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Tumor-targeting micelles based on folic acid and α-tocopherol succinate conjugated hyaluronic acid for paclitaxel delivery
Colloids and Surfaces B: Biointerfaces 177 (2019) 11–18
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Tumor-targeting micelles based on folic acid and α-tocopherol succinate conjugated hyaluronic acid for paclitaxel delivery ⁎⁎
Xin Zhanga, Na Liangb, , Xianfeng Gonga, Yoshiaki Kawashimac, Fude Cuid, Shaoping Suna,
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a
Department of Pharmaceutical Engineering, School of Chemistry and Material Science, Heilongjiang University, Harbin 150080, China Key Laboratory of Photochemical Biomaterials and Energy Storage Materials, Heilongjiang Province, College of Chemistry & Chemical Engineering, Harbin Normal University, Harbin 150025, China c Department of Pharmaceutical Engineering, School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650, Japan d School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hyaluronic acid α-Tocopherol succinate Folic acid Micelles Paclitaxel
Tumor-targeting micelles for the delivery of paclitaxel (PTX) were developed based on folic acid and α-tocopherol succinate conjugated hyaluronic acid (FA-HA-TOS). The conjugate FA-HA-TOS was synthesized by an esterification reaction and was characterized by proton nuclear magnetic resonance (1H NMR) and Fourier transform infrared (FT-IR) analysis. The conjugate self-assembles into nanosized micelles in aqueous medium with a critical micellar concentration (CMC) of 1.12 × 10−2 mg/mL. The FA-HA-TOS micelles demonstrated high drug loading and entrapment efficiency for PTX, with respective values of 21.37% and 90.48%. The physicochemical properties of the micelles were measured by DLS, TEM and XRD. Moreover, in vitro and in vivo evaluations were performed to demonstrate the superior antitumor activity of the PTX-loaded micelles. It was suggested that the FA-HA-TOS micelle system represents a promising nanocarrier for targeted delivery of PTX.
1. Introduction The treatment of cancer is a challenging endeavor worldwide. Traditional drug delivery systems are incapable of distinguishing tumor cells from normal cells, which leads to serious toxic side effects. In recent years, targeted cancer chemotherapy based on nanocarriers has received widespread attention. Due to their specific size, the nanoparticles are able to passively accumulate within tumor sites by the enhanced permeability and retention (EPR) effect [1]. However, the nonspecific delivery and lack of selective tumor uptake continues to hamper chemotherapeutic efficacy. To further enhance the targeting effect, active targeting strategies that introduced tumor-targeting moieties to the nanoparticles have been investigated [2,3]. The active targeting nanodrug delivery system could significantly improve the concentration of therapeutic drugs within tumor sites. Recently, polymeric micelles have gained increasing attention as nanocarriers [4,5]. Their specific core-shell structure can effectively encapsulate the hydrophobic drug into the hydrophobic core by physical embedding or chemical bonding, thus enhancing the water solubility of the drug [6,7]. Furthermore, micelles offer several unique advantages such as drug accumulation in tumor sites via EPR effect,
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sustained drug release behavior, and prolonged drug circulation time by avoiding rapid renal clearance [8,9]. It is feasible to conjugate targeting ligands onto the surface of the micelles in order to develop more effective site-specific drug delivery systems [10]. Hyaluronic acid (HA) has been extensively applied in the pharmaceutical and biomedical fields due to its excellent biocompatibility, biodegradability, low toxicity and low immunogenicity [11,12]. With functional groups of carboxylic acid and hydroxyl moieties, HA could be chemically modified by crosslinking or conjugation reactions. More importantly, HA is closely related to angiogenesis in many types of tumors that overexpress HA receptors on the surface (CD44 and RHAMM). For these reasons, HA has been explored as a targeting vehicle for drug delivery [13]. To further improve the targeting efficiency toward cancer cells, a dual-targeting micelle system could be exploited. Folic acid (FA) is a targeting agent which has shown a great prospect in targeted drug delivery systems because of its strong binding with folate receptors [14,15]. The folate receptors are highly expressed on the surface of many cancer cells, but they are present in low or undetectable levels in most normal cells. The introduction of FA could improve the therapeutic efficiency and avoid injury to normal tissues. Moreover, after conjugation with drug carriers, FA still retained its high
Corresponding author at: School of Chemistry and Material Science, Heilongjiang University, No. 74, Xuefu Street, Harbin 150080, China. Corresponding author at: College of Chemistry & Chemical Engineering, Harbin Normal University, No. 1, Shida Road, Harbin 150025, China. E-mail addresses: [email protected] (N. Liang), [email protected] (S. Sun).
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https://doi.org/10.1016/j.colsurfb.2019.01.044 Received 12 October 2018; Received in revised form 17 January 2019; Accepted 19 January 2019 Available online 23 January 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Schematic diagram of the preparation procedure and tumor-targeting properties of PTX-loaded FA-HA-TOS micelles.
HA. In brief, the water-soluble HA (100 mg) was dissolved in anhydrous formamide (5 mL) and stirred for 3 h. TOS (32 mg) was dissolved in N,N-dimethylformamide (DMF) (3 mL) with the addition of EDC (13 mg) and NHS (9 mg) to activate the carboxylic acid groups. The activated TOS solution was then added in a dropwise fashion into the HA solution under vigorous stirring, and the mixture was stirred for 36 h at ambient temperature in darkness. Afterwards, the resultant mixture was purified by dialysis against an excess amount of distilled water for three days to remove the unreacted reagent. The product HATOS was isolated as a sponge by lyophilization. FA-HA-TOS was then synthesized via the ester linkage between the hydroxyl group of HA-TOS and the carboxyl group of FA. A typical synthesis procedure was as follows: FA (28 mg) was activated for 4 h under consistent stirring in the presence of EDC (11 mg) and NHS (7 mg) in DMF. The activated FA solution was added to the HA-TOS (with substitution degree of 7.5%) in DMF solution in a dropwise manner. The reaction was allowed to proceed for 36 h under magnetic stirring in the dark. The product was dialyzed against distilled water (MWCO of 7 kDa) for purification and then lyophilized to obtain a yellow powder. The formation of FA-HA-TOS was confirmed by proton nuclear magnetic resonance (1H NMR) analysis at 400 MHz (AV-400, Bruker, Switzerland) and Fourier transform infrared (FT-IR) analysis in the range of 4000–400 cm−1 (Tensor II, Bruker, Switzerland). The substitution degree of FA was determined using an ultraviolet-visible (UV–vis) spectrophotometer (UV mini-1240, Shimadzu, Japan) at 350 nm.
binding affinity to the folate receptors [16]. The α-tocopherol succinate (TOS) is a well-known vitamin E derivative composed of three domains, including the hydrophobic domain, signaling domain and functional domain. The excellent lipophilic nature makes it a good solvent for many hydrophobic drugs [17]. Grafting of TOS onto the hydrophilic materials may produce an amphiphilic polymer which could self-assemble into micelles to enhance the solubility of hydrophobic drugs. For the reasons above, a tumor-targeting micellar system based on FA and TOS conjugated HA (FA-HA-TOS) was designed for PTX delivery in this study. With respect to the hydrophilicity of HA, the introduction of hydrophobic TOS could induce the conjugate self-assembly into micelles in aqueous medium. Furthermore, PTX was encapsulated into the micelles. Taking advantage of CD44 and folate receptor-mediated targeting, the developed PTX-loaded FA-HA-TOS micelles could improve the delivery accuracy of the drug to the target sites, thus enhancing the antitumor activity of this system and reducing the toxicity to normal tissues. The main scheme of the present study is shown in Fig. 1. Herein, the synthesis of FA-HA-TOS conjugate, the fabrication and characterization of PTX-loaded FA-HA-TOS micelles as well as the in vitro and in vivo antitumor efficacy were studied in detail. 2. Materials and methods 2.1. Materials Paclitaxel (PTX) was purchased from Natural Field Biological Technology Co., Ltd., Xi’an, China. Sodium hyaluronate (Mw = 9.0 kDa) was provided by Freda Biochem Co., Ltd., Shangdong, China. α-Tocopherol succinate (TOS) was gained from Xinchang Pharmaceutical Co., Ltd., Zhejiang, China. 1-Ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were obtained from Shanghai Medpep Co., Ltd., Shanghai, China. Folic acid (FA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and pyrene (purity > 99%) were obtained from Sigma Chemical Co., St. Louis, USA. Cremophor EL was a kind gift from BASF Corp., Ludwigshafen, Germany. Dulbecco’s Modified Eagle’s medium (DMEM), penicillin-streptomycin mixture and fetal bovine serum (FBS) were gained from Gibco BRL, Carlsbad, USA. All other chemicals and solvents were of analytical or chromatographic grade and used without further purification. Distilled water was used in all experiments.
2.3. Determination of critical micelle concentration (CMC) The CMC of FA-HA-TOS conjugate was determined by fluorescence spectroscopy, as described previously, using pyrene as the hydrophobic fluorescence probe [18]. Briefly, 100 μL of pyrene/acetone solution with the concentration of 6.0 × 10−5 mol/L was added into a series of 10 mL volumetric flasks. After acetone was fully evaporated, 10 mL of FA-HA-TOS solution with different concentrations was respectively added to each volumetric flask. The final concentration of pyrene was controlled at 6.0 × 10−7 mol/L. After overnight incubation, the fluorescence property of pyrene was studied using a spectrofluorophotometer (F-2500 FL Spectrophotometer, Hitachi Ltd., Japan), with the emission spectra recorded from 300 to 450 nm under the excitation wavelength of 335 nm. From the emission spectra, the intensity ratio of the third peak to the first peak (I384/I373) was analyzed for the calculation of CMC.
2.2. Synthesis of FA-HA-TOS First, the HA-TOS conjugate was synthesized by coupling TOS with 12
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PTX-loaded micelles against the red blood cells (RBCs). First, the RBCs were prepared from rabbit whole blood by centrifugation at 3000 rpm for 10 min, with plasma and buffy coat removed. The RBCs were washed with normal saline 3 times and then diluted to a 2% suspension (v/ v). For determination, 2.5 mL of the erythrocyte suspension was added into each tube, which contained different amounts of PTX-loaded nanoparticles and Cremophor EL-based formulations, respectively. Then, 0.9% NaCl was added to obtain a final volume of 5 mL. The final PTX concentration in each tube ranged from 10 μg/mL to 200 μg/mL. All of the samples were incubated in a 37 °C water bath for 2 h, and then centrifuged at 3000 rpm for 10 min. The absorbance of the supernatant was measured by spectrophotometric determination at 540 nm (UV mini-1240, Shimadzu, Japan). Distilled water and normal saline served as the positive control and negative control, respectively. The Cremophor EL-based formulation (Taxol®) was also tested in the same way as a comparison. The hemolysis (%) was calculated using the following equation.
2.4. Preparation of PTX-loaded FA-HA-TOS micelles The PTX-loaded FA-HA-TOS micelles were prepared by a probe-type ultrasonic method, as described in previous study [19]. FA-HA-TOS conjugate was dissolved in distilled water. The PTX-acetone solution with the concentration of 0.2 mg/mL was added slowly under ultrasonication at 400 W for 3 min (pulse on 2.0 s and pulse off 2.0 s) (JY92II, Ningbo Scientz Biotechnology Co., Ltd., China). The above solution was placed in an ice bath to prevent overheating. To remove unloaded PTX, the mixture was dialyzed against an excess amount of distilled water for 4 h with a dialysis bag (MWCO of 7 kDa). Subsequently, the resultant product was lyophilized to obtain the PTX-loaded FA-HA-TOS micelle powder. The blank micelles were prepared by the same procedure without PTX added. 2.5. Characterization of PTX-loaded FA-HA-TOS micelles The particle size and zeta potential of the PTX-loaded FA-HA-TOS micelles were determined by a dynamic light scattering (DLS) method using a Malvern Zetasizer Nano-ZS90 system (Malvern Instruments, UK). The morphology of the prepared micelles was observed by transmission electron microscopy (TEM) (H-7650, Hitachi Ltd., Japan). For observation, the sample was placed on a copper grid and negatively stained with phosphotungstic acid (2%, w/v). To elucidate the existence form of PTX in the micelles, the crystallization behavior of PTX in the PTX-loaded FA-HA-TOS micelles was investigated by X-ray diffraction (XRD) analysis using an X-ray diffractometer (Geigerflex, Rigaku Co., Japan) with Cu Kα radiation in the range of 5–50° (2θ) at 30 kV and 30 mA. Samples were scanned at a scanning speed of 4°/min, and the step size was 0.02°.
Hemolysis (%) = (Asample − Anegative) / (Apositive − Anegative) × 100% (3) where Asample, Anegative and Apositive were the absorbance values of the sample, negative control and positive control, respectively. 2.8. Cell cultures MCF-7 cells (human breast cancer cells) and H22 cells (mouse hepatocellular carcinoma cells) were kindly donated by the Department of Pharmacology, Harbin Medical University. Cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin solution at 37 °C in a humidified atmosphere containing 5% CO2. 2.9. In vitro cytotoxicity
2.6. Reverse-phase HPLC analysis of PTX
The in vitro cytotoxicity of PTX-loaded FA-HA-TOS micelles was determined by MTT assay in comparison with the Cremophor EL-based formulation. Briefly, MCF-7 cells were seeded in a 96-well plate at the density of 2 × 104 cells per well. When the cell confluence reached 75%, the cells were incubated with blank micelles, free PTX, the Cremophor EL-based formulation and PTX-loaded micelles in a concentration gradient. After 24 h of incubation, 10 μL of MTT solution was added and the cells were incubated for another 4 h. At a predetermined time, the unreacted MTT was aspirated away and 100 μL of DMSO was added to dissolve the formazan crystals. The cell viability was analyzed by a BioRad microplate reader (Bio-Rad 680, Bio-Rad Laboratories, USA) at 490 nm. Untreated cells were taken as the control representing 100% viability. Cell viability was calculated as follows:
2.6.1. Measurement of PTX concentration The concentration of PTX was analyzed using a reverse-phase HPLC system, which was equipped with a mobile phase delivery pump (LC10ATVP, Shimadzu, Japan) and a UV detector (SPD-10A UV/Vis detector, Shimadzu, Japan). For separation, a Diamonsil™ C18 reversephase column (200 mm × 4.6 mm, 5 μm, Dikma Technologies Inc., China) was used. The mobile phase consisting of acetonitrile and water at the ratio of 70:30 (v/v) was freshly prepared and degassed before use. The column temperature was maintained at 30 °C, and the flow rate and detection wavelength were set at 0.8 mL/min and 227 nm, respectively. The sample solution was injected at a volume of 20 μL for HPLC analysis.
Cell viability (%) = Absorbance of cells exposed to the sample / Absorbance of untreated cells × 100% (4)
2.6.2. Determination of drug encapsulation efficiency and drug loading The PTX incorporated into FA-HA-TOS micelles was measured as follows: a certain amount of freeze-dried PTX-loaded FA-HA-TOS micelle powder was dispersed in acetonitrile and then ultrasonicated at 180 W for 5 min to destroy the structure of micelles, to extract PTX from the micelles. After filtered through a 0.45 μm microfiltration membrane, the PTX content was determined by the abovementioned HPLC method. The drug encapsulation efficiency (EE%) and drug loading (DL %) of the PTX-loaded micelles were calculated by the following equations.
2.10. In vivo antitumor efficacy Specific pathogen-free male Kunming mice weighing 20 ± 2 g were supplied by the Laboratory Animal Center of Harbin Medical University, Harbin, China. The mice were maintained at a constant temperature of 22 ± 2 °C and humidity of 50 ± 10% under a 12 h light/dark cycle with free access to murine chow and water. The animal experiments were approved by the Animal Ethics Committee of Harbin Medical University. The in vivo antitumor efficacy evaluation was performed, referring to the procedure described previously [20]. Briefly, an H22 cell suspension with 1 × 106 cells in 0.1 mL of normal saline was subcutaneously injected into the right flanks of the mice. Three days after inoculation, the mice were randomly divided into three groups (n = 6) and treated with normal saline, the Cremophor EL-based formulation (10 mg/kg) and PTX-loaded FA-HA-TOS micelles (10 mg/kg),
EE% = weight of PTX in micelles / weight of PTX fed initially × 100% (1) DL% = weight of PTX in micelles / weight of PTX-loaded micelles × 100% (2)
2.7. Hemolysis evaluation Hemolysis assay was performed to test the hemolytic activity of 13
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Fig. 2. Scheme of the synthesis of FA-HA-TOS.
respectively. The samples were injected via the tail vein once every three days, for a total of four injections. After 12 days of treatment, the mice were sacrificed and the tumors were excised. The antitumor efficacy was evaluated by tumor inhibition rate (TIR), which was calculated from the following equation.
performed using the Student’s t-test with p < 0.05 as the significance level.
TIR (%) = (Tumor weight of the model group − Tumor weight of the treated group) / Tumor weight of the model group × 100% (5)
3.1. Preparation and characterization of FA-HA-TOS
3. Results and discussion
The synthetic scheme of FA-HA-TOS is shown in Fig. 2. Briefly, this amphiphilic conjugate was prepared by a two-step reaction. First, TOS was covalently linked to the backbones of HA to form HA-TOS via the ester bond formation between the eCOOH of TOS and the eOH of HA. FA was then introduced to prepare FA-HA-TOS through the esterification reaction between the eCOOH of FA and the eOH of HA. Both
2.11. Statistical analysis Each experiment was performed in triplicate. Values were expressed as the mean ± standard deviation (SD). Statistical data analysis was 14
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Fig. 3. Characterization of FA-HA-TOS. (A) 1H NMR spectra of (a) HA, (b) HA-TOS and (c) FA-HA-TOS. (B) FT-IR spectra of (a) HA, (b) HA-TOS and (c) FA-HA-TOS.
0.83–1.21 ppm in the spectrum of HA-TOS were attributed to the methyl and methylene hydrogens (eCH3 and eCH2−) of TOS. Furthermore, the characteristic signal due to the methyl hydrogen on the benzene ring of TOS was detected at 2.8 ppm. These signals demonstrated the successful conjugation of TOS to HA. In the spectrum of FAHA-TOS, signals due to the protons on the benzene ring and heterocyclic ring of FA were observed at 6.65, 7.55 and 8.65 ppm, respectively. These results confirmed the successful synthesis of FA-HA-TOS.
reactions were performed in the presence of EDC and NHS, which were used to active the eCOOH of TOS and FA. Throughout the synthesis process, the carboxyl groups of HA did not participate in any chemical reaction, which was beneficial to retain the tumor-targeting property of HA. It was reported that the carboxyl groups in HA played a critical role in CD44-mediated targeting [21]. 1 H NMR spectra were used to confirm the conjugate formation. As shown in Fig. 3A, when compared with HA, the newly emerged peaks at 15
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3.3. Preparation of PTX-loaded FA-HA-TOS micelles
The grafting of TOS and FA to HA was further confirmed by FT-IR analysis. As illustrated in Fig. 3B, the absorption band at 3374 cm−1 was attributed to the eOH of HA. Peaks at 1617 and 1417 cm−1 were assigned to the asymmetric stretching vibration and symmetric stretching vibration of eCOOH in HA, respectively. Compared with HA, HA-TOS exhibited new peaks at 2936 and 2867 cm−1, which were assigned to the stretching vibrations of eCH3 and eCH2− of TOS, respectively. The peak at 1754 cm−1 was attributed to the C]O stretching vibration of the ester, and the peak at 1658 cm−1 was ascribed to the CeO stretching vibration of the carboxyl. For FA-HA-TOS, the absorption peak at 1770 cm−1 corresponded to the C]O stretching vibration of the newly formed ester, and the band at 1710 cm−1 was attributed to the C]O stretching vibration of the α-carboxylic acid group of FA. Furthermore, the intensity decrease of eOH in curve b and curve c was due to the binding of carboxyl groups of FA and TOS to the hydroxyl groups of HA. These differences indicated the formation of FAHA-TOS. The degree of substitution (DS) of HA was defined as the number of grafted reagent molecules per 100 sugar units of HA. To calculate the number of TOS molecules grafted onto HA, the integration unit ratio of the NMR peak attributed to the methyl group of TOS (0.83 ppm [12H, eCH3]) to the characteristic peak of the N-acetyl group in HA (1.9 ppm [3H, eCOCH3−]) was determined, and the DS of TOS was calculated as 7.5%. The DS of FA was determined by an ultraviolet-visible spectrophotometer, with the absorbance of FA recorded at 350 nm. The amount of grafted FA was calculated from a calibration curve, which was constructed using a series of solutions with increasing amounts of FA. The DS of FA was calculated as 11.3%.
The FA-HA-TOS conjugate included the hydrophobic segments of TOS and hydrophilic segments of HA. Due to the amphiphilic nature, the conjugate could self-assemble to form thermodynamically stable micelles in aqueous medium. For PTX-loaded micelles, the PTX was encapsulated in the hydrophobic core of micelles, and the HA segments served as the hydrophilic shell to stabilize the micelles. For the optimized PTX-loaded FA-HA-TOS micelles, the drug encapsulation efficiency reached up to 90.48%, and the drug loading was as high as 21.37%. 3.4. Characterization of PTX-loaded FA-HA-TOS micelles 3.4.1. Particle size and zeta potential The diameters of the blank and drug-loaded micelles were measured by DLS method. The mean sizes of the bare and PTX-loaded FA-HA-TOS micelles were 96 nm and 135 nm, respectively. It is commonly known that nanoparticles with sizes below 200 nm can preferentially accumulate within tumor sites via the EPR effect. The zeta potential values of the blank and drug-loaded micelles were measured as -22.7 mV and −33.41 mV. It was obvious that the negative zeta potential was due to the presence of the carboxylic acid groups of HA. It was reported that positively charged nanoparticles were easily cleared by the RES and presented a strong interaction with serum components, which resulted in severe aggregation and short blood circulation half-life [22,23]. In contrast, the negatively charged micelles solved these problems to a certain degree. Compared with the blank micelles, PTX-loaded micelles exhibited a much lower zeta potential, which was probably due to the introduction of the drug.
3.2. CMC determination
3.4.2. TEM observation TEM was used to directly visualize the size and morphology of the PTX-loaded micelles. As shown in Fig. 5A, the micelles exhibited near spherical shape, and the particle size revealed by TEM was smaller than that measured by the DLS method, which could be explained by the difference between the dried state and the hydrated state of the particles.
CMC was used to investigate the stability of the micelles. The CMC of FA-HA-TOS was measured by a pyrene fluorescence probe method. At low copolymer concentrations, the fluorescence intensity remained constant. When the concentration increased to the CMC, micelles formed and pyrene was incorporated into the micelles, which lead to the increase of fluorescence intensity. Furthermore, the intensity of the third peak increased more dramatically than that of the first peak. Therefore, the ratio increased abruptly. The plot of the intensity ratio of I384/I374 against the logarithm of polymer concentration is shown in Fig. 4. The CMC could be determined from the intersection of two straight lines. The CMC of FA-HA-TOS was approximately 1.12 × 10−2 mg/mL. The low value suggested the stability of FA-HA-TOS micelles in the dilute condition.
3.4.3. XRD analysis To describe the existence state of PTX in the drug-loaded micelles, XRD analysis was performed on PTX, blank micelles, PTX-loaded micelles and the physical mixture of PTX and blank micelles. As shown in Fig. 5B, PTX showed intense peaks at 2θ of 5.53°, 8.87°, 11.06° and 12.42°, and there were numerous small peaks in the range of 13°–30°. The typical crystal peaks of PTX were still observed in the pattern of the physical mixture of PTX and blank micelles, albeit with weakened intensity, but not in that of the PTX-loaded micelles. The PTX-loaded micelles exhibited a similar spectrum with blank micelles. It was implied that PTX was either molecularly dispersed in the polymer or distributed in the micelles in an amorphous state. 3.5. Hemolysis evaluation Blood compatibility is an important factor for drug delivery systems that are designed for intravenous administration. In general, the system should have minimized nonspecific interactions with RBC to ensure the safety of injection. It was observed that the PTX-loaded micelles exhibited no hemolytic phenomenon within the concentration range studied and that the hemolysis was no more than 5%. This could be explained by the excellent biocompatibility of HA as a polymer for drug delivery. Moreover, HA possessed negative charge due to the carboxyl group, and it demonstrated negligible interaction with the same charged cell membrane of RBC. As a comparison, the Cremophor ELbased formulation induced hemolysis of up to 11%, which could be attributed to the use of ethanol and Cremophor EL in the formulation. It
Fig. 4. Plot of the fluorescence intensity ratio of I3/I1 from pyrene excitation spectra against the logarithm of FA-HA-TOS concentration. 16
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Fig. 5. Characterization of PTX-loaded FA-HA-TOS micelles. (A) TEM image of PTX-loaded FA-HA-TOS micelles. (B) XRD patterns of (a) blank micelles, (b) PTXloaded micelles, (c) physical mixture of PTX and blank micelles and (d) PTX.
dual-targeting property of the system. For MCF-7 cells, which overexpressed CD44 receptors and folate receptors on the cell membrane [24,25], it was speculated that more PTX-loaded FA-HA-TOS micelles were internalized into MCF-7 cells via receptor-mediated endocytosis.
was speculated that the PTX-loaded FA-HA-TOS micelles might be safe for intravenous injection.
3.6. In vitro cytotoxicity study 3.7. In vivo antitumor efficacy study
For antitumor drug delivery systems, the in vitro cytotoxicity was a significant predictor of the in vivo antitumor activity of the system. As shown in Fig. 6A, the bare FA-HA-TOS micelles exhibited negligible cytotoxicity, which indicated the nontoxic nature and desirable safety of FA-HA-TOS micelles. Excitingly, the PTX-loaded FA-HA-TOS micelles exhibited slightly higher cytotoxicity than the Cremophor EL-based formulation and free PTX in DMSO. This might be attributed to the
To further evaluate the antitumor activity of the dual-targeting PTXloaded FA-HA-TOS micelles, H22 tumor-bearing Kunming mouse models were established [26]. As shown in Fig. 6B, the tumor sizes were much smaller in the treatment groups than those in the model group, with TIR of 63.2% and 51.5% for the micelle group and the Cremophor
Fig. 6. Antitumor efficacy of PTX-loaded FA-HA-TOS micelles. (A) In vitro cytotoxicity of PTX-loaded FA-HA-TOS micelles against MCF-7 cells. (B) In vivo antitumor efficacy of PTX-loaded FA-HA-TOS micelles. 17
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EL-based formulation group, respectively. The superior antitumor efficacy of PTX-loaded FA-HA-TOS micelles might be explained by the following facts: the PTX-loaded FA-HA-TOS micelles exhibited significant negative charge, which could enhance the stability of the system and avoid the rapid clearance by RES, to increase the blood circulation half-life. Furthermore, the mean particle size in the range of 100–200 nm could lead to the accumulation of the micelles in the tumor sites via the EPR effect. Most important of all, the dual targeting property of the micelles from HA and FA could significantly increase the internalization into tumor cells via CD44 receptor and folate receptor-mediated active targeting. Based on the above evidence, the PTX-loaded FA-HA-TOS micelles represent a promising carrier system for tumor therapy.
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4. Conclusions In this study, a tumor-targeting micellar system for anticancer drug delivery was developed based on FA-HA-TOS conjugates. The FA-HATOS was synthesized by linking TOS and FA to the backbones of HA via ester bonds. With an amphiphilic structure, FA-HA-TOS could self-assemble into micelles in aqueous milieu and solubilize PTX within the hydrophobic core. The PTX-loaded micelles exerted excellent anticancer effects against MCF-7 cells in vitro and against H22 cells in vivo due to enhanced active targeting via the combined use of HA and FA. The tumor-targeting FA-HA-TOS micelle system represents a promising nanocarrier for anticancer drug delivery. Disclosures The authors report no conflicts of interest in this work. Acknowledgments This work was funded by the National Natural Science Foundation of China (No. 51403057), Heilongjiang Natural Science Foundation (No. E2018052), the Research and Development Project of Scientific and Technological Achievements for Colleges and Universities of Heilongjiang Province (No. TSTAU-R2018023), Harbin Science and Technology Innovation Talents Special Fund Project (No. 2016RQQXJ097, No. 2016RQQXJ131), and the Doctoral Scientific Research Startup Foundation of Harbin Normal University (No. XKB201304). References [1] H. Maeda, H. Nakamura, J. Fang, The EPR effect for macromolecular drug delivery to solid tumors: improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo, Adv. Drug Deliv. Rev. 65 (2013) 71–79. [2] K. Seidi, H.A. Neubauer, R. Moriggl, R. Jahanban-Esfahlan, T. Javaheri, Tumor target amplification: implications for nano drug delivery systems, J. Control. Release 275 (2018) 142–161. [3] K. Zhang, P. Li, Y. He, X. Bo, X. Li, D. Li, H. Chen, H. Xu, Synergistic retention strategy of RGD active targeting and radiofrequency-enhanced permeability for intensified RF & chemotherapy synergistic tumor treatment, Biomaterials 99 (2016) 34–46.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Tumor-targeting micelles based on folic acid and α-tocopherol succinate conjugated hyaluronic acid for paclitaxel delivery ⁎⁎
Xin Zhanga, Na Liangb, , Xianfeng Gonga, Yoshiaki Kawashimac, Fude Cuid, Shaoping Suna,
T
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a
Department of Pharmaceutical Engineering, School of Chemistry and Material Science, Heilongjiang University, Harbin 150080, China Key Laboratory of Photochemical Biomaterials and Energy Storage Materials, Heilongjiang Province, College of Chemistry & Chemical Engineering, Harbin Normal University, Harbin 150025, China c Department of Pharmaceutical Engineering, School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650, Japan d School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hyaluronic acid α-Tocopherol succinate Folic acid Micelles Paclitaxel
Tumor-targeting micelles for the delivery of paclitaxel (PTX) were developed based on folic acid and α-tocopherol succinate conjugated hyaluronic acid (FA-HA-TOS). The conjugate FA-HA-TOS was synthesized by an esterification reaction and was characterized by proton nuclear magnetic resonance (1H NMR) and Fourier transform infrared (FT-IR) analysis. The conjugate self-assembles into nanosized micelles in aqueous medium with a critical micellar concentration (CMC) of 1.12 × 10−2 mg/mL. The FA-HA-TOS micelles demonstrated high drug loading and entrapment efficiency for PTX, with respective values of 21.37% and 90.48%. The physicochemical properties of the micelles were measured by DLS, TEM and XRD. Moreover, in vitro and in vivo evaluations were performed to demonstrate the superior antitumor activity of the PTX-loaded micelles. It was suggested that the FA-HA-TOS micelle system represents a promising nanocarrier for targeted delivery of PTX.
1. Introduction The treatment of cancer is a challenging endeavor worldwide. Traditional drug delivery systems are incapable of distinguishing tumor cells from normal cells, which leads to serious toxic side effects. In recent years, targeted cancer chemotherapy based on nanocarriers has received widespread attention. Due to their specific size, the nanoparticles are able to passively accumulate within tumor sites by the enhanced permeability and retention (EPR) effect [1]. However, the nonspecific delivery and lack of selective tumor uptake continues to hamper chemotherapeutic efficacy. To further enhance the targeting effect, active targeting strategies that introduced tumor-targeting moieties to the nanoparticles have been investigated [2,3]. The active targeting nanodrug delivery system could significantly improve the concentration of therapeutic drugs within tumor sites. Recently, polymeric micelles have gained increasing attention as nanocarriers [4,5]. Their specific core-shell structure can effectively encapsulate the hydrophobic drug into the hydrophobic core by physical embedding or chemical bonding, thus enhancing the water solubility of the drug [6,7]. Furthermore, micelles offer several unique advantages such as drug accumulation in tumor sites via EPR effect,
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sustained drug release behavior, and prolonged drug circulation time by avoiding rapid renal clearance [8,9]. It is feasible to conjugate targeting ligands onto the surface of the micelles in order to develop more effective site-specific drug delivery systems [10]. Hyaluronic acid (HA) has been extensively applied in the pharmaceutical and biomedical fields due to its excellent biocompatibility, biodegradability, low toxicity and low immunogenicity [11,12]. With functional groups of carboxylic acid and hydroxyl moieties, HA could be chemically modified by crosslinking or conjugation reactions. More importantly, HA is closely related to angiogenesis in many types of tumors that overexpress HA receptors on the surface (CD44 and RHAMM). For these reasons, HA has been explored as a targeting vehicle for drug delivery [13]. To further improve the targeting efficiency toward cancer cells, a dual-targeting micelle system could be exploited. Folic acid (FA) is a targeting agent which has shown a great prospect in targeted drug delivery systems because of its strong binding with folate receptors [14,15]. The folate receptors are highly expressed on the surface of many cancer cells, but they are present in low or undetectable levels in most normal cells. The introduction of FA could improve the therapeutic efficiency and avoid injury to normal tissues. Moreover, after conjugation with drug carriers, FA still retained its high
Corresponding author at: School of Chemistry and Material Science, Heilongjiang University, No. 74, Xuefu Street, Harbin 150080, China. Corresponding author at: College of Chemistry & Chemical Engineering, Harbin Normal University, No. 1, Shida Road, Harbin 150025, China. E-mail addresses: [email protected] (N. Liang), [email protected] (S. Sun).
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https://doi.org/10.1016/j.colsurfb.2019.01.044 Received 12 October 2018; Received in revised form 17 January 2019; Accepted 19 January 2019 Available online 23 January 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Schematic diagram of the preparation procedure and tumor-targeting properties of PTX-loaded FA-HA-TOS micelles.
HA. In brief, the water-soluble HA (100 mg) was dissolved in anhydrous formamide (5 mL) and stirred for 3 h. TOS (32 mg) was dissolved in N,N-dimethylformamide (DMF) (3 mL) with the addition of EDC (13 mg) and NHS (9 mg) to activate the carboxylic acid groups. The activated TOS solution was then added in a dropwise fashion into the HA solution under vigorous stirring, and the mixture was stirred for 36 h at ambient temperature in darkness. Afterwards, the resultant mixture was purified by dialysis against an excess amount of distilled water for three days to remove the unreacted reagent. The product HATOS was isolated as a sponge by lyophilization. FA-HA-TOS was then synthesized via the ester linkage between the hydroxyl group of HA-TOS and the carboxyl group of FA. A typical synthesis procedure was as follows: FA (28 mg) was activated for 4 h under consistent stirring in the presence of EDC (11 mg) and NHS (7 mg) in DMF. The activated FA solution was added to the HA-TOS (with substitution degree of 7.5%) in DMF solution in a dropwise manner. The reaction was allowed to proceed for 36 h under magnetic stirring in the dark. The product was dialyzed against distilled water (MWCO of 7 kDa) for purification and then lyophilized to obtain a yellow powder. The formation of FA-HA-TOS was confirmed by proton nuclear magnetic resonance (1H NMR) analysis at 400 MHz (AV-400, Bruker, Switzerland) and Fourier transform infrared (FT-IR) analysis in the range of 4000–400 cm−1 (Tensor II, Bruker, Switzerland). The substitution degree of FA was determined using an ultraviolet-visible (UV–vis) spectrophotometer (UV mini-1240, Shimadzu, Japan) at 350 nm.
binding affinity to the folate receptors [16]. The α-tocopherol succinate (TOS) is a well-known vitamin E derivative composed of three domains, including the hydrophobic domain, signaling domain and functional domain. The excellent lipophilic nature makes it a good solvent for many hydrophobic drugs [17]. Grafting of TOS onto the hydrophilic materials may produce an amphiphilic polymer which could self-assemble into micelles to enhance the solubility of hydrophobic drugs. For the reasons above, a tumor-targeting micellar system based on FA and TOS conjugated HA (FA-HA-TOS) was designed for PTX delivery in this study. With respect to the hydrophilicity of HA, the introduction of hydrophobic TOS could induce the conjugate self-assembly into micelles in aqueous medium. Furthermore, PTX was encapsulated into the micelles. Taking advantage of CD44 and folate receptor-mediated targeting, the developed PTX-loaded FA-HA-TOS micelles could improve the delivery accuracy of the drug to the target sites, thus enhancing the antitumor activity of this system and reducing the toxicity to normal tissues. The main scheme of the present study is shown in Fig. 1. Herein, the synthesis of FA-HA-TOS conjugate, the fabrication and characterization of PTX-loaded FA-HA-TOS micelles as well as the in vitro and in vivo antitumor efficacy were studied in detail. 2. Materials and methods 2.1. Materials Paclitaxel (PTX) was purchased from Natural Field Biological Technology Co., Ltd., Xi’an, China. Sodium hyaluronate (Mw = 9.0 kDa) was provided by Freda Biochem Co., Ltd., Shangdong, China. α-Tocopherol succinate (TOS) was gained from Xinchang Pharmaceutical Co., Ltd., Zhejiang, China. 1-Ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were obtained from Shanghai Medpep Co., Ltd., Shanghai, China. Folic acid (FA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and pyrene (purity > 99%) were obtained from Sigma Chemical Co., St. Louis, USA. Cremophor EL was a kind gift from BASF Corp., Ludwigshafen, Germany. Dulbecco’s Modified Eagle’s medium (DMEM), penicillin-streptomycin mixture and fetal bovine serum (FBS) were gained from Gibco BRL, Carlsbad, USA. All other chemicals and solvents were of analytical or chromatographic grade and used without further purification. Distilled water was used in all experiments.
2.3. Determination of critical micelle concentration (CMC) The CMC of FA-HA-TOS conjugate was determined by fluorescence spectroscopy, as described previously, using pyrene as the hydrophobic fluorescence probe [18]. Briefly, 100 μL of pyrene/acetone solution with the concentration of 6.0 × 10−5 mol/L was added into a series of 10 mL volumetric flasks. After acetone was fully evaporated, 10 mL of FA-HA-TOS solution with different concentrations was respectively added to each volumetric flask. The final concentration of pyrene was controlled at 6.0 × 10−7 mol/L. After overnight incubation, the fluorescence property of pyrene was studied using a spectrofluorophotometer (F-2500 FL Spectrophotometer, Hitachi Ltd., Japan), with the emission spectra recorded from 300 to 450 nm under the excitation wavelength of 335 nm. From the emission spectra, the intensity ratio of the third peak to the first peak (I384/I373) was analyzed for the calculation of CMC.
2.2. Synthesis of FA-HA-TOS First, the HA-TOS conjugate was synthesized by coupling TOS with 12
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PTX-loaded micelles against the red blood cells (RBCs). First, the RBCs were prepared from rabbit whole blood by centrifugation at 3000 rpm for 10 min, with plasma and buffy coat removed. The RBCs were washed with normal saline 3 times and then diluted to a 2% suspension (v/ v). For determination, 2.5 mL of the erythrocyte suspension was added into each tube, which contained different amounts of PTX-loaded nanoparticles and Cremophor EL-based formulations, respectively. Then, 0.9% NaCl was added to obtain a final volume of 5 mL. The final PTX concentration in each tube ranged from 10 μg/mL to 200 μg/mL. All of the samples were incubated in a 37 °C water bath for 2 h, and then centrifuged at 3000 rpm for 10 min. The absorbance of the supernatant was measured by spectrophotometric determination at 540 nm (UV mini-1240, Shimadzu, Japan). Distilled water and normal saline served as the positive control and negative control, respectively. The Cremophor EL-based formulation (Taxol®) was also tested in the same way as a comparison. The hemolysis (%) was calculated using the following equation.
2.4. Preparation of PTX-loaded FA-HA-TOS micelles The PTX-loaded FA-HA-TOS micelles were prepared by a probe-type ultrasonic method, as described in previous study [19]. FA-HA-TOS conjugate was dissolved in distilled water. The PTX-acetone solution with the concentration of 0.2 mg/mL was added slowly under ultrasonication at 400 W for 3 min (pulse on 2.0 s and pulse off 2.0 s) (JY92II, Ningbo Scientz Biotechnology Co., Ltd., China). The above solution was placed in an ice bath to prevent overheating. To remove unloaded PTX, the mixture was dialyzed against an excess amount of distilled water for 4 h with a dialysis bag (MWCO of 7 kDa). Subsequently, the resultant product was lyophilized to obtain the PTX-loaded FA-HA-TOS micelle powder. The blank micelles were prepared by the same procedure without PTX added. 2.5. Characterization of PTX-loaded FA-HA-TOS micelles The particle size and zeta potential of the PTX-loaded FA-HA-TOS micelles were determined by a dynamic light scattering (DLS) method using a Malvern Zetasizer Nano-ZS90 system (Malvern Instruments, UK). The morphology of the prepared micelles was observed by transmission electron microscopy (TEM) (H-7650, Hitachi Ltd., Japan). For observation, the sample was placed on a copper grid and negatively stained with phosphotungstic acid (2%, w/v). To elucidate the existence form of PTX in the micelles, the crystallization behavior of PTX in the PTX-loaded FA-HA-TOS micelles was investigated by X-ray diffraction (XRD) analysis using an X-ray diffractometer (Geigerflex, Rigaku Co., Japan) with Cu Kα radiation in the range of 5–50° (2θ) at 30 kV and 30 mA. Samples were scanned at a scanning speed of 4°/min, and the step size was 0.02°.
Hemolysis (%) = (Asample − Anegative) / (Apositive − Anegative) × 100% (3) where Asample, Anegative and Apositive were the absorbance values of the sample, negative control and positive control, respectively. 2.8. Cell cultures MCF-7 cells (human breast cancer cells) and H22 cells (mouse hepatocellular carcinoma cells) were kindly donated by the Department of Pharmacology, Harbin Medical University. Cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin solution at 37 °C in a humidified atmosphere containing 5% CO2. 2.9. In vitro cytotoxicity
2.6. Reverse-phase HPLC analysis of PTX
The in vitro cytotoxicity of PTX-loaded FA-HA-TOS micelles was determined by MTT assay in comparison with the Cremophor EL-based formulation. Briefly, MCF-7 cells were seeded in a 96-well plate at the density of 2 × 104 cells per well. When the cell confluence reached 75%, the cells were incubated with blank micelles, free PTX, the Cremophor EL-based formulation and PTX-loaded micelles in a concentration gradient. After 24 h of incubation, 10 μL of MTT solution was added and the cells were incubated for another 4 h. At a predetermined time, the unreacted MTT was aspirated away and 100 μL of DMSO was added to dissolve the formazan crystals. The cell viability was analyzed by a BioRad microplate reader (Bio-Rad 680, Bio-Rad Laboratories, USA) at 490 nm. Untreated cells were taken as the control representing 100% viability. Cell viability was calculated as follows:
2.6.1. Measurement of PTX concentration The concentration of PTX was analyzed using a reverse-phase HPLC system, which was equipped with a mobile phase delivery pump (LC10ATVP, Shimadzu, Japan) and a UV detector (SPD-10A UV/Vis detector, Shimadzu, Japan). For separation, a Diamonsil™ C18 reversephase column (200 mm × 4.6 mm, 5 μm, Dikma Technologies Inc., China) was used. The mobile phase consisting of acetonitrile and water at the ratio of 70:30 (v/v) was freshly prepared and degassed before use. The column temperature was maintained at 30 °C, and the flow rate and detection wavelength were set at 0.8 mL/min and 227 nm, respectively. The sample solution was injected at a volume of 20 μL for HPLC analysis.
Cell viability (%) = Absorbance of cells exposed to the sample / Absorbance of untreated cells × 100% (4)
2.6.2. Determination of drug encapsulation efficiency and drug loading The PTX incorporated into FA-HA-TOS micelles was measured as follows: a certain amount of freeze-dried PTX-loaded FA-HA-TOS micelle powder was dispersed in acetonitrile and then ultrasonicated at 180 W for 5 min to destroy the structure of micelles, to extract PTX from the micelles. After filtered through a 0.45 μm microfiltration membrane, the PTX content was determined by the abovementioned HPLC method. The drug encapsulation efficiency (EE%) and drug loading (DL %) of the PTX-loaded micelles were calculated by the following equations.
2.10. In vivo antitumor efficacy Specific pathogen-free male Kunming mice weighing 20 ± 2 g were supplied by the Laboratory Animal Center of Harbin Medical University, Harbin, China. The mice were maintained at a constant temperature of 22 ± 2 °C and humidity of 50 ± 10% under a 12 h light/dark cycle with free access to murine chow and water. The animal experiments were approved by the Animal Ethics Committee of Harbin Medical University. The in vivo antitumor efficacy evaluation was performed, referring to the procedure described previously [20]. Briefly, an H22 cell suspension with 1 × 106 cells in 0.1 mL of normal saline was subcutaneously injected into the right flanks of the mice. Three days after inoculation, the mice were randomly divided into three groups (n = 6) and treated with normal saline, the Cremophor EL-based formulation (10 mg/kg) and PTX-loaded FA-HA-TOS micelles (10 mg/kg),
EE% = weight of PTX in micelles / weight of PTX fed initially × 100% (1) DL% = weight of PTX in micelles / weight of PTX-loaded micelles × 100% (2)
2.7. Hemolysis evaluation Hemolysis assay was performed to test the hemolytic activity of 13
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Fig. 2. Scheme of the synthesis of FA-HA-TOS.
respectively. The samples were injected via the tail vein once every three days, for a total of four injections. After 12 days of treatment, the mice were sacrificed and the tumors were excised. The antitumor efficacy was evaluated by tumor inhibition rate (TIR), which was calculated from the following equation.
performed using the Student’s t-test with p < 0.05 as the significance level.
TIR (%) = (Tumor weight of the model group − Tumor weight of the treated group) / Tumor weight of the model group × 100% (5)
3.1. Preparation and characterization of FA-HA-TOS
3. Results and discussion
The synthetic scheme of FA-HA-TOS is shown in Fig. 2. Briefly, this amphiphilic conjugate was prepared by a two-step reaction. First, TOS was covalently linked to the backbones of HA to form HA-TOS via the ester bond formation between the eCOOH of TOS and the eOH of HA. FA was then introduced to prepare FA-HA-TOS through the esterification reaction between the eCOOH of FA and the eOH of HA. Both
2.11. Statistical analysis Each experiment was performed in triplicate. Values were expressed as the mean ± standard deviation (SD). Statistical data analysis was 14
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Fig. 3. Characterization of FA-HA-TOS. (A) 1H NMR spectra of (a) HA, (b) HA-TOS and (c) FA-HA-TOS. (B) FT-IR spectra of (a) HA, (b) HA-TOS and (c) FA-HA-TOS.
0.83–1.21 ppm in the spectrum of HA-TOS were attributed to the methyl and methylene hydrogens (eCH3 and eCH2−) of TOS. Furthermore, the characteristic signal due to the methyl hydrogen on the benzene ring of TOS was detected at 2.8 ppm. These signals demonstrated the successful conjugation of TOS to HA. In the spectrum of FAHA-TOS, signals due to the protons on the benzene ring and heterocyclic ring of FA were observed at 6.65, 7.55 and 8.65 ppm, respectively. These results confirmed the successful synthesis of FA-HA-TOS.
reactions were performed in the presence of EDC and NHS, which were used to active the eCOOH of TOS and FA. Throughout the synthesis process, the carboxyl groups of HA did not participate in any chemical reaction, which was beneficial to retain the tumor-targeting property of HA. It was reported that the carboxyl groups in HA played a critical role in CD44-mediated targeting [21]. 1 H NMR spectra were used to confirm the conjugate formation. As shown in Fig. 3A, when compared with HA, the newly emerged peaks at 15
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3.3. Preparation of PTX-loaded FA-HA-TOS micelles
The grafting of TOS and FA to HA was further confirmed by FT-IR analysis. As illustrated in Fig. 3B, the absorption band at 3374 cm−1 was attributed to the eOH of HA. Peaks at 1617 and 1417 cm−1 were assigned to the asymmetric stretching vibration and symmetric stretching vibration of eCOOH in HA, respectively. Compared with HA, HA-TOS exhibited new peaks at 2936 and 2867 cm−1, which were assigned to the stretching vibrations of eCH3 and eCH2− of TOS, respectively. The peak at 1754 cm−1 was attributed to the C]O stretching vibration of the ester, and the peak at 1658 cm−1 was ascribed to the CeO stretching vibration of the carboxyl. For FA-HA-TOS, the absorption peak at 1770 cm−1 corresponded to the C]O stretching vibration of the newly formed ester, and the band at 1710 cm−1 was attributed to the C]O stretching vibration of the α-carboxylic acid group of FA. Furthermore, the intensity decrease of eOH in curve b and curve c was due to the binding of carboxyl groups of FA and TOS to the hydroxyl groups of HA. These differences indicated the formation of FAHA-TOS. The degree of substitution (DS) of HA was defined as the number of grafted reagent molecules per 100 sugar units of HA. To calculate the number of TOS molecules grafted onto HA, the integration unit ratio of the NMR peak attributed to the methyl group of TOS (0.83 ppm [12H, eCH3]) to the characteristic peak of the N-acetyl group in HA (1.9 ppm [3H, eCOCH3−]) was determined, and the DS of TOS was calculated as 7.5%. The DS of FA was determined by an ultraviolet-visible spectrophotometer, with the absorbance of FA recorded at 350 nm. The amount of grafted FA was calculated from a calibration curve, which was constructed using a series of solutions with increasing amounts of FA. The DS of FA was calculated as 11.3%.
The FA-HA-TOS conjugate included the hydrophobic segments of TOS and hydrophilic segments of HA. Due to the amphiphilic nature, the conjugate could self-assemble to form thermodynamically stable micelles in aqueous medium. For PTX-loaded micelles, the PTX was encapsulated in the hydrophobic core of micelles, and the HA segments served as the hydrophilic shell to stabilize the micelles. For the optimized PTX-loaded FA-HA-TOS micelles, the drug encapsulation efficiency reached up to 90.48%, and the drug loading was as high as 21.37%. 3.4. Characterization of PTX-loaded FA-HA-TOS micelles 3.4.1. Particle size and zeta potential The diameters of the blank and drug-loaded micelles were measured by DLS method. The mean sizes of the bare and PTX-loaded FA-HA-TOS micelles were 96 nm and 135 nm, respectively. It is commonly known that nanoparticles with sizes below 200 nm can preferentially accumulate within tumor sites via the EPR effect. The zeta potential values of the blank and drug-loaded micelles were measured as -22.7 mV and −33.41 mV. It was obvious that the negative zeta potential was due to the presence of the carboxylic acid groups of HA. It was reported that positively charged nanoparticles were easily cleared by the RES and presented a strong interaction with serum components, which resulted in severe aggregation and short blood circulation half-life [22,23]. In contrast, the negatively charged micelles solved these problems to a certain degree. Compared with the blank micelles, PTX-loaded micelles exhibited a much lower zeta potential, which was probably due to the introduction of the drug.
3.2. CMC determination
3.4.2. TEM observation TEM was used to directly visualize the size and morphology of the PTX-loaded micelles. As shown in Fig. 5A, the micelles exhibited near spherical shape, and the particle size revealed by TEM was smaller than that measured by the DLS method, which could be explained by the difference between the dried state and the hydrated state of the particles.
CMC was used to investigate the stability of the micelles. The CMC of FA-HA-TOS was measured by a pyrene fluorescence probe method. At low copolymer concentrations, the fluorescence intensity remained constant. When the concentration increased to the CMC, micelles formed and pyrene was incorporated into the micelles, which lead to the increase of fluorescence intensity. Furthermore, the intensity of the third peak increased more dramatically than that of the first peak. Therefore, the ratio increased abruptly. The plot of the intensity ratio of I384/I374 against the logarithm of polymer concentration is shown in Fig. 4. The CMC could be determined from the intersection of two straight lines. The CMC of FA-HA-TOS was approximately 1.12 × 10−2 mg/mL. The low value suggested the stability of FA-HA-TOS micelles in the dilute condition.
3.4.3. XRD analysis To describe the existence state of PTX in the drug-loaded micelles, XRD analysis was performed on PTX, blank micelles, PTX-loaded micelles and the physical mixture of PTX and blank micelles. As shown in Fig. 5B, PTX showed intense peaks at 2θ of 5.53°, 8.87°, 11.06° and 12.42°, and there were numerous small peaks in the range of 13°–30°. The typical crystal peaks of PTX were still observed in the pattern of the physical mixture of PTX and blank micelles, albeit with weakened intensity, but not in that of the PTX-loaded micelles. The PTX-loaded micelles exhibited a similar spectrum with blank micelles. It was implied that PTX was either molecularly dispersed in the polymer or distributed in the micelles in an amorphous state. 3.5. Hemolysis evaluation Blood compatibility is an important factor for drug delivery systems that are designed for intravenous administration. In general, the system should have minimized nonspecific interactions with RBC to ensure the safety of injection. It was observed that the PTX-loaded micelles exhibited no hemolytic phenomenon within the concentration range studied and that the hemolysis was no more than 5%. This could be explained by the excellent biocompatibility of HA as a polymer for drug delivery. Moreover, HA possessed negative charge due to the carboxyl group, and it demonstrated negligible interaction with the same charged cell membrane of RBC. As a comparison, the Cremophor ELbased formulation induced hemolysis of up to 11%, which could be attributed to the use of ethanol and Cremophor EL in the formulation. It
Fig. 4. Plot of the fluorescence intensity ratio of I3/I1 from pyrene excitation spectra against the logarithm of FA-HA-TOS concentration. 16
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Fig. 5. Characterization of PTX-loaded FA-HA-TOS micelles. (A) TEM image of PTX-loaded FA-HA-TOS micelles. (B) XRD patterns of (a) blank micelles, (b) PTXloaded micelles, (c) physical mixture of PTX and blank micelles and (d) PTX.
dual-targeting property of the system. For MCF-7 cells, which overexpressed CD44 receptors and folate receptors on the cell membrane [24,25], it was speculated that more PTX-loaded FA-HA-TOS micelles were internalized into MCF-7 cells via receptor-mediated endocytosis.
was speculated that the PTX-loaded FA-HA-TOS micelles might be safe for intravenous injection.
3.6. In vitro cytotoxicity study 3.7. In vivo antitumor efficacy study
For antitumor drug delivery systems, the in vitro cytotoxicity was a significant predictor of the in vivo antitumor activity of the system. As shown in Fig. 6A, the bare FA-HA-TOS micelles exhibited negligible cytotoxicity, which indicated the nontoxic nature and desirable safety of FA-HA-TOS micelles. Excitingly, the PTX-loaded FA-HA-TOS micelles exhibited slightly higher cytotoxicity than the Cremophor EL-based formulation and free PTX in DMSO. This might be attributed to the
To further evaluate the antitumor activity of the dual-targeting PTXloaded FA-HA-TOS micelles, H22 tumor-bearing Kunming mouse models were established [26]. As shown in Fig. 6B, the tumor sizes were much smaller in the treatment groups than those in the model group, with TIR of 63.2% and 51.5% for the micelle group and the Cremophor
Fig. 6. Antitumor efficacy of PTX-loaded FA-HA-TOS micelles. (A) In vitro cytotoxicity of PTX-loaded FA-HA-TOS micelles against MCF-7 cells. (B) In vivo antitumor efficacy of PTX-loaded FA-HA-TOS micelles. 17
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EL-based formulation group, respectively. The superior antitumor efficacy of PTX-loaded FA-HA-TOS micelles might be explained by the following facts: the PTX-loaded FA-HA-TOS micelles exhibited significant negative charge, which could enhance the stability of the system and avoid the rapid clearance by RES, to increase the blood circulation half-life. Furthermore, the mean particle size in the range of 100–200 nm could lead to the accumulation of the micelles in the tumor sites via the EPR effect. Most important of all, the dual targeting property of the micelles from HA and FA could significantly increase the internalization into tumor cells via CD44 receptor and folate receptor-mediated active targeting. Based on the above evidence, the PTX-loaded FA-HA-TOS micelles represent a promising carrier system for tumor therapy.
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4. Conclusions In this study, a tumor-targeting micellar system for anticancer drug delivery was developed based on FA-HA-TOS conjugates. The FA-HATOS was synthesized by linking TOS and FA to the backbones of HA via ester bonds. With an amphiphilic structure, FA-HA-TOS could self-assemble into micelles in aqueous milieu and solubilize PTX within the hydrophobic core. The PTX-loaded micelles exerted excellent anticancer effects against MCF-7 cells in vitro and against H22 cells in vivo due to enhanced active targeting via the combined use of HA and FA. The tumor-targeting FA-HA-TOS micelle system represents a promising nanocarrier for anticancer drug delivery. Disclosures The authors report no conflicts of interest in this work. Acknowledgments This work was funded by the National Natural Science Foundation of China (No. 51403057), Heilongjiang Natural Science Foundation (No. E2018052), the Research and Development Project of Scientific and Technological Achievements for Colleges and Universities of Heilongjiang Province (No. TSTAU-R2018023), Harbin Science and Technology Innovation Talents Special Fund Project (No. 2016RQQXJ097, No. 2016RQQXJ131), and the Doctoral Scientific Research Startup Foundation of Harbin Normal University (No. XKB201304). References [1] H. Maeda, H. Nakamura, J. Fang, The EPR effect for macromolecular drug delivery to solid tumors: improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo, Adv. Drug Deliv. Rev. 65 (2013) 71–79. [2] K. Seidi, H.A. Neubauer, R. Moriggl, R. Jahanban-Esfahlan, T. Javaheri, Tumor target amplification: implications for nano drug delivery systems, J. Control. Release 275 (2018) 142–161. [3] K. Zhang, P. Li, Y. He, X. Bo, X. Li, D. Li, H. Chen, H. Xu, Synergistic retention strategy of RGD active targeting and radiofrequency-enhanced permeability for intensified RF & chemotherapy synergistic tumor treatment, Biomaterials 99 (2016) 34–46.
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