed a negative surface charge of −16.8 and −9.5 mV, respectively. HA-CM4 exhibited a somewhat lower critical aggregation concentration (CAC) than HA-CM9 (9.1 versus 15.8 mg/L) (Table 1).
Fig. 2. The DOX (A) and CM (B) release profiles of DOX-loaded HA-CM4 nanogels at 37 ◦ C in PB after UV irradiation (100 mW/cm2 ). The DOX (C) and CM (D) release profiles of DOX-loaded HA-CM4 nanogels at 37 ◦ C in PB after NIR irradiation (785 nm, 2 W/cm2 ). Data are presented as the average ± standard deviation (n = 3).
552
C. Hang et al. / Colloids and Surfaces B: Biointerfaces 158 (2017) 547–555
Fig. 3. CLSM images of MCF-7 cells incubated with DOX-loaded HA-CM4 nanogels after NIR irradiation (A), free HA (5 L, 5 mg/mL) pretreated MCF-7 cells with NIR irradiation (B), and MCF-7 cells without NIR irradiation (C). The cells were incubated with nanogels for 4 h, and culture medium was replaced with fresh medium. Then the cells were irradiated 60 min by NIR (780 nm, 2 W/cm2 ) and further incubated for 8 h. [DOX] = 10 g/mL. The scale bars represent 20 m.
HA-CM nanogels were stable for at least one week on storage and in PBS in the dark at r.t. However, upon UV irradiation HA-CM4 nanogels swelled quickly over time (Fig. 1C), which is likely due to cleavage of the urethane bonds that link CM to HA-Lys (Scheme S2) [38–40]. The size of HA-CM4 nanogels increased from 150 nm
to ca. 460, 780 and 960 nm at 12 h following 2, 5 and 10 min UV irradiation, respectively. TEM micrographs displayed fewer particles with much smaller size (Fig. S3) after light irradiation. As comparison, HA-CM9 nanogels showed less size increase following UV irradiation (Fig. S4). Notably, Zhao et al. reported that coumarin
Fig. 4. MTT assays. (A) The antitumor activity of DOX-loaded HA-CM nanogels with or without NIR irradiation to MCF-7 and U87MG cells. Cells pretreated with free HA (5 L, 5 mg/mL), blank cells irradiated with NIR, DOX-loaded HA-CM nanogels and free DOX without NIR irradiation were used as controls ([DOX] = 10 g/mL). The cells were incubated with nanogels for 4 h, medium was replaced with fresh culture medium followed by NIR irradiation (780 nm, 2 W/cm2 ) and incubation for 72 h (Student’s t-test, **p < 0.01, ***p < 0.001). (B) The cytotoxicity of HA-CM4 nanogels following 24 h incubation. Data are presented as the average ± standard deviation (n = 4).
C. Hang et al. / Colloids and Surfaces B: Biointerfaces 158 (2017) 547–555
553
Table 2 Characteristics of DOX-loaded HA-CM nanogels (0.3 mg/mL). Entry
nanogels
Feed ratio (wt.%)
sizea (nm)
PDIa
DLCb (wt.%)
DLEb (%)
1 2 3 4
HA-CM4
5 10 5 10
138.0 149.7 141.4 147.8
0.23 0.26 0.24 0.21
3.4 5.2 4.0 6.1
71.5 57.4 83.3 67.6
a b
HA-CM9 Determined by DLS at 25 ◦ C in PB (pH 7.4, 10 mM). Determined using fluorometry.
derivatives have two photo absorption property and can respond to both UV and NIR [35]. To investigate whether the present nanogels are also NIR-sensitive, HA-CM4 nanogels were irradiated by NIR light (785 nm, 2 W/cm2 ). DLS measurements displayed that HACM4 nanogels enlarged from 150 to 235 nm in 12 h following 1 h NIR irradiation (Fig. 1D). Fig. S5 shows that NIR irradiation induced less size increase of HA-CM9 nanogels.
3.3. DOX loading and light-triggered DOX release DOX could be conveniently encapsulated into HA-CM nanogels. DLS results illustrated that DOX-loaded nanogels had similar sizes to the blank nanogels and decent drug loading efficiency (DLE) of 57.4%–83.3% at 5 and 10 wt.% DOX in feed (Table 2). HA-CM4 nanogels had slightly lower DOX loading capacity than HA-CM9, due to a lower CM content. The photo irradiation would induce cleavage of urethane bonds that link CM to HA-Lys (Scheme S2) [38–40], leading to accelerated drug release. Here, we investigated the in vitro release of DOX and CM from DOX-loaded HA-CM nanogels upon UV or NIR irradiation. Fig. 2A shows that 2 and 5 min UV irradiation resulted in 43.5% and 74.9% DOX release, respectively, from DOX-loaded HACM4 nanogels in 48 h. In contrast, less than 15% DOX was released when UV was not applied. Notably, UV irradiation also led to an accelerated CM release from nanogels (Fig. 2B). For example, 31.4% and 49.5% CM was released in 48 h from DOX-loaded HA-CM4 nanogels at 2 and 5 min UV irradiation, respectively, confirming that UV triggers the cleavage of the urethane bonds and CM release, which in turn brings about nanogel destabilization and DOX release. Zhao et al. reported that almost 100% o-nitrobenzyl (ONB) bonds in PEO-b-PNBM micelles were cleaved after UV irradiation (365 nm, 2 W/cm2 ) for 20 min [49]. Johnson et al. reported that in PEG-NBOCDOX the ONB bonds were chopped and 70% DOX was released upon UV irradiation (365 nm, 2 W/cm2 ) [50]. It was shown that DOX and CM release from DOX-loaded HA-CM9 nanogels could also be triggered by UV irradiation (Fig. S6), though less pronounced than for DOX-loaded HA-CM4 nanogels. Fig. 2C&D show that NIR irradiation could also induce coumarin and DOX release from DOX-loaded HA-CM4 nanogels. For instances, DOX-loaded HA-CM4 nanogels with 60 min NIR irradiation released 51.7% DOX and 30.0% coumarin in 48 h. Similar to results obtained with UV irradiation, DOX-loaded HA-CM9 nanogels displayed less CM and DOX release than DOX-loaded HA-CM4 nanogels (Fig. S7). It was noted that the drug release of DOX-HA-CM4 triggered by NIR, though much slower than that by UV, is fast as compared to previous reports [18,36]. For example, Zhao et al. reported that after pulse NIR laser irradiation (794 nm, sapphire) for 220 min, ca. 50% of paclitaxel was released from coumarin based polypeptide micelles within 145 h [18]. The fast NIR-responsive drug release of DOX-loaded HA-CM nanogels is likely due to their highly porous structure and fast drug diffusion. HA-CM4 nanogels with better stability and photo-sensitivity than HA-CM9 nanogels were selected for the following cell studies. Moreover, as NIR light (650–1000 nm) has a deeper tissue penetration (several centimeters) and better cell compatibility [10,51],
here we investigated triggered drug release in cancer cells by NIR irradiation. 3.4. Cellular uptake and intracellular release of DOX upon NIR irradiation Since DOX and coumarin are inherently fluorescent, we utilized CLSM to study their intracellular release behaviors from DOXloaded HA-CM4 nanogels in CD44 over-expressing (CD44+) MCF-7 cells, either with or without NIR irradiation. It was noted that cell morphology did not changed after NIR irradiation (Fig. S8). Fig. 3A shows that DOX-loaded HA-CM4 nanogels combined with NIR irradiation efficiently delivered DOX to the nuclei of MCF-7 cells. CM in or released from the nanogels mostly remained in the cytosol. In comparison, both fluorescence of DOX and CM were very low and mainly located in the cytosol when MCF-7 cells were pretreated with free HA or without NIR irradiation (Fig. 3B &C), confirming that the nanogels are internalized by CD44+ MCF-7 cells via receptormediated endocytosis, and intracellular DOX release is effectively triggered by NIR irradiation. Moreover, the coumarin fluorescence followed the same trend as DOX, showing much more intense fluorescence in NIR treated MCF-7 cells than those without NIR treatment. 3.5. CD44 targeted delivery of DOX by HA-CM nanogels The antitumor effect of DOX-loaded HA-CM4 nanogels with or without NIR irradiation was explored using MTT assays in MCF-7 cells. U87 MG cells that express low level of CD44 (CD44-) were used as controls. The results showed that DOX-loaded HA-CM4 nanogels combined with 60 min NIR irradiation had significantly higher growth inhibition of MCF-7 cells than without NIR irradiation (cell viabilities: ca. 41% versus 65%) following 48 h incubation (Fig. 4A). The antitumor effect of DOX-loaded HA-CM4 nanogels to MCF-7 cells was noticeably reduced by pretreating the cells with free HA, possibly due to the blockage of CD44 on the cell surface by free HA that was reported to have high specificity to CD44. In contrast, significantly lower cytotoxicity was observed for DOXloaded HA-CM4 nanogels in CD44- U87 MG cells, either with or without NIR irradiation, likely due to inefficient cellular uptake of DOX-loaded HA-CM4 nanogels. These results confirm that HA-CM nanogels are taken up by CD44+ MCF-7 cells by a ligand-receptor mediated endocytosis pathway, and NIR effectively triggers intracellular DOX release. It should further be noted that NIR (785 nm, 2 W/cm2 ) irradiation for 60 min did not cause significant cell death. Moreover, empty HA-CM4 nanogels was virtually nontoxic to both MCF-7 and U87 MG cells at tested concentrations up to 1.0 mg/mL (Fig. 4B). Both degradation products, HA-Lys polymer and free CM, were shown to be nontoxic to cells (Fig. S9). 4. Conclusions We have demonstrated that simple hyaluronic acid-(7-N,Ndiethylamino-4- hydroxymethylcoumarin) (HA-CM) conjugates form robust, nano-sized and negatively charged nanogels. Notably,
554
C. Hang et al. / Colloids and Surfaces B: Biointerfaces 158 (2017) 547–555
these multifunctional nanogels show decent loading of doxorubicin (DOX), active targeting ability to CD44 over-expressing MCF-7 cells and remotely triggered intracellular DOX release by either NIR or UV irradiation. The in vitro studies reveal that DOX-loaded HA-CM nanogels combined with NIR irradiation can cause significantly higher antitumor effect to MCF-7 cells than to U87 MG cells (low CD44 expression) under otherwise the same condition as well as to MCF-7 cells without NIR irradiation. Tumor-targeted HA-CM nanogels with easy preparation and photo-controlled intracellular drug release have a great potential for cancer chemotherapy. Acknowledgements This work is financially supported by research grants from the National Natural Science Foundation of China (NSFC 51473111, 51633005, 51561135010) and the Major Program of the Natural Science Foundation of Jiangsu Province (14KJA150008). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2017.07. 041. References [1] V.P. Torchilin, Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery, Nat. Rev. Drug Discov. 13 (2014) 813–827. [2] S. Mura, J. Nicolas, P. Couvreur, Stimuli-responsive nanocarriers for drug delivery, Nat. Mater. 12 (2013) 991–1003. [3] F.H. Meng, R. Cheng, C. Deng, Z.Y. Zhong, Intracellular drug release nanosystems, Mater. Today 15 (2012) 436–442. [4] R. Cheng, F.H. Meng, C. Deng, H.-A. Klok, Z.Y. Zhong, Dual and multi-stimuli responsive polymeric nanoparticles for programmed site-specific drug delivery, Biomaterials 34 (2013) 3647–3657. [5] M. Han, Q. Lv, X.-J. Tang, Y.-L. Hu, D.-H. Xu, F.-Z. Li, W.-Q. Liang, J.-Q. Gao, Overcoming drug resistance of MCF-7/ADR cells by altering intracellular distribution of doxorubicin via MVP knockdown with a novel siRNA polyamidoamine-hyaluronic acid complex, J. Control. Release 163 (2012) 136–144. [6] L. Qiu, M. Qiao, Q. Chen, C. Tian, M. Long, M. Wang, Z. Li, W. Hu, G. Li, L. Cheng, L. Cheng, H. Hu, X. Zhao, D. Chen, Enhanced effect of pH-sensitive mixed copolymer micelles for overcoming multidrug resistance of doxorubicin, Biomaterials 35 (2014) 9877–9887. [7] L. Xiao, X. Xiong, X. Sun, Y. Zhu, H. Yang, H. Chen, L. Gan, H. Xu, X. Yang, Role of cellular uptake in the reversal of multidrug resistance by PEG-b-PLA polymeric micelles, Biomaterials 32 (2011) 5148–5157. [8] Y. Guo, W. He, S. Yang, D. Zhao, Z. Li, Y. Luan, Co-delivery of docetaxel and verapamil by reduction-sensitive PEG-PLGA-SS-DTX conjugate micelles to reverse the multi-drug resistance of breast cancer, Colloid. Surface. B 151 (2017) 119–127. [9] F.H. Meng, Y. Zhong, R. Cheng, C. Deng, Z. Zhong, pH-sensitive polymeric nanoparticles for tumor-targeting doxorubicin delivery: concept and recent advances, Nanomedicine 9 (2014) 487–499. [10] B.P. Timko, T. Dvir, D.S. Kohane, Remotely triggerable drug delivery systems, Adv. Mater. 22 (2010) 4925–4943. [11] A. Bansal, Y. Zhang, Photocontrolled nanoparticle delivery systems for biomedical applications, Accounts Chem. Res. 47 (2014) 3052–3060. [12] A.Y. Rwei, W. Wang, D.S. Kohane, Photoresponsive nanoparticles for drug delivery, Nano Today 10 (2015) 451–467. [13] Y. Yang, Y. Yang, X. Xie, X. Cai, Z. Wang, W. Gong, H. Zhang, Y. Li, X. Mei, A near-infrared two-photon-sensitive peptide-mediated liposomal delivery system, Colloid. Surface. B 128 (2015) 427–438. [14] T. Dvir, M.R. Banghart, B.P. Timko, R. Langer, D.S. Kohane, Photo-targeted nanoparticles, Nano Lett. 10 (2010) 250–254. [15] R. Tong, H.D. Hemmati, R. Langer, D.S. Kohane, Photoswitchable nanoparticles for triggered tissue penetration and drug delivery, J. Am. Chem. Soc. 134 (2012) 8848–8855. [16] L.L. Meng, W. Huang, D. Wang, X. Huang, X. Zhu, D. Yan, Chitosan-based nanocarriers with pH and light dual response for anticancer drug delivery, Biomacromolecules 14 (2013) 2601–2610. [17] G.-Y. Liu, C.-J. Chen, D.-D. Li, S.-S. Wang, J. Ji, Near-infrared light-sensitive micelles for enhanced intracellular drug delivery, J. Mater. Chem. 22 (2012) 16865–16871. [18] S. Kumar, J.-F. Allard, D. Morris, Y.L. Dory, M. Lepage, Y. Zhao, Near-infrared light sensitive polypeptide block copolymer micelles for drug delivery, J. Mater. Chem. 22 (2012) 7252–7257.
[19] C. Chen, G. Liu, X. Liu, S. Pang, C. Zhu, L. Lv, J. Ji, Photo-responsive, biocompatible polymeric micelles self-assembled from hyperbranched polyphosphate-based polymers, Polym. Chem. 2 (2011) 1389–1397. [20] E. Mabrouk, S. Bonneau, L. Jia, D. Cuvelier, M.H. Li, P. Nassoy, Photosensitization of polymer vesicles: a multistep chemical process deciphered by micropipette manipulation, Soft Matter 6 (2010) 4863–4875. [21] E. Cabane, V. Malinova, S. Menon, C.G. Palivan, W. Meier, Photoresponsive polymersomes as smart, triggerable nanocarriers, Soft Matter 7 (2011) 9167–9176. [22] J.F. Lovell, C.S. Jin, E. Huynh, H. Jin, C. Kim, J.L. Rubinstein, W.C.W. Chan, W. Cao, L.V. Wang, G. Zheng, Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents, Nat. Mater. 10 (2011) 324–332. [23] R. Huschka, J. Zuloaga, M.W. Knight, L.V. Brown, P. Nordlander, N.J. Halas, Light-Induced release of DNA from gold nanoparticles: nanoshells and nanorods, J. Am. Chem. Soc. 133 (2011) 12247–12255. [24] Y.N. Zhong, C. Wang, R. Cheng, L. Cheng, F.H. Meng, Z. Liu, Z.Y. Zhong, cRGD-directed, NIR-responsive and robust AuNR/PEG-PCL hybrid nanoparticles for targeted chemotherapy of glioblastoma in vivo, J. Control. Release 195 (2014) 63–71. [25] W. Zhang, F. Wang, Y. Wang, J. Wang, Y. Yu, S. Guo, R. Chen, D. Zhou, pH and near-infrared light dual-stimuli responsive drug delivery using DNA-conjugated gold nanorods for effective treatment of multidrug resistant cancer cells, J. Control. Release 232 (2016) 9–19. [26] T.-Y. Hsieh, W.-C. Huang, Y.-D. Kang, C.-Y. Chu, W.-L. Liao, Y.-Y. Chen, S.-Y. Chen, Neurotensin-conjugated reduced graphene oxide with multi-stage near-infrared-triggered synergic targeted neuron gene transfection in vitro and In vivo for neurodegenerative disease therapy, Adv. Healthc. Mater. 5 (2016) 3016–3026. [27] Q. He, D.O. Kiesewetter, Y. Qu, X. Fu, J. Fan, P. Huang, Y. Liu, G. Zhu, Y. Liu, Z. Qian, X. Chen, NIR-responsive on-demand release of CO from metal carbonyl-caged graphene oxide nanomedicine, Adv. Mater. 27 (2015) 6741–6746. [28] M.K.G. Jayakumar, N.M. Idris, Y. Zhang, Remote activation of biomolecules in deep tissues using near-infrared-to-UV upconversion nanotransducers, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 8483–8488. [29] J.-M. Schumers, J.-F. Gohy, C.-A. Fustin, A versatile strategy for the synthesis of block copolymers bearing a photocleavable junction, Polym. Chem. 1 (2010) 161–163. [30] V. Yesilyurt, R. Ramireddy, S. Thayumanavan, Photoregulated release of noncovalent guests from dendritic amphiphilic nanocontainers, Angew. Chem. Int. Edit. 50 (2011) 3038–3042. [31] D. Han, X. Tong, Y. Zhao, Fast photodegradable block copolymer micelles for burst release, Macromolecules 44 (2011) 437–439. [32] S.S. Agasti, A. Chompoosor, C.-C. You, P. Ghosh, C.K. Kim, V.M. Rotello, Photoregulated release of caged anticancer drugs from gold nanoparticles, J. Am. Chem. Soc. 131 (2009) 5728-. [33] D.S. Achilleos, T.A. Hatton, M. Vamvakaki, Light-regulated supramolecular engineering of polymeric nanocapsules, J. Am. Chem. Soc. 134 (2012) 5726–5729. [34] Q. Lin, Q. Huang, C. Li, C. Bao, Z. Liu, F. Li, L. Zhu, Anticancer drug release from a mesoporous silica based nanophotocage regulated by either a one- or two-photon process, J. Am. Chem. Soc. 132 (2010) 10645–10647. [35] J. Babin, M. Pelletier, M. Lepage, J.-F. Allard, D. Morris, Y. Zhao, A new two-photon-sensitive block copolymer nanocarrier, Angew. Chem.-Int. Ed. 48 (2009) 3329–3332. [36] C.d.G. Lux, C.L. McFearin, S. Joshi-Barr, J. Sankaranarayanan, N. Fomina, A. Almutairi, Single UV or near IR triggering event leads to polymer degradation into small molecules, Acs Macro Lett. 1 (2012) 922–926. [37] N. Fomina, C.L. McFearin, M. Sermsakdi, J.M. Morachis, A. Almutairi, Low power, biologically benign NIR light triggers polymer disassembly, Macromolecules 44 (2011) 8590–8597. [38] V. Hagen, B. Dekowski, N. Kotzur, R. Lechler, B. Wiesner, B. Briand, M. Beyermann, {7- bis(carboxymethyl)amino coumarin-4-yl}methoxycarbonyl derivatives for photorelease of carboxylic acids alcohols/phenols, thioalcohols/thiophenols, and amines, Chem. Eur. J. 14 (2008) 1621–1627. [39] T. Furuta, S.S.H. Wang, J.L. Dantzker, T.M. Dore, W.J. Bybee, E.M. Callaway, W. Denk, R.Y. Tsien, Brominated 7-hydroxycoumarin-4-ylmethyls: photolabile protecting groups with biologically useful cross-sections for two photon photolysis, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 1193–1200. [40] V. Hagen, B. Dekowski, V. Nache, R. Schmidt, D. Geissler, D. Lorenz, J. Eichhorst, S. Keller, H. Kaneko, K. Benndorf, B. Wiesner, Coumarinylmethyl esters for ultrafast release of high concentrations of cyclic nucleotides upon one- and two-photon photolysis, Angew. Chem.-Int. Ed. 44 (2005) 7887–7891. [41] K. Katayama, S. Tsukiji, T. Furuta, T. Nagamune, A bromocoumarin-based linker for synthesis of photocleavable peptidoconjugates with high photosensitivity, Chem. Commun. 539 (2008) 5399–5401. [42] Q. Lin, C. Bao, S. Cheng, Y. Yang, W. Ji, L. Zhu, Target-activated coumarin phototriggers specifically switch on fluorescence and photocleavage upon bonding to thiol-Bearing protein, J. Am. Chem. Soc. 134 (2012) 5052–5055. [43] C.d.G. Lux, J. Lux, G. Collet, S. He, M. Chan, J. Olejniczak, A. Foucault-Collett, A. Almutairi, Short soluble coumarin crosslinkers for light-controlled release of cells and proteins from hydrogels, Biomacromolecules 16 (2015) 3286–3296. [44] K.Y. Choi, E.J. Jeon, H.Y. Yoon, B.S. Lee, J.H. Na, K.H. Min, S.Y. Kim, S.-J. Myung, S. Lee, X. Chen, I.C. Kwon, K. Choi, S.Y. Jeong, K. Kim, J.H. Park, Theranostic
C. Hang et al. / Colloids and Surfaces B: Biointerfaces 158 (2017) 547–555 nanoparticles based on PEGylated hyaluronic acid for the diagnosis, therapy and monitoring of colon cancer, Biomaterials 33 (2012) 6186–6193. [45] J.-H. Park, H.-J. Cho, H.Y. Yoon, I.-S. Yoon, S.-H. Ko, J.-S. Shim, J.-H. Cho, J.H. Park, K. Kim, I.C. Kwon, D.-D. Kim, Hyaluronic acid derivative-coated nanohybrid liposomes for cancer imaging and drug delivery, J. Control. Release 174 (2014) 98–108. [46] Y.N. Zhong, J. Zhang, R. Cheng, C. Deng, F.H. Meng, F. Xie, Z.Y. Zhong, Reversibly crosslinked hyaluronic acid nanoparticles for active targeting and intelligent delivery of doxorubicin to drug resistant CD44 + human breast tumor xenografts, J. Control. Release 205 (2015) 144–154. [47] S. Ganesh, A.K. Iyer, D.V. Morrissey, M.M. Amiji, Hyaluronic acid based self-assembling nanosystems for CD44 target mediated siRNA delivery to solid tumors, Biomaterials 34 (2013) 3489–3502.
555
[48] Y. Zou, Y. Song, W.J. Yang, F.H. Meng, H.Y. Liu, Z.Y. Zhong, Galactose-installed photo-crosslinked pH-sensitive degradable micelles for active targeting chemotherapy of hepatocellular carcinoma in mice, J. Control. Release 193 (2014) 154–161. [49] J.Q. Jiang, X. Tong, D. Morris, Y. Zhao, Toward photocontrolled release using light-dissociable block copolymer micelles, Macromolecules 39 (2006) 4633–4640. [50] J.A. Johnson, Y.Y. Lu, A.O. Burts, Y.-H. Lim, M.G. Finn, J.T. Koberstein, N.J. Turro, D.A. Tirrell, R.H. Grubbs, Core-clickable PEG-branch-azide bivalent-bottle-brush polymers by ROMP: grafting-through and clicking-to, J. Am. Chem. Soc. 133 (2011) 559–566. [51] Z. Meng, F. Wei, R. Wang, M. Xia, Z. Chen, H. Wang, M. Zhu, NIR-laser-switched In vivo smart nanocapsules for synergic photothermal and chemotherapy of tumors, Adv. Mater. 28 (2016) 245–253.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
NIR and UV-responsive degradable hyaluronic acid nanogels for CD44-targeted and remotely triggered intracellular doxorubicin delivery Chunfeng Hang 1 , Yan Zou 1 , Yinan Zhong, Zhiyuan Zhong ∗ , Fenghua Meng ∗ Biomedical Polymers Laboratory, and Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, PR China
a r t i c l e
i n f o
Article history: Received 6 May 2017 Received in revised form 11 July 2017 Accepted 16 July 2017 Available online 18 July 2017 Keywords: Nanogels Light-sensitive Targeted delivery Anticancer drug Cancer chemotherapy
a b s t r a c t Hyaluronic acid (HA) is an endogenous polysaccharide that shows intrinsic targetability to CD44+ cancer cells. Here, we developed NIR and UV-responsive degradable nanogels from hyaluronic acidg-7-N,N-diethylamino-4-hydroxymethylcoumarin (HA-CM) for CD44 targeted and remotely controlled intracellular doxorubicin (DOX) delivery. Nanometer-sized HA-CM nanogels could readily load DOX, and both NIR and UV irradiation could significantly enhance DOX release from the nanogels, resulting from light-triggered cleavage of urethane bonds that connect CM to HA. MTT assays showed that DOX-loaded HA-CM nanogels combined with NIR irradiation induced much higher antitumor activity to MCF-7 cells (CD44+) than to U-87MG cells (CD44-) and free HA pretreated MCF-7 cells. CLSM observations confirmed that DOX-loaded HA-CM nanogels were internalized by CD44+ cells via receptor mediated endocytosis mechanism, and intracellular DOX release was triggered by NIR. These HA-CM nanogels with easy preparation, CD44 targetability and photo-controlled intracellular drug release are interesting for cancer chemotherapy. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Stimuli-responsive nanosystems can achieve better control over drug release leading to not only better therapeutic effects but also reduced adverse effects [1,2]. In the past years, nanosystems responding to varying internal stimuli (e.g. redox potential, pH, enzymes) and external stimuli (e.g. light, temperature, magnetic field) have been designed and explored for drug delivery [3,4]. Fast intracellular drug release of stimuli-responsive nanosystems has been reported to effectively overcome multidrug resistance (MDR) [5–8]. The internal stimulus-responsive nanosystems would achieve accelerated drug release only when arriving at the right compartments [9]. For example, to take effect, reduction-sensitive nanoparticles need to be delivered to the cytosols of cancer cells. Reduction-sensitivity can hardly work if the nanotherapeutics can’t escape from endo/lysosomal compartments. The other issue with
∗ Corresponding authors. E-mail addresses: [email protected] (Z. Zhong), [email protected] (F. Meng). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.colsurfb.2017.07.041 0927-7765/© 2017 Elsevier B.V. All rights reserved.
internal stimulus-responsive nanosystems is that there is limited control over rate of drug release. In contrast, external stimulus-responsive nanosystems have the advantages of superior spatiotemporal control over drug release. These nanosystems enabling repeated on-demand fast dosing allow multiple dosages from a single administration and can be compliant to the patients’ schedule [10]. Light is an attractive stimulus for constructing responsive nanosystems [11,12]. Various light responsive nanosystems including liposomes [13], nanoparticles [14–16], micelles [17–19] and polymersomes [20–22] have been reported for controlled release of different drugs. Light responsive nanosystems are usually prepared by using inorganic nanoparticles such as gold nanorods or nanoshells [23–25], graphene oxides [26,27] and up-conversion nanoparticles [28], or by incorporating photochromic groups such as o-nitrobenzyl (ONB) [29–32], spiropyran [33], 2-diazo-1,2-naphthoquinone [19] and coumarin [34,35] into polymer backbone or side chains. Coumarin-based nanosystems are particularly interesting in that coumarin has an adequately high two-photon absorption cross section thus suitable for NIR or UV triggered drug release [34–37]. Various coumarin derivatives were able to undergo photo-triggered cleavage [38–40], which has been employed for controlled activation or release of
548
C. Hang et al. / Colloids and Surfaces B: Biointerfaces 158 (2017) 547–555
Scheme 1. Light-responsive HA-CM nanogels for CD44 targeted and remotely controlled DOX delivery. (i) Receptor-mediated endocytosis, and (ii) nanogel swelling and drug release upon light irradiation.
biomacromolecules like peptides [41], proteins [42] and cells [43]. However, most reported systems involve either complicated synthesis or non-degradable materials, which restricts their potential biomedical applications. Hyaluronic acid (HA) is an endogenous polysaccharide that is highly water soluble, biocompatible and biodegradable and moreover shows intrinsic targetability to cancer cells over-expressing CD44 [44,45]. For example, we have reported that reversibly crosslinked HA nanoparticles actively targeted and delivered doxorubicin to CD44 over-expressing, drug resistant human breast tumor xenografts in mice [46]. Mansoor et al. reported that HA-based nanosystem encapsulated SSB/PLK1 siRNA exhibited selective gene knockdown in solid as well as metastatic tumor models [47]. Here, we designed and developed NIR and UV-responsive degradable HA nanogels from HA-(7-N,N-diethylamino-4hydroxymethylcoumarin) (HA-CM) conjugates for CD44 targeted and triggered intracellular doxorubicin (DOX) delivery (Scheme 1). Notably, HA-CM4 with only four CM molecules per HA chain could form small-sized, negatively charged and stable nanogels. Moreover, HA-CM is easy to synthesize. The in vitro studies demonstrate that DOX-loaded HA-CM4 nanogels possess active targeting ability to CD44 positive MCF-7 cells and intracellular DOX release can be triggered by NIR irradiation. 2. Experimental section 2.1. Synthesis of 7-N,N-diethylamino-4-hydroxymethylcoumarin (CM-OH) CM-OH was synthesized according to a reported procedure [35]. Briefly, to 7-N,N-diethylamino-4-methylcoumarin (20 g, 0.864 mmol) dissolved in hot xylene (150 mL) was added selenium dioxide (13.4 g, 0.12 mol). After refluxing the mixture for 16 h, the insoluble selenium dioxide was filtrated off. The filtrate was
concentrated and dried by rotary evaporation. The solid residue was dissolved in 150 mL of dry ethanol/THF (1/1, v/v) mixture. NaBH4 (3.86 g, 8.64 mmol) was added slowly under stirring. After 4 h, remaining NaBH4 was hydrolyzed with 1 M HCl (20 mL). The residues following evaporating organic solvent were dissolved in dichloromethane (DCM) and washed with K2 CO3 aqueous solution (×3). The organic phase was desiccated over magnesium sulfate, filtered and dried by evaporation under vacuum. CM-OH was recovered by chromatography on silica gel (CH2 Cl2 /acetone 5:1). Yield: 22%. 1 H NMR (400 MHz, CDCl3 , ppm): ı 7.30 (-CH-CH-C-), 6.57 (-NC-CH-CH-), 6.52 (-C-CH-CO-), 6.25 (-N-C-CH-C-), 4.83 (-C-CH2 -OH), 3.40 (N-CH2 -CH3 ), and 1.20 (-CH2 -CH3 ). 2.2. Synthesis of [7-(diethylamino)coumarin-4-yl]methyl 4-nitrophenyl carbonate (CM-NPC) Under a nitrogen flow, to a stirred solution of CM-OH (0.5 g, 2 mmol) in anhydrous DCM (30 mL) were added triethylamine (0.85 mL, 6 mmol), pyridine (0.16 g, 2 mmol) and NPC (0.49 g, 2.4 mmol). The mixture was constantly stirred for 16 h at r.t. CMNPC was recovered by adding 15-fold ice cold n-hexane, filtration, washing with anhydrous diethyl ether (×3) and vacuum drying. Yield: 90%. 1 H NMR (400 MHz, CDCl3 , ppm): ı 8.35 (-CH-CH-CNO2 ), 7.40 (-O-C-CH-CH-), 7.30 (-CH-CH-C-), 6.57 (-N-C-CH-CH-), 6.52 (-C-CH-CO-), 6.25 (-N-C-CH-C-), 5.44 (-C-CH2 -O-CO-), 3.40 (N-CH2 -CH3 ), and 1.20 (-CH2 -CH3 ). 2.3. Synthesis of HA-Lys HA-Lys was synthesized according to a previously reported procedure [46] with slight modifications. Into 10 mL methanol solution of H-lys(Boc)-OMe·HCl (0.40 g, 1.35 mmol) was added triethylamine (0.14 g, 1.35 mmol). The mixture was stirred for 30 min at r.t. Then an aqueous solution (pH 8.5) containing HA (molecular weight: 8.9 kDa, 0.50 g, 1.32 mmol carboxyl groups), EDC (0.77 g,
C. Hang et al. / Colloids and Surfaces B: Biointerfaces 158 (2017) 547–555
4.03 mmol) and NHS (0.23 g, 2.02 mmol) were added at r.t. The reaction proceeded for 48 h. The resulting solution was extensively dialyzed (MWCO 3500) against water followed by lyophilization. The obtained HA-Lys(Boc) was deprotected by adding diluted hydrochloric acid (10 mL, 1 M) and TFA (1/1 v/v). After stirring for 6 h, HA-Lys was obtained after dialysis (MWCO 3500) against water (pH 7.0) followed by lyophilization. Yield: 91%. 1 H NMR (400 MHz, D2 O, ppm): ı 4.41 (-CH-CH-CO-NH-), 3.84–3.28 (HA main chain), 3.12 (-CH-CH2 -CH2 -), 2.84 (-CH-CH2 -NH2 ), 1.96 (-NH-CO-CH3 ), and 1.14 (-CH-CH2 -CH2 -NH2 ). At Lys/COOH molar ratio of 1:2 and 1:1, HA-Lys conjugates with 5 and 12 Lys residues per HA chain were obtained, denoted as HA-Lys5 and HA-Lys12, respectively. 2.4. Synthesis of HA-CM Under nitrogen atmosphere, into 10 mL dry methanamide solution of HA-Lys5 (0.15 g, 0.08 mmol amines) was added 2 mL DMF solution of CM-NPC (65.5 mg, 0.16 mmol) and DMAP (19.3 mg, 0.16 mmol). The mixture was stirred for 24 h. HA-CM was recovered by extensive dialysis (MWCO 3500) against methanamide/DMF (1/1, v/v) for 4 times and then against deionized water for 3 times followed by lyophilization. Yield: 85%. UV spectroscopy showed that thus-obtained HA-CM had 4 CM molecules per HA chain (denoted as HA-CM4). In a similar way, HA-CM9 was obtained from HA-Lys12.
549
range of 315–400 nm (100 mW/cm2 ), or a near-infrared semiconductor laser (Changchun Femtosecond Technology Co., Ltd.) operated at 785 nm (2 W/cm2 ). After irradiation for predetermined time, the dispersions were stirred and incubated at 37 ◦ C (200 rpm). The changes in size, size distribution or morphology of nanogels with time were evaluated using DLS and TEM, respectively. 2.7. Loading and light-triggered release of DOX of HA-CM nanogels DOX was loaded into the nanogels by dropwise addition of 1.8 mL PB (10 mM, pH 7.4) to a mixture of 0.2 mL of HA-CM in methanamide/DMF (3 mg/mL) and 12 L DOX in DMSO (5 mg/mL) under stirring at 25 ◦ C, followed by dialysis (MWCO 3500) for 24 h with five times change of dialysis medium. The whole procedure was performed in the dark. To determine drug loading content (DLC), the obtained dispersions were diluted with 5- fold DMSO and analyzed using fluorometry (ex. 480 nm and em. 560 nm) based on a calibration curve obtained with solutions of known DOX concentrations. DLC and DLE (drug loading efficiency) were calculated according to the following formula: DLC(wt.%) = (weight of loaded DOX/weight of polymer and DOX) × 100
2.5. Formation and characterization of nanogels HA-CM nanogels were prepared by dropwise addition of 1.8 mL phosphate buffer (PB, 10 mM, pH 7.4, Na2 HPO4 ·12H2 O: 2.9 g/L, NaH2 PO4 ·2H2 O: 0.3 g/L) to 0.2 mL of HA-CM solution (3 mg/mL) in methanamide/DMF (1/1, v/v) under stirring at 25 ◦ C followed by extensive dialysis (MWCO 3500) against PB for 24 h. The size and size distribution of the nanogels were determined by DLS, and CAC was determined by fluorometry [24,48]. 2.6. Light-triggered destabilization of HA-CM nanogels 0.5 mL of HA-CM nanogels in PB (10 mM, pH 7.4) were irradiated by a UVITRON INTELLI-RAY-400 system operated at a wavelength
DLE(%) = (weight of loaded DOX/weight of DOX in feed) × 100 The release profiles of DOX from HA-CM nanogels in PB were studied using a dialysis tube (MWCO 12000) at 37 ◦ C either under dark or irradiated by UV (100 mW/cm2 ) or NIR (785 nm, 2 W/cm2 ). In order to acquire sink conditions, the study was conducted at low DLCs (ca. 1.0 wt.%) with 0.7 mL of nanogels dialyzed in 25 mL PB. At predetermined time points, 6 mL release medium was withdrawn and 6 mL fresh medium was added. The DOX and coumarin released in the medium were quantified using a fluorometry (DOX: ex. 480 nm and em. 560 nm; coumarin: ex. 380 nm, em. 430 nm). The release experiments were conducted in triplicate, and the results presented are the average ± standard deviations (SD).
Scheme 2. Synthesis of HA-CM. Conditions: (i) H-lys(Boc)-OMe·HCl, Et3 N, methanol, r.t., 0.5 h; EDC, NHS, water, r.t., 2 d; (ii) hydrochloric acid (1 M), TFA, r.t., 6 h; (iii) CM-NPC, DMAP, DMF, r.t., 24 h.
550
C. Hang et al. / Colloids and Surfaces B: Biointerfaces 158 (2017) 547–555
Fig. 1. The size distribution profiles of HA-CM nanogels (0.3 mg/mL) in PB (10 mM, pH 7.4) determined by DLS (A) and TEM (B). The changes in size of HA-CM4 nanogels after (C) UV irradiation (100 mW/cm2 ) and (D) NIR irradiation (785 nm, 2 W/cm2 ) followed by 48 h incubation. Data are presented as the average ± standard deviation (n = 3).
2.8. Cell viability assays MCF-7 cells (CD44 positive) were plated in a 384-well plate (2 × 103 cells/well) using DMEM medium supplemented with FBS (10%), glutamate (1%), antibiotic penicillin (100 IU/mL) and streptomycin (100 g/mL) for 24 h. The medium was removed and fresh medium was added followed by addition of 5 L DOX loaded HACM4 nanogels (25 g/mL) or PBS. After incubation for 4 h, medium was replaced with fresh medium. MCF-7 cells were subjected to NIR light irradiation (785 nm, 2 W/cm2 ) for 60 min and further incubated for 72 h. The medium was aspirated and cells were subjected to MTT assays as described previously [46]. MCF-7 cells incubated with DOX loaded HA-CM4 without NIR irradiation or free DOX were used as controls. Low CD44 expressing U-87MG cells were used as negative controls. To study competition binding to CD44, free HA (5 mg/mL, 5 L) were applied to pretreat MCF-7 and U-87MG cells for 4 h before addition of DOX loaded HA-CM4 and NIR irradiation. The cytotoxicity of empty HA-CM nanogels and the prepolymer HA-Lys12 with or without 32.5 M coumarin (equivalent amount of coumarin from full degradation of 1.0 mg/mL HA-CM9 nanogels) was investigated using MCF-7 and U87MG cells following 24 h incubation.
2.9. Cellular uptake and intracellular DOX release using CLSM MCF-7 cells were seeded in 8-well glass-bottomed Lab-TekTM Chamber Slide System (∼5 × 104 cells/well) for 24 h. 20 L of DOXloaded HA-CM4 nanogels or free DOX in PB (10 g DOX/mL) were added. After 4 h incubation, culture medium was replaced with
fresh medium, and the cells were illuminated by NIR (785 nm, 2 W/cm2 ) for 60 min followed by 8 h incubation. DOX loaded HACM4 nanogels without NIR irradiation, and DOX loaded HA-CM4 incubated with free HA (5 mg/mL, 5 L, 4 h) pre-treated MCF-7 cells with NIR irradiation were used as controls. The cells were fixed using formaldehyde, stained with 4 ,6-diamidino-2-phenylindole (DAPI) and washed with PBS, before subject to CLSM observation (TCS SP5). 2.10. Statistical analyses Data were expressed as mean ± SD. Differences between groups were assessed using the paired, two-sided Student’s t-test. *p < 0.05 was considered significant, and **p < 0.01, ***p < 0.001 were considered highly significant. 3. Results and discussion 3.1. Synthesis of HA-CM conjugates HA-CM conjugates were readily synthesized by reacting 4-nitrophenyl carbonate (NPC) activated coumarin (CM-NPC) with HA-lysine (HA-Lys) conjugate (Scheme 2). Here CMNPC was obtained in two steps (Scheme S1). Firstly, 7diethylamino-4-methylcoumarin (CM) was oxidized by SeO2 and then reduced using NaBH4 to yield 7-N,N-diethylamino4-hydroxymethylcoumarin (CM-OH) [35]. 1 H NMR spectrum of CM-OH displayed appearance of a peak assignable to methylene protons neighboring hydroxyl group (ı 4.83) (Fig. S1A). The sig-
C. Hang et al. / Colloids and Surfaces B: Biointerfaces 158 (2017) 547–555
551
Table 1 Characteristics of HA-CM nanogels. Entry
Polymer
Sizea (nm)
PDIa
Zeta potentialb (mV)
CACc (mg/L)
1 2
HA-CM4 HA-CM9
147.2 ± 5.8 165.4 ± 7.2
0.17 ± 0.03 0.18 ± 0.04
−16.8 ± 1.2 −9.5 ± 0.8
9.1 15.8
a b c
Determined by DLS. Determined in PB (10 mM, pH 7.4); data are presented as the average ± standard deviation (n = 3). Using pyrene as a fluorescent probe.
nals at ı 4.83, 3.40 and 1.20 had an integral ratio close to 1:2:3, confirming that CM-OH has been successfully synthesized [35]. Then, the hydroxyl group of CM-OH was activated using NPC to get CM-NPC. 1 H NMR spectrum showed complete shift of methylene protons from ␦ 4.83–5.44 while all other peaks of CM moieties remained unchanged (Fig. S1B). The peaks assignable to the NPC were discerned at ı 8.29 and 7.39. The signals at ı 8.29, 5.44 and 3.40 demonstrated an integral ratio close to 1:1:2, confirming the successful synthesis of CM-NPC. HA-Lys was prepared according to our previous report [46]. 1 H NMR spectrum showed characteristic peaks of HA at ı 4.41 (anomeric methine proton) and 1.98 (methyl proton) and lysine at ı 3.12 (methine proton) and 2.84 (methylene proton) (Fig. S2). By varying Lys(Boc)-OMe·HCl/carboxyl group of HA ratios from 1:2 to 1:1, we have obtained HA-Lys conjugates with 5 and 12 Lys per HA chain (denoted as HA-Lys5 and HA-Lys12, respectively), as determined by comparing the integral ratio of signals at ␦ 3.12–4.41. The treatment of HA-Lys5 and HA-Lys12 with CM-NPC gave HA-CM conjugates. The number of CM per HA chain was quantified using
UV spectroscopy at 380 nm using the formula A = 22.452 × C + 0.14 (C: in mg/mL). The results showed that HA-CM conjugates with 4 and 9 CM per HA chain (i.e. HA-CM4 and HA-CM9) were obtained from HA-Lys5 and HA-Lys12, respectively. It should be noted that CM was linked to HA-Lys via a urethane bond, which can be cleaved upon photo irradiation [38–40]. 3.2. Preparation and light-responsiveness of HA-CM nanogels HA-CM conjugates readily formed nanogels in aqueous solutions. DLS showed that HA-CM4 and HA-CM9 nanogels had hydrodynamic sizes of 147.2 and 165.4 nm, respectively (Fig. 1A). TEM micrograph displayed an average size of 40–80 nm for HA-CM4 nanogels (Fig. 1B). The much smaller size than the hydrodynamic size measured using DLS was mainly owing to nanogel dehydration and shrinkage upon drying. HA-CM4 and HA-CM9 nanogels revealed a negative surface charge of −16.8 and −9.5 mV, respectively. HA-CM4 exhibited a somewhat lower critical aggregation concentration (CAC) than HA-CM9 (9.1 versus 15.8 mg/L) (Table 1).
Fig. 2. The DOX (A) and CM (B) release profiles of DOX-loaded HA-CM4 nanogels at 37 ◦ C in PB after UV irradiation (100 mW/cm2 ). The DOX (C) and CM (D) release profiles of DOX-loaded HA-CM4 nanogels at 37 ◦ C in PB after NIR irradiation (785 nm, 2 W/cm2 ). Data are presented as the average ± standard deviation (n = 3).
552
C. Hang et al. / Colloids and Surfaces B: Biointerfaces 158 (2017) 547–555
Fig. 3. CLSM images of MCF-7 cells incubated with DOX-loaded HA-CM4 nanogels after NIR irradiation (A), free HA (5 L, 5 mg/mL) pretreated MCF-7 cells with NIR irradiation (B), and MCF-7 cells without NIR irradiation (C). The cells were incubated with nanogels for 4 h, and culture medium was replaced with fresh medium. Then the cells were irradiated 60 min by NIR (780 nm, 2 W/cm2 ) and further incubated for 8 h. [DOX] = 10 g/mL. The scale bars represent 20 m.
HA-CM nanogels were stable for at least one week on storage and in PBS in the dark at r.t. However, upon UV irradiation HA-CM4 nanogels swelled quickly over time (Fig. 1C), which is likely due to cleavage of the urethane bonds that link CM to HA-Lys (Scheme S2) [38–40]. The size of HA-CM4 nanogels increased from 150 nm
to ca. 460, 780 and 960 nm at 12 h following 2, 5 and 10 min UV irradiation, respectively. TEM micrographs displayed fewer particles with much smaller size (Fig. S3) after light irradiation. As comparison, HA-CM9 nanogels showed less size increase following UV irradiation (Fig. S4). Notably, Zhao et al. reported that coumarin
Fig. 4. MTT assays. (A) The antitumor activity of DOX-loaded HA-CM nanogels with or without NIR irradiation to MCF-7 and U87MG cells. Cells pretreated with free HA (5 L, 5 mg/mL), blank cells irradiated with NIR, DOX-loaded HA-CM nanogels and free DOX without NIR irradiation were used as controls ([DOX] = 10 g/mL). The cells were incubated with nanogels for 4 h, medium was replaced with fresh culture medium followed by NIR irradiation (780 nm, 2 W/cm2 ) and incubation for 72 h (Student’s t-test, **p < 0.01, ***p < 0.001). (B) The cytotoxicity of HA-CM4 nanogels following 24 h incubation. Data are presented as the average ± standard deviation (n = 4).
C. Hang et al. / Colloids and Surfaces B: Biointerfaces 158 (2017) 547–555
553
Table 2 Characteristics of DOX-loaded HA-CM nanogels (0.3 mg/mL). Entry
nanogels
Feed ratio (wt.%)
sizea (nm)
PDIa
DLCb (wt.%)
DLEb (%)
1 2 3 4
HA-CM4
5 10 5 10
138.0 149.7 141.4 147.8
0.23 0.26 0.24 0.21
3.4 5.2 4.0 6.1
71.5 57.4 83.3 67.6
a b
HA-CM9 Determined by DLS at 25 ◦ C in PB (pH 7.4, 10 mM). Determined using fluorometry.
derivatives have two photo absorption property and can respond to both UV and NIR [35]. To investigate whether the present nanogels are also NIR-sensitive, HA-CM4 nanogels were irradiated by NIR light (785 nm, 2 W/cm2 ). DLS measurements displayed that HACM4 nanogels enlarged from 150 to 235 nm in 12 h following 1 h NIR irradiation (Fig. 1D). Fig. S5 shows that NIR irradiation induced less size increase of HA-CM9 nanogels.
3.3. DOX loading and light-triggered DOX release DOX could be conveniently encapsulated into HA-CM nanogels. DLS results illustrated that DOX-loaded nanogels had similar sizes to the blank nanogels and decent drug loading efficiency (DLE) of 57.4%–83.3% at 5 and 10 wt.% DOX in feed (Table 2). HA-CM4 nanogels had slightly lower DOX loading capacity than HA-CM9, due to a lower CM content. The photo irradiation would induce cleavage of urethane bonds that link CM to HA-Lys (Scheme S2) [38–40], leading to accelerated drug release. Here, we investigated the in vitro release of DOX and CM from DOX-loaded HA-CM nanogels upon UV or NIR irradiation. Fig. 2A shows that 2 and 5 min UV irradiation resulted in 43.5% and 74.9% DOX release, respectively, from DOX-loaded HACM4 nanogels in 48 h. In contrast, less than 15% DOX was released when UV was not applied. Notably, UV irradiation also led to an accelerated CM release from nanogels (Fig. 2B). For example, 31.4% and 49.5% CM was released in 48 h from DOX-loaded HA-CM4 nanogels at 2 and 5 min UV irradiation, respectively, confirming that UV triggers the cleavage of the urethane bonds and CM release, which in turn brings about nanogel destabilization and DOX release. Zhao et al. reported that almost 100% o-nitrobenzyl (ONB) bonds in PEO-b-PNBM micelles were cleaved after UV irradiation (365 nm, 2 W/cm2 ) for 20 min [49]. Johnson et al. reported that in PEG-NBOCDOX the ONB bonds were chopped and 70% DOX was released upon UV irradiation (365 nm, 2 W/cm2 ) [50]. It was shown that DOX and CM release from DOX-loaded HA-CM9 nanogels could also be triggered by UV irradiation (Fig. S6), though less pronounced than for DOX-loaded HA-CM4 nanogels. Fig. 2C&D show that NIR irradiation could also induce coumarin and DOX release from DOX-loaded HA-CM4 nanogels. For instances, DOX-loaded HA-CM4 nanogels with 60 min NIR irradiation released 51.7% DOX and 30.0% coumarin in 48 h. Similar to results obtained with UV irradiation, DOX-loaded HA-CM9 nanogels displayed less CM and DOX release than DOX-loaded HA-CM4 nanogels (Fig. S7). It was noted that the drug release of DOX-HA-CM4 triggered by NIR, though much slower than that by UV, is fast as compared to previous reports [18,36]. For example, Zhao et al. reported that after pulse NIR laser irradiation (794 nm, sapphire) for 220 min, ca. 50% of paclitaxel was released from coumarin based polypeptide micelles within 145 h [18]. The fast NIR-responsive drug release of DOX-loaded HA-CM nanogels is likely due to their highly porous structure and fast drug diffusion. HA-CM4 nanogels with better stability and photo-sensitivity than HA-CM9 nanogels were selected for the following cell studies. Moreover, as NIR light (650–1000 nm) has a deeper tissue penetration (several centimeters) and better cell compatibility [10,51],
here we investigated triggered drug release in cancer cells by NIR irradiation. 3.4. Cellular uptake and intracellular release of DOX upon NIR irradiation Since DOX and coumarin are inherently fluorescent, we utilized CLSM to study their intracellular release behaviors from DOXloaded HA-CM4 nanogels in CD44 over-expressing (CD44+) MCF-7 cells, either with or without NIR irradiation. It was noted that cell morphology did not changed after NIR irradiation (Fig. S8). Fig. 3A shows that DOX-loaded HA-CM4 nanogels combined with NIR irradiation efficiently delivered DOX to the nuclei of MCF-7 cells. CM in or released from the nanogels mostly remained in the cytosol. In comparison, both fluorescence of DOX and CM were very low and mainly located in the cytosol when MCF-7 cells were pretreated with free HA or without NIR irradiation (Fig. 3B &C), confirming that the nanogels are internalized by CD44+ MCF-7 cells via receptormediated endocytosis, and intracellular DOX release is effectively triggered by NIR irradiation. Moreover, the coumarin fluorescence followed the same trend as DOX, showing much more intense fluorescence in NIR treated MCF-7 cells than those without NIR treatment. 3.5. CD44 targeted delivery of DOX by HA-CM nanogels The antitumor effect of DOX-loaded HA-CM4 nanogels with or without NIR irradiation was explored using MTT assays in MCF-7 cells. U87 MG cells that express low level of CD44 (CD44-) were used as controls. The results showed that DOX-loaded HA-CM4 nanogels combined with 60 min NIR irradiation had significantly higher growth inhibition of MCF-7 cells than without NIR irradiation (cell viabilities: ca. 41% versus 65%) following 48 h incubation (Fig. 4A). The antitumor effect of DOX-loaded HA-CM4 nanogels to MCF-7 cells was noticeably reduced by pretreating the cells with free HA, possibly due to the blockage of CD44 on the cell surface by free HA that was reported to have high specificity to CD44. In contrast, significantly lower cytotoxicity was observed for DOXloaded HA-CM4 nanogels in CD44- U87 MG cells, either with or without NIR irradiation, likely due to inefficient cellular uptake of DOX-loaded HA-CM4 nanogels. These results confirm that HA-CM nanogels are taken up by CD44+ MCF-7 cells by a ligand-receptor mediated endocytosis pathway, and NIR effectively triggers intracellular DOX release. It should further be noted that NIR (785 nm, 2 W/cm2 ) irradiation for 60 min did not cause significant cell death. Moreover, empty HA-CM4 nanogels was virtually nontoxic to both MCF-7 and U87 MG cells at tested concentrations up to 1.0 mg/mL (Fig. 4B). Both degradation products, HA-Lys polymer and free CM, were shown to be nontoxic to cells (Fig. S9). 4. Conclusions We have demonstrated that simple hyaluronic acid-(7-N,Ndiethylamino-4- hydroxymethylcoumarin) (HA-CM) conjugates form robust, nano-sized and negatively charged nanogels. Notably,
554
C. Hang et al. / Colloids and Surfaces B: Biointerfaces 158 (2017) 547–555
these multifunctional nanogels show decent loading of doxorubicin (DOX), active targeting ability to CD44 over-expressing MCF-7 cells and remotely triggered intracellular DOX release by either NIR or UV irradiation. The in vitro studies reveal that DOX-loaded HA-CM nanogels combined with NIR irradiation can cause significantly higher antitumor effect to MCF-7 cells than to U87 MG cells (low CD44 expression) under otherwise the same condition as well as to MCF-7 cells without NIR irradiation. Tumor-targeted HA-CM nanogels with easy preparation and photo-controlled intracellular drug release have a great potential for cancer chemotherapy. Acknowledgements This work is financially supported by research grants from the National Natural Science Foundation of China (NSFC 51473111, 51633005, 51561135010) and the Major Program of the Natural Science Foundation of Jiangsu Province (14KJA150008). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2017.07. 041. References [1] V.P. Torchilin, Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery, Nat. Rev. Drug Discov. 13 (2014) 813–827. [2] S. Mura, J. Nicolas, P. Couvreur, Stimuli-responsive nanocarriers for drug delivery, Nat. Mater. 12 (2013) 991–1003. [3] F.H. Meng, R. Cheng, C. Deng, Z.Y. Zhong, Intracellular drug release nanosystems, Mater. Today 15 (2012) 436–442. [4] R. Cheng, F.H. Meng, C. Deng, H.-A. Klok, Z.Y. Zhong, Dual and multi-stimuli responsive polymeric nanoparticles for programmed site-specific drug delivery, Biomaterials 34 (2013) 3647–3657. [5] M. Han, Q. Lv, X.-J. Tang, Y.-L. Hu, D.-H. Xu, F.-Z. Li, W.-Q. Liang, J.-Q. Gao, Overcoming drug resistance of MCF-7/ADR cells by altering intracellular distribution of doxorubicin via MVP knockdown with a novel siRNA polyamidoamine-hyaluronic acid complex, J. Control. Release 163 (2012) 136–144. [6] L. Qiu, M. Qiao, Q. Chen, C. Tian, M. Long, M. Wang, Z. Li, W. Hu, G. Li, L. Cheng, L. Cheng, H. Hu, X. Zhao, D. Chen, Enhanced effect of pH-sensitive mixed copolymer micelles for overcoming multidrug resistance of doxorubicin, Biomaterials 35 (2014) 9877–9887. [7] L. Xiao, X. Xiong, X. Sun, Y. Zhu, H. Yang, H. Chen, L. Gan, H. Xu, X. Yang, Role of cellular uptake in the reversal of multidrug resistance by PEG-b-PLA polymeric micelles, Biomaterials 32 (2011) 5148–5157. [8] Y. Guo, W. He, S. Yang, D. Zhao, Z. Li, Y. Luan, Co-delivery of docetaxel and verapamil by reduction-sensitive PEG-PLGA-SS-DTX conjugate micelles to reverse the multi-drug resistance of breast cancer, Colloid. Surface. B 151 (2017) 119–127. [9] F.H. Meng, Y. Zhong, R. Cheng, C. Deng, Z. Zhong, pH-sensitive polymeric nanoparticles for tumor-targeting doxorubicin delivery: concept and recent advances, Nanomedicine 9 (2014) 487–499. [10] B.P. Timko, T. Dvir, D.S. Kohane, Remotely triggerable drug delivery systems, Adv. Mater. 22 (2010) 4925–4943. [11] A. Bansal, Y. Zhang, Photocontrolled nanoparticle delivery systems for biomedical applications, Accounts Chem. Res. 47 (2014) 3052–3060. [12] A.Y. Rwei, W. Wang, D.S. Kohane, Photoresponsive nanoparticles for drug delivery, Nano Today 10 (2015) 451–467. [13] Y. Yang, Y. Yang, X. Xie, X. Cai, Z. Wang, W. Gong, H. Zhang, Y. Li, X. Mei, A near-infrared two-photon-sensitive peptide-mediated liposomal delivery system, Colloid. Surface. B 128 (2015) 427–438. [14] T. Dvir, M.R. Banghart, B.P. Timko, R. Langer, D.S. Kohane, Photo-targeted nanoparticles, Nano Lett. 10 (2010) 250–254. [15] R. Tong, H.D. Hemmati, R. Langer, D.S. Kohane, Photoswitchable nanoparticles for triggered tissue penetration and drug delivery, J. Am. Chem. Soc. 134 (2012) 8848–8855. [16] L.L. Meng, W. Huang, D. Wang, X. Huang, X. Zhu, D. Yan, Chitosan-based nanocarriers with pH and light dual response for anticancer drug delivery, Biomacromolecules 14 (2013) 2601–2610. [17] G.-Y. Liu, C.-J. Chen, D.-D. Li, S.-S. Wang, J. Ji, Near-infrared light-sensitive micelles for enhanced intracellular drug delivery, J. Mater. Chem. 22 (2012) 16865–16871. [18] S. Kumar, J.-F. Allard, D. Morris, Y.L. Dory, M. Lepage, Y. Zhao, Near-infrared light sensitive polypeptide block copolymer micelles for drug delivery, J. Mater. Chem. 22 (2012) 7252–7257.
[19] C. Chen, G. Liu, X. Liu, S. Pang, C. Zhu, L. Lv, J. Ji, Photo-responsive, biocompatible polymeric micelles self-assembled from hyperbranched polyphosphate-based polymers, Polym. Chem. 2 (2011) 1389–1397. [20] E. Mabrouk, S. Bonneau, L. Jia, D. Cuvelier, M.H. Li, P. Nassoy, Photosensitization of polymer vesicles: a multistep chemical process deciphered by micropipette manipulation, Soft Matter 6 (2010) 4863–4875. [21] E. Cabane, V. Malinova, S. Menon, C.G. Palivan, W. Meier, Photoresponsive polymersomes as smart, triggerable nanocarriers, Soft Matter 7 (2011) 9167–9176. [22] J.F. Lovell, C.S. Jin, E. Huynh, H. Jin, C. Kim, J.L. Rubinstein, W.C.W. Chan, W. Cao, L.V. Wang, G. Zheng, Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents, Nat. Mater. 10 (2011) 324–332. [23] R. Huschka, J. Zuloaga, M.W. Knight, L.V. Brown, P. Nordlander, N.J. Halas, Light-Induced release of DNA from gold nanoparticles: nanoshells and nanorods, J. Am. Chem. Soc. 133 (2011) 12247–12255. [24] Y.N. Zhong, C. Wang, R. Cheng, L. Cheng, F.H. Meng, Z. Liu, Z.Y. Zhong, cRGD-directed, NIR-responsive and robust AuNR/PEG-PCL hybrid nanoparticles for targeted chemotherapy of glioblastoma in vivo, J. Control. Release 195 (2014) 63–71. [25] W. Zhang, F. Wang, Y. Wang, J. Wang, Y. Yu, S. Guo, R. Chen, D. Zhou, pH and near-infrared light dual-stimuli responsive drug delivery using DNA-conjugated gold nanorods for effective treatment of multidrug resistant cancer cells, J. Control. Release 232 (2016) 9–19. [26] T.-Y. Hsieh, W.-C. Huang, Y.-D. Kang, C.-Y. Chu, W.-L. Liao, Y.-Y. Chen, S.-Y. Chen, Neurotensin-conjugated reduced graphene oxide with multi-stage near-infrared-triggered synergic targeted neuron gene transfection in vitro and In vivo for neurodegenerative disease therapy, Adv. Healthc. Mater. 5 (2016) 3016–3026. [27] Q. He, D.O. Kiesewetter, Y. Qu, X. Fu, J. Fan, P. Huang, Y. Liu, G. Zhu, Y. Liu, Z. Qian, X. Chen, NIR-responsive on-demand release of CO from metal carbonyl-caged graphene oxide nanomedicine, Adv. Mater. 27 (2015) 6741–6746. [28] M.K.G. Jayakumar, N.M. Idris, Y. Zhang, Remote activation of biomolecules in deep tissues using near-infrared-to-UV upconversion nanotransducers, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 8483–8488. [29] J.-M. Schumers, J.-F. Gohy, C.-A. Fustin, A versatile strategy for the synthesis of block copolymers bearing a photocleavable junction, Polym. Chem. 1 (2010) 161–163. [30] V. Yesilyurt, R. Ramireddy, S. Thayumanavan, Photoregulated release of noncovalent guests from dendritic amphiphilic nanocontainers, Angew. Chem. Int. Edit. 50 (2011) 3038–3042. [31] D. Han, X. Tong, Y. Zhao, Fast photodegradable block copolymer micelles for burst release, Macromolecules 44 (2011) 437–439. [32] S.S. Agasti, A. Chompoosor, C.-C. You, P. Ghosh, C.K. Kim, V.M. Rotello, Photoregulated release of caged anticancer drugs from gold nanoparticles, J. Am. Chem. Soc. 131 (2009) 5728-. [33] D.S. Achilleos, T.A. Hatton, M. Vamvakaki, Light-regulated supramolecular engineering of polymeric nanocapsules, J. Am. Chem. Soc. 134 (2012) 5726–5729. [34] Q. Lin, Q. Huang, C. Li, C. Bao, Z. Liu, F. Li, L. Zhu, Anticancer drug release from a mesoporous silica based nanophotocage regulated by either a one- or two-photon process, J. Am. Chem. Soc. 132 (2010) 10645–10647. [35] J. Babin, M. Pelletier, M. Lepage, J.-F. Allard, D. Morris, Y. Zhao, A new two-photon-sensitive block copolymer nanocarrier, Angew. Chem.-Int. Ed. 48 (2009) 3329–3332. [36] C.d.G. Lux, C.L. McFearin, S. Joshi-Barr, J. Sankaranarayanan, N. Fomina, A. Almutairi, Single UV or near IR triggering event leads to polymer degradation into small molecules, Acs Macro Lett. 1 (2012) 922–926. [37] N. Fomina, C.L. McFearin, M. Sermsakdi, J.M. Morachis, A. Almutairi, Low power, biologically benign NIR light triggers polymer disassembly, Macromolecules 44 (2011) 8590–8597. [38] V. Hagen, B. Dekowski, N. Kotzur, R. Lechler, B. Wiesner, B. Briand, M. Beyermann, {7- bis(carboxymethyl)amino coumarin-4-yl}methoxycarbonyl derivatives for photorelease of carboxylic acids alcohols/phenols, thioalcohols/thiophenols, and amines, Chem. Eur. J. 14 (2008) 1621–1627. [39] T. Furuta, S.S.H. Wang, J.L. Dantzker, T.M. Dore, W.J. Bybee, E.M. Callaway, W. Denk, R.Y. Tsien, Brominated 7-hydroxycoumarin-4-ylmethyls: photolabile protecting groups with biologically useful cross-sections for two photon photolysis, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 1193–1200. [40] V. Hagen, B. Dekowski, V. Nache, R. Schmidt, D. Geissler, D. Lorenz, J. Eichhorst, S. Keller, H. Kaneko, K. Benndorf, B. Wiesner, Coumarinylmethyl esters for ultrafast release of high concentrations of cyclic nucleotides upon one- and two-photon photolysis, Angew. Chem.-Int. Ed. 44 (2005) 7887–7891. [41] K. Katayama, S. Tsukiji, T. Furuta, T. Nagamune, A bromocoumarin-based linker for synthesis of photocleavable peptidoconjugates with high photosensitivity, Chem. Commun. 539 (2008) 5399–5401. [42] Q. Lin, C. Bao, S. Cheng, Y. Yang, W. Ji, L. Zhu, Target-activated coumarin phototriggers specifically switch on fluorescence and photocleavage upon bonding to thiol-Bearing protein, J. Am. Chem. Soc. 134 (2012) 5052–5055. [43] C.d.G. Lux, J. Lux, G. Collet, S. He, M. Chan, J. Olejniczak, A. Foucault-Collett, A. Almutairi, Short soluble coumarin crosslinkers for light-controlled release of cells and proteins from hydrogels, Biomacromolecules 16 (2015) 3286–3296. [44] K.Y. Choi, E.J. Jeon, H.Y. Yoon, B.S. Lee, J.H. Na, K.H. Min, S.Y. Kim, S.-J. Myung, S. Lee, X. Chen, I.C. Kwon, K. Choi, S.Y. Jeong, K. Kim, J.H. Park, Theranostic
C. Hang et al. / Colloids and Surfaces B: Biointerfaces 158 (2017) 547–555 nanoparticles based on PEGylated hyaluronic acid for the diagnosis, therapy and monitoring of colon cancer, Biomaterials 33 (2012) 6186–6193. [45] J.-H. Park, H.-J. Cho, H.Y. Yoon, I.-S. Yoon, S.-H. Ko, J.-S. Shim, J.-H. Cho, J.H. Park, K. Kim, I.C. Kwon, D.-D. Kim, Hyaluronic acid derivative-coated nanohybrid liposomes for cancer imaging and drug delivery, J. Control. Release 174 (2014) 98–108. [46] Y.N. Zhong, J. Zhang, R. Cheng, C. Deng, F.H. Meng, F. Xie, Z.Y. Zhong, Reversibly crosslinked hyaluronic acid nanoparticles for active targeting and intelligent delivery of doxorubicin to drug resistant CD44 + human breast tumor xenografts, J. Control. Release 205 (2015) 144–154. [47] S. Ganesh, A.K. Iyer, D.V. Morrissey, M.M. Amiji, Hyaluronic acid based self-assembling nanosystems for CD44 target mediated siRNA delivery to solid tumors, Biomaterials 34 (2013) 3489–3502.
555
[48] Y. Zou, Y. Song, W.J. Yang, F.H. Meng, H.Y. Liu, Z.Y. Zhong, Galactose-installed photo-crosslinked pH-sensitive degradable micelles for active targeting chemotherapy of hepatocellular carcinoma in mice, J. Control. Release 193 (2014) 154–161. [49] J.Q. Jiang, X. Tong, D. Morris, Y. Zhao, Toward photocontrolled release using light-dissociable block copolymer micelles, Macromolecules 39 (2006) 4633–4640. [50] J.A. Johnson, Y.Y. Lu, A.O. Burts, Y.-H. Lim, M.G. Finn, J.T. Koberstein, N.J. Turro, D.A. Tirrell, R.H. Grubbs, Core-clickable PEG-branch-azide bivalent-bottle-brush polymers by ROMP: grafting-through and clicking-to, J. Am. Chem. Soc. 133 (2011) 559–566. [51] Z. Meng, F. Wei, R. Wang, M. Xia, Z. Chen, H. Wang, M. Zhu, NIR-laser-switched In vivo smart nanocapsules for synergic photothermal and chemotherapy of tumors, Adv. Mater. 28 (2016) 245–253.