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Biodegradable reduction-responsive polymeric micelles for enhanced delivery of melphalan to retinoblastoma cells
International Journal of Biological Macromolecules 141 (2019) 997–1003
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Biodegradable reduction-responsive polymeric micelles for enhanced delivery of melphalan to retinoblastoma cells Jia Li ⇑,1, Jihong Wang 1, Xuetong Zhang, Xin Xia, Chenchen Zhang Department of Ophthalmology, Affiliated Hospital of Jiangnan University, Wuxi 214062, China
a r t i c l e
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Article history: Received 18 June 2019 Received in revised form 3 September 2019 Accepted 11 September 2019 Available online 12 September 2019 Keywords: Retinoblastoma Melphalan Reduction-responsive Polymeric micelles Drug delivery
a b s t r a c t Melphalan (MEL) is an effective chemotherapeutic agent for treatment of retinoblastoma (Rb) which is the most common childhood malignancy. However, the inherent cardiopulmonary toxicity and hazardous integration limit its therapeutic effect on RB. N-Acetylheparosan (AH), a natural heparin-like polysaccharide in mammals with long circulation effect and good biocompatibility, was linked by d-a-tocopherol acid succinate (VES) via and cystamine (CYS) to synthesize reduction-responsive N-acetylheparosan-CYS-Vitamin E succinate (AHV) copolymers. In addition, CYS was replaced by adipic acid dihydrazide (ADH) to obtain a control of non-reduction-responsive polymers N-acetylheparosanADH-Vitamin E succinate (ADV). MEL-loaded AHV micelles (MEL/AHV) as well as ADV micelles (MEL/ ADV) were prepared with small particle size and high drug loading content. In vitro drug release showed that MEL/AHV micelles presented obvious reduction-triggered release behavior compared with MEL/ADV. In vitro antitumor effects were investigated using WERI-Rb-1 retinoblastoma cells. Cytotoxicity experiments showed that the IC50 of MEL/AHV was significantly lower than that of free MEL and MEL/ADV, suggesting that MEL/AHV enhanced the cytotoxicity against retinoblastoma cells. Furthermore, MEL/AHV micelles were more easily uptaken by multiple pathways compared with MEL/ADV and free MEL. Therefore, MEL/AHV might be a potential delivery system for enhanced delivery of melphalan to Rb cells. Ó 2019 Elsevier B.V. All rights reserved.
1. Introduction As the most common ocular tumor in children, retinoblastoma (Rb) has attracted widespread attention. According to statistics, one in every 15,000 to 20,000 children suffers from the disease, and most of them are children under 5 years old [1,2]. Although retinoblastoma is the first tumor that raises concerns about the genetic etiology of cancer, the mortality of retinoblastoma still reaches 70%, especially in low- and middle-income countries [3]. Usually, the occurrence of the tumor is accompanied by the loss of retinoblastoma gene 1 (RB1) [4]. Although therapy of Rb has excellent curing prospect, patients often suffer from loss of vision and second malignancies [5,6]. Nowadays, the most common method of treating Rb is the intra-arterial or intravitreal injection of chemotherapeutic drugs. But it has been reported that intraarterial injection of melphalan (MEL) can cause lung toxicity, arrhythmia and other cytotoxic side effects [7,8]. Therefore, in order to reduce the side effects and improve patient survival
⇑ Corresponding author. 1
E-mail address: [email protected] (J. Li). These authors contributed equally to this work.
https://doi.org/10.1016/j.ijbiomac.2019.09.085 0141-8130/Ó 2019 Elsevier B.V. All rights reserved.
chance, it is necessary to develop new treatment strategies for MEL. In cancer chemotherapy, some biological barriers often hinder the transport and accumulation of chemotherapeutic drugs in tumor cells. Antitumor drugs loaded in polymer micelles produce high therapeutic efficiency for solid tumors, but the cargoes need to be released from micelles into tumor cells on time [9,10]. In recent years, therefore, environmentally stimuli-responsive polymer micelles have been widely used for targeted delivery and controlled release of antitumor drugs. This method can significantly promote the internalization of micelles through receptormediated endocytosis and subsequently enhance the responsiveness of the nanocarriers in the cell for drug release [11–15]. In the past decade, in order to increase the release of drugs in tumor cells, intelligent vectors responding to different tumor microenvironments have been developed. Stimulating sensitive drug carriers can be triggered by pH, temperature, enzymes, redox potential, light, magnetic fields and ultrasound [14–20] to rapidly release the entrapped drugs. In these stimuli-responsive drug delivery systems, reduction-responsive drug carriers have been extensively studied because of the difference in redox condition between tumor cell fluid and extracellular [21–23]. It is worth noting that
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the concentration of glutathione (GSH), a thiol tripeptide capable of reducing disulfide bonds, in many tumor cells is higher than that of normal cells [24]. The difference in the GSH concentration can cause the rapid release of active substances such as drugs, genes, and proteins from intracellular reduction-responsive drug carriers [25–27]. So, reduction-responsive polymer micelles can be widely used in drug delivery depending on the large difference between extracellular fluid and intracellular environment. Polymer micelles containing a disulfide bond (-S-S-) are stable in body fluids, but when they arrive at tumor cells, the disulfide bond is cleaved into a sulfhydryl group by GSH, causing destruction of the micelle structure resulting in fast drug release. As a natural heparin-like polysaccharide in vivo, N-acetylheparosan (AH) cannot be degraded by heparanase because of the unsulfated oxygen site, but it will be degraded to normal monosaccharides in lysosomes, which make AH has good long circulation effect and biocompatibility [28]. Herein, AH and d-a-tocopherol acid succinate (VES) were linked by cystamine (CYS) to synthesize reduction-responsive N-acetylheparosan-CYSVitamin E succinate (AHV) copolymers for delivery of melphalan to retinoblastoma cells. The preparation and antitumor effect of MEL-loaded micelles are shown in Fig. 1. The properties of melphalan-loaded micelles (MEL/AHV) were investigated, and the cytotoxicity and intracellular uptake of the MEL/AHV micelles against WERI-Rb-1 retinoblastoma cells were evaluated. Furthermore, the internalization pathway of MEL/AHV micelles was further tested.
(Shanghai, China). Melphalan (MEL) was obtained from Macklin Biochemical (Shanghai, China). Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS) and RPMI-1640 medium were obtained from Gibco BRL (Maryland, USA). 4,6-Diamidino-2phenylindole (DAPI) was purchased from Shanghai Beyotime Biotechnology (Shanghai, China). Cell Counting Kit-8 (CCK-8), RIPA Lysis Buffer and Enhanced BCA Protein Assay Kit were purchased from Beyotime Biotechnology (Shanghai, China). All other reagents were of analytical grade. 2.2. Synthesis of AHV copolymers Cystamine dihydrochloride was pretreated with sodium hydroxide solution through acid-base neutralization before it’s used [30]. Firstly, N-acetylheparosan (3 104moL) was dissolved in formamide at room temperature. NHS (6 104 moL) and EDC (6 104 moL) were added to activate carboxylic groups. After being stirred for 2 h, 10 mL of cystamine (3 104 moL) solution was added dropwise and stirred for 48 h under a nitrogen atmosphere at room temperature. The mixture was purified by dialyzing (MWCO 3500) with distilled water for 48 h and then freeze-dried to obtain AH-cystamine product. Secondly, VES (3 104 moL) was dissolved in DMF and activated using the same method. Then it was added into the AH-cystamine (3 104 moL) solution and stirred under a nitrogen atmosphere at room temperature. After 48 h, the solution was dialyzed (MWCO 3500) against 25% ethanol solution and distilled water, and then freeze-dried to obtain AHV copolymers.
2. Experimental section 2.1. Materials D-a-Tocopherol acid succinate (VES), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were provided by Aladdin (Shanghai, China). Cystamine dihydrochloride (CYS) and Adipodihydrazide (ADH) were purchased from Energy Chemical Reagent (Shanghai, China). N-acetylheparosan (AH, Mw = 5.437 104) was gifted by Jiangnan university (Wuxi, China). Dithiothreitol (DTT), Dimethyl sulfoxide (DSMO), penicillin-streptomycin solution and pancreatin were provided by Shanghai Songan Biotech (Shanghai, China). Dimethylformamide (DMF), formamide, triethylamine (TEA) and tetrahydrofuran (THF) were obtained from Shanghai Reagent Chemical
2.3. Synthesis of ADV copolymers The N-acetylheparosan (1.25 104moL) was dissolved in formamide, and activated by EDC (2.5 104 moL) and NHS (2.5 104 moL). Then adipic acid dihydrazide (7.25 104 moL) was added and the pH was adjusted to 4.75 using 0.1 M hydrochloric acid. After 1 h, 0.1 M sodium hydroxide was added to the mixture solution to end the reaction. The solution was then dialyzed (MWCO 3500) against distilled water for 48 h and freeze-dried to obtain AH-ADH. Then NHS and EDC were added to the VES (1.5 104 moL) solution to activate the carboxylic groups. AH-ADH (1.5 104 moL) was dropped into the mixed solution and stirred for 48 h under a nitrogen atmosphere at room temperature. The mixed solution was dialyzed (MWCO 3500) and then freeze-dried to obtain ADV copolymers. 2.4. Preparation and characterization of MEL-loaded micelles
Fig. 1. Construction and intracellular delivery of the reduction-responsive MEL/ AHV micelles.
AH-based copolymer (10 mg) was sufficiently dissolved in PBS (pH 7.4, 10 mL). Then 2 mL MEL solution was slowly dropped into the copolymer solution and stirred overnight in dark. MEL-loaded AH-based copolymer micelles (MEL/AHV and MEL/ADV) were obtained after ultrasonic 30 min in an ice bath. The average particle size, polydispersity index (PDI) and zeta potential of the micelles were measured by Zetasizer Nano ZS apparatus (Malvern Instruments, UK) at 37 °C. The MEL-loaded micelles were dropped onto the copper grid and negatively stained with 2% phosphotungstic acid. Then, the morphology of the micelles was observed by JEM-2100 transmission electron microscope (TEM) with 80 kV of acceleration voltage (JEO, Japan). Drug loading content (DL) and encapsulation efficiency (EE) were measured by a UV/vis spectrophotometer (UV-2550, Shimadzu, Japan) at a wavelength of 301 nm. Free MEL was separated by ultrafiltration, and the micellar structure was destroyed by dimethyl sulfoxide to test the total drug content.
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2.5. Serum stability of MEL-loaded micelles The serum stability of the MEL/AHV and MEL/ADV micelles were tested by dynamic light scattering spectrophotometry (Zetasizer Nano ZS apparatus, Malvern Instruments, UK). Briefly, the MEL-loaded micelles were dispersed in pH 7.4 PBS solution mixed with DMEM solution containing 10% FBS, and the particle size were measured at different times to monitor the stability of the micelles. 2.6. In vitro reduction-triggered release The in vitro release behaviors of MEL from the micelles were measured using the dialysis method. 2 mL of MEL-loaded micelles was placed into the dialysis bag (MWCO 1000) which was immersed in 20.0 mL PBS (pH 7.4) or GSH-containing PBS (10 mM, pH 7.0) at 37 °C under shaking (100 r/min). 2 mL of release medium was sampled and the fresh PBS replaced with the same volume at different time. The amount of MEL was determined using UV/vis absorbance at 301 nm. The cumulative release of MEL as follows:
Erð%Þ ¼
P VO Cn þ Ve 1n1 Ci 100% mMEL
where Er is the cumulative release of MEL, mMEL represents the total amount of MEL in the micelles. V0 and Ve are the total volume and replacement volume of the release medium, respectively. Ci is the MEL concentration released at the ith sample. 2.7. Cell culture Human retinoblastoma cancer cells (WERI-Rb-1) was purchased from Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS), 100 mg/mL streptomycin sulfate and 100 U/mL penicillin G sodium. The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. 2.8. Cytotoxicity evaluation WERI-Rb-1 cells were seeded into 96-well plates with 5 103 cells per well to preculture for 12 h. Then a fresh medium containing various concentration of blank micelles or drug-loaded micelles was added to each well. After incubation for 24 h, 20 lL of CCK-8 solution was added and the cell viability was measured using Multiskan MK3 microplate reader (Thermo, USA).
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inhibitors and their experimental concentration were as follows: genistein (GS, 10 lgmL1) [29], chlorpromazine (CPZ, 10 lgmL1) [30], colchicine (CC, 40 lgmL1) [31] and indomethacin (IDM, 10 lgmL1) [29,32]. WERI-Rb-1 cells were seeded into 96-well plates at a density of 5 103 cells/well for incubation of 24 h. The viability of cells incubated with each inhibitor was measured according to the above described. WERI-Rb-1 cells were seeded into 6-well plates at a density of 5 105cells/well. Then, the inhibitors were added to replace the old medium, and drug-loaded micelles (MEL concentration of 5 lgmL1) were incubated for 1 h. The content of MEL was measured by UV/vis. The control group treated without inhibitors at 37 °C was set to 100%. 2.11. Statistical analysis All experiments were operated at least three times independently and the data were presented as the mean ± standard deviation (SD). The one-way ANOVA-LSD and IndependentSamples t-Test were adopted to determine the statistical significance of differences. p < 0.05, p < 0.01 and p < 0.001 were indicated statistical significance, high significance and extreme significance, respectively, while p > 0.05 was indicated no significant difference. 3. Results and discussion 3.1. Synthesis of reduction-responsive AHV copolymers The synthetic scheme and 1H NMR spectrum of AHV copolymers are presented in Fig. 2. The carboxyl groups on the VES and AH polysaccharide were attached to the amino groups of CYS, respectively. The characteristic peaks of glucosamine on AH polysaccharide were shown at 3.63 ppm, 3.83 ppm and 5.31 ppm and the typical peak of -CO-CH3 on AH polysaccharide was shown at 1.98 ppm. The typical peaks of glucuronic acid on AH polysaccharide were shown at 3.29 ppm, 3.72 ppm, and 4.54 ppm. The typical peaks of -CH2- and –CH3 on the alkanes of VES were shown at 1.05–1.35 ppm and 0.85 ppm, and -CH3 on the phenyl ring was shown at 2.63 ppm. The characteristic peaks of -CONH- were shown between 8.05 and 8.11 ppm. Based on these results, it can be concluded that AHV was successfully synthesized. 3.2. Preparation and characterization of AH-based micelles
The cellular uptake of free MEL, MEL/AHV or MEL/ADV micelles in WERI-Rb-1 cells was measured by UV–vis spectrophotometer (UV-2550, Shimadzu, Japan). The cells were seeded into 6-well plates with 5 105 cells per well for preculture 12 h, and the medium containing free MEL or drug-loaded micelles (MEL concentration of 5.0 lgmL1) was added into each well. After incubation for different times, the cells were collected by centrifugation and lysed by RIPA Lysis Buffer. The cell lysate was diluted with PBS and centrifuged. Then the content of MEL was determined by UV/vis. The content of protein in each well was determined by the Enhanced BCA Protein Assay Kit. The uptake of MEL was calculated as the amount of MEL (lg)/the amount of cells protein (mg).
The blank micelles and MEL-loaded micelles were prepared by ultrasonic method. The micelles are formed because of the amphipathic nature of AHV copolymers which contain both hydrophilic regions of AH as well as hydrophobic regions of VES. Their physicochemical properties were characterized and shown in Table 1. Although the particle size of micelles slightly increased after entrapment of MEL, all the particle sizes of the micelles were below 150 nm with a narrow size distribution (Fig. 3A). This indicates that the micelles can be well targeted to the tumor site by the EPR effect. The negative charge resulting from the electronegative AH shell of the micelles could ensure its stability in the blood. TEM showed that the micelles had a nearly spherical morphology and good dispersibility (Fig. 3B). Furthermore, the drug loading of micelles was 13–16%, and the encapsulation efficiency was above 85%, suggesting that the micelles had a good drug encapsulation capacity.
2.10. Cellular uptake mechanism studies
3.3. Serum stability of MEL-loaded micelles
The cytotoxicity of each inhibitor against WERI-Rb-1 cells at the experimental concentration was firstly tested by CCK-8 assays. The
The stability of MEL/AHV and MEL/ADV micelles was studied in pH 7.4 PBS solution mixed with DMEM solution containing 10%
2.9. Cell uptake analysis
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Fig. 2. (A) Synthetic scheme of AHV copolymers and 1H NMR spectra of (B) AH polysaccharide and (C) AHV copolymers.
Table 1 Characterization of blank micelles and drug-loaded micelles (n = 3). Formulations
Particle size (nm)
PDI
Zeta potential (mV)
EE (%)
DL (%)
AHV ADV MEL/AHV MEL/ADV
100.4 ± 6.3 94.3 ± 5.7 120.4 ± 6.3 114.3 ± 5.7
0.175 ± 0.05 0.158 ± 0.09 0.142 ± 0.03 0.173 ± 0.07
24.31 ± 3.1 23.57 ± 2.7 21.39 ± 1.9 19.56 ± 2.8
– – 86.37 ± 3.11 89.25 ± 2.32
– – 14.38 ± 1.55 15.67 ± 1.49
Fig. 3. (A) Particle size distribution and (B) TEM image of MEL/AHV micelles.
J. Li et al. / International Journal of Biological Macromolecules 141 (2019) 997–1003
FBS. The average size of the protein in FBS is 10 nm, which don’t affect the measurement of the micellar particle size. As shown in Fig. 4A, no significant change in particle size was observed in MEL/AHV as well as and MEL/ADV after incubation with FBS for 48 h. Obviously, no precipitates and visible particles were found, and there was no significant increase in the micelles particle size after incubation with FBS. The result indicates that only a small amount of proteins were adsorbed on the surface of micelles. Therefore, MEL/AHV micelles will not aggregate into larger particles by absorbing serum proteins after intravenous injection probably due to the generation of a hydrophilic shell.
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and MEL/AHV decreased from 16.63% to 3.42% as the concentration increased from 6.25 lg/mL to 200 lg/mL. In other words, the gap was gradually narrowed. Moreover, there were significant differences between MEL/AHV and MEL/ADV at concentration of 6.25–50 lg/mL (P < 0.001) and 100 lg/mL (p < 0.05), respectively, while no significant difference was seen at concentration of 200 lg/mL (p > 0.05). We speculated that this might be that though more MEL could be released from redox-sensitive MEL/AHV than MEL/ADV in tumor cells, the amount of released drug from both of the micelles with high concentration (e.g. 200 lg/mL) had reached a certain concentration to kill most of the cells, which seemed to diminish the effect of redox- sensitive releases.
3.4. In vitro drug release studies 3.6. Cellular uptake studies To study the redox-sensitive release property of the micelles, the release behaviors of MEL from MEL/AHV and MEL/ADV micelles were investigated at different concentrations of GSH. As shown in Fig. 4B, the cumulative release of MEL from MEL/AHV and MEL/ADV micelles was about 30% at pH 7.4, suggesting that the micelles maintained good stability in the normal physiological condition. However, the release of MEL from MEL/AHV micelles in 10 mM GSH was above 60%, which significantly higher than that of pH 7.4 condition and MEL/ADV micelles (p < 0.001). It is well known that disulfide bonds are unstable in reductive conditions. So, MEL/AHV micelles containing disulfide linkages are depolymerized in response to the high GSH, leading to change in the amphipathic balance and thus release of the drugs. These results indicate that the reduction-responsive MEL/ADV micelles can be depolymerized to rapidly release MEL in the tumor microenvironment. 3.5. In vitro cytotoxicity assay The cytotoxicity of free MEL, MEL/AHV and MEL/ADV was evaluated by determining cellular viability through the CCK-8 assay. At a relatively high concentration (500 lgmL1) of blank micelles, the viabilities of WERI-Rb-1 cells were still >90% (Fig. 5A), suggesting the blank micelles were non-toxic. As shown in Fig. 5B, however, MEL/AHV was toxic to WERI-Rb-1 cells in a dose-dependent manner. The cells died significantly with drug concentration increasing (Fig. 5C). The median inhibitory concentrations (IC50) of MEL/AHV, MEL/ADV, and free MEL were (10.91 ± 0.56) lgmL1, (26.01 ± 1.42) lgmL1 and (48.15 ± 1.89) lgmL1, respectively. There was a significant difference between MEL/AHV and the other groups (P < 0.001), indicating that the reductionresponsive micelles could be used as effective drug nanocarriers. However, the difference value of cell viability between MEL/ADV
The cellular uptake of free MEL, MEL/AHV and MEL/ADV micelles was evaluated by UV/vis spectrophotometry. The protein standard was tested firstly for the determination of the protein content of each group (Fig. 6A). From the results, the uptake of all the formulations increased significantly with time as shown in Fig. 6B. It was worth noting that the uptake of micelles was much higher than that of free MEL (P < 0.001), which indicated that the micelles could be easily uptaken by tumor cells. Furthermore, there was a significant difference in cellular uptake between MEL/AHV and MEL/ADV, indicating that the structure of reduction-responsive MEL/AHV was easily disassembled to release drugs in tumor cells. 3.7. The cellular uptake mechanism WERI-Rb-1 cells were treated with various uptake inhibitors in order to explore the potential endocytic pathway of MEL/AHV micelles. Generally, CPZ and IDM are inhibitors of clathrinmediated endocytosis and caveolae-mediated endocytosis, respectively, while CC and GS are inhibitors of macropinocytosis and clathrin/caveolae-independent endocytosis, respectively. As shown in Fig. 7A, it could be found that the inhibitors were non-toxic against WERI-Rb-1 cells at the experimental concentrations due to the high cell viability. Then the uptake of MEL/AHV micelles in tumor cells treated with various inhibitors is shown in Fig. 7B. Compared with control group, the cell uptake of HA-based micelles decreased significantly at low temperatures (p < 0.001). This indicates that the cellular uptake of micelles is energy-dependent. The experimental results showed that the cellular uptake of the micelles after treatment with CC and CPZ was both decreased (p < 0.001), and the former was reduced more significantly. How-
Fig. 4. (A) Serum stability of and (B) in vitro release behaviors of MEL-loaded micelles (n = 3).
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Fig. 5. Cytotoxicity of blank micelles (A) and drug-loaded micelles (B) at various concentrations (n = 3). (C) Cell viabilities of MEL/AHV at 0 lg/mL (C-1), 6.25 lg/mL (C-2), 50 lg/mL (C-3) and 200 lg/mL (C-4) (scale bar: 100 lm).
Fig. 6. (A) The standard curve for determination of protein content and (B) the cellular uptake of free MEL, MEL/AHV and MEL/ADV (n = 3).
Fig. 7. (A) Cytotoxicity of the endocytosis inhibitors at the experimental concentration, and (B) effects of endocytosis inhibitors on uptake of MEL/AHV micelles (n = 3).
J. Li et al. / International Journal of Biological Macromolecules 141 (2019) 997–1003
ever, no significant decline was observed when the cells were treated with IDM and GS. These results indicate that though macropinocytosis and clathrin-mediated endocytosis both affect cellular uptake of the MEL/AHV micelle, macropinocytosis plays a major role.
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Biodegradable reduction-responsive polymeric micelles for enhanced delivery of melphalan to retinoblastoma cells Jia Li ⇑,1, Jihong Wang 1, Xuetong Zhang, Xin Xia, Chenchen Zhang Department of Ophthalmology, Affiliated Hospital of Jiangnan University, Wuxi 214062, China
a r t i c l e
i n f o
Article history: Received 18 June 2019 Received in revised form 3 September 2019 Accepted 11 September 2019 Available online 12 September 2019 Keywords: Retinoblastoma Melphalan Reduction-responsive Polymeric micelles Drug delivery
a b s t r a c t Melphalan (MEL) is an effective chemotherapeutic agent for treatment of retinoblastoma (Rb) which is the most common childhood malignancy. However, the inherent cardiopulmonary toxicity and hazardous integration limit its therapeutic effect on RB. N-Acetylheparosan (AH), a natural heparin-like polysaccharide in mammals with long circulation effect and good biocompatibility, was linked by d-a-tocopherol acid succinate (VES) via and cystamine (CYS) to synthesize reduction-responsive N-acetylheparosan-CYS-Vitamin E succinate (AHV) copolymers. In addition, CYS was replaced by adipic acid dihydrazide (ADH) to obtain a control of non-reduction-responsive polymers N-acetylheparosanADH-Vitamin E succinate (ADV). MEL-loaded AHV micelles (MEL/AHV) as well as ADV micelles (MEL/ ADV) were prepared with small particle size and high drug loading content. In vitro drug release showed that MEL/AHV micelles presented obvious reduction-triggered release behavior compared with MEL/ADV. In vitro antitumor effects were investigated using WERI-Rb-1 retinoblastoma cells. Cytotoxicity experiments showed that the IC50 of MEL/AHV was significantly lower than that of free MEL and MEL/ADV, suggesting that MEL/AHV enhanced the cytotoxicity against retinoblastoma cells. Furthermore, MEL/AHV micelles were more easily uptaken by multiple pathways compared with MEL/ADV and free MEL. Therefore, MEL/AHV might be a potential delivery system for enhanced delivery of melphalan to Rb cells. Ó 2019 Elsevier B.V. All rights reserved.
1. Introduction As the most common ocular tumor in children, retinoblastoma (Rb) has attracted widespread attention. According to statistics, one in every 15,000 to 20,000 children suffers from the disease, and most of them are children under 5 years old [1,2]. Although retinoblastoma is the first tumor that raises concerns about the genetic etiology of cancer, the mortality of retinoblastoma still reaches 70%, especially in low- and middle-income countries [3]. Usually, the occurrence of the tumor is accompanied by the loss of retinoblastoma gene 1 (RB1) [4]. Although therapy of Rb has excellent curing prospect, patients often suffer from loss of vision and second malignancies [5,6]. Nowadays, the most common method of treating Rb is the intra-arterial or intravitreal injection of chemotherapeutic drugs. But it has been reported that intraarterial injection of melphalan (MEL) can cause lung toxicity, arrhythmia and other cytotoxic side effects [7,8]. Therefore, in order to reduce the side effects and improve patient survival
⇑ Corresponding author. 1
E-mail address: [email protected] (J. Li). These authors contributed equally to this work.
https://doi.org/10.1016/j.ijbiomac.2019.09.085 0141-8130/Ó 2019 Elsevier B.V. All rights reserved.
chance, it is necessary to develop new treatment strategies for MEL. In cancer chemotherapy, some biological barriers often hinder the transport and accumulation of chemotherapeutic drugs in tumor cells. Antitumor drugs loaded in polymer micelles produce high therapeutic efficiency for solid tumors, but the cargoes need to be released from micelles into tumor cells on time [9,10]. In recent years, therefore, environmentally stimuli-responsive polymer micelles have been widely used for targeted delivery and controlled release of antitumor drugs. This method can significantly promote the internalization of micelles through receptormediated endocytosis and subsequently enhance the responsiveness of the nanocarriers in the cell for drug release [11–15]. In the past decade, in order to increase the release of drugs in tumor cells, intelligent vectors responding to different tumor microenvironments have been developed. Stimulating sensitive drug carriers can be triggered by pH, temperature, enzymes, redox potential, light, magnetic fields and ultrasound [14–20] to rapidly release the entrapped drugs. In these stimuli-responsive drug delivery systems, reduction-responsive drug carriers have been extensively studied because of the difference in redox condition between tumor cell fluid and extracellular [21–23]. It is worth noting that
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the concentration of glutathione (GSH), a thiol tripeptide capable of reducing disulfide bonds, in many tumor cells is higher than that of normal cells [24]. The difference in the GSH concentration can cause the rapid release of active substances such as drugs, genes, and proteins from intracellular reduction-responsive drug carriers [25–27]. So, reduction-responsive polymer micelles can be widely used in drug delivery depending on the large difference between extracellular fluid and intracellular environment. Polymer micelles containing a disulfide bond (-S-S-) are stable in body fluids, but when they arrive at tumor cells, the disulfide bond is cleaved into a sulfhydryl group by GSH, causing destruction of the micelle structure resulting in fast drug release. As a natural heparin-like polysaccharide in vivo, N-acetylheparosan (AH) cannot be degraded by heparanase because of the unsulfated oxygen site, but it will be degraded to normal monosaccharides in lysosomes, which make AH has good long circulation effect and biocompatibility [28]. Herein, AH and d-a-tocopherol acid succinate (VES) were linked by cystamine (CYS) to synthesize reduction-responsive N-acetylheparosan-CYSVitamin E succinate (AHV) copolymers for delivery of melphalan to retinoblastoma cells. The preparation and antitumor effect of MEL-loaded micelles are shown in Fig. 1. The properties of melphalan-loaded micelles (MEL/AHV) were investigated, and the cytotoxicity and intracellular uptake of the MEL/AHV micelles against WERI-Rb-1 retinoblastoma cells were evaluated. Furthermore, the internalization pathway of MEL/AHV micelles was further tested.
(Shanghai, China). Melphalan (MEL) was obtained from Macklin Biochemical (Shanghai, China). Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS) and RPMI-1640 medium were obtained from Gibco BRL (Maryland, USA). 4,6-Diamidino-2phenylindole (DAPI) was purchased from Shanghai Beyotime Biotechnology (Shanghai, China). Cell Counting Kit-8 (CCK-8), RIPA Lysis Buffer and Enhanced BCA Protein Assay Kit were purchased from Beyotime Biotechnology (Shanghai, China). All other reagents were of analytical grade. 2.2. Synthesis of AHV copolymers Cystamine dihydrochloride was pretreated with sodium hydroxide solution through acid-base neutralization before it’s used [30]. Firstly, N-acetylheparosan (3 104moL) was dissolved in formamide at room temperature. NHS (6 104 moL) and EDC (6 104 moL) were added to activate carboxylic groups. After being stirred for 2 h, 10 mL of cystamine (3 104 moL) solution was added dropwise and stirred for 48 h under a nitrogen atmosphere at room temperature. The mixture was purified by dialyzing (MWCO 3500) with distilled water for 48 h and then freeze-dried to obtain AH-cystamine product. Secondly, VES (3 104 moL) was dissolved in DMF and activated using the same method. Then it was added into the AH-cystamine (3 104 moL) solution and stirred under a nitrogen atmosphere at room temperature. After 48 h, the solution was dialyzed (MWCO 3500) against 25% ethanol solution and distilled water, and then freeze-dried to obtain AHV copolymers.
2. Experimental section 2.1. Materials D-a-Tocopherol acid succinate (VES), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were provided by Aladdin (Shanghai, China). Cystamine dihydrochloride (CYS) and Adipodihydrazide (ADH) were purchased from Energy Chemical Reagent (Shanghai, China). N-acetylheparosan (AH, Mw = 5.437 104) was gifted by Jiangnan university (Wuxi, China). Dithiothreitol (DTT), Dimethyl sulfoxide (DSMO), penicillin-streptomycin solution and pancreatin were provided by Shanghai Songan Biotech (Shanghai, China). Dimethylformamide (DMF), formamide, triethylamine (TEA) and tetrahydrofuran (THF) were obtained from Shanghai Reagent Chemical
2.3. Synthesis of ADV copolymers The N-acetylheparosan (1.25 104moL) was dissolved in formamide, and activated by EDC (2.5 104 moL) and NHS (2.5 104 moL). Then adipic acid dihydrazide (7.25 104 moL) was added and the pH was adjusted to 4.75 using 0.1 M hydrochloric acid. After 1 h, 0.1 M sodium hydroxide was added to the mixture solution to end the reaction. The solution was then dialyzed (MWCO 3500) against distilled water for 48 h and freeze-dried to obtain AH-ADH. Then NHS and EDC were added to the VES (1.5 104 moL) solution to activate the carboxylic groups. AH-ADH (1.5 104 moL) was dropped into the mixed solution and stirred for 48 h under a nitrogen atmosphere at room temperature. The mixed solution was dialyzed (MWCO 3500) and then freeze-dried to obtain ADV copolymers. 2.4. Preparation and characterization of MEL-loaded micelles
Fig. 1. Construction and intracellular delivery of the reduction-responsive MEL/ AHV micelles.
AH-based copolymer (10 mg) was sufficiently dissolved in PBS (pH 7.4, 10 mL). Then 2 mL MEL solution was slowly dropped into the copolymer solution and stirred overnight in dark. MEL-loaded AH-based copolymer micelles (MEL/AHV and MEL/ADV) were obtained after ultrasonic 30 min in an ice bath. The average particle size, polydispersity index (PDI) and zeta potential of the micelles were measured by Zetasizer Nano ZS apparatus (Malvern Instruments, UK) at 37 °C. The MEL-loaded micelles were dropped onto the copper grid and negatively stained with 2% phosphotungstic acid. Then, the morphology of the micelles was observed by JEM-2100 transmission electron microscope (TEM) with 80 kV of acceleration voltage (JEO, Japan). Drug loading content (DL) and encapsulation efficiency (EE) were measured by a UV/vis spectrophotometer (UV-2550, Shimadzu, Japan) at a wavelength of 301 nm. Free MEL was separated by ultrafiltration, and the micellar structure was destroyed by dimethyl sulfoxide to test the total drug content.
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2.5. Serum stability of MEL-loaded micelles The serum stability of the MEL/AHV and MEL/ADV micelles were tested by dynamic light scattering spectrophotometry (Zetasizer Nano ZS apparatus, Malvern Instruments, UK). Briefly, the MEL-loaded micelles were dispersed in pH 7.4 PBS solution mixed with DMEM solution containing 10% FBS, and the particle size were measured at different times to monitor the stability of the micelles. 2.6. In vitro reduction-triggered release The in vitro release behaviors of MEL from the micelles were measured using the dialysis method. 2 mL of MEL-loaded micelles was placed into the dialysis bag (MWCO 1000) which was immersed in 20.0 mL PBS (pH 7.4) or GSH-containing PBS (10 mM, pH 7.0) at 37 °C under shaking (100 r/min). 2 mL of release medium was sampled and the fresh PBS replaced with the same volume at different time. The amount of MEL was determined using UV/vis absorbance at 301 nm. The cumulative release of MEL as follows:
Erð%Þ ¼
P VO Cn þ Ve 1n1 Ci 100% mMEL
where Er is the cumulative release of MEL, mMEL represents the total amount of MEL in the micelles. V0 and Ve are the total volume and replacement volume of the release medium, respectively. Ci is the MEL concentration released at the ith sample. 2.7. Cell culture Human retinoblastoma cancer cells (WERI-Rb-1) was purchased from Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS), 100 mg/mL streptomycin sulfate and 100 U/mL penicillin G sodium. The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. 2.8. Cytotoxicity evaluation WERI-Rb-1 cells were seeded into 96-well plates with 5 103 cells per well to preculture for 12 h. Then a fresh medium containing various concentration of blank micelles or drug-loaded micelles was added to each well. After incubation for 24 h, 20 lL of CCK-8 solution was added and the cell viability was measured using Multiskan MK3 microplate reader (Thermo, USA).
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inhibitors and their experimental concentration were as follows: genistein (GS, 10 lgmL1) [29], chlorpromazine (CPZ, 10 lgmL1) [30], colchicine (CC, 40 lgmL1) [31] and indomethacin (IDM, 10 lgmL1) [29,32]. WERI-Rb-1 cells were seeded into 96-well plates at a density of 5 103 cells/well for incubation of 24 h. The viability of cells incubated with each inhibitor was measured according to the above described. WERI-Rb-1 cells were seeded into 6-well plates at a density of 5 105cells/well. Then, the inhibitors were added to replace the old medium, and drug-loaded micelles (MEL concentration of 5 lgmL1) were incubated for 1 h. The content of MEL was measured by UV/vis. The control group treated without inhibitors at 37 °C was set to 100%. 2.11. Statistical analysis All experiments were operated at least three times independently and the data were presented as the mean ± standard deviation (SD). The one-way ANOVA-LSD and IndependentSamples t-Test were adopted to determine the statistical significance of differences. p < 0.05, p < 0.01 and p < 0.001 were indicated statistical significance, high significance and extreme significance, respectively, while p > 0.05 was indicated no significant difference. 3. Results and discussion 3.1. Synthesis of reduction-responsive AHV copolymers The synthetic scheme and 1H NMR spectrum of AHV copolymers are presented in Fig. 2. The carboxyl groups on the VES and AH polysaccharide were attached to the amino groups of CYS, respectively. The characteristic peaks of glucosamine on AH polysaccharide were shown at 3.63 ppm, 3.83 ppm and 5.31 ppm and the typical peak of -CO-CH3 on AH polysaccharide was shown at 1.98 ppm. The typical peaks of glucuronic acid on AH polysaccharide were shown at 3.29 ppm, 3.72 ppm, and 4.54 ppm. The typical peaks of -CH2- and –CH3 on the alkanes of VES were shown at 1.05–1.35 ppm and 0.85 ppm, and -CH3 on the phenyl ring was shown at 2.63 ppm. The characteristic peaks of -CONH- were shown between 8.05 and 8.11 ppm. Based on these results, it can be concluded that AHV was successfully synthesized. 3.2. Preparation and characterization of AH-based micelles
The cellular uptake of free MEL, MEL/AHV or MEL/ADV micelles in WERI-Rb-1 cells was measured by UV–vis spectrophotometer (UV-2550, Shimadzu, Japan). The cells were seeded into 6-well plates with 5 105 cells per well for preculture 12 h, and the medium containing free MEL or drug-loaded micelles (MEL concentration of 5.0 lgmL1) was added into each well. After incubation for different times, the cells were collected by centrifugation and lysed by RIPA Lysis Buffer. The cell lysate was diluted with PBS and centrifuged. Then the content of MEL was determined by UV/vis. The content of protein in each well was determined by the Enhanced BCA Protein Assay Kit. The uptake of MEL was calculated as the amount of MEL (lg)/the amount of cells protein (mg).
The blank micelles and MEL-loaded micelles were prepared by ultrasonic method. The micelles are formed because of the amphipathic nature of AHV copolymers which contain both hydrophilic regions of AH as well as hydrophobic regions of VES. Their physicochemical properties were characterized and shown in Table 1. Although the particle size of micelles slightly increased after entrapment of MEL, all the particle sizes of the micelles were below 150 nm with a narrow size distribution (Fig. 3A). This indicates that the micelles can be well targeted to the tumor site by the EPR effect. The negative charge resulting from the electronegative AH shell of the micelles could ensure its stability in the blood. TEM showed that the micelles had a nearly spherical morphology and good dispersibility (Fig. 3B). Furthermore, the drug loading of micelles was 13–16%, and the encapsulation efficiency was above 85%, suggesting that the micelles had a good drug encapsulation capacity.
2.10. Cellular uptake mechanism studies
3.3. Serum stability of MEL-loaded micelles
The cytotoxicity of each inhibitor against WERI-Rb-1 cells at the experimental concentration was firstly tested by CCK-8 assays. The
The stability of MEL/AHV and MEL/ADV micelles was studied in pH 7.4 PBS solution mixed with DMEM solution containing 10%
2.9. Cell uptake analysis
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Fig. 2. (A) Synthetic scheme of AHV copolymers and 1H NMR spectra of (B) AH polysaccharide and (C) AHV copolymers.
Table 1 Characterization of blank micelles and drug-loaded micelles (n = 3). Formulations
Particle size (nm)
PDI
Zeta potential (mV)
EE (%)
DL (%)
AHV ADV MEL/AHV MEL/ADV
100.4 ± 6.3 94.3 ± 5.7 120.4 ± 6.3 114.3 ± 5.7
0.175 ± 0.05 0.158 ± 0.09 0.142 ± 0.03 0.173 ± 0.07
24.31 ± 3.1 23.57 ± 2.7 21.39 ± 1.9 19.56 ± 2.8
– – 86.37 ± 3.11 89.25 ± 2.32
– – 14.38 ± 1.55 15.67 ± 1.49
Fig. 3. (A) Particle size distribution and (B) TEM image of MEL/AHV micelles.
J. Li et al. / International Journal of Biological Macromolecules 141 (2019) 997–1003
FBS. The average size of the protein in FBS is 10 nm, which don’t affect the measurement of the micellar particle size. As shown in Fig. 4A, no significant change in particle size was observed in MEL/AHV as well as and MEL/ADV after incubation with FBS for 48 h. Obviously, no precipitates and visible particles were found, and there was no significant increase in the micelles particle size after incubation with FBS. The result indicates that only a small amount of proteins were adsorbed on the surface of micelles. Therefore, MEL/AHV micelles will not aggregate into larger particles by absorbing serum proteins after intravenous injection probably due to the generation of a hydrophilic shell.
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and MEL/AHV decreased from 16.63% to 3.42% as the concentration increased from 6.25 lg/mL to 200 lg/mL. In other words, the gap was gradually narrowed. Moreover, there were significant differences between MEL/AHV and MEL/ADV at concentration of 6.25–50 lg/mL (P < 0.001) and 100 lg/mL (p < 0.05), respectively, while no significant difference was seen at concentration of 200 lg/mL (p > 0.05). We speculated that this might be that though more MEL could be released from redox-sensitive MEL/AHV than MEL/ADV in tumor cells, the amount of released drug from both of the micelles with high concentration (e.g. 200 lg/mL) had reached a certain concentration to kill most of the cells, which seemed to diminish the effect of redox- sensitive releases.
3.4. In vitro drug release studies 3.6. Cellular uptake studies To study the redox-sensitive release property of the micelles, the release behaviors of MEL from MEL/AHV and MEL/ADV micelles were investigated at different concentrations of GSH. As shown in Fig. 4B, the cumulative release of MEL from MEL/AHV and MEL/ADV micelles was about 30% at pH 7.4, suggesting that the micelles maintained good stability in the normal physiological condition. However, the release of MEL from MEL/AHV micelles in 10 mM GSH was above 60%, which significantly higher than that of pH 7.4 condition and MEL/ADV micelles (p < 0.001). It is well known that disulfide bonds are unstable in reductive conditions. So, MEL/AHV micelles containing disulfide linkages are depolymerized in response to the high GSH, leading to change in the amphipathic balance and thus release of the drugs. These results indicate that the reduction-responsive MEL/ADV micelles can be depolymerized to rapidly release MEL in the tumor microenvironment. 3.5. In vitro cytotoxicity assay The cytotoxicity of free MEL, MEL/AHV and MEL/ADV was evaluated by determining cellular viability through the CCK-8 assay. At a relatively high concentration (500 lgmL1) of blank micelles, the viabilities of WERI-Rb-1 cells were still >90% (Fig. 5A), suggesting the blank micelles were non-toxic. As shown in Fig. 5B, however, MEL/AHV was toxic to WERI-Rb-1 cells in a dose-dependent manner. The cells died significantly with drug concentration increasing (Fig. 5C). The median inhibitory concentrations (IC50) of MEL/AHV, MEL/ADV, and free MEL were (10.91 ± 0.56) lgmL1, (26.01 ± 1.42) lgmL1 and (48.15 ± 1.89) lgmL1, respectively. There was a significant difference between MEL/AHV and the other groups (P < 0.001), indicating that the reductionresponsive micelles could be used as effective drug nanocarriers. However, the difference value of cell viability between MEL/ADV
The cellular uptake of free MEL, MEL/AHV and MEL/ADV micelles was evaluated by UV/vis spectrophotometry. The protein standard was tested firstly for the determination of the protein content of each group (Fig. 6A). From the results, the uptake of all the formulations increased significantly with time as shown in Fig. 6B. It was worth noting that the uptake of micelles was much higher than that of free MEL (P < 0.001), which indicated that the micelles could be easily uptaken by tumor cells. Furthermore, there was a significant difference in cellular uptake between MEL/AHV and MEL/ADV, indicating that the structure of reduction-responsive MEL/AHV was easily disassembled to release drugs in tumor cells. 3.7. The cellular uptake mechanism WERI-Rb-1 cells were treated with various uptake inhibitors in order to explore the potential endocytic pathway of MEL/AHV micelles. Generally, CPZ and IDM are inhibitors of clathrinmediated endocytosis and caveolae-mediated endocytosis, respectively, while CC and GS are inhibitors of macropinocytosis and clathrin/caveolae-independent endocytosis, respectively. As shown in Fig. 7A, it could be found that the inhibitors were non-toxic against WERI-Rb-1 cells at the experimental concentrations due to the high cell viability. Then the uptake of MEL/AHV micelles in tumor cells treated with various inhibitors is shown in Fig. 7B. Compared with control group, the cell uptake of HA-based micelles decreased significantly at low temperatures (p < 0.001). This indicates that the cellular uptake of micelles is energy-dependent. The experimental results showed that the cellular uptake of the micelles after treatment with CC and CPZ was both decreased (p < 0.001), and the former was reduced more significantly. How-
Fig. 4. (A) Serum stability of and (B) in vitro release behaviors of MEL-loaded micelles (n = 3).
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Fig. 5. Cytotoxicity of blank micelles (A) and drug-loaded micelles (B) at various concentrations (n = 3). (C) Cell viabilities of MEL/AHV at 0 lg/mL (C-1), 6.25 lg/mL (C-2), 50 lg/mL (C-3) and 200 lg/mL (C-4) (scale bar: 100 lm).
Fig. 6. (A) The standard curve for determination of protein content and (B) the cellular uptake of free MEL, MEL/AHV and MEL/ADV (n = 3).
Fig. 7. (A) Cytotoxicity of the endocytosis inhibitors at the experimental concentration, and (B) effects of endocytosis inhibitors on uptake of MEL/AHV micelles (n = 3).
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ever, no significant decline was observed when the cells were treated with IDM and GS. These results indicate that though macropinocytosis and clathrin-mediated endocytosis both affect cellular uptake of the MEL/AHV micelle, macropinocytosis plays a major role.
[15]
[16]
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