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Hyaluronic acid-coated solid lipid nanoparticles to overcome drug-resistance in tumor cells
Journal of Drug Delivery Science and Technology 50 (2019) 365–371
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Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Hyaluronic acid-coated solid lipid nanoparticles to overcome drugresistance in tumor cells
T
Sang-Eun Leea,1, Chang Doo Leeb,1, Jung Bin Ahna, Dong-Hyun Kima, Jung Kyu Leeb, Ji-Yun Leeb, Jin-Seok Choia,c, Jeong-Sook Parka,∗ a
College of Pharmacy, Chungnam National University, Daejeon, 34134, Republic of Korea College of Pharmacy, Chung-Ang University, Seoul, 06974, Republic of Korea c Department of Medical Management, Chodang University, Muan-gun, Jeollanam-do, 58530, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hyaluronic acid Solid lipid nanoparticles CD44s targeted drug delivery system
In this study, we aimed to prepare hyaluronic acid (HA)-coated, docetaxel (DTX)-loaded, solid lipid nanoparticles (HA-SLN) to target and overcome drug-resistant tumor cells. SLN were prepared with stearic acid (SA), hexadecyltrimethylammonium bromide (CTAB), soy phosphatidylcholine (PC), and DTX by the lipid film method. Next, the lipid nanoparticles were coated with HA via electrostatic attraction. SLN and HA-SLN were characterized and evaluated. An in vitro drug release assay was performed in phosphate-buffered saline for 72 h. The extent of CD44 expression in MCF7, MDA-MB-231, and MCF7/ADR cells was investigated using western blotting. Next, cellular uptake and cytotoxicity were compared. The particle sizes of SLN and HA-SLN were 109.5 ± 8.2 and 224.3 ± 15.9 nm, respectively. SLN had a positive surface charge of 32.5 ± 3.7 mV, whereas HA-SLN had a negative charge of −17.1 ± 0.7 mV. A considerable amount of CD44 expression was detected in MCF7/ADR cells. The cellular uptake and cytotoxicity of HA-SLN were higher in MCF7/ADR cells than in the other cells, showing sufficient targetability and efficacy to control tumor resistance. Therefore, HA-SLN are an effective, targeted drug delivery system for delivering DTX to overcome drug-resistant tumors.
1. Introduction Current chemotherapeutic agents and targeted therapies have increased the survival rate and the quality of life of cancer patients [1]. However, despite good initial therapeutic response, after continuous use of an anticancer drug, cancer cells that have developed resistance to this drug can lead to chemotherapy failure [2]. This is one of the reasons for difficulty in conquering cancer through successful cancer treatments. Solid lipid nanoparticles (SLN) could be an ideal choice for delivering anticancer agents to overcome drug resistance. SLN present attractive advantages over other colloidal carriers; these advantages include the feasibility of incorporating lipophilic and hydrophilic drugs, biocompatibility of carriers, and possibility of controlled drug release and drug targeting. Especially, SLN can be easily surface-modified to introduce diverse targeting moieties [3–5]. Hyaluronic acid (HA) is a negatively charged natural polysaccharide, composed of repeating glucuronic acid and glucosamine disaccharide units. Owing to features such as biocompatibility,
biodegradability, non-toxicity, non-immunogenicity, and presence of many functional groups for modification, HA is an interesting choice for targeted drug delivery systems [6–8]. In addition, the relationship between CD44, an HA receptor, and cancer resistance has recently been explored. Many recent reports have shown that HA and CD44 play an important role in the manifestation of resistance in cancer cells. Toole et al. proposed that hyaluronan, CD44, and emmprin are important coregulators of various activities crucial to drug resistance [9]. Moreover, CD44 promotes multidrug resistance by protecting P-glycoprotein [10]. Some investigators discovered that CD44 could be used as a surface biomarker for drug resistance [11]. CD44 is also overexpressed in melanoma, lymphoma, colorectal, breast, and lung tumor cells [12,13]. However, CD44 exhibits low expression in normal tissues and cells, and these cells do not directly contact the bloodstream [14]. Therefore, application of HA in drug delivery systems will possibly allow us to target and overcome drug-resistant tumors for chemotherapy. In this study, we aimed to prepare HA-coated, docetaxel (DTX)loaded SLN (HA-SLN) to target and overcome drug-resistant tumor
∗
Corresponding author. College of Pharmacy, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon, 34134, Republic of Korea. E-mail address: [email protected] (J.-S. Park). 1 Both authors contributed equally to this work. https://doi.org/10.1016/j.jddst.2019.01.042 Received 2 October 2018; Received in revised form 22 January 2019; Accepted 30 January 2019 Available online 04 February 2019 1773-2247/ © 2019 Elsevier B.V. All rights reserved.
Journal of Drug Delivery Science and Technology 50 (2019) 365–371
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Scheme. 1. Schematic illustrations for preparation of HA-SLN and target delivery of HA-SLN to CD44-expressing cancer cells. Table 1 Physicochemical properties of SLN and HA-SLN (mean ± s.d., n = 3).
SLN HA-SLN
Size (nm)
PdI
Zeta potential (mV)
Encapsulation efficiency (%)
110 ± 8 224 ± 16
0.26 ± 0.059 0.11 ± 0.031
32.5 ± 3.67 −17.1 ± 0.73
12.0 ± 2.76 10.5 ± 3.92
cells. SLN were fabricated and then coated with HA via electrostatic attraction. To confirm the physicochemical properties of the nanocarriers, particle size, zeta potential, drug loading efficiency, and differential scanning calorimetry were evaluated. An in vitro drug release assay was also performed. We have conducted a series of cell experiments to determine if this drug delivery system can target and is effective against drug-resistance tumors. The extent of CD44 expression in MCF7, MDA-MB-231, and MCF7/ADR cell extracts was investigated using western blotting. Next, cellular uptake and cytotoxicity were compared with respect to CD44 expression.
Fig. 2. DSC thermograms of stearic acid, CTAB, soy PC, DTX, physical mixture, SLN, and HA-SLN.
Fig. 1. Size distribution and TEM images of (a) SLN and (b) HA-SLN. Scale bar, 100 nm. 366
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purchased from Avanti Polar Lipids (Alabaster, AL, USA). DTX and Taxotere® were gifted by SamYang Corporation (Seoul, Korea). HA (molecular weght: ∼100 kDa) was obtained from Bioland (Cheonan, Korea). Methanol and acetonitrile for high-performance liquid chromatography (HPLC) were obtained from SKYSOLTECH® (SK Chemicals, Seongnam, Korea). Fetal bovine serum, antibiotics, and RPMI 1640 medium were obtained from Hyclone™ (Logan, UT, USA). All other chemicals were reagent-grade and used without further purification. Distilled and deionized water were used after sterilization. 2.2. Preparation of docetaxel-loaded SLN and HA-SLN DTX-loaded SLN were prepared by the modified lipid film method [15]. Stearic acid, PC, CTAB, and DTX were mixed at a weight ratio of 10:10:10:3 (total weight, 66 mg), and then dissolved in chloroform (20 mL). The organic solvent was removed on a rotary evaporator (RE 47; Yamato Scientific, Tokyo, Japan). The dried lipid film was placed in a desiccator at 40 °C overnight to remove traces of organic solvent. The film was hydrated with distilled water. Next, the hydrated lipid solution was sonicated with a probe-type sonicator (KFS-300 N, Ultrasonic Processor; Korea Process Technology, Seoul, Korea) for 10 min at 300 W. To remove crystal drug and over-sized particle aggregates (i.e., unencapsulated drug) from the solution, the lipid solution was filtered through a syringe filter (pore size, 1 μm; diameter, 25 mm; Whatman, Florham Park, NJ, USA). SLN were collected via centrifugation at 12,000 rpm for 20 min, and then dispersed in phosphate-buffered saline (PBS). To fabricate HA-SLN, equal volumes of HA solution (at 0.3 mg/mL) were added to the drug-loaded SLN suspension with stirring. After incubation at room temperature for 1 h, the suspensions were centrifuged at 12,000 rpm for 20 min to remove uncoated HAs. To analyze of cellular uptake, Coumarin 6-loaded lipid nanoparticles (SLN and HA-SLN) were also fabricated by the same method as described above. Instead of DTX, coumarin 6 (1 mg) was dissolved in ethanol (10 mL) and mixed with the oil phase.
Fig. 3. Release profiles of DTX from Taxotere®, SLN, and HA-SLN in PBS (pH 7.4) at 37 °C (dialysis method). Bars represent means ± s.d. (n = 3).
2.3. Characterization of SLN and HA-SLN The particle size and zeta potential of the lipid dispersions were measured using a dynamic light scattering (DLS) spectrophotometer (Zetasizer, Malvern, UK) at ambient temperature. The samples were diluted in deionized water to obtain adequate signal. DTX-loaded SLN were diluted to 10 times their volume with ethanol, and the drug content was analyzed with an HPLC system, as reported in our previous study [15]. Briefly, amount of DTX was determined using an SPˆLC HPLC system (Shiseido, Tokyo, Japan). Separation was performed on a Capcell Pak C18 column (150 mm × 4.6 mm I.D., 5 μm particle size; Shiseido) maintained at 30 °C. The mobile phase was acetonitrile:methanol:distilled water (50:30:20, v/v/v) and was delivered at a flow rate 0.5 mL/min. The mobile phase was premixed, filtered through a 0.45-μm polyvinylidene fluoride (PVDF) filter, and degassed before use. The injection volume was 25 μL. Samples were detected at a wavelength of 230 nm. Particle size and polydispersity of the nanoparticles were measured to investigate their aqueous stability. Samples were stored at 4 °C and analyzed for 4 weeks. Morphological analysis of liposomes was performed through transmission electron microscopy (TEM). Diluted sample (10 μL) was placed on a carbon-coated grid and air-dried at ambient temperature. The fixed sample was assessed with a TEM instrument (Tecnai G2 F30 FEI; Thermo Fisher Scientific, Waltham, MA, USA) after staining with uranyl acetate (1%, w/v). Thermal analysis of SA, CTAB, lecithin, DTX, HA, physical mixtures, SLN, and HA-SLN was performed using differential scanning calorimetry (DSC, S-650; SCINCO, Seoul, Korea). Samples (2 mg) were placed in aluminum pans. Thermal behavior of the samples was investigated at a scanning rate of 20 °C/min from 25 to 400 °C.
Fig. 4. (a) Expression of CD44 in MCF7, MCF7/ADR, and MDA-MB-231 cells. The expression of CD44 was evaluated by western blotting using β-actin as an internal control. (b) Quantitative graph of CD44 expression in breast cancer cells. Bars represent means ± s.d. (n = 3). In all analyses, p < 0.01 (**) and p < 0.05 (*) indicate statistical significance.
2. Materials and methods 2.1. Materials Stearic acid (SA), hexadecyltrimethylammonium bromide (CTAB), chloroform, dimethyl sulfoxide (DMSO), and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Soy phosphatidylcholine (PC) was 367
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Fig. 5. Cellular uptake of coumarin 6-loaded formulations in breast cancer cells. (a) Confocal images after cellular uptake of SLN and HA-SLN in MCF7, MDA-MB231, and MCF7/ADR cells for 2 h. Green and blue colors represent coumarin-6-loaded SLN and HA-SLN (fluorescein isothiocyanate (FITC) filter), and the cell nuclei (4′,6-diamidino-2-phenylindole (DAPI) filter), respectively. (b) Quantification of SLN and HA-SLN. HA-dependent cellular uptake in MCF7, MDA-MB-231, and MCF7/ ADR cells after 2 h. Results are expressed as means ± s.d. (n = 3). p < 0.01 (**) and p < 0.05 (*)indicate statistical significance. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
RIPA buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1.0% NP-40; 2 mM EDTA; 5 mM NaF; 1 mM phenylmethylsulfonyl fluoride; 1 mM sodium orthovanadate; 0.5% sodium deoxycholate; and 0.1% sodium dodecyl sulfate). Lysates were incubated at 4 °C for 30 min and then centrifuged at 8000×g for 10 min at 4 °C. Concentration of extracted protein was determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). Cell lysates (15 μg of protein) were subjected to 13.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred electrophoretically onto polyvinylidene fluoride membranes (ATTO Corp., Tokyo, Japan) at 200 mA for 1.5 h. The blots were blocked with 5% bovine serum albumin in TBS-T (20 mM Tris; 137 mM NaCl, pH 7.4; 0.05% Tween® 20) at 4 °C for 1.5 h, and then incubated with anti-CD44 or β-actin primary antibodies at 4 °C overnight. After washing the blots, the blots were subsequently incubated with an appropriate secondary antibody. Protein bands were detected using an ECL kit (ATTO Corp.), and the band intensities were quantified using the Quantity One software (Bio-Rad, Hercules, CA, USA).
2.4. In vitro drug release assay In vitro release of DTX from Taxotere®, SLN, and HA-SLN was evaluated by the dialysis method. Taxotere® is mainly composed of ethanol and Tween® 80 to increase the solubility of DTX. Briefly, 1 mL of Taxotere® solution (40 μg/mL, diluted in PBS), 1 mL of DTX-loaded SLN dispersion (40 μg/mL, diluted in PBS), and 1 mL of HA-SLN dispersion (40 μg/mL, diluted in PBS) were added to a dialysis bag, separately. Next, the dialysis bags were incubated with 20 mL of release medium (PBS 0.5% containing Tween® 80, pH 7.4) at 37 °C with stirring at 100 rpm. At predetermined time intervals, 1 mL of the dialysate was removed, and replenished with the same volume of release medium. Amount of DTX in dialysate samples was determined by the HPLC method, as described in section 2.3. 2.5. Western blotting analysis Human breast adenocarcinoma cells, MCF7, MCF7/ADR, and MDAMB-231 cells, were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified incubator supplied with 5% CO2. Cells were washed twice in ice-cold PBS and lysed with
2.6. Cellular uptake studies For quantitative estimation of uptake, cells were seeded in 12-well plates at a density of 2 × 105 cells/well. Upon reaching 70–80% 368
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normalized to total protein content. Confocal microscopy was also performed to study the cellular uptake of SLN and HA-SLN in MCF7, MCF7/ADR, and MDA-MB-231 cells. In detail, 5 × 105 cells/well were cultured in 12-well plates containing a sterilized glass coverslip at the bottom of each well, and then incubated overnight. The cells were treated with 0.1 μg/mL coumarin 6loaded SLN or HA-SLN for 2 h. Next, the cells were washed three times with PBS and then fixed with 4% paraformaldehyde for 15 min. The glass coverslip with attached cells was then removed from the well and placed on a glass microscope slide in the presence of mounting agent. Confocal analysis was performed on an LSM5 Live Configuration Variotwo VRGB (Zeiss, Oberkochen, Germany). 2.7. In vitro cytotoxicity studies The cytotoxicity of Taxotere®, drug-loaded lipid nanoparticles, and unloaded lipid nanoparticles was determined against MCF7, MCF7/ ADR, and MDA-MBA-231 cells. MTT is a yellow compound that is reduced in the mitochondria of viable cells to a blue formazan product with a maximum absorbance at 570 nm. Breast cancer cells were seeded at 1 × 104 cells/well in 96-well plates. After overnight incubation, the cells were exposed to the formulations in medium for 24 h. Next, the medium was removed and 100 μL of MTT-containing medium (5 mg/ mL) was added to the wells. Following 4 h of incubation at 37 °C, the MTT-containing medium was aspirated carefully to avoid disturbing any formazan crystals that had formed, and 100 μL DMSO was added to each well. The plates were incubated at ambient temperature for 30 min and absorbance at 570 nm was measured using a microplate reader (Sunrise™; Tecan Trading, Switzerland). Cell viability was expressed as a percentage of the untreated control cells. 2.8. Statistical analysis Statistical analysis was performed by the paired t-test using SigmaPlot (ver. 12.5; SYSTAT, Inc., Chicago, IL, USA). Data are expressed as means ± standard deviation (s.d.). In all analyses, p < 0.01 (**) and p < 0.05 (*) indicated statistical significance. 3. Results and discussion 3.1. Characterization of HA-coated lipid nanoparticles HA-SLN were prepared using electrostatic attraction between the negatively charged HA and the cationic surfactant, CTAB. In this study, high-molecular-weight HA (∼100 kDa) was selected in accordance with a report that high-molecular-weight HA-coated particles are more effective against cancerous cells expressing CD44 [16]. The schematic illustration is shown in Scheme 1. The physicochemical characteristics of the lipid carriers are summarized in Table 1. The particle size of the plain lipid nanoparticles were approximately 100 nm, whereas those of the HA-coated formulations tended to increase. The polydispersity index of HA-SLN was lower than that of SLN, because the electronic interaction between HA and the cationic surface might produce a more compact structure of lipid nanoparticles [17]. To confirm the results of DLS, TEM was performed. TEM images show nano-sized particles that were consistent with the DLS results (Fig. 1). The HA coating was also observed through TEM images. SLN showed a spherical shape, and HA corona surrounding SLN was observed in HA-SLN (Fig. 1). It was confirmed that HA could well coat the cationic SLN through electrostatic coupling. The results of comparative analysis of zeta potentials before and after HA coating showed that the positive surface charge (32.5 ± 3.67 mV) of SLN had changed to negative (−17.1 ± 0.73 mV) after HA coating. Plain lipid nanoparticles possess CTAB cationic surfactants in their structures, which have positive charges, imparting a net positive charge to the plain lipid particles.
Fig. 6. Cytotoxicity of DTX-loaded formulations against (a) MCF7, (b) MDAMB-231, and (c) MCF7/ADR cells. Bars represent means ± s.d. (n = 6).
confluence, the cells were incubated with coumarin-6-loaded SLN and HA-SLN (0.1 μg/mL coumarin-6). After incubation for 0.5, 1, or 2 h, the cells were washed twice with cold PBS. Subsequently, the cells were lysed through addition of Triton™ X-100 (0.5%, w/v). Fluorescence intensities were measured using a multimode microplate reader (Synergy H1 Hybrid Multi-Mode Microplate Reader; BioTek, Winooski, VT, USA) at excitation and emission wavelengths of 440 and 520 nm, respectively. Protein was quantified using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific), and fluorescence intensities were 369
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3.4. Cellular uptake
After coating SLN with HA, the surface charges of the lipid nanoparticles became negative because HA molecules have negatively charged carboxylic acid groups in their structure. This change in the surface charge of HA-SLN showed that HA molecules are bound to the surface of plain lipid nanoparticles [18]. To characterize the drug–lipid interactions, DSC was performed for lipid alone, drug alone, formulations, and other excipients. As shown in Fig. 2, the DSC thermogram of stearic acid shows a sharp melting peak at 71 °C. Moreover, CTAB has melting peaks at 105 °C and 269 °C, and DTX exhibits a melting peak at 170 °C. However, no peaks for DTX are shown in the thermograms of the formulations, indicating that DTX, being molecularly dispersed within the solid dispersion, does not show crystallinity [19]. Many studies have reported that HA coating enhances the stability of nanoparticles [20–22]. In this study, the mean particle size and polydispersity of SLN and HA-SLN were evaluated to investigate the effect of HA coating on the stability of SLN. The size and polydispersity index (PdI) of SLN slightly increased from 109.5 ± 8.21 to 128.5 ± 15.84 nm after 4 weeks. In contrast, the mean particle diameter and the degree of dispersion of HA-SLN were maintained steadily during the observation period, as shown in Fig. S1 (see supplementary information). Therefore, coating with natural hydrophilic polymers appeared to enhance nanoparticle stability in aqueous solution.
In this study, tumor cells expressing CD44 were used. Two control groups were used for comparison, MDA-MB-231 cells (highly expressing CD44, as positive controls) and MCF7 cells (barely expressing CD44, as negative controls). To evaluate the intracellular uptake of the formulation and the role of CD44 expression on the cell surface, coumarin 6 was encapsulated in the nanoparticles and its fluorescence intensity was measured by fluorescence and confocal microscopy. There was no significant difference in uptake between SLN and HASLN in MCF7 cells (Fig. 5). However, the uptake of HA-SLN in MCF7/ ADR and MDA-MB-231 cells was higher than that of SLN. This could occur because CD44 receptor-mediated endocytosis significantly increased the cellular uptake of HA-coated nanocarriers. Therefore, in MCF7 cells, which express few CD44 receptors, the uptake of both formulations (i.e., SLN and HA-SLN) was expected to be similar. After the addition of free HA to the culture medium, the uptake of HA-SLN dropped to the level observed with SLN in MDA-MB-231 cells. In MCF7/ ADR cells, the uptake of HA-SLN decreased, but it was still higher than that of SLN. The reduction could be due to the competitive binding of free HA molecules to CD44, which inhibits CD44 receptor-mediated nanoparticle endocytosis on the surface of MCF7/ADR and MDA-MB231 cells. MCF7/ADR cells express diverse MDR proteins, including MDR1 [25]. These resistant proteins will inhibit cellular uptake of the nanoparticles, but the HA target moiety could overcome this resistance and increase the accumulation of the drug that has been encapsulated in lipid nanoparticles.
3.2. In vitro drug release The DTX release profiles of SLN and HA-SLN are shown in Fig. 3. There was a burst release within 4 h, with nearly 40% of DTX was released from the lipid formulations. This probably occurred a significant amount of drug is distributed in the outer shell of nanoparticles [5]. The total amount of DTX released from SLN after 24, 48 and 72 h was higher than that from HA-SLN (58.9 ± 3.4% and 64.0 ± 3.8% after 24 h; 65.3 ± 1.4% and 59.3 ± 2.0% after 48 h; 68.1 ± 1.2% and 62.8 ± 1.5% after 72 h, respectively). There was no statistically significant difference in the cumulative amount from DTX released from nanoparticles after 24 h (p < 0.5; paired t-test), but there were significant differences in those after 48 and 72 h (p < 0.05 and p < 0.01, respectively; paired t-test). The profiles showed that HA prolonged the release of DTX from SLN. The HA coating could decrease the permeability of SLN. Moreover, HA molecules produce a dense hydrophilic matrix around lipid nanoparticles, which decreases the release rate of a hydrophobic drug [17,23].
3.5. Cytotoxicity The dose–response curves for Taxotere®, drug-loaded formulation, and unloaded formulation are shown in Fig. 6 and Fig. S2 (see supplementary information). As many previous researchers have reported, cationic particles have much more toxic to cells [26]. Similar results were obtained in this study. Blank SLN showed greater cytotoxicity against breast cancer cells than other blank nanoparticles (Fig. S2). As mentioned above, the SLN are surrounded by CTAB and the surface is positively charged. Because of the role of the cationic surfactant, it would have caused more damage to the cells. In contrast, blank HA-SLN were almost completely nontoxic (Fig. S2). The HA coating might prevent the cationic surfactants in the lipid nanoparticles from directly contacting the cells. In addition, HA is well known to be biocompatible and nontoxic natural polymer. For this reason, it seems that the blank SLN shows little toxicity against breast cancer cells. HA-SLN (73.4 ± 9.4 and 86.1 ± 4.4%) exhibited the higher cytotoxicity than SLN (45.0 ± 5.4 and 56.5 ± 3.4%) and Taxotere® (17.6 ± 4.3 and 27.7 ± 8.9%) against MCF7/ADR and MDA-MB231 cells at a DTX concentration of 10 μg/mL (paired t-test; p < 0.05) (Fig. 6b and c). This result is in agreement with the results of the uptake studies. HA-SLN have a high binding affinity to CD44 receptors; therefore, the specific recognition of HA-SLN by CD44 surface receptors results in CD44-mediated endocytosis. HA-modified nanoparticles have been studied as targets for delivering chemotherapeutic agents to cancer cells to overcome MDR and reduce effective dose [20]. In this study, it also could be concluded that the active targeting of DTX carriers by HA ligands to CD44 receptors leads to a higher cytotoxic activity and selectivity toward MCF7/ADR and MDA-MBC-231 cells.
3.3. CD44 expression Recent studies have reported the association of drug resistance with HA. HA and CD44 play an important role in resistance to anticancer drugs [9,12,16]. CD44 increases in many malignant and resistant cancers, and this can be categorized as a functional strategy used by cancer cells for self-preservation. Increased CD44 on the cell surface increases HA levels, thereby increasing the flow pressure and decreasing the inflow of foreign substances [16]. In addition, CD44 can enhance the drug resistance of cancer cells by protecting P-glycoprotein [24]. To compare the effect of HA-SLN on breast cancer cells, we measured the expression of CD44 on four cell lines. The expression level of CD44 in each breast cancer cell is shown in Fig. 4. MDA-MB-231 cells (118.25 ± 2.44%) are well-known to highly express CD44 protein which was confirmed in the results of our study. CD44 expression was approximately 7-fold higher in MCF7/ADR cells (1.34 ± 0.24%) than in MCF7 cells (9.48 ± 0.44%). This result in MCF7/ADR cells also showed that CD44 expression levels are elevated for cell defense. This leads to the expectation that HA-containing formulations will be effective in targeting resistant cancer cells.
4. Conclusion In this study, a DTX-containing, HA-coated lipid formulation was prepared and characterized. HA-SLN were nano-sized particles that had higher aqueous stability than uncoated SLN over 4 weeks. The targeting moiety of HA-SLN was evaluated with the CD44-expressing breast cancer cells, MCF7/ADR and MDA-MB-231. HA-SLN showed higher uptake and cytotoxicity against CD44-expressing cell lines than 370
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untargeted lipid formulation. HA-SLN also worked effectively, especially against drug-resistant MCF7/ADR cells owing to the HA-target moiety. Therefore, HA-SLN could be a potent drug delivery system that can achieve both target and overcome drug resistance.
[11]
[12]
Declarations of interest [13]
None.
[14]
Acknowledgement [15]
This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Korea government, MSIP (No. 2015R1A2A1A10051596) and by the Chung-Ang University Graduate Research Scholarship in 2017.
[16]
[17]
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jddst.2019.01.042.
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Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Hyaluronic acid-coated solid lipid nanoparticles to overcome drugresistance in tumor cells
T
Sang-Eun Leea,1, Chang Doo Leeb,1, Jung Bin Ahna, Dong-Hyun Kima, Jung Kyu Leeb, Ji-Yun Leeb, Jin-Seok Choia,c, Jeong-Sook Parka,∗ a
College of Pharmacy, Chungnam National University, Daejeon, 34134, Republic of Korea College of Pharmacy, Chung-Ang University, Seoul, 06974, Republic of Korea c Department of Medical Management, Chodang University, Muan-gun, Jeollanam-do, 58530, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hyaluronic acid Solid lipid nanoparticles CD44s targeted drug delivery system
In this study, we aimed to prepare hyaluronic acid (HA)-coated, docetaxel (DTX)-loaded, solid lipid nanoparticles (HA-SLN) to target and overcome drug-resistant tumor cells. SLN were prepared with stearic acid (SA), hexadecyltrimethylammonium bromide (CTAB), soy phosphatidylcholine (PC), and DTX by the lipid film method. Next, the lipid nanoparticles were coated with HA via electrostatic attraction. SLN and HA-SLN were characterized and evaluated. An in vitro drug release assay was performed in phosphate-buffered saline for 72 h. The extent of CD44 expression in MCF7, MDA-MB-231, and MCF7/ADR cells was investigated using western blotting. Next, cellular uptake and cytotoxicity were compared. The particle sizes of SLN and HA-SLN were 109.5 ± 8.2 and 224.3 ± 15.9 nm, respectively. SLN had a positive surface charge of 32.5 ± 3.7 mV, whereas HA-SLN had a negative charge of −17.1 ± 0.7 mV. A considerable amount of CD44 expression was detected in MCF7/ADR cells. The cellular uptake and cytotoxicity of HA-SLN were higher in MCF7/ADR cells than in the other cells, showing sufficient targetability and efficacy to control tumor resistance. Therefore, HA-SLN are an effective, targeted drug delivery system for delivering DTX to overcome drug-resistant tumors.
1. Introduction Current chemotherapeutic agents and targeted therapies have increased the survival rate and the quality of life of cancer patients [1]. However, despite good initial therapeutic response, after continuous use of an anticancer drug, cancer cells that have developed resistance to this drug can lead to chemotherapy failure [2]. This is one of the reasons for difficulty in conquering cancer through successful cancer treatments. Solid lipid nanoparticles (SLN) could be an ideal choice for delivering anticancer agents to overcome drug resistance. SLN present attractive advantages over other colloidal carriers; these advantages include the feasibility of incorporating lipophilic and hydrophilic drugs, biocompatibility of carriers, and possibility of controlled drug release and drug targeting. Especially, SLN can be easily surface-modified to introduce diverse targeting moieties [3–5]. Hyaluronic acid (HA) is a negatively charged natural polysaccharide, composed of repeating glucuronic acid and glucosamine disaccharide units. Owing to features such as biocompatibility,
biodegradability, non-toxicity, non-immunogenicity, and presence of many functional groups for modification, HA is an interesting choice for targeted drug delivery systems [6–8]. In addition, the relationship between CD44, an HA receptor, and cancer resistance has recently been explored. Many recent reports have shown that HA and CD44 play an important role in the manifestation of resistance in cancer cells. Toole et al. proposed that hyaluronan, CD44, and emmprin are important coregulators of various activities crucial to drug resistance [9]. Moreover, CD44 promotes multidrug resistance by protecting P-glycoprotein [10]. Some investigators discovered that CD44 could be used as a surface biomarker for drug resistance [11]. CD44 is also overexpressed in melanoma, lymphoma, colorectal, breast, and lung tumor cells [12,13]. However, CD44 exhibits low expression in normal tissues and cells, and these cells do not directly contact the bloodstream [14]. Therefore, application of HA in drug delivery systems will possibly allow us to target and overcome drug-resistant tumors for chemotherapy. In this study, we aimed to prepare HA-coated, docetaxel (DTX)loaded SLN (HA-SLN) to target and overcome drug-resistant tumor
∗
Corresponding author. College of Pharmacy, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon, 34134, Republic of Korea. E-mail address: [email protected] (J.-S. Park). 1 Both authors contributed equally to this work. https://doi.org/10.1016/j.jddst.2019.01.042 Received 2 October 2018; Received in revised form 22 January 2019; Accepted 30 January 2019 Available online 04 February 2019 1773-2247/ © 2019 Elsevier B.V. All rights reserved.
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Scheme. 1. Schematic illustrations for preparation of HA-SLN and target delivery of HA-SLN to CD44-expressing cancer cells. Table 1 Physicochemical properties of SLN and HA-SLN (mean ± s.d., n = 3).
SLN HA-SLN
Size (nm)
PdI
Zeta potential (mV)
Encapsulation efficiency (%)
110 ± 8 224 ± 16
0.26 ± 0.059 0.11 ± 0.031
32.5 ± 3.67 −17.1 ± 0.73
12.0 ± 2.76 10.5 ± 3.92
cells. SLN were fabricated and then coated with HA via electrostatic attraction. To confirm the physicochemical properties of the nanocarriers, particle size, zeta potential, drug loading efficiency, and differential scanning calorimetry were evaluated. An in vitro drug release assay was also performed. We have conducted a series of cell experiments to determine if this drug delivery system can target and is effective against drug-resistance tumors. The extent of CD44 expression in MCF7, MDA-MB-231, and MCF7/ADR cell extracts was investigated using western blotting. Next, cellular uptake and cytotoxicity were compared with respect to CD44 expression.
Fig. 2. DSC thermograms of stearic acid, CTAB, soy PC, DTX, physical mixture, SLN, and HA-SLN.
Fig. 1. Size distribution and TEM images of (a) SLN and (b) HA-SLN. Scale bar, 100 nm. 366
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purchased from Avanti Polar Lipids (Alabaster, AL, USA). DTX and Taxotere® were gifted by SamYang Corporation (Seoul, Korea). HA (molecular weght: ∼100 kDa) was obtained from Bioland (Cheonan, Korea). Methanol and acetonitrile for high-performance liquid chromatography (HPLC) were obtained from SKYSOLTECH® (SK Chemicals, Seongnam, Korea). Fetal bovine serum, antibiotics, and RPMI 1640 medium were obtained from Hyclone™ (Logan, UT, USA). All other chemicals were reagent-grade and used without further purification. Distilled and deionized water were used after sterilization. 2.2. Preparation of docetaxel-loaded SLN and HA-SLN DTX-loaded SLN were prepared by the modified lipid film method [15]. Stearic acid, PC, CTAB, and DTX were mixed at a weight ratio of 10:10:10:3 (total weight, 66 mg), and then dissolved in chloroform (20 mL). The organic solvent was removed on a rotary evaporator (RE 47; Yamato Scientific, Tokyo, Japan). The dried lipid film was placed in a desiccator at 40 °C overnight to remove traces of organic solvent. The film was hydrated with distilled water. Next, the hydrated lipid solution was sonicated with a probe-type sonicator (KFS-300 N, Ultrasonic Processor; Korea Process Technology, Seoul, Korea) for 10 min at 300 W. To remove crystal drug and over-sized particle aggregates (i.e., unencapsulated drug) from the solution, the lipid solution was filtered through a syringe filter (pore size, 1 μm; diameter, 25 mm; Whatman, Florham Park, NJ, USA). SLN were collected via centrifugation at 12,000 rpm for 20 min, and then dispersed in phosphate-buffered saline (PBS). To fabricate HA-SLN, equal volumes of HA solution (at 0.3 mg/mL) were added to the drug-loaded SLN suspension with stirring. After incubation at room temperature for 1 h, the suspensions were centrifuged at 12,000 rpm for 20 min to remove uncoated HAs. To analyze of cellular uptake, Coumarin 6-loaded lipid nanoparticles (SLN and HA-SLN) were also fabricated by the same method as described above. Instead of DTX, coumarin 6 (1 mg) was dissolved in ethanol (10 mL) and mixed with the oil phase.
Fig. 3. Release profiles of DTX from Taxotere®, SLN, and HA-SLN in PBS (pH 7.4) at 37 °C (dialysis method). Bars represent means ± s.d. (n = 3).
2.3. Characterization of SLN and HA-SLN The particle size and zeta potential of the lipid dispersions were measured using a dynamic light scattering (DLS) spectrophotometer (Zetasizer, Malvern, UK) at ambient temperature. The samples were diluted in deionized water to obtain adequate signal. DTX-loaded SLN were diluted to 10 times their volume with ethanol, and the drug content was analyzed with an HPLC system, as reported in our previous study [15]. Briefly, amount of DTX was determined using an SPˆLC HPLC system (Shiseido, Tokyo, Japan). Separation was performed on a Capcell Pak C18 column (150 mm × 4.6 mm I.D., 5 μm particle size; Shiseido) maintained at 30 °C. The mobile phase was acetonitrile:methanol:distilled water (50:30:20, v/v/v) and was delivered at a flow rate 0.5 mL/min. The mobile phase was premixed, filtered through a 0.45-μm polyvinylidene fluoride (PVDF) filter, and degassed before use. The injection volume was 25 μL. Samples were detected at a wavelength of 230 nm. Particle size and polydispersity of the nanoparticles were measured to investigate their aqueous stability. Samples were stored at 4 °C and analyzed for 4 weeks. Morphological analysis of liposomes was performed through transmission electron microscopy (TEM). Diluted sample (10 μL) was placed on a carbon-coated grid and air-dried at ambient temperature. The fixed sample was assessed with a TEM instrument (Tecnai G2 F30 FEI; Thermo Fisher Scientific, Waltham, MA, USA) after staining with uranyl acetate (1%, w/v). Thermal analysis of SA, CTAB, lecithin, DTX, HA, physical mixtures, SLN, and HA-SLN was performed using differential scanning calorimetry (DSC, S-650; SCINCO, Seoul, Korea). Samples (2 mg) were placed in aluminum pans. Thermal behavior of the samples was investigated at a scanning rate of 20 °C/min from 25 to 400 °C.
Fig. 4. (a) Expression of CD44 in MCF7, MCF7/ADR, and MDA-MB-231 cells. The expression of CD44 was evaluated by western blotting using β-actin as an internal control. (b) Quantitative graph of CD44 expression in breast cancer cells. Bars represent means ± s.d. (n = 3). In all analyses, p < 0.01 (**) and p < 0.05 (*) indicate statistical significance.
2. Materials and methods 2.1. Materials Stearic acid (SA), hexadecyltrimethylammonium bromide (CTAB), chloroform, dimethyl sulfoxide (DMSO), and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Soy phosphatidylcholine (PC) was 367
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Fig. 5. Cellular uptake of coumarin 6-loaded formulations in breast cancer cells. (a) Confocal images after cellular uptake of SLN and HA-SLN in MCF7, MDA-MB231, and MCF7/ADR cells for 2 h. Green and blue colors represent coumarin-6-loaded SLN and HA-SLN (fluorescein isothiocyanate (FITC) filter), and the cell nuclei (4′,6-diamidino-2-phenylindole (DAPI) filter), respectively. (b) Quantification of SLN and HA-SLN. HA-dependent cellular uptake in MCF7, MDA-MB-231, and MCF7/ ADR cells after 2 h. Results are expressed as means ± s.d. (n = 3). p < 0.01 (**) and p < 0.05 (*)indicate statistical significance. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
RIPA buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1.0% NP-40; 2 mM EDTA; 5 mM NaF; 1 mM phenylmethylsulfonyl fluoride; 1 mM sodium orthovanadate; 0.5% sodium deoxycholate; and 0.1% sodium dodecyl sulfate). Lysates were incubated at 4 °C for 30 min and then centrifuged at 8000×g for 10 min at 4 °C. Concentration of extracted protein was determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). Cell lysates (15 μg of protein) were subjected to 13.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred electrophoretically onto polyvinylidene fluoride membranes (ATTO Corp., Tokyo, Japan) at 200 mA for 1.5 h. The blots were blocked with 5% bovine serum albumin in TBS-T (20 mM Tris; 137 mM NaCl, pH 7.4; 0.05% Tween® 20) at 4 °C for 1.5 h, and then incubated with anti-CD44 or β-actin primary antibodies at 4 °C overnight. After washing the blots, the blots were subsequently incubated with an appropriate secondary antibody. Protein bands were detected using an ECL kit (ATTO Corp.), and the band intensities were quantified using the Quantity One software (Bio-Rad, Hercules, CA, USA).
2.4. In vitro drug release assay In vitro release of DTX from Taxotere®, SLN, and HA-SLN was evaluated by the dialysis method. Taxotere® is mainly composed of ethanol and Tween® 80 to increase the solubility of DTX. Briefly, 1 mL of Taxotere® solution (40 μg/mL, diluted in PBS), 1 mL of DTX-loaded SLN dispersion (40 μg/mL, diluted in PBS), and 1 mL of HA-SLN dispersion (40 μg/mL, diluted in PBS) were added to a dialysis bag, separately. Next, the dialysis bags were incubated with 20 mL of release medium (PBS 0.5% containing Tween® 80, pH 7.4) at 37 °C with stirring at 100 rpm. At predetermined time intervals, 1 mL of the dialysate was removed, and replenished with the same volume of release medium. Amount of DTX in dialysate samples was determined by the HPLC method, as described in section 2.3. 2.5. Western blotting analysis Human breast adenocarcinoma cells, MCF7, MCF7/ADR, and MDAMB-231 cells, were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified incubator supplied with 5% CO2. Cells were washed twice in ice-cold PBS and lysed with
2.6. Cellular uptake studies For quantitative estimation of uptake, cells were seeded in 12-well plates at a density of 2 × 105 cells/well. Upon reaching 70–80% 368
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normalized to total protein content. Confocal microscopy was also performed to study the cellular uptake of SLN and HA-SLN in MCF7, MCF7/ADR, and MDA-MB-231 cells. In detail, 5 × 105 cells/well were cultured in 12-well plates containing a sterilized glass coverslip at the bottom of each well, and then incubated overnight. The cells were treated with 0.1 μg/mL coumarin 6loaded SLN or HA-SLN for 2 h. Next, the cells were washed three times with PBS and then fixed with 4% paraformaldehyde for 15 min. The glass coverslip with attached cells was then removed from the well and placed on a glass microscope slide in the presence of mounting agent. Confocal analysis was performed on an LSM5 Live Configuration Variotwo VRGB (Zeiss, Oberkochen, Germany). 2.7. In vitro cytotoxicity studies The cytotoxicity of Taxotere®, drug-loaded lipid nanoparticles, and unloaded lipid nanoparticles was determined against MCF7, MCF7/ ADR, and MDA-MBA-231 cells. MTT is a yellow compound that is reduced in the mitochondria of viable cells to a blue formazan product with a maximum absorbance at 570 nm. Breast cancer cells were seeded at 1 × 104 cells/well in 96-well plates. After overnight incubation, the cells were exposed to the formulations in medium for 24 h. Next, the medium was removed and 100 μL of MTT-containing medium (5 mg/ mL) was added to the wells. Following 4 h of incubation at 37 °C, the MTT-containing medium was aspirated carefully to avoid disturbing any formazan crystals that had formed, and 100 μL DMSO was added to each well. The plates were incubated at ambient temperature for 30 min and absorbance at 570 nm was measured using a microplate reader (Sunrise™; Tecan Trading, Switzerland). Cell viability was expressed as a percentage of the untreated control cells. 2.8. Statistical analysis Statistical analysis was performed by the paired t-test using SigmaPlot (ver. 12.5; SYSTAT, Inc., Chicago, IL, USA). Data are expressed as means ± standard deviation (s.d.). In all analyses, p < 0.01 (**) and p < 0.05 (*) indicated statistical significance. 3. Results and discussion 3.1. Characterization of HA-coated lipid nanoparticles HA-SLN were prepared using electrostatic attraction between the negatively charged HA and the cationic surfactant, CTAB. In this study, high-molecular-weight HA (∼100 kDa) was selected in accordance with a report that high-molecular-weight HA-coated particles are more effective against cancerous cells expressing CD44 [16]. The schematic illustration is shown in Scheme 1. The physicochemical characteristics of the lipid carriers are summarized in Table 1. The particle size of the plain lipid nanoparticles were approximately 100 nm, whereas those of the HA-coated formulations tended to increase. The polydispersity index of HA-SLN was lower than that of SLN, because the electronic interaction between HA and the cationic surface might produce a more compact structure of lipid nanoparticles [17]. To confirm the results of DLS, TEM was performed. TEM images show nano-sized particles that were consistent with the DLS results (Fig. 1). The HA coating was also observed through TEM images. SLN showed a spherical shape, and HA corona surrounding SLN was observed in HA-SLN (Fig. 1). It was confirmed that HA could well coat the cationic SLN through electrostatic coupling. The results of comparative analysis of zeta potentials before and after HA coating showed that the positive surface charge (32.5 ± 3.67 mV) of SLN had changed to negative (−17.1 ± 0.73 mV) after HA coating. Plain lipid nanoparticles possess CTAB cationic surfactants in their structures, which have positive charges, imparting a net positive charge to the plain lipid particles.
Fig. 6. Cytotoxicity of DTX-loaded formulations against (a) MCF7, (b) MDAMB-231, and (c) MCF7/ADR cells. Bars represent means ± s.d. (n = 6).
confluence, the cells were incubated with coumarin-6-loaded SLN and HA-SLN (0.1 μg/mL coumarin-6). After incubation for 0.5, 1, or 2 h, the cells were washed twice with cold PBS. Subsequently, the cells were lysed through addition of Triton™ X-100 (0.5%, w/v). Fluorescence intensities were measured using a multimode microplate reader (Synergy H1 Hybrid Multi-Mode Microplate Reader; BioTek, Winooski, VT, USA) at excitation and emission wavelengths of 440 and 520 nm, respectively. Protein was quantified using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific), and fluorescence intensities were 369
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3.4. Cellular uptake
After coating SLN with HA, the surface charges of the lipid nanoparticles became negative because HA molecules have negatively charged carboxylic acid groups in their structure. This change in the surface charge of HA-SLN showed that HA molecules are bound to the surface of plain lipid nanoparticles [18]. To characterize the drug–lipid interactions, DSC was performed for lipid alone, drug alone, formulations, and other excipients. As shown in Fig. 2, the DSC thermogram of stearic acid shows a sharp melting peak at 71 °C. Moreover, CTAB has melting peaks at 105 °C and 269 °C, and DTX exhibits a melting peak at 170 °C. However, no peaks for DTX are shown in the thermograms of the formulations, indicating that DTX, being molecularly dispersed within the solid dispersion, does not show crystallinity [19]. Many studies have reported that HA coating enhances the stability of nanoparticles [20–22]. In this study, the mean particle size and polydispersity of SLN and HA-SLN were evaluated to investigate the effect of HA coating on the stability of SLN. The size and polydispersity index (PdI) of SLN slightly increased from 109.5 ± 8.21 to 128.5 ± 15.84 nm after 4 weeks. In contrast, the mean particle diameter and the degree of dispersion of HA-SLN were maintained steadily during the observation period, as shown in Fig. S1 (see supplementary information). Therefore, coating with natural hydrophilic polymers appeared to enhance nanoparticle stability in aqueous solution.
In this study, tumor cells expressing CD44 were used. Two control groups were used for comparison, MDA-MB-231 cells (highly expressing CD44, as positive controls) and MCF7 cells (barely expressing CD44, as negative controls). To evaluate the intracellular uptake of the formulation and the role of CD44 expression on the cell surface, coumarin 6 was encapsulated in the nanoparticles and its fluorescence intensity was measured by fluorescence and confocal microscopy. There was no significant difference in uptake between SLN and HASLN in MCF7 cells (Fig. 5). However, the uptake of HA-SLN in MCF7/ ADR and MDA-MB-231 cells was higher than that of SLN. This could occur because CD44 receptor-mediated endocytosis significantly increased the cellular uptake of HA-coated nanocarriers. Therefore, in MCF7 cells, which express few CD44 receptors, the uptake of both formulations (i.e., SLN and HA-SLN) was expected to be similar. After the addition of free HA to the culture medium, the uptake of HA-SLN dropped to the level observed with SLN in MDA-MB-231 cells. In MCF7/ ADR cells, the uptake of HA-SLN decreased, but it was still higher than that of SLN. The reduction could be due to the competitive binding of free HA molecules to CD44, which inhibits CD44 receptor-mediated nanoparticle endocytosis on the surface of MCF7/ADR and MDA-MB231 cells. MCF7/ADR cells express diverse MDR proteins, including MDR1 [25]. These resistant proteins will inhibit cellular uptake of the nanoparticles, but the HA target moiety could overcome this resistance and increase the accumulation of the drug that has been encapsulated in lipid nanoparticles.
3.2. In vitro drug release The DTX release profiles of SLN and HA-SLN are shown in Fig. 3. There was a burst release within 4 h, with nearly 40% of DTX was released from the lipid formulations. This probably occurred a significant amount of drug is distributed in the outer shell of nanoparticles [5]. The total amount of DTX released from SLN after 24, 48 and 72 h was higher than that from HA-SLN (58.9 ± 3.4% and 64.0 ± 3.8% after 24 h; 65.3 ± 1.4% and 59.3 ± 2.0% after 48 h; 68.1 ± 1.2% and 62.8 ± 1.5% after 72 h, respectively). There was no statistically significant difference in the cumulative amount from DTX released from nanoparticles after 24 h (p < 0.5; paired t-test), but there were significant differences in those after 48 and 72 h (p < 0.05 and p < 0.01, respectively; paired t-test). The profiles showed that HA prolonged the release of DTX from SLN. The HA coating could decrease the permeability of SLN. Moreover, HA molecules produce a dense hydrophilic matrix around lipid nanoparticles, which decreases the release rate of a hydrophobic drug [17,23].
3.5. Cytotoxicity The dose–response curves for Taxotere®, drug-loaded formulation, and unloaded formulation are shown in Fig. 6 and Fig. S2 (see supplementary information). As many previous researchers have reported, cationic particles have much more toxic to cells [26]. Similar results were obtained in this study. Blank SLN showed greater cytotoxicity against breast cancer cells than other blank nanoparticles (Fig. S2). As mentioned above, the SLN are surrounded by CTAB and the surface is positively charged. Because of the role of the cationic surfactant, it would have caused more damage to the cells. In contrast, blank HA-SLN were almost completely nontoxic (Fig. S2). The HA coating might prevent the cationic surfactants in the lipid nanoparticles from directly contacting the cells. In addition, HA is well known to be biocompatible and nontoxic natural polymer. For this reason, it seems that the blank SLN shows little toxicity against breast cancer cells. HA-SLN (73.4 ± 9.4 and 86.1 ± 4.4%) exhibited the higher cytotoxicity than SLN (45.0 ± 5.4 and 56.5 ± 3.4%) and Taxotere® (17.6 ± 4.3 and 27.7 ± 8.9%) against MCF7/ADR and MDA-MB231 cells at a DTX concentration of 10 μg/mL (paired t-test; p < 0.05) (Fig. 6b and c). This result is in agreement with the results of the uptake studies. HA-SLN have a high binding affinity to CD44 receptors; therefore, the specific recognition of HA-SLN by CD44 surface receptors results in CD44-mediated endocytosis. HA-modified nanoparticles have been studied as targets for delivering chemotherapeutic agents to cancer cells to overcome MDR and reduce effective dose [20]. In this study, it also could be concluded that the active targeting of DTX carriers by HA ligands to CD44 receptors leads to a higher cytotoxic activity and selectivity toward MCF7/ADR and MDA-MBC-231 cells.
3.3. CD44 expression Recent studies have reported the association of drug resistance with HA. HA and CD44 play an important role in resistance to anticancer drugs [9,12,16]. CD44 increases in many malignant and resistant cancers, and this can be categorized as a functional strategy used by cancer cells for self-preservation. Increased CD44 on the cell surface increases HA levels, thereby increasing the flow pressure and decreasing the inflow of foreign substances [16]. In addition, CD44 can enhance the drug resistance of cancer cells by protecting P-glycoprotein [24]. To compare the effect of HA-SLN on breast cancer cells, we measured the expression of CD44 on four cell lines. The expression level of CD44 in each breast cancer cell is shown in Fig. 4. MDA-MB-231 cells (118.25 ± 2.44%) are well-known to highly express CD44 protein which was confirmed in the results of our study. CD44 expression was approximately 7-fold higher in MCF7/ADR cells (1.34 ± 0.24%) than in MCF7 cells (9.48 ± 0.44%). This result in MCF7/ADR cells also showed that CD44 expression levels are elevated for cell defense. This leads to the expectation that HA-containing formulations will be effective in targeting resistant cancer cells.
4. Conclusion In this study, a DTX-containing, HA-coated lipid formulation was prepared and characterized. HA-SLN were nano-sized particles that had higher aqueous stability than uncoated SLN over 4 weeks. The targeting moiety of HA-SLN was evaluated with the CD44-expressing breast cancer cells, MCF7/ADR and MDA-MB-231. HA-SLN showed higher uptake and cytotoxicity against CD44-expressing cell lines than 370
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untargeted lipid formulation. HA-SLN also worked effectively, especially against drug-resistant MCF7/ADR cells owing to the HA-target moiety. Therefore, HA-SLN could be a potent drug delivery system that can achieve both target and overcome drug resistance.
[11]
[12]
Declarations of interest [13]
None.
[14]
Acknowledgement [15]
This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Korea government, MSIP (No. 2015R1A2A1A10051596) and by the Chung-Ang University Graduate Research Scholarship in 2017.
[16]
[17]
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jddst.2019.01.042.
[18]
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