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Chitosan-coated magnetic solid lipid nanoparticles for controlled release of letrozole
Journal of Drug Delivery Science and Technology 57 (2020) 101621
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Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Chitosan-coated magnetic solid lipid nanoparticles for controlled release of letrozole
T
Zeynab Ahmadifarda, Ahmad Ahmedab, Mahsa Rasekhiand, Sajad Moradic, Elham Arkanc,∗∗ a
Student Research Committee, Kermanshah University of Medical Sciences, Kermanshah, Iran Department of Basic Medical Sciences, College of Medicine, QU Health, Qatar University, Doha, Qatar c Nano Drug Delivery Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran d Pharmaceutical Sciences Research Center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Chitosan Letrozole Solid lipid nanoparticles Magnetic drug delivery Low frequency pulsed magnetic field Controlled release
In present study, magnetic solid lipid nanoparticles (MSLNs) with chitosan (CHI) coating were defined, and the rapid delivery of Letrozole (LTZ) to cancer cells by both active and passive targeting were evaluated. LTZ loaded MSLNs were fabricated using the modified solvent evaporation-ultrasonic combination method. The physicochemical parameters were optimized to achieve a suitable drug delivery system. Afterward Fourier transform infrared spectroscopy (FTIR), differential scanning calorimeter (DSC) and X-ray diffraction (XRD) used as characteristic analyses. DPPH free radical scavenging test was carried out to evaluate the antioxidant capacities of LEC (Lecithin), MSLNs, LTZ, LTZ + MSLNs, and BHT (Butylated hydroxytoluene). DPPH test indicated similar antioxidant activities for LTZ + MSLNs and BHT. For the determining of anti-breast cancer potentials of LEC, MNPs, LTZ, and LTZ + MSLNs, MTT assay was used on the normal cell line (HUVEC), breast adenocarcinoma (MCF7), breast carcinoma (Hs 578Bst), infiltrating ductal cell carcinoma (Hs 319.T), infiltrating lobular carcinoma of breast (UACC-3133), inflammatory carcinoma of the breast (UACC-732), and metastatic carcinoma (MDA-MB-453) cell lines. The desired and favorable particle size and surface charge nanocarrier for targeting cancer cells was obtained. The effect of LFPMF on drug release of the drug delivery system distinctly demonstrated. The findings showed that MSLN loaded with LTZ increased chemotherapy efficiency. LTZ encapsulated MSLN can act as a suitable and promising candidate for targeted and controlled therapy for cancer.
1. Introduction Though it is done advances in cancer treatment recently, it is still one of the most mortal diseases that annually capture the lives of millions of people around the world [1] [1] [1]. Chemotherapy is one of the common procedures for cancer treatment, which due to their nonspecificity and in the case of non-targeted delivery systems the applying drugs cause severe side effects on healthy cells and body. Therefore, the development of targeted and controlled drug release systems has recently become more important in clinical research. In this case, nanocarrier systems are suggested that in contrast to conventional chemotherapy, offer many unique properties, like as enhanced permeability and retention (EPR), improved delivery of insoluble drugs, drug protection from harsh environments, controlled release of drugs, and so on [2,3]. Solid lipid nanoparticles (SLNs) are nano drug delivery systems whose outstanding properties including the possibility of controlled
∗
release and drug targeting, good compatibility, high drug loading capacity, high physical stability, the ability to protect unstable drugs from degradation, not using of toxic solvent during the synthesis of these nanoparticles and the ease of production on a large scale are considerable. In fact, SLN is an alternate carrier system for conventional colloidal carriers such as liposomes, emulsions, and polymeric nanoparticles. In order to increase the uptake rate and improve the SLN delivery efficiency, surface modification and bio-polymer coating have recently been used [4–7]. Chitosan is naturally cationic copolymers composed of N-acetyl glucosamine and D-glucosamine in a random and because of its unique properties such as lack of toxicity, biocompatibility, and high biodegradability, increased bio adhesive and permeability properties and anti-oxidant activity are widely used in drug delivery systems and the transfer of anticancer agents [8–10]. Iron oxide nanoparticles have advantages including low toxicity, great biocompatibility, excellent magnetic properties and the reactive
Corresponding author. E-mail addresses: [email protected], [email protected] (E. Arkan).
https://doi.org/10.1016/j.jddst.2020.101621 Received 24 September 2019; Received in revised form 5 January 2020; Accepted 23 February 2020 Available online 28 February 2020 1773-2247/ © 2020 Elsevier B.V. All rights reserved.
Journal of Drug Delivery Science and Technology 57 (2020) 101621
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Scheme 1. A schematic of the step-by-step preparation of LTZ-loaded magnetic solid lipid nanoparticles with chitosan coating; Abbreviations: magnetic nanoparticles (MNPs), dichloromethane (DCM), Dioctyl sulfosuccinate sodium salt (AOT) and magnetic solid lipid nanoparticles (MSLNs).
2.2. Preparation of magnetic nanoparticles coated with lecithin
level that can be easily reformed with biocompatible coatings, and because of these properties, they can be utilized as therapeutic and diagnostic agents. In fact, the magnetic delivery system is targeted by using magnetic nanoparticle carriers by magnetic fields to place them into the tumor, and then the drug can be released by alterations in physiological stimulants such as pH and temperature or enzymatic acting from the carriers and uptaking by tumor cells. Magnetic nanoparticles tend to be agglomerated, so these nanoparticles are generally coated with polymers or surfactants to reduce particle agglomeration [10–13]. Letrozole is one of the most effective third-generation competitive inhibitors of aromatase enzyme that prevents excessive biosynthesis of estrogen in the body. The drug has serious side effects such as flushing, breast tenderness, and headache in addition to needs to frequent administration because of short half-life. For this reason, it is suitable for a targeted and controlled drug delivery system to increase the therapeutic efficacy and reduce the harmful effects of LTZ [14,15]. In the present study, using magnetic nanoparticles, a spatiotemporal controlled nanocarrier of chitosan-coated solid lipid nanoparticles have been developed by solvent evaporation-ultra sonication technique for LTZ delivery. The characterization of the produced Nano carriers was performed by PCS, FTIR, SEM, XRD and DSC and their cytotoxicity were investigated by MTT test. The encapsulated efficiency of the magnetic delivery system and the effect of magnetic nanoparticles on the drug release profile were studied also in vitro.
Magnetic nanoparticles were synthesized by the standard method of chemical co-precipitation [16]. Briefly, a mixture of iron salts II (FeCl2.4H2O, 1 g) and iron salts III (FeCl3.6H2O, 3 g) was prepared in 50 mL of distilled water under the nitrogen condition. During the stirring, the temperature was brought to 75 °C and the NaOH solution was slowly added to the reaction in order to adjust the pH = 10. By increasing the pH, the color of the solution became black that indicating the formation of magnetic nanoparticles. The nanoparticles were collected with a permanent magnet followed by several times of washing with deionized water. To prepare the magnetic nanoparticles coated with lecithin [17], the obtained nanoparticles were poured into a lecithin solution (50 mg lecithin in 5 cc chloroform) and stirred for 1 h. In order to ensure the lecithin is coated around the magnetic nanoparticles, a mixture of chloroform: water (50%:50%) was used to separate the coated particles from uncoated ones.
2.3. Preparation of magnetic solid lipid nanoparticles (MSLNs) Solid lipid nanoparticles loaded with LTZ and hydrophobic magnetic nanoparticles were prepared by novel combined methods of solvent evaporation-ultrasonic [18]. Lipids (stearic acid (5 mg), tripalmitin glycerol (5 mg)), letrozole (6 mg), lecithin-coated magnetic nanoparticles (2 mg) and AOT (as a surfactant) were dissolved in 1 mL of Dichloromethane. The lipid phase was then well dispersed in the aqueous phase (20 mL distilled water) using a probe sonicator (Sonopuls Ultrasonic Homogenizer HD 2070, Berlin), for 4 min. Then for preparation of solid lipid nanoparticles, the organic solvent was evaporated by stirring for 45 min at a speed of 900 rpm. In order to better homogenization, SLNs were sonicated by bath sonication for 4 min. Anionic solid lipid nanoparticles were easily coated with cationic polymer of chitosan [19]. Chitosan solution (0.1%, W/V) was prepared in distilled water. The prepared solid lipid nanoparticles were gradually added to the polymer solution and continued by slow stirring for half an hour (Scheme 1).
2. Material and methods 2.1. Materials Letrozole was obtained from Iran Hormone Pharmaceutical Co. (I. R. Iran). Glycerol tripalmitate (TPG) was provided by Alfa Aesar (Germany). CHI (low molecular weight, by the degree of deacetylation ≥ 75%), stearic acid (SA), soy-lecithin, Dioctyl sulfosuccinate sodium salt (AOT), FeCl3. 6H2O and FeCl2. 4H2O were purchased from Merck Co. (Darmstadt, Germany). Other reagents and chemicals were of the analytical grade available. 2
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2.4. Characterization of MSLNs
DPPH radical was calculated using the following formula:
The DLS method was used for determination of the size, polydispersity index (PDI) and zeta potential of nanoparticles by nanosizer instrument (Malvern, England). Precise size measurement of nanoparticles was done using scanning electron microscopy (SEM) (MIRA3TESCAN-XMU microscope, Germany). The FTIR spectra in the range of 400–4000 cm−1 were scanned by a Shimadzu IR2000 (Japan) and a scan rate of 8/cm for 100 scans. The DSC thermograms were shown at 10 °C to 240 °C with the temperature ramp of 10 °C/min by DSC tool (SDT Q600, TA, and USA). The XRD pattern of CHI-SLNs was scanned from 10 to 80° (2 θ) with a step size of 0.05° and a time per step of 1 s using X-ray diffractometer (PW1730, 40 kV, 30 mA, Philips, Netherlands).
I%=
where I = DPPH inhibition (%), A0 = absorbance of control sample (t = 0 h) and A = absorbance of a tested sample at the end of the reaction (t = 1 h). The amount of sample necessary to decrease the absorbance of DPPH by 50% (IC50) was calculated graphically for LEC, MSLNs, LTZ, and LTZ + MSLNs in different concentrations [20]. 2.9. Measurement of cell toxicity In this experiment, the following cell lines have been used for investing the cytotoxicity effects of the LEC, MSLNs, LTZ, and LTZ + MSLNs using an MTT assay:
2.5. Determination of the encapsulation efficiency (EE) and drug loading (DL)
a) b) c) d) e) f) g)
The concentration of LTZ was measured by Ultra Violet (UV) spectrophotometry at a wavelength of 240 nm. The content of free drugs exists in the supernatant obtained by centrifugation (20 min, 15,000 rpm) was detected. The EE and DL was performed using the following equations:
EE (%) =
Wt − Wf × 100 Wt
DL (%) =
Wt − Wf × 100 Ws
A0 − A × 100 A0
Normal cell line: HUVEC. Breast adenocarcinoma cell line: MCF7. Breast carcinoma cell line: Hs 578Bst. Infiltrating ductal cell carcinoma cell line: Hs 319.T. Infiltrating lobular carcinoma of breast cell line: UACC-3133. Inflammatory carcinoma of the breast cell line: UACC-732. Metastatic carcinoma cell line: MDA-MB-453.
(1) These cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% (w/v) FBS, 100U/mL penicillin, and 100 μg/mL streptomycin. Then, cells were distributed at 10,000 cells/well in 96well plates. The cells were grown under a humidified incubator with 5% CO2 at 37 °C until reaching confluency (typically after 24 h). The cells were treated with LEC, MSLNs, LTZ, and LTZ + MSLNs at concentrations of 0, 1, 2, 3, 7, 15, 31, 62, 125, 250, 500, and 1000 μg/mL and subsequently incubated for 2 and 24 h. LEC, MSLNs, LTZ, and LTZ + MSLNs were sterilized using UV radiation for 1 h. Finally, the MTT solution (5 mg/mL in PBS) was added to each well and incubated for 4 h at 37 °C. The medium with MTT was removed and the formazan crystals formed in the living cells were dissolved in 100 μL DMSO per well. All tests were run in the triplicates. The relative viability (%) was calculated based on the absorbance at λ = 570 nm determined using a microplate reader [21].
(2)
where the Wt, Wf and Ws are the initial amount, the free drug measured in the supernatant, and the total weight of the nanoparticulate system respectively. 2.6. Investigation of drug release The release profile of Nano delivery system was evaluated using a dialysis bag with the cut off of 3500 KD. The samplings were done in different time intervals up to 3 days. A low frequency pulsed magnetic field (LFPMF) of 50 Hz was applied to drug loaded MSLNs. 2.7. Applying magnetic field
Percentage of cell viability (%) = (Sample absorbance/Control absorbance) × 100
A copper coil with a lacquered coating and 900 rounds around the iron core was used to generate a 50 Hz magnetic field. An alternating current adapter device was used to generate a sine wave with pulse width 5 ms. The field strength was measured by a Teslameter device (MG-3003SD Lutron, Japan) at about 8 mT. For the drug release test, polymethyl methacrylate bases were used, to fix the coil at a specified distance from the dialysis bag, and then the magnetic field was performed.
The half-maximal cell viability concentration (IC50) was calculated by linear regression analysis and expressed as a mean of three determinations. 2.10. Data management and statistical analysis The data on absorbance measures were entered in the Microsoft® Excel spreadsheet one-word program, where it was organized and then exported to Minitab statistical software for analysis. This was found to conform to assumptions of parametric data and expressed as Means ± Standard Deviations (SD). All statistical analyses were performed using Minitab (Minitab 17.1 Version, NC, USA). The analyzed data was presented in Figures.
2.8. Assessment of the antioxidant activity The antioxidant activity was measured in terms of hydrogen donating or radical scavenging ability using the stable radical DPPH. Experiments were carried out according to the method of previous studies [20] with a slight modification. The reduction of the radical is followed by a decrease in the absorbance at 517 nm. A volume of 2 mL of LEC, MSLNs, LTZ, and LTZ + MSLNs was put into test tubes and 2 mL of 1 mM DPPH solution was added. The tubes were covered with parafilm and kept again in the dark for 1 h. Absorbance at 517 nm was measured with a spectrophotometer UV–Vis and compared to an (Butylated hydroxytoluene (BHT)) calibration curve. A mixture of methanol and DPPH solution served as a blank while a reaction mixture of methanol, DPPH and standard BHT served as the positive control. Each assay was carried out in triplicate. The percentage inhibition of the
3. Results and discussion 3.1. MSLNs characterization Herein a chitosan-coated magnetic nano solid lipid carrier containing the anticancer drug of LTZ was prepared and was characterized. Its release kinetic along with anticancer activity was evaluated. The size and distribution of particle sizes are the main parameters 3
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Table 1 Physicochemical characteristics and dynamic light scattering data of uncoatedSLN and coated-SLN loaded with LTZ. Formulations
Size (nm)
Charge (mV)
PDI
EE%
DL%
Uncoated SLN CHI-LTZ-SLN
158.2 193.2
42.520.2-
0.188 0.200
– 90.1
– 26.20
CHI: Chitosan; LTZ: Letrozole; SLN: Solid lipid nanoparticle; Drug loading; EE: Encapsulation efficiency; PDI: Polydispersity index.
DL:
for the stability of the system and passive targeting of therapeutic agents into tumors with taking advantages of EPR effect [20]. As shown in Table 1, the hydrodynamic particle sizes of chitosan uncoated and coated MSLN nanoparticles were achieved 158.2, 193.2 nm and PDI value of 0.188 and 0.200 respectively. The results were indicated that these particles are suitable for accumulation in the target tumor tissue. A low PDI of the system was proved a good size monodispersity which assures the nanoparticle solution stability (Table 1). Researches have shown that nanoparticles with the negative charge tend to stay longer in bloodstream than nanoparticles positively charged and their clearance by the immune system is slower [21–23]. Negative nanoparticles showed less cytotoxicity as well [21]. Therefore, in the synthesis of formulation, we used a smaller amount of chitosan to have both suitable particle size and negative surface charge with the help to nanoparticles become hydrophilic. The zeta potential of uncoated and coated nanoparticles with chitosan was −42.5 and −20.2, respectively. The observed difference in charge of uncoated and coated nanoparticles indicates that a thin layer of chitosan has covered the SLN nanoparticles. However, the chitosan was added to the delivery system and lead to hydrophilic, reducing the removal of macrophage and decrement negative charge of lipid nanoparticles. The high negative charge of nanoparticles act as a barrier during passing through the vessels and entering the cells. Chitosan-coated nanoparticles were used for other analyzes. The image of SEM of CHI-MSLN was showed the precise particle size of the drug loaded nanocarrier that is about 50 nm (Fig. 1). Some differences between the SEM and DLS results are related to the differences in hydrodynamic diameter and those of the particle itself. The FTIR spectrum shown in Fig. 2 accurately demonstrates that chitosan has covered MSLN loaded with LTZ. According to the chitosan index peaks in wavelengths 3437, 2854 and 2920, 1639, 1103 cm−1 related to O–H and N–H stretch, C–H stretch, C–N–H of amide I and C–O–C vibration respectively that exist in the CHI-MSLN sample, it confirms the coating of nanoparticles with chitosan as can be compared
Fig. 2. The FT-IR spectra of lipids, chitosan, MNPs, CHI-MSLNs loaded with LTZ.
with chitosan spectrum. Several peaks of the lipids in the MSLN structure are observed at wavelengths of 1562 and 1415 and 1694 cm−1 affiliated to C]O stretching bands, shifting these peaks to the right can be due to interactions of lipids with chitosan. Since the LTZ characteristic peak in the wavelength 2250 cm−1 hasn't appeared, it indicates that LTZ has been entirely encapsulated into the nanoparticles [24]. The same result was obtained for MNPs with lecithin coating due to the index peak decay of MNPs in wavelength of around 600 cm−1 [25]. The DSC is a standard method to obtain detailed information about any changes in the system's enthalpy become from any endothermic or exothermic phenomenon take place in the system. Then the information can be interpreted to achieve some viewpoint about for example changes in molecular and supramolecular conformation or interactions etc. There are an exothermic and two broad endothermic peaks placed in 40, 67 and 133 °c respectively (Fig. 3). The first sharp exothermic peak in 40 °c is related to movement and interactions of residual water molecules with the nanoparticles [26]. The peak present in 67 °c according to literatures belongs to the phase transition of lipids in their melting point [27]. The latest and broadest peak can be related to corrosion of chitosan from the surface of SLNs. The results also confirm the good stability of the system in conditions close to body temperature
Fig. 1. Scanning electron microscopy (SEM) image of CHI-MSLNs. 4
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Fig. 3. Differential scanning calorimetry (DSC) thermogram of CHI-SLN. Fig. 6. The antioxidant properties of LEC, MSLNs, LTZ, LTZ + MSLNs, and BHT against DPPH. LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles. BHT: Butylated hydroxytoluene.
regarding the Bragg's law which points that the proximate sheets the higher degrees of 2θ can be because of existence of some pressure on crystals and confirming SLN coating by chitosan. A peak in 2θ of 32° belongs to magnetic nanoparticles and due to its strong crystal structure there is no significant shift in it. 3.2. In vitro drug release study The characteristic of cancer cells whose extracellular pH is more acidic than the pH of healthy cells can be used for target delivery system. PH-sensitive polymers are an adequate choice for such a purpose and many studies have been done on them [30]. Chitosan has been extensively investigated in pH-sensitive drug delivery systems due to the protonation of amine groups in the acidic pH, which is in its main chain [3,19]. According to the results of other researchers, the release of the drug was more stable and faster at acidic pH, so in this work, a pH of 6 for the release medium was used. Since pH of the initial endosomes is acidic, the above nano carrier system can be used for intracellular drug delivery [19,31]. The magnetic nanoparticles in nano drug delivery systems are
Fig. 4. X-ray diffractogram of CHI-MSLNs.
and storing in room temperature. The X-Ray Diffraction results confirm lipid crystallization and chitosan coating on MSLNs. As can be seen in Fig. 4, there are several peaks in 2θ of 17°, 20°, 30° which according to previous literature are related to the orthorhombic lamellar lattice structure of lipid's hydrocarbon tail [28]. Also compare to Lukowski et al. reported [29], a distinct right shift in the related peaks has observed. This shifts
Fig. 5. Drug release profile of CHI-MSLN at (A) non-LFPMF, (B) applied LFPMF. (n = 3). 5
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Table 2 The IC50 of LEC, MSLNs, LTZ, LTZ + MSLNs, and BHT in antioxidant test.
IC50 against DPPH
LEC (μg/mL)
MSLNs (μg/mL)
LTZ (μg/mL)
LTZ + MSLNs (μg/mL)
BHT (μg/mL)
–
576
342
198
251
LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles. BHT: Butylated hydroxytoluene.
Fig. 7. The cytotoxicity properties of LEC, MSLNs, LTZ, and LTZ + MSLNs against HUVEC cell line. LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles. HUVECs: Human umbilical vein endothelial cells.
Fig. 9. The anti-breast cancer properties of LEC, MSLNs, LTZ, and LTZ + MSLNs against breast carcinoma (Hs 578Bst) cell line. LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles.
Fig. 10. The anti-breast cancer properties of LEC, MSLNs, LTZ, and LTZ + MSLNs against infiltrating ductal cell carcinoma (Hs 319.T) cell line. LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles
Fig. 8. The anti-breast cancer properties of LEC, MSLNs, LTZ, and LTZ + MSLNs against breast adenocarcinoma (MCF7) cell line. LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles.
steer and momentum force on the nanoparticle within the nanocarrier, it exerts pressure on the drug and expels it from the system. Typically, in magnetic delivery systems, an alternating and highfrequency magnetic field is used to increase the local heating of magnetic nanoparticles. Consequently, by increasing the temperature and eddy current induced in the magnetic field, can damage healthy cells.
important in several aspects. On one hand, using a constant magnetic field at a specific point of the body, the nanosystem could be accumulated there. Actually, by applying alternating magnetic fields either high frequency for heat generation or low frequency for mechanical pressure, it can trigger the drug release from the system. After inducing 6
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Fig. 11. The anti-breast cancer properties of LEC, MSLNs, LTZ, and LTZ + MSLNs against infiltrating lobular carcinoma of breast (UACC-3133) cell line. LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles.
Fig. 13. The anti-breast cancer properties of LEC, MSLNs, LTZ, and LTZ + MSLNs against metastatic carcinoma (MDA-MB-453) cell line. LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles.
delivery system, the drug can be targeted to the desired location by a controlled and successful release and transfer a large amount of the drug to the cancer cell over a short period of time. It should be noted that the applied LFPMF value was low, meanwhile, with a favorable result. The LFPMF was eventually applied for 1 h. In fact, LFPMF cannot enhance enough temperature as much as system degradation. Herein, the temperature of the system was measured after applying 1 h of LFPMF and it was specified that the temperature increased only 1–1.5 °c.
3.3. Antioxidant assay Humans have evolved a complex and sophisticated antioxidant system to maintain the redox homeostasis and protect cells and organ systems against free radicals. Components of antioxidant defense systems are of either exogenous or endogenous in origin which functions synergistically and interactively to neutralize free radicals. These components include dietary antioxidants like ascorbic acid (vitamin C), tocotrienols (vitamin E), glutathione, β-carotene, uric acid, and α-tocopherol [20]. The antioxidant enzymes such as catalase, glutathione reductase, superoxide dismutase, and glutathione peroxidase also catalyze free radical scavenging activities. Other antioxidant components include metal-binding proteins (like lactoferrin, albumin, ceruloplasmin, and ferritin) which sequester free copper ions and/or free iron that catalyze oxidative reactions [20]. In the present experiment, LEC, MSLNs, LTZ, and LTZ + MSLNs similar to BHT indicated a notable concentration-dependent DPPH radical scavenging effect. The interaction between the MSLNs, LTZ, and LTZ + MSLNs and DPPH might have occurred through the transfer of electrons and hydrogen ions to 2,2-diphenyl-1-picrylhydrazyl radical to form a stable2,2-diphenyl-1-picrylhydrazine molecule (DPPH) [20]. The IC50 values of MSLNs, LTZ, LTZ + MSLNs, and BHT were 576, 342, 198, and 251, respectively (Fig. 6; Table 2). The antioxidant activity exhibited by MSLNs, LTZ, and LTZ + MSLNs can be attributed to the presence of various molecules that are thought to function interactively and synergistically to neutralize reactive oxygen species (ROS) and reactive nitrogen species (RNS) [20].
Fig. 12. The anti-breast cancer properties of LEC, MSLNs, LTZ, and LTZ + MSLNs against inflammatory carcinoma of the breast (UACC-732) cell line. LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles.
Therefore, low frequency pulsed magnetic fields (LFPMF) are used to increase the release of drugs from the system with mechanical movements and without increasing the temperature [32–34]. So we used the magnetic nanoparticles, not only accumulating the nanocarrier to the desired target site, but also by applying an LFPMF, causing a faster release of the drug without creating a significant change in temperature. The release of LTZ from the MSLN Nano carrier was investigated by the dialysis method in two states, applying an LFPMF and non-LFPMF [35,36]. As shown in Fig. 5, in the non-LFPMF state after 12 h, 50% of the drug is released, but by applying LFPMF the same amount of LTZ is released in 1 h. Given these results, the effect of the LFPMF on magnetic nanoparticles is clearly observable. Admittedly, utilizing this drug 7
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Table 3 The IC50 of LEC, MSLNs, LTZ, and LTZ + MSLNs in cytotoxicity test.
IC50 IC50 IC50 IC50 IC50 IC50 IC50
against against against against against against against
HUVEC MCF7 Hs 578Bst Hs 319.T UACC-3133 UACC-732 MDA-MB-453
LEC (μg/mL)
MSLNs (μg/mL)
LTZ (μg/mL)
LTZ + MSLNs (μg/mL)
– – – – – – –
– 687 551 469 448 356 754
– 345 332 311 265 236 399
– 221 215 208 170 161 247
LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles.
LFPMF during 1 h, which was faster and more controlled than the nonLFPMF applying conditions (the same amount of drug released during 12 h). DPPH free radical scavenging test was carried out to evaluate the antioxidant capacities of LEC, MSLNs, LTZ, LTZ + MSLNs, and BHT. DPPH test indicated similar antioxidant activities for LTZ + MSLNs and BHT that indicated a notable concentration-dependent DPPH radical scavenging effect. MTT assay was used on HUVEC, MCF7, Hs 578Bst, Hs 319.T, UACC-3133, UACC-732, and MDA-MB-453 cell lines and the best result of cytotoxicity property of LTZ + MSLNs against the above cell lines was seen in the case of the UACC-732 cell line. This magnetic lipid drug delivery system offers promising potential for the targeted and controlled treatment of chemotherapy agents-resistant cancers.
3.4. Cytotoxicity assay In the present study, the treated cells with various concentrations of the present LEC, MSLNs, LTZ, and LTZ + MSLNs were examined by MTT test for 48 h regarding the cytotoxicity property on the normal cell line (HUVEC), breast adenocarcinoma (MCF7), breast carcinoma (Hs 578Bst), infiltrating ductal cell carcinoma (Hs 319.T), infiltrating lobular carcinoma of breast (UACC-3133), inflammatory carcinoma of the breast (UACC-732), and metastatic carcinoma (MDA-MB-453) cell lines (Figs. 7–13; Table 3). The absorbance rate was determined at 570 nm, which revealed extraordinary viability on normal cell line (HUVEC) even up to 1000 μg/mL for LEC, MSLNs, LTZ, and LTZ + MSLNs. In the case of breast cancer cell lines, the viability of them decreased dose-dependently in the presence of LEC, MSLNs, LTZ, and LTZ + MSLNs. The IC50 of MSLNs, LTZ, and LTZ + MSLNs against MCF7 cell line were 687, 345, and 221 μg/mL, respectively; against Hs 578Bst cell line were 551, 332, and 215 μg/mL, respectively; against Hs 319.T cell line were 469, 311 and 208 μg/mL, respectively; against UACC-3133 cell line were 448, 265, and 170 μg/mL, respectively; against UACC732 cell line were 356, 236, and 161 μg/mL, respectively; against MDAMB-453 cell line were 754, 399 and 247 μg/mL, respectively. The best result of cytotoxicity property of LTZ + MSLNs against the above cell lines was seen in the case of the UACC-732 cell line. Probably the anti-breast cancer potentials of LTZ + MSLNs are related to their antioxidant activities. The previous studies have reported that antioxidant compounds such as nanomaterial's as single electron donors can stabilize and scavenge the free radicals, which in conditions of oxidative stress may begin angiogenesis or carcinogenesis [21]. Free radical-induced development of cancer involves malignant transformation due to DNA mutations and changed gene expression through epigenetic mechanisms which in turn causes the uncontrolled proliferation of cancerous cells [21]. Many researchers reported a significant role of antioxidant compounds such as medicinal plants and silver nanoparticles in growth inhibition of prostate, breast, ovary, endometrium, colon, and lung cancer cells with removing free radicals [21].
Funding This work was supported by the research council of Kermanshah University of Medical Sciences (Grant Number. 97720). CRediT authorship contribution statement Zeynab Ahmadifard: Writing - original draft, Investigation, Formal analysis. Ahmad Ahmeda: Formal analysis, Software. Mahsa Rasekhian: Supervision. Sajad Moradi: Writing - review & editing, Project administration. Declaration of competing interest The authors all confirm there is no conflict of interest. Acknowledgments The authors gratefully acknowledge the research council of Kermanshah University of Medical Sciences. This work was performed in partial fulfillment of the requirement for M.Sc. Nanomedicine of Zeynab Ahmadifard, in the faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran. References [1] D. Peer, J.M. Karp, S. Hong, O.C. Farokhzad, R. Margalit, R. Langer, Nanocarriers as an emerging platform for cancer therapy, Nat. Nanotechnol. 2 (12) (2007) 751. [2] I.I. Lungu, M. Radulescu, G.D. Mogosanu, A.M. Grumezescu, pH sensitive core-shell magnetic nanoparticles for targeted drug delivery in cancer therapy, Rom. J. Morphol. Embryol. 57 (1) (2016) 23–32. [3] G. Lv, L. Qiu, G. Liu, W. Wang, K. Li, X. Zhao, et al., pH sensitive chitosan-mesoporous silica nanoparticles for targeted delivery of a ruthenium complex with enhanced anticancer effects, Dalton Trans. 45 (45) (2016) 18147–18155. [4] Y. Luo, Z. Teng, Y. Li, Q. Wang, Solid lipid nanoparticles for oral drug delivery: chitosan coating improves stability, controlled delivery, mucoadhesion and cellular uptake, Carbohydr. Polym. 122 (2015) 221–229. [5] M. Zhou, J. Hou, Z. Zhong, N. Hao, Y. Lin, C. Li, Targeted delivery of hyaluronic acid-coated solid lipid nanoparticles for rheumatoid arthritis therapy, Drug Deliv. 25 (1) (2018) 716–722. [6] H. Yuan, J. Miao, Y.-Z. Du, J. You, F.-Q. Hu, S. Zeng, Cellular uptake of solid lipid nanoparticles and cytotoxicity of encapsulated paclitaxel in A549 cancer cells, Int.
4. Conclusion In the present study, a LTZ nano delivery system was formulated with an easy method for targeted and controlled release. The magnetic nanoparticles and LTZ were loaded into solid lipid nanoparticles with chitosan natural polymer coating. MSLNs with chitosan coating were successfully characterized so that the nanoparticles with size, PDI and surface charge of 193.2, 0.2 and −20.2, and with EE and DL of 90.1 and 26.2 were obtained respectively. FTIR analysis showed that coating of nanoparticles was done by chitosan. DSC and XRD analyses confirmed FTIR results. The release profile of the drug that was investigated comparatively, showed that 50% of the drug was exerted by applying 8
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Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Chitosan-coated magnetic solid lipid nanoparticles for controlled release of letrozole
T
Zeynab Ahmadifarda, Ahmad Ahmedab, Mahsa Rasekhiand, Sajad Moradic, Elham Arkanc,∗∗ a
Student Research Committee, Kermanshah University of Medical Sciences, Kermanshah, Iran Department of Basic Medical Sciences, College of Medicine, QU Health, Qatar University, Doha, Qatar c Nano Drug Delivery Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran d Pharmaceutical Sciences Research Center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Chitosan Letrozole Solid lipid nanoparticles Magnetic drug delivery Low frequency pulsed magnetic field Controlled release
In present study, magnetic solid lipid nanoparticles (MSLNs) with chitosan (CHI) coating were defined, and the rapid delivery of Letrozole (LTZ) to cancer cells by both active and passive targeting were evaluated. LTZ loaded MSLNs were fabricated using the modified solvent evaporation-ultrasonic combination method. The physicochemical parameters were optimized to achieve a suitable drug delivery system. Afterward Fourier transform infrared spectroscopy (FTIR), differential scanning calorimeter (DSC) and X-ray diffraction (XRD) used as characteristic analyses. DPPH free radical scavenging test was carried out to evaluate the antioxidant capacities of LEC (Lecithin), MSLNs, LTZ, LTZ + MSLNs, and BHT (Butylated hydroxytoluene). DPPH test indicated similar antioxidant activities for LTZ + MSLNs and BHT. For the determining of anti-breast cancer potentials of LEC, MNPs, LTZ, and LTZ + MSLNs, MTT assay was used on the normal cell line (HUVEC), breast adenocarcinoma (MCF7), breast carcinoma (Hs 578Bst), infiltrating ductal cell carcinoma (Hs 319.T), infiltrating lobular carcinoma of breast (UACC-3133), inflammatory carcinoma of the breast (UACC-732), and metastatic carcinoma (MDA-MB-453) cell lines. The desired and favorable particle size and surface charge nanocarrier for targeting cancer cells was obtained. The effect of LFPMF on drug release of the drug delivery system distinctly demonstrated. The findings showed that MSLN loaded with LTZ increased chemotherapy efficiency. LTZ encapsulated MSLN can act as a suitable and promising candidate for targeted and controlled therapy for cancer.
1. Introduction Though it is done advances in cancer treatment recently, it is still one of the most mortal diseases that annually capture the lives of millions of people around the world [1] [1] [1]. Chemotherapy is one of the common procedures for cancer treatment, which due to their nonspecificity and in the case of non-targeted delivery systems the applying drugs cause severe side effects on healthy cells and body. Therefore, the development of targeted and controlled drug release systems has recently become more important in clinical research. In this case, nanocarrier systems are suggested that in contrast to conventional chemotherapy, offer many unique properties, like as enhanced permeability and retention (EPR), improved delivery of insoluble drugs, drug protection from harsh environments, controlled release of drugs, and so on [2,3]. Solid lipid nanoparticles (SLNs) are nano drug delivery systems whose outstanding properties including the possibility of controlled
∗
release and drug targeting, good compatibility, high drug loading capacity, high physical stability, the ability to protect unstable drugs from degradation, not using of toxic solvent during the synthesis of these nanoparticles and the ease of production on a large scale are considerable. In fact, SLN is an alternate carrier system for conventional colloidal carriers such as liposomes, emulsions, and polymeric nanoparticles. In order to increase the uptake rate and improve the SLN delivery efficiency, surface modification and bio-polymer coating have recently been used [4–7]. Chitosan is naturally cationic copolymers composed of N-acetyl glucosamine and D-glucosamine in a random and because of its unique properties such as lack of toxicity, biocompatibility, and high biodegradability, increased bio adhesive and permeability properties and anti-oxidant activity are widely used in drug delivery systems and the transfer of anticancer agents [8–10]. Iron oxide nanoparticles have advantages including low toxicity, great biocompatibility, excellent magnetic properties and the reactive
Corresponding author. E-mail addresses: [email protected], [email protected] (E. Arkan).
https://doi.org/10.1016/j.jddst.2020.101621 Received 24 September 2019; Received in revised form 5 January 2020; Accepted 23 February 2020 Available online 28 February 2020 1773-2247/ © 2020 Elsevier B.V. All rights reserved.
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Scheme 1. A schematic of the step-by-step preparation of LTZ-loaded magnetic solid lipid nanoparticles with chitosan coating; Abbreviations: magnetic nanoparticles (MNPs), dichloromethane (DCM), Dioctyl sulfosuccinate sodium salt (AOT) and magnetic solid lipid nanoparticles (MSLNs).
2.2. Preparation of magnetic nanoparticles coated with lecithin
level that can be easily reformed with biocompatible coatings, and because of these properties, they can be utilized as therapeutic and diagnostic agents. In fact, the magnetic delivery system is targeted by using magnetic nanoparticle carriers by magnetic fields to place them into the tumor, and then the drug can be released by alterations in physiological stimulants such as pH and temperature or enzymatic acting from the carriers and uptaking by tumor cells. Magnetic nanoparticles tend to be agglomerated, so these nanoparticles are generally coated with polymers or surfactants to reduce particle agglomeration [10–13]. Letrozole is one of the most effective third-generation competitive inhibitors of aromatase enzyme that prevents excessive biosynthesis of estrogen in the body. The drug has serious side effects such as flushing, breast tenderness, and headache in addition to needs to frequent administration because of short half-life. For this reason, it is suitable for a targeted and controlled drug delivery system to increase the therapeutic efficacy and reduce the harmful effects of LTZ [14,15]. In the present study, using magnetic nanoparticles, a spatiotemporal controlled nanocarrier of chitosan-coated solid lipid nanoparticles have been developed by solvent evaporation-ultra sonication technique for LTZ delivery. The characterization of the produced Nano carriers was performed by PCS, FTIR, SEM, XRD and DSC and their cytotoxicity were investigated by MTT test. The encapsulated efficiency of the magnetic delivery system and the effect of magnetic nanoparticles on the drug release profile were studied also in vitro.
Magnetic nanoparticles were synthesized by the standard method of chemical co-precipitation [16]. Briefly, a mixture of iron salts II (FeCl2.4H2O, 1 g) and iron salts III (FeCl3.6H2O, 3 g) was prepared in 50 mL of distilled water under the nitrogen condition. During the stirring, the temperature was brought to 75 °C and the NaOH solution was slowly added to the reaction in order to adjust the pH = 10. By increasing the pH, the color of the solution became black that indicating the formation of magnetic nanoparticles. The nanoparticles were collected with a permanent magnet followed by several times of washing with deionized water. To prepare the magnetic nanoparticles coated with lecithin [17], the obtained nanoparticles were poured into a lecithin solution (50 mg lecithin in 5 cc chloroform) and stirred for 1 h. In order to ensure the lecithin is coated around the magnetic nanoparticles, a mixture of chloroform: water (50%:50%) was used to separate the coated particles from uncoated ones.
2.3. Preparation of magnetic solid lipid nanoparticles (MSLNs) Solid lipid nanoparticles loaded with LTZ and hydrophobic magnetic nanoparticles were prepared by novel combined methods of solvent evaporation-ultrasonic [18]. Lipids (stearic acid (5 mg), tripalmitin glycerol (5 mg)), letrozole (6 mg), lecithin-coated magnetic nanoparticles (2 mg) and AOT (as a surfactant) were dissolved in 1 mL of Dichloromethane. The lipid phase was then well dispersed in the aqueous phase (20 mL distilled water) using a probe sonicator (Sonopuls Ultrasonic Homogenizer HD 2070, Berlin), for 4 min. Then for preparation of solid lipid nanoparticles, the organic solvent was evaporated by stirring for 45 min at a speed of 900 rpm. In order to better homogenization, SLNs were sonicated by bath sonication for 4 min. Anionic solid lipid nanoparticles were easily coated with cationic polymer of chitosan [19]. Chitosan solution (0.1%, W/V) was prepared in distilled water. The prepared solid lipid nanoparticles were gradually added to the polymer solution and continued by slow stirring for half an hour (Scheme 1).
2. Material and methods 2.1. Materials Letrozole was obtained from Iran Hormone Pharmaceutical Co. (I. R. Iran). Glycerol tripalmitate (TPG) was provided by Alfa Aesar (Germany). CHI (low molecular weight, by the degree of deacetylation ≥ 75%), stearic acid (SA), soy-lecithin, Dioctyl sulfosuccinate sodium salt (AOT), FeCl3. 6H2O and FeCl2. 4H2O were purchased from Merck Co. (Darmstadt, Germany). Other reagents and chemicals were of the analytical grade available. 2
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2.4. Characterization of MSLNs
DPPH radical was calculated using the following formula:
The DLS method was used for determination of the size, polydispersity index (PDI) and zeta potential of nanoparticles by nanosizer instrument (Malvern, England). Precise size measurement of nanoparticles was done using scanning electron microscopy (SEM) (MIRA3TESCAN-XMU microscope, Germany). The FTIR spectra in the range of 400–4000 cm−1 were scanned by a Shimadzu IR2000 (Japan) and a scan rate of 8/cm for 100 scans. The DSC thermograms were shown at 10 °C to 240 °C with the temperature ramp of 10 °C/min by DSC tool (SDT Q600, TA, and USA). The XRD pattern of CHI-SLNs was scanned from 10 to 80° (2 θ) with a step size of 0.05° and a time per step of 1 s using X-ray diffractometer (PW1730, 40 kV, 30 mA, Philips, Netherlands).
I%=
where I = DPPH inhibition (%), A0 = absorbance of control sample (t = 0 h) and A = absorbance of a tested sample at the end of the reaction (t = 1 h). The amount of sample necessary to decrease the absorbance of DPPH by 50% (IC50) was calculated graphically for LEC, MSLNs, LTZ, and LTZ + MSLNs in different concentrations [20]. 2.9. Measurement of cell toxicity In this experiment, the following cell lines have been used for investing the cytotoxicity effects of the LEC, MSLNs, LTZ, and LTZ + MSLNs using an MTT assay:
2.5. Determination of the encapsulation efficiency (EE) and drug loading (DL)
a) b) c) d) e) f) g)
The concentration of LTZ was measured by Ultra Violet (UV) spectrophotometry at a wavelength of 240 nm. The content of free drugs exists in the supernatant obtained by centrifugation (20 min, 15,000 rpm) was detected. The EE and DL was performed using the following equations:
EE (%) =
Wt − Wf × 100 Wt
DL (%) =
Wt − Wf × 100 Ws
A0 − A × 100 A0
Normal cell line: HUVEC. Breast adenocarcinoma cell line: MCF7. Breast carcinoma cell line: Hs 578Bst. Infiltrating ductal cell carcinoma cell line: Hs 319.T. Infiltrating lobular carcinoma of breast cell line: UACC-3133. Inflammatory carcinoma of the breast cell line: UACC-732. Metastatic carcinoma cell line: MDA-MB-453.
(1) These cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% (w/v) FBS, 100U/mL penicillin, and 100 μg/mL streptomycin. Then, cells were distributed at 10,000 cells/well in 96well plates. The cells were grown under a humidified incubator with 5% CO2 at 37 °C until reaching confluency (typically after 24 h). The cells were treated with LEC, MSLNs, LTZ, and LTZ + MSLNs at concentrations of 0, 1, 2, 3, 7, 15, 31, 62, 125, 250, 500, and 1000 μg/mL and subsequently incubated for 2 and 24 h. LEC, MSLNs, LTZ, and LTZ + MSLNs were sterilized using UV radiation for 1 h. Finally, the MTT solution (5 mg/mL in PBS) was added to each well and incubated for 4 h at 37 °C. The medium with MTT was removed and the formazan crystals formed in the living cells were dissolved in 100 μL DMSO per well. All tests were run in the triplicates. The relative viability (%) was calculated based on the absorbance at λ = 570 nm determined using a microplate reader [21].
(2)
where the Wt, Wf and Ws are the initial amount, the free drug measured in the supernatant, and the total weight of the nanoparticulate system respectively. 2.6. Investigation of drug release The release profile of Nano delivery system was evaluated using a dialysis bag with the cut off of 3500 KD. The samplings were done in different time intervals up to 3 days. A low frequency pulsed magnetic field (LFPMF) of 50 Hz was applied to drug loaded MSLNs. 2.7. Applying magnetic field
Percentage of cell viability (%) = (Sample absorbance/Control absorbance) × 100
A copper coil with a lacquered coating and 900 rounds around the iron core was used to generate a 50 Hz magnetic field. An alternating current adapter device was used to generate a sine wave with pulse width 5 ms. The field strength was measured by a Teslameter device (MG-3003SD Lutron, Japan) at about 8 mT. For the drug release test, polymethyl methacrylate bases were used, to fix the coil at a specified distance from the dialysis bag, and then the magnetic field was performed.
The half-maximal cell viability concentration (IC50) was calculated by linear regression analysis and expressed as a mean of three determinations. 2.10. Data management and statistical analysis The data on absorbance measures were entered in the Microsoft® Excel spreadsheet one-word program, where it was organized and then exported to Minitab statistical software for analysis. This was found to conform to assumptions of parametric data and expressed as Means ± Standard Deviations (SD). All statistical analyses were performed using Minitab (Minitab 17.1 Version, NC, USA). The analyzed data was presented in Figures.
2.8. Assessment of the antioxidant activity The antioxidant activity was measured in terms of hydrogen donating or radical scavenging ability using the stable radical DPPH. Experiments were carried out according to the method of previous studies [20] with a slight modification. The reduction of the radical is followed by a decrease in the absorbance at 517 nm. A volume of 2 mL of LEC, MSLNs, LTZ, and LTZ + MSLNs was put into test tubes and 2 mL of 1 mM DPPH solution was added. The tubes were covered with parafilm and kept again in the dark for 1 h. Absorbance at 517 nm was measured with a spectrophotometer UV–Vis and compared to an (Butylated hydroxytoluene (BHT)) calibration curve. A mixture of methanol and DPPH solution served as a blank while a reaction mixture of methanol, DPPH and standard BHT served as the positive control. Each assay was carried out in triplicate. The percentage inhibition of the
3. Results and discussion 3.1. MSLNs characterization Herein a chitosan-coated magnetic nano solid lipid carrier containing the anticancer drug of LTZ was prepared and was characterized. Its release kinetic along with anticancer activity was evaluated. The size and distribution of particle sizes are the main parameters 3
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Table 1 Physicochemical characteristics and dynamic light scattering data of uncoatedSLN and coated-SLN loaded with LTZ. Formulations
Size (nm)
Charge (mV)
PDI
EE%
DL%
Uncoated SLN CHI-LTZ-SLN
158.2 193.2
42.520.2-
0.188 0.200
– 90.1
– 26.20
CHI: Chitosan; LTZ: Letrozole; SLN: Solid lipid nanoparticle; Drug loading; EE: Encapsulation efficiency; PDI: Polydispersity index.
DL:
for the stability of the system and passive targeting of therapeutic agents into tumors with taking advantages of EPR effect [20]. As shown in Table 1, the hydrodynamic particle sizes of chitosan uncoated and coated MSLN nanoparticles were achieved 158.2, 193.2 nm and PDI value of 0.188 and 0.200 respectively. The results were indicated that these particles are suitable for accumulation in the target tumor tissue. A low PDI of the system was proved a good size monodispersity which assures the nanoparticle solution stability (Table 1). Researches have shown that nanoparticles with the negative charge tend to stay longer in bloodstream than nanoparticles positively charged and their clearance by the immune system is slower [21–23]. Negative nanoparticles showed less cytotoxicity as well [21]. Therefore, in the synthesis of formulation, we used a smaller amount of chitosan to have both suitable particle size and negative surface charge with the help to nanoparticles become hydrophilic. The zeta potential of uncoated and coated nanoparticles with chitosan was −42.5 and −20.2, respectively. The observed difference in charge of uncoated and coated nanoparticles indicates that a thin layer of chitosan has covered the SLN nanoparticles. However, the chitosan was added to the delivery system and lead to hydrophilic, reducing the removal of macrophage and decrement negative charge of lipid nanoparticles. The high negative charge of nanoparticles act as a barrier during passing through the vessels and entering the cells. Chitosan-coated nanoparticles were used for other analyzes. The image of SEM of CHI-MSLN was showed the precise particle size of the drug loaded nanocarrier that is about 50 nm (Fig. 1). Some differences between the SEM and DLS results are related to the differences in hydrodynamic diameter and those of the particle itself. The FTIR spectrum shown in Fig. 2 accurately demonstrates that chitosan has covered MSLN loaded with LTZ. According to the chitosan index peaks in wavelengths 3437, 2854 and 2920, 1639, 1103 cm−1 related to O–H and N–H stretch, C–H stretch, C–N–H of amide I and C–O–C vibration respectively that exist in the CHI-MSLN sample, it confirms the coating of nanoparticles with chitosan as can be compared
Fig. 2. The FT-IR spectra of lipids, chitosan, MNPs, CHI-MSLNs loaded with LTZ.
with chitosan spectrum. Several peaks of the lipids in the MSLN structure are observed at wavelengths of 1562 and 1415 and 1694 cm−1 affiliated to C]O stretching bands, shifting these peaks to the right can be due to interactions of lipids with chitosan. Since the LTZ characteristic peak in the wavelength 2250 cm−1 hasn't appeared, it indicates that LTZ has been entirely encapsulated into the nanoparticles [24]. The same result was obtained for MNPs with lecithin coating due to the index peak decay of MNPs in wavelength of around 600 cm−1 [25]. The DSC is a standard method to obtain detailed information about any changes in the system's enthalpy become from any endothermic or exothermic phenomenon take place in the system. Then the information can be interpreted to achieve some viewpoint about for example changes in molecular and supramolecular conformation or interactions etc. There are an exothermic and two broad endothermic peaks placed in 40, 67 and 133 °c respectively (Fig. 3). The first sharp exothermic peak in 40 °c is related to movement and interactions of residual water molecules with the nanoparticles [26]. The peak present in 67 °c according to literatures belongs to the phase transition of lipids in their melting point [27]. The latest and broadest peak can be related to corrosion of chitosan from the surface of SLNs. The results also confirm the good stability of the system in conditions close to body temperature
Fig. 1. Scanning electron microscopy (SEM) image of CHI-MSLNs. 4
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Fig. 3. Differential scanning calorimetry (DSC) thermogram of CHI-SLN. Fig. 6. The antioxidant properties of LEC, MSLNs, LTZ, LTZ + MSLNs, and BHT against DPPH. LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles. BHT: Butylated hydroxytoluene.
regarding the Bragg's law which points that the proximate sheets the higher degrees of 2θ can be because of existence of some pressure on crystals and confirming SLN coating by chitosan. A peak in 2θ of 32° belongs to magnetic nanoparticles and due to its strong crystal structure there is no significant shift in it. 3.2. In vitro drug release study The characteristic of cancer cells whose extracellular pH is more acidic than the pH of healthy cells can be used for target delivery system. PH-sensitive polymers are an adequate choice for such a purpose and many studies have been done on them [30]. Chitosan has been extensively investigated in pH-sensitive drug delivery systems due to the protonation of amine groups in the acidic pH, which is in its main chain [3,19]. According to the results of other researchers, the release of the drug was more stable and faster at acidic pH, so in this work, a pH of 6 for the release medium was used. Since pH of the initial endosomes is acidic, the above nano carrier system can be used for intracellular drug delivery [19,31]. The magnetic nanoparticles in nano drug delivery systems are
Fig. 4. X-ray diffractogram of CHI-MSLNs.
and storing in room temperature. The X-Ray Diffraction results confirm lipid crystallization and chitosan coating on MSLNs. As can be seen in Fig. 4, there are several peaks in 2θ of 17°, 20°, 30° which according to previous literature are related to the orthorhombic lamellar lattice structure of lipid's hydrocarbon tail [28]. Also compare to Lukowski et al. reported [29], a distinct right shift in the related peaks has observed. This shifts
Fig. 5. Drug release profile of CHI-MSLN at (A) non-LFPMF, (B) applied LFPMF. (n = 3). 5
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Table 2 The IC50 of LEC, MSLNs, LTZ, LTZ + MSLNs, and BHT in antioxidant test.
IC50 against DPPH
LEC (μg/mL)
MSLNs (μg/mL)
LTZ (μg/mL)
LTZ + MSLNs (μg/mL)
BHT (μg/mL)
–
576
342
198
251
LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles. BHT: Butylated hydroxytoluene.
Fig. 7. The cytotoxicity properties of LEC, MSLNs, LTZ, and LTZ + MSLNs against HUVEC cell line. LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles. HUVECs: Human umbilical vein endothelial cells.
Fig. 9. The anti-breast cancer properties of LEC, MSLNs, LTZ, and LTZ + MSLNs against breast carcinoma (Hs 578Bst) cell line. LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles.
Fig. 10. The anti-breast cancer properties of LEC, MSLNs, LTZ, and LTZ + MSLNs against infiltrating ductal cell carcinoma (Hs 319.T) cell line. LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles
Fig. 8. The anti-breast cancer properties of LEC, MSLNs, LTZ, and LTZ + MSLNs against breast adenocarcinoma (MCF7) cell line. LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles.
steer and momentum force on the nanoparticle within the nanocarrier, it exerts pressure on the drug and expels it from the system. Typically, in magnetic delivery systems, an alternating and highfrequency magnetic field is used to increase the local heating of magnetic nanoparticles. Consequently, by increasing the temperature and eddy current induced in the magnetic field, can damage healthy cells.
important in several aspects. On one hand, using a constant magnetic field at a specific point of the body, the nanosystem could be accumulated there. Actually, by applying alternating magnetic fields either high frequency for heat generation or low frequency for mechanical pressure, it can trigger the drug release from the system. After inducing 6
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Fig. 11. The anti-breast cancer properties of LEC, MSLNs, LTZ, and LTZ + MSLNs against infiltrating lobular carcinoma of breast (UACC-3133) cell line. LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles.
Fig. 13. The anti-breast cancer properties of LEC, MSLNs, LTZ, and LTZ + MSLNs against metastatic carcinoma (MDA-MB-453) cell line. LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles.
delivery system, the drug can be targeted to the desired location by a controlled and successful release and transfer a large amount of the drug to the cancer cell over a short period of time. It should be noted that the applied LFPMF value was low, meanwhile, with a favorable result. The LFPMF was eventually applied for 1 h. In fact, LFPMF cannot enhance enough temperature as much as system degradation. Herein, the temperature of the system was measured after applying 1 h of LFPMF and it was specified that the temperature increased only 1–1.5 °c.
3.3. Antioxidant assay Humans have evolved a complex and sophisticated antioxidant system to maintain the redox homeostasis and protect cells and organ systems against free radicals. Components of antioxidant defense systems are of either exogenous or endogenous in origin which functions synergistically and interactively to neutralize free radicals. These components include dietary antioxidants like ascorbic acid (vitamin C), tocotrienols (vitamin E), glutathione, β-carotene, uric acid, and α-tocopherol [20]. The antioxidant enzymes such as catalase, glutathione reductase, superoxide dismutase, and glutathione peroxidase also catalyze free radical scavenging activities. Other antioxidant components include metal-binding proteins (like lactoferrin, albumin, ceruloplasmin, and ferritin) which sequester free copper ions and/or free iron that catalyze oxidative reactions [20]. In the present experiment, LEC, MSLNs, LTZ, and LTZ + MSLNs similar to BHT indicated a notable concentration-dependent DPPH radical scavenging effect. The interaction between the MSLNs, LTZ, and LTZ + MSLNs and DPPH might have occurred through the transfer of electrons and hydrogen ions to 2,2-diphenyl-1-picrylhydrazyl radical to form a stable2,2-diphenyl-1-picrylhydrazine molecule (DPPH) [20]. The IC50 values of MSLNs, LTZ, LTZ + MSLNs, and BHT were 576, 342, 198, and 251, respectively (Fig. 6; Table 2). The antioxidant activity exhibited by MSLNs, LTZ, and LTZ + MSLNs can be attributed to the presence of various molecules that are thought to function interactively and synergistically to neutralize reactive oxygen species (ROS) and reactive nitrogen species (RNS) [20].
Fig. 12. The anti-breast cancer properties of LEC, MSLNs, LTZ, and LTZ + MSLNs against inflammatory carcinoma of the breast (UACC-732) cell line. LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles.
Therefore, low frequency pulsed magnetic fields (LFPMF) are used to increase the release of drugs from the system with mechanical movements and without increasing the temperature [32–34]. So we used the magnetic nanoparticles, not only accumulating the nanocarrier to the desired target site, but also by applying an LFPMF, causing a faster release of the drug without creating a significant change in temperature. The release of LTZ from the MSLN Nano carrier was investigated by the dialysis method in two states, applying an LFPMF and non-LFPMF [35,36]. As shown in Fig. 5, in the non-LFPMF state after 12 h, 50% of the drug is released, but by applying LFPMF the same amount of LTZ is released in 1 h. Given these results, the effect of the LFPMF on magnetic nanoparticles is clearly observable. Admittedly, utilizing this drug 7
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Table 3 The IC50 of LEC, MSLNs, LTZ, and LTZ + MSLNs in cytotoxicity test.
IC50 IC50 IC50 IC50 IC50 IC50 IC50
against against against against against against against
HUVEC MCF7 Hs 578Bst Hs 319.T UACC-3133 UACC-732 MDA-MB-453
LEC (μg/mL)
MSLNs (μg/mL)
LTZ (μg/mL)
LTZ + MSLNs (μg/mL)
– – – – – – –
– 687 551 469 448 356 754
– 345 332 311 265 236 399
– 221 215 208 170 161 247
LEC: Lecithin. LTZ: Letrozole. MSLNs: Magnetic solid lipid nanoparticles.
LFPMF during 1 h, which was faster and more controlled than the nonLFPMF applying conditions (the same amount of drug released during 12 h). DPPH free radical scavenging test was carried out to evaluate the antioxidant capacities of LEC, MSLNs, LTZ, LTZ + MSLNs, and BHT. DPPH test indicated similar antioxidant activities for LTZ + MSLNs and BHT that indicated a notable concentration-dependent DPPH radical scavenging effect. MTT assay was used on HUVEC, MCF7, Hs 578Bst, Hs 319.T, UACC-3133, UACC-732, and MDA-MB-453 cell lines and the best result of cytotoxicity property of LTZ + MSLNs against the above cell lines was seen in the case of the UACC-732 cell line. This magnetic lipid drug delivery system offers promising potential for the targeted and controlled treatment of chemotherapy agents-resistant cancers.
3.4. Cytotoxicity assay In the present study, the treated cells with various concentrations of the present LEC, MSLNs, LTZ, and LTZ + MSLNs were examined by MTT test for 48 h regarding the cytotoxicity property on the normal cell line (HUVEC), breast adenocarcinoma (MCF7), breast carcinoma (Hs 578Bst), infiltrating ductal cell carcinoma (Hs 319.T), infiltrating lobular carcinoma of breast (UACC-3133), inflammatory carcinoma of the breast (UACC-732), and metastatic carcinoma (MDA-MB-453) cell lines (Figs. 7–13; Table 3). The absorbance rate was determined at 570 nm, which revealed extraordinary viability on normal cell line (HUVEC) even up to 1000 μg/mL for LEC, MSLNs, LTZ, and LTZ + MSLNs. In the case of breast cancer cell lines, the viability of them decreased dose-dependently in the presence of LEC, MSLNs, LTZ, and LTZ + MSLNs. The IC50 of MSLNs, LTZ, and LTZ + MSLNs against MCF7 cell line were 687, 345, and 221 μg/mL, respectively; against Hs 578Bst cell line were 551, 332, and 215 μg/mL, respectively; against Hs 319.T cell line were 469, 311 and 208 μg/mL, respectively; against UACC-3133 cell line were 448, 265, and 170 μg/mL, respectively; against UACC732 cell line were 356, 236, and 161 μg/mL, respectively; against MDAMB-453 cell line were 754, 399 and 247 μg/mL, respectively. The best result of cytotoxicity property of LTZ + MSLNs against the above cell lines was seen in the case of the UACC-732 cell line. Probably the anti-breast cancer potentials of LTZ + MSLNs are related to their antioxidant activities. The previous studies have reported that antioxidant compounds such as nanomaterial's as single electron donors can stabilize and scavenge the free radicals, which in conditions of oxidative stress may begin angiogenesis or carcinogenesis [21]. Free radical-induced development of cancer involves malignant transformation due to DNA mutations and changed gene expression through epigenetic mechanisms which in turn causes the uncontrolled proliferation of cancerous cells [21]. Many researchers reported a significant role of antioxidant compounds such as medicinal plants and silver nanoparticles in growth inhibition of prostate, breast, ovary, endometrium, colon, and lung cancer cells with removing free radicals [21].
Funding This work was supported by the research council of Kermanshah University of Medical Sciences (Grant Number. 97720). CRediT authorship contribution statement Zeynab Ahmadifard: Writing - original draft, Investigation, Formal analysis. Ahmad Ahmeda: Formal analysis, Software. Mahsa Rasekhian: Supervision. Sajad Moradi: Writing - review & editing, Project administration. Declaration of competing interest The authors all confirm there is no conflict of interest. Acknowledgments The authors gratefully acknowledge the research council of Kermanshah University of Medical Sciences. This work was performed in partial fulfillment of the requirement for M.Sc. Nanomedicine of Zeynab Ahmadifard, in the faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran. References [1] D. Peer, J.M. Karp, S. Hong, O.C. Farokhzad, R. Margalit, R. Langer, Nanocarriers as an emerging platform for cancer therapy, Nat. Nanotechnol. 2 (12) (2007) 751. [2] I.I. Lungu, M. Radulescu, G.D. Mogosanu, A.M. Grumezescu, pH sensitive core-shell magnetic nanoparticles for targeted drug delivery in cancer therapy, Rom. J. Morphol. Embryol. 57 (1) (2016) 23–32. [3] G. Lv, L. Qiu, G. Liu, W. Wang, K. Li, X. Zhao, et al., pH sensitive chitosan-mesoporous silica nanoparticles for targeted delivery of a ruthenium complex with enhanced anticancer effects, Dalton Trans. 45 (45) (2016) 18147–18155. [4] Y. Luo, Z. Teng, Y. Li, Q. Wang, Solid lipid nanoparticles for oral drug delivery: chitosan coating improves stability, controlled delivery, mucoadhesion and cellular uptake, Carbohydr. Polym. 122 (2015) 221–229. [5] M. Zhou, J. Hou, Z. Zhong, N. Hao, Y. Lin, C. Li, Targeted delivery of hyaluronic acid-coated solid lipid nanoparticles for rheumatoid arthritis therapy, Drug Deliv. 25 (1) (2018) 716–722. [6] H. Yuan, J. Miao, Y.-Z. Du, J. You, F.-Q. Hu, S. Zeng, Cellular uptake of solid lipid nanoparticles and cytotoxicity of encapsulated paclitaxel in A549 cancer cells, Int.
4. Conclusion In the present study, a LTZ nano delivery system was formulated with an easy method for targeted and controlled release. The magnetic nanoparticles and LTZ were loaded into solid lipid nanoparticles with chitosan natural polymer coating. MSLNs with chitosan coating were successfully characterized so that the nanoparticles with size, PDI and surface charge of 193.2, 0.2 and −20.2, and with EE and DL of 90.1 and 26.2 were obtained respectively. FTIR analysis showed that coating of nanoparticles was done by chitosan. DSC and XRD analyses confirmed FTIR results. The release profile of the drug that was investigated comparatively, showed that 50% of the drug was exerted by applying 8
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