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Fabrication of quercetin-loaded PLGA nanoparticles via electrohydrodynamic atomization for cardiovascular disease
Available online at www.sciencedirect.com
ScienceDirect Materials Today: Proceedings 5 (2018) 15998–16005
www.materialstoday.com/proceedings
NN17
Fabrication of quercetin-loaded PLGA nanoparticles via electrohydrodynamic atomization for cardiovascular disease M. Giannouli1, V. Karagkiozaki1,2, F.Pappa1, I. Moutsios1, C.Gravalidis1*, S. Logothetidis1 1. Nanotechnology Lab LTFN, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece 2. BL Nanobiomed PC, Vasilisis Olgas 141, 54645, Thessaloniki, Greece
Abstract
Cardiovascular diseases constitute a major public health concern in industrialized nations. Oxidative stress induced free radicals play a critical role in cellular processes implicated in atherosclerosis and many other heart diseases. Quercetin (Qu) is an antioxidant drug which is shown that effectively protects against cardiovascular diseases (CVDs). Encapsulation of drugs in polymeric NPs are widely used in producing sustained and controllable drug release, or to avoid degradation of non-released drugs. In this current work, a novel system of polymeric PLGA NPs loaded with Qu, was fabricated via electrohydrodynamic atomization process (EHDA) in order to improve poor aqueous solubility and stability of the drug with the aim of preventing atherosclerosis. The fabricated nanoparticles collected in a stable glass substrate. The results of atomic force microscopy (AFM) analysis and the scanning electron microscope (SEM), confirmed the fabrication of spherical polymeric nanoparticles with diameter ranging from 300nm to350 nm, narrow size distribution and smooth surface. The release profile of quercetin from the particles was investigated by determining the drug amount released at specific intervals for by luminescence. Furthermore, XRD analysis was used to determine the physical status of Qu encapsulated in NPs compared with that of pure Qu. The information obtained from this study facilitates the design and fabrication of polymeric nanoparticles as possible delivery systems for encapsulation, protection and controlled release of the flavonoid quercetin which is aiming to protect against CVDs. © 2018 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of 14th International Conference on Nanosciences & Nanotechnologies (NN17). Keywords: Electrohydrodynamic atomization (EHDA), Drug Delivery system, Quercetin, Poly (lactic-co-glycolic acid), Oxidative stress
* Corresponding author. Tel.: +302310998850; fax: +302310998390. E-mail address: [email protected] 2214-7853 © 2018 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of 14th International Conference on Nanosciences & Nanotechnologies (NN17).
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1. Introduction Cardiovascular diseases (CVD) are still the first and foremost cause of in both men and women. According to 2011 World Health Organization (WHO) report "Global atlas on cardiovascular disease prevention and control" CVD are referred as the leading cause of death and ailment reaching a presentence of 31 % worldwide [1]. The dominant processes involved in cardiovascular diseases is atherosclerosis. Atherosclerosis is a systemic, chronic and inflammatory disease that is due to endothelial malfunctions and the progressive establishment is mainly due to oxidative stress and other key parameters like lipid deposition and monocyte recruitment [12]. CVD and atherosclerosis formulate a composite outcome affected by numerous risk factors including high cholesterol, hypertension, smoking, diabetes, unhealthy diet, family history of early myocardial infarction and stress. These risk factors are directly associated with oxidative stress caused by high levels of Reactive Oxygen Spices (ROS) and free radicals [2]. ROS consists of Superoxide anion (O2⋅−O2⋅−), hydrogen peroxide (H2O2), and hydroxyl (⋅HO⋅HO). They created through physiological processes of the human body and play key role in the control of homeostasis of cells. Nevertheless, the existence of ROS in an unregulated quantity contributes to the pathogenesis of cardiovascular diseases. [13] (Figure 1) Generally, increased production of ROS may lead to atherosclerosis through four main mechanisms: endothelial cell dysfunction, vascular smooth muscle cells (VSMC) growth, monocyte migration and oxidation of LDLs [3]. Also, oxidative stress be related to diabetic cardiomyopathy, congestive cardiomyopathy, and hypertensive heart disease [17]. Thus, it is obvious to say that reduction of oxidative stress may lead to prevention and treatment of cardiovascular diseases. Antioxidants are the key to reducing the oxidative stress. As reported by many epidemiological studies increasing in antioxidant levels restricts the clinical expression of coronary artery disease [16]. In this way, polyphenolic compounds, such as Quercetin, raised interest due to their valuable antioxidant properties. Quercetin, 3,3’,4’,5’-7-pentahydroxy flavone (Figure 2) is one of the most plentiful flavonoid in plants with important pharmacological properties including antioxidant, neurological, antiviral, anticancer, cardiovascular, antimicrobial, anti-inflammatory and hepatoprotective [4,5]. The antioxidant activity of this flavonoid is higher than other popular antioxidant molecules like ascorbyl, trolox and rutin, due to the number and position of the free hydroxyl groups in its molecule [4]. Antioxidant mechanism of Quercetin involves capturing free radicals and inhibition over matrix metalloproteinases (MMP) activity, which leads to the end of oxidation. The mechanism of suppression of free radical processes through Quercetin is done in three stages: the formation of superoxide anion, the production of hydroxyl radicals and the formation of lipid peroxy radicals [2,5]. However, the use of the dietary flavonoid is limited due to its poor water solubility and instability either in physiological medium (pH, enzymes, presence of other molecules), or in the pharmaceutical products processing, or during storage (temperature, light, pH) [6]. These properties of quercetin result in poor bioavailability and permeability and may cause indefinite or poor pharmacological effect. One way to overcome these problems is to entrap the molecule into nanocarriers. Carriers that used in nanomedicine differ in their chemical properties and they can be liposomes, polymeric nanoparticles, dendrimers, magnetic nanoparticles, quantum dots, nanotubes or mixtures of them [12]. Polymeric nanoparticulate drug delivery systems are the dominant approach of nanocarrier-based drug delivery system. They can improve the bioavailability of poorly absorbed drugs, extend their residence time in the body, and provide a sustained drug release model. Ιn desirable characteristics of nanocarriers included: biocompatibility, drug compatibility, reduction of side effects, stability in physiological body fluids, high residence time in the systemic circulation and decreased toxicity [14]. To this end, the most commonly used polymer for manufacturing polymeric nanoparticles is PLGA or poly (lactic-co-glycolic acid). This polymeric material is biocompatible, biodegradable, non-immunogenic and it is hydrolyzed in the body to produce the biodegradable monomers, lactic acid and glycolic acid [18,7]. Τhe monomers are metabolized through Krebs cycle and eliminated easily by the human body [15]. Furthermore, PLGA is approved by the Food and Drug Administration (FDA) and European Medicine Agency (EMA) in several drug delivery systems [7]. Numerous techniques for preparing polymeric nanoparticulate drug-delivery systems have been already broadly used, for example emulsion solvent evaporation, nanoprecipitation, spray drying, interfacial polymerization etc. Nevertheless, these techniques exhibit some drawbacks including low drug-loading efficiency, fabrication of nanoparticles larger than 100 nm with high polydispersity and inability for large scale fabrication [10]. In recent years, electrospraying or electrohydrodynamic atomization (EHDA) is employed for fabrication of micro- and nanosized particles and there are many publications relating to the development of electrosprayed drug delivery systems [8,9]. Compared with other nanofabrication techniques, electrospraying is simpler, more flexible, automated, and
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has the ability to generate nanoparticles with high drug loaded efficiency [11]. Electrospraying is a one-step technique based on liquid atomization induced by electrical forces to achieve micro- and nano- particles. In its simplest form, during the electrospraying process a high voltage is applied to a liquid fitted in a syringe pump with a metal or a glass capillary. The fluid deforms into Taylor cone at the edge of the capillary due to the applied voltage, and a jet emits from the cone and breaks up into charged droplets. Only when the droplets are separated from the cone, the solvent starts to evaporate and generates particles promoted to the collector [8,9]. Therefore, the aim of this study is to encapsulate Quercetin within PLGA nanoparticles via electrohydrodynamic atomization process, in order to improve the poor aqueous solubility and stability of the drug, which in turn will help in the prevention of atherosclerosis.
Fig.1: Risk factors implicated in atherosclerosis through ROS.
Fig.2: Chemical structure of flavonoid Quercetin 2. MATERIALS AND METHODS 2.1 Materials Poly (DL-lactic-co-glycolic-acid)-PLGA (lactide: glycolide (75:25), MW = 66.000-107.000 g/mol ), Quercetin solid (assay >95%, HPLC), Acetone (for HPLC, purity > 98.5%) and Phosphate Buffer Saline Solution (PBS, 1×) were obtained by Sigma – Aldrich. Ultrapure (Type 1) water purchased from Merck Millipore, Direct-Q, 3UV was used during the study.
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2.2 Particle preparation PLGA-quercetin nanoparticles were prepared by using single-nozzle electrospraying with esprayer™ ES-2000S Electrospray Deposition (ESD) equipment. Electrospraying applies electrostatic forces to produce nanoparticles of a polymeric solution fitted in the syringe. Solutions were prepared by dissolving appropriate amounts of PLGA and Quercetin in acetone. Specifically, PLGA was dissolved in acetone after overnight stirring in order to achieve a clear solution. Then, Quercetin was added in the acetone solution and mixed until a homogeneous solution was formed. For the fabrication of blank nanoparticles, we used the acetone solution of PLGA without drug. The solutions after preparation were placed in a glass syringe with a metal nozzle and the nozzle was positioned vertically relative to the ground electrode. The two electrodes were adjusted at a distance approximately 60 mm separately. Other parameters (Voltage, Flow rate) were fixed in such a way that the system works in the cone-jet mode. Blank or Quercetin loaded PLGA nanoparticles that prepared during the electrospraying process were then collected on stable glass slides (1cm x 1cm) which were located on the ground electrode under the nozzle. 2.3 Particle Size and Morphology properties Size and morphology of blank and Quercetin loaded nanoparticles were investigated through usage of Atomic Force Microscopy (AFM) (NT – MDT solver, Scanning Probe Microscope with anti – vibration system MICRO 40 by Halcyonics) and Scanning Electron Microscopy(SEM) (JOEL, JSM – 840A) methods. From the AFM images of each sample, 100 particles were measured using the software IMAGEJ and the mean diameter as well as the standard deviation were calculated. 2.4 In vitro degradation study 1cm X 1cm glass samples with electrosprayed nanoparticles were set in well plates and the degradation study took place in Phosphate Buffered Saline at pH 7.2. The temperature was kept constant at 37 o C as samples were kept in an incubator (Galaxy 1705 incubator) during the study. The degradation study was conducted for both blank and Quercetin loaded PLGA nanoparticles. The morphology of degraded particles has been examined by Atomic Force Microscopy (AFM) (NT – MDT solver, Scanning Probe Microscope with anti – vibration system MICRO 40 by Halcyonics). and the p H of the degradation medium was measured by p H meter. 2.5 In vitro drug release study Quercetin loaded PLGA particulates that immobilized on glass (1 cm X 1 cm) were put in well plates and incubated (with use of Galaxy 1705 incubator) in PBS at p H 7.2 and constant temperature 37 o C. Drug release kinetics was investigated by determining the drug amount released at specific intervals for over a month by luminescence using the Luminometer Promega CLOMAX MULTI DETECTION SYSTEM, at 369 nm for Quercetin. 2.6 X-Ray Diffraction (XRD) X-Ray diffraction was conducted to determine the physical status and the crystallinity of pure Quercetin powder, blank PLGA nanoparticles and Quercetin loaded PLGA nanoparticles. XRD patterns were recorded using an X-ray diffractometer (Siemens D5000). 3. RESULTS AND DISCUSSION 3.1 Size and morphology of blank and Quercetin loaded PLGA nanoparticles Size and morphology of electrosprayed PLGA nanoparticles were studied using AFM for blank and a combination of AFM and SEM for drug loaded nanoparticles. The height, the phase and the 3D depiction of blank PLGA
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nanoparticles that immobilized on glass are shown in figure 3 (a, b, c). Generally, fabricated particles were approximately spherical in shape with smooth surface and uniform morphology. Their mean particle size was 343.5 nm and the standard deviation was ±25.4 nm. Also, blank PLGA nanoparticles had narrow dispersion as depicted in histogram of size distribution (figure 3d). Morphological properties of Quercetin loaded PLGA nanoparticles are shown at AFM (fig 4a) and SEM images (fig 4b). Analogous to blank nanoparticles case, drug loaded PLGA nanoparticles were approximately spherical with smooth surface and narrow dispersion as illustrated in histogram of size distribution (fig 4c). The mean diameter was 384.9 nm and the standard deviation was ±26.3 nm. In contrast to blank PLGA nanoparticles, Quercetin loaded PLGA nanoparticles were showed to have an increase in the mean size due to the presence of the drug.
a.
b.
c.
d. Fig.3:AFM images of blank PLGA nanoparticles. Height(a), Phase(b), 3D depiction(c) and size distribution(d)
a. b. c. Fig.4: AFM(a), SEM(b) images of Quercetin loaded PLGA nanoparticles and size distribution of them(c) 3.2 In vitro degradation study of blank and Quercetin loaded PLGA nanoparticles. Blank PLGA nanoparticles had almost fully degraded within a period of 20 days in PBS as a degradation medium. Morphological properties of degraded nanoparticles during the study, are shown at AFM images (figure 5). After the first day of the study, nanoparticles had lost their spherical shape (figure 5b) and they had been agglomerated (fig 5b, c). From the seventh day they had started creating films (figure 5d) and after the eleventh up to the twentieth day blank nanoparticles had degraded (figure 5e, f). From the figure 6 it can be seen that during the time of degradation the p H of PBS decreases because of the presence of acidic degradation products of PLGA. Specifically, the initial pH value of degradation medium (PBS) was 7.4 and during the time of degradation was reduced until the value 6.1
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(the fifteenth day) due to accumulation of glycolic acid and lactic acid. The pH reduction confirms the PLGA nanoparticles degradation. In the case of Quercetin loaded nanoparticles duration of degradation in PBS was fifty days. Morphological properties of degraded nanoparticles during the study, are shown at AFM images (figure 7). Analogous to the blank nanoparticles and the Quercetin loaded nanoparticles after 5 days had lost their spherical shape and they agglomerated (figure 7b,7c). After the twenty-fifth day of the degradation study were created films and as from the thirtieth fifth day up to the fiftieth had degraded. The longer period of degradation of quercetin-loaded nanoparticles is due to the existence of the drug. Results of the degradation study of drug loaded PLGA nanoparticles accordance with the results of the in vitro drug release study presented in section 3.3
Fig.5: AFM images of blank PLGA nanoparticles after 0 day (a), 1 day (b), 4 days (c), 7 days (d), 11 days (e) and 15 days(f) of in vitro degradation study.
Fig.6: Changes in the pH of the degradation medium at specific times during the degradation study
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Fig.7: AFM images of Quercetin loaded PLGA nanoparticles after 0 days (a), 5 days (b), 15 days (c), 25 days (d), 35 days (e) and 50 days(f) of degradation study 3.3 In vitro drug release study The in vitro release behavior of Quercetin from PLGA nanoparticles is shown in figure 8. As can be seen, Quercetin release from nanoparticles followed a biphasic release model with a primary release conducted in the first 24hrs followed by a sustained release for 59 days. The burst drug release of PLGA nanoparticles was climbed at 27% due to the fact of the existence of quercetin molecules on or near the surface of polymeric nanoparticles. Simultaneously, the sustained release was shown to reach a 96.1% caused by the degradation of the polymeric matrix carried out in more than 50 days as shown in session 3.2.
Fig.8: In vitro drug release profile of quercetin loaded PLGA nanoparticles 3.4 Physical status of Quercetin in PLGA NPs In order to determine the physical status of drug molecule, polymer and polymeric nanoparticles we used the X-ray diffraction. The XRD patterns for pure Quercetin powder (black diagram), blank PLGA nanoparticles (red diagram) and quercetin-loaded PLGA nanoparticles (green diagram) are shown in figure 9. For pure drug, the XRD pattern showed characteristic peaks because of its crystalline structure. For blank PLGA nanoparticles, the XRD pattern
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showed no peak, suggesting that polymeric blank nanoparticles are amorphous. For quercetin-loaded PLGA nanoparticles the XRD pattern also showed no peak claim that Quercetin exists dispersed in a non-crystalline state within the PLGA polymeric matrix.
Fig.9: XRD patterns of Quercetin powder, blank PLGA nanoparticles and Quercetin loaded PLGA nanoparticles. 4. CONCLUSIONS Electrohydrodynamic Atomization process is a suitable technique for fabrication of polymeric nanoparticles that used in drug delivery systems. PLGA nanoparticles were prepared successfully and used to encapsulate Quercetin to protect the drug and control pharmacokinetics properties. Morphology analysis (AFM and SEM measurements) illustrated that PLGA nanoparticles are spherical with an average size of 343 nm for PLGA nanoparticles and 384 nm for Quercetin loaded PLGA nanoparticles. Quercetin encapsulated within the polymeric matrix in an noncrystalline state. Quercetin loaded nanoparticles exhibited a biphasic release model of the drug with a rapid release over a few hours and a sustained slow release over 59 days. Therefore, this kind of drug delivery system of the antioxidant drug Quercetin, has great potential to be used in prevention of atherosclerosis and other relative cardiovascular diseases. 5. REFERENCES [1] World Heart Organization global atlas on cardiovascular disease prevention and control, Geneva 2011 [2] Parul Lakhanpal et al., Role of quercetin in cardiovascular diseases”, Internet Journal of Medical Update, Vol. 3, No. 1, Jan-Jun 2008 [3] Alexander RW,”Hypertension and the pathogenesis of atherosclerosis: oxidative stress and the mediation of arterial inflammatory response: a new perspective.”Hypertension , pp:155-61,1995 [4] Avnesh Kumari et al., Colloids and Surfaces B: Biointerfaces, 80 (2010) 184-192 2010 [5] Aneela Maalik et al., Tropical Journal of Pharmaceutical Research, 13 (2014) 1561-1566 [6] Hector Pool et al., “Antioxidant Effects of Quercetin and Catechin Encapsulated into PLGA Nanoparticles”, Hindawi Publishing Corporation Journal of Nanomaterials Vol 2012 [7] Lucia Ruxandra Tefas et. al., Clujul Medical 88 (2015) no. 2 [8] B. T. Midhun et al., Journal of Biomaterials Science, 22 (2011) 2431–2444 [9] Alejandro Sosnik, J. Biomed. Nanotechnol, 10 (2014) 9 [10] Maedeh Zamani et. al., International Journal of Nanomedicine 8 (2013) 2997–3017 [11] Leilei Zhang et al., HHS Author Manuscripts, pp 595–612,2012 [12] Varvara Karagkiozaki et al., J. Biomed. Nanotechnol. 11 (2015) 191–210 [13] Keyvan Karimi Galougahi et al., European Heart Journal 36 (2015) 1576–1582 [14] Varvara Karagkiozaki, “Horizons in clinical nanomedicine” [15] Fatemeh Sadat et al., Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 [16] S.L. Nuttall et al., Q J Med 92 (1999) 239–244 [17] FredricJ.Pashkow et al., , International Journal of Inflammation Vol. 2011, 2011 [18] Magdalena Stevanović and Dragan Uskoković, “Influence of Different Degradation Medium on Release of Ascorbic Acid from PLGA Nano ans Microspheres”, in proc. of “Physical Chemistry 2008. 9th International Conference on Fundamental and Applied Aspects of Physical Chemistry, Belgrade, September 24-26, 200”
ScienceDirect Materials Today: Proceedings 5 (2018) 15998–16005
www.materialstoday.com/proceedings
NN17
Fabrication of quercetin-loaded PLGA nanoparticles via electrohydrodynamic atomization for cardiovascular disease M. Giannouli1, V. Karagkiozaki1,2, F.Pappa1, I. Moutsios1, C.Gravalidis1*, S. Logothetidis1 1. Nanotechnology Lab LTFN, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece 2. BL Nanobiomed PC, Vasilisis Olgas 141, 54645, Thessaloniki, Greece
Abstract
Cardiovascular diseases constitute a major public health concern in industrialized nations. Oxidative stress induced free radicals play a critical role in cellular processes implicated in atherosclerosis and many other heart diseases. Quercetin (Qu) is an antioxidant drug which is shown that effectively protects against cardiovascular diseases (CVDs). Encapsulation of drugs in polymeric NPs are widely used in producing sustained and controllable drug release, or to avoid degradation of non-released drugs. In this current work, a novel system of polymeric PLGA NPs loaded with Qu, was fabricated via electrohydrodynamic atomization process (EHDA) in order to improve poor aqueous solubility and stability of the drug with the aim of preventing atherosclerosis. The fabricated nanoparticles collected in a stable glass substrate. The results of atomic force microscopy (AFM) analysis and the scanning electron microscope (SEM), confirmed the fabrication of spherical polymeric nanoparticles with diameter ranging from 300nm to350 nm, narrow size distribution and smooth surface. The release profile of quercetin from the particles was investigated by determining the drug amount released at specific intervals for by luminescence. Furthermore, XRD analysis was used to determine the physical status of Qu encapsulated in NPs compared with that of pure Qu. The information obtained from this study facilitates the design and fabrication of polymeric nanoparticles as possible delivery systems for encapsulation, protection and controlled release of the flavonoid quercetin which is aiming to protect against CVDs. © 2018 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of 14th International Conference on Nanosciences & Nanotechnologies (NN17). Keywords: Electrohydrodynamic atomization (EHDA), Drug Delivery system, Quercetin, Poly (lactic-co-glycolic acid), Oxidative stress
* Corresponding author. Tel.: +302310998850; fax: +302310998390. E-mail address: [email protected] 2214-7853 © 2018 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of 14th International Conference on Nanosciences & Nanotechnologies (NN17).
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1. Introduction Cardiovascular diseases (CVD) are still the first and foremost cause of in both men and women. According to 2011 World Health Organization (WHO) report "Global atlas on cardiovascular disease prevention and control" CVD are referred as the leading cause of death and ailment reaching a presentence of 31 % worldwide [1]. The dominant processes involved in cardiovascular diseases is atherosclerosis. Atherosclerosis is a systemic, chronic and inflammatory disease that is due to endothelial malfunctions and the progressive establishment is mainly due to oxidative stress and other key parameters like lipid deposition and monocyte recruitment [12]. CVD and atherosclerosis formulate a composite outcome affected by numerous risk factors including high cholesterol, hypertension, smoking, diabetes, unhealthy diet, family history of early myocardial infarction and stress. These risk factors are directly associated with oxidative stress caused by high levels of Reactive Oxygen Spices (ROS) and free radicals [2]. ROS consists of Superoxide anion (O2⋅−O2⋅−), hydrogen peroxide (H2O2), and hydroxyl (⋅HO⋅HO). They created through physiological processes of the human body and play key role in the control of homeostasis of cells. Nevertheless, the existence of ROS in an unregulated quantity contributes to the pathogenesis of cardiovascular diseases. [13] (Figure 1) Generally, increased production of ROS may lead to atherosclerosis through four main mechanisms: endothelial cell dysfunction, vascular smooth muscle cells (VSMC) growth, monocyte migration and oxidation of LDLs [3]. Also, oxidative stress be related to diabetic cardiomyopathy, congestive cardiomyopathy, and hypertensive heart disease [17]. Thus, it is obvious to say that reduction of oxidative stress may lead to prevention and treatment of cardiovascular diseases. Antioxidants are the key to reducing the oxidative stress. As reported by many epidemiological studies increasing in antioxidant levels restricts the clinical expression of coronary artery disease [16]. In this way, polyphenolic compounds, such as Quercetin, raised interest due to their valuable antioxidant properties. Quercetin, 3,3’,4’,5’-7-pentahydroxy flavone (Figure 2) is one of the most plentiful flavonoid in plants with important pharmacological properties including antioxidant, neurological, antiviral, anticancer, cardiovascular, antimicrobial, anti-inflammatory and hepatoprotective [4,5]. The antioxidant activity of this flavonoid is higher than other popular antioxidant molecules like ascorbyl, trolox and rutin, due to the number and position of the free hydroxyl groups in its molecule [4]. Antioxidant mechanism of Quercetin involves capturing free radicals and inhibition over matrix metalloproteinases (MMP) activity, which leads to the end of oxidation. The mechanism of suppression of free radical processes through Quercetin is done in three stages: the formation of superoxide anion, the production of hydroxyl radicals and the formation of lipid peroxy radicals [2,5]. However, the use of the dietary flavonoid is limited due to its poor water solubility and instability either in physiological medium (pH, enzymes, presence of other molecules), or in the pharmaceutical products processing, or during storage (temperature, light, pH) [6]. These properties of quercetin result in poor bioavailability and permeability and may cause indefinite or poor pharmacological effect. One way to overcome these problems is to entrap the molecule into nanocarriers. Carriers that used in nanomedicine differ in their chemical properties and they can be liposomes, polymeric nanoparticles, dendrimers, magnetic nanoparticles, quantum dots, nanotubes or mixtures of them [12]. Polymeric nanoparticulate drug delivery systems are the dominant approach of nanocarrier-based drug delivery system. They can improve the bioavailability of poorly absorbed drugs, extend their residence time in the body, and provide a sustained drug release model. Ιn desirable characteristics of nanocarriers included: biocompatibility, drug compatibility, reduction of side effects, stability in physiological body fluids, high residence time in the systemic circulation and decreased toxicity [14]. To this end, the most commonly used polymer for manufacturing polymeric nanoparticles is PLGA or poly (lactic-co-glycolic acid). This polymeric material is biocompatible, biodegradable, non-immunogenic and it is hydrolyzed in the body to produce the biodegradable monomers, lactic acid and glycolic acid [18,7]. Τhe monomers are metabolized through Krebs cycle and eliminated easily by the human body [15]. Furthermore, PLGA is approved by the Food and Drug Administration (FDA) and European Medicine Agency (EMA) in several drug delivery systems [7]. Numerous techniques for preparing polymeric nanoparticulate drug-delivery systems have been already broadly used, for example emulsion solvent evaporation, nanoprecipitation, spray drying, interfacial polymerization etc. Nevertheless, these techniques exhibit some drawbacks including low drug-loading efficiency, fabrication of nanoparticles larger than 100 nm with high polydispersity and inability for large scale fabrication [10]. In recent years, electrospraying or electrohydrodynamic atomization (EHDA) is employed for fabrication of micro- and nanosized particles and there are many publications relating to the development of electrosprayed drug delivery systems [8,9]. Compared with other nanofabrication techniques, electrospraying is simpler, more flexible, automated, and
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has the ability to generate nanoparticles with high drug loaded efficiency [11]. Electrospraying is a one-step technique based on liquid atomization induced by electrical forces to achieve micro- and nano- particles. In its simplest form, during the electrospraying process a high voltage is applied to a liquid fitted in a syringe pump with a metal or a glass capillary. The fluid deforms into Taylor cone at the edge of the capillary due to the applied voltage, and a jet emits from the cone and breaks up into charged droplets. Only when the droplets are separated from the cone, the solvent starts to evaporate and generates particles promoted to the collector [8,9]. Therefore, the aim of this study is to encapsulate Quercetin within PLGA nanoparticles via electrohydrodynamic atomization process, in order to improve the poor aqueous solubility and stability of the drug, which in turn will help in the prevention of atherosclerosis.
Fig.1: Risk factors implicated in atherosclerosis through ROS.
Fig.2: Chemical structure of flavonoid Quercetin 2. MATERIALS AND METHODS 2.1 Materials Poly (DL-lactic-co-glycolic-acid)-PLGA (lactide: glycolide (75:25), MW = 66.000-107.000 g/mol ), Quercetin solid (assay >95%, HPLC), Acetone (for HPLC, purity > 98.5%) and Phosphate Buffer Saline Solution (PBS, 1×) were obtained by Sigma – Aldrich. Ultrapure (Type 1) water purchased from Merck Millipore, Direct-Q, 3UV was used during the study.
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2.2 Particle preparation PLGA-quercetin nanoparticles were prepared by using single-nozzle electrospraying with esprayer™ ES-2000S Electrospray Deposition (ESD) equipment. Electrospraying applies electrostatic forces to produce nanoparticles of a polymeric solution fitted in the syringe. Solutions were prepared by dissolving appropriate amounts of PLGA and Quercetin in acetone. Specifically, PLGA was dissolved in acetone after overnight stirring in order to achieve a clear solution. Then, Quercetin was added in the acetone solution and mixed until a homogeneous solution was formed. For the fabrication of blank nanoparticles, we used the acetone solution of PLGA without drug. The solutions after preparation were placed in a glass syringe with a metal nozzle and the nozzle was positioned vertically relative to the ground electrode. The two electrodes were adjusted at a distance approximately 60 mm separately. Other parameters (Voltage, Flow rate) were fixed in such a way that the system works in the cone-jet mode. Blank or Quercetin loaded PLGA nanoparticles that prepared during the electrospraying process were then collected on stable glass slides (1cm x 1cm) which were located on the ground electrode under the nozzle. 2.3 Particle Size and Morphology properties Size and morphology of blank and Quercetin loaded nanoparticles were investigated through usage of Atomic Force Microscopy (AFM) (NT – MDT solver, Scanning Probe Microscope with anti – vibration system MICRO 40 by Halcyonics) and Scanning Electron Microscopy(SEM) (JOEL, JSM – 840A) methods. From the AFM images of each sample, 100 particles were measured using the software IMAGEJ and the mean diameter as well as the standard deviation were calculated. 2.4 In vitro degradation study 1cm X 1cm glass samples with electrosprayed nanoparticles were set in well plates and the degradation study took place in Phosphate Buffered Saline at pH 7.2. The temperature was kept constant at 37 o C as samples were kept in an incubator (Galaxy 1705 incubator) during the study. The degradation study was conducted for both blank and Quercetin loaded PLGA nanoparticles. The morphology of degraded particles has been examined by Atomic Force Microscopy (AFM) (NT – MDT solver, Scanning Probe Microscope with anti – vibration system MICRO 40 by Halcyonics). and the p H of the degradation medium was measured by p H meter. 2.5 In vitro drug release study Quercetin loaded PLGA particulates that immobilized on glass (1 cm X 1 cm) were put in well plates and incubated (with use of Galaxy 1705 incubator) in PBS at p H 7.2 and constant temperature 37 o C. Drug release kinetics was investigated by determining the drug amount released at specific intervals for over a month by luminescence using the Luminometer Promega CLOMAX MULTI DETECTION SYSTEM, at 369 nm for Quercetin. 2.6 X-Ray Diffraction (XRD) X-Ray diffraction was conducted to determine the physical status and the crystallinity of pure Quercetin powder, blank PLGA nanoparticles and Quercetin loaded PLGA nanoparticles. XRD patterns were recorded using an X-ray diffractometer (Siemens D5000). 3. RESULTS AND DISCUSSION 3.1 Size and morphology of blank and Quercetin loaded PLGA nanoparticles Size and morphology of electrosprayed PLGA nanoparticles were studied using AFM for blank and a combination of AFM and SEM for drug loaded nanoparticles. The height, the phase and the 3D depiction of blank PLGA
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nanoparticles that immobilized on glass are shown in figure 3 (a, b, c). Generally, fabricated particles were approximately spherical in shape with smooth surface and uniform morphology. Their mean particle size was 343.5 nm and the standard deviation was ±25.4 nm. Also, blank PLGA nanoparticles had narrow dispersion as depicted in histogram of size distribution (figure 3d). Morphological properties of Quercetin loaded PLGA nanoparticles are shown at AFM (fig 4a) and SEM images (fig 4b). Analogous to blank nanoparticles case, drug loaded PLGA nanoparticles were approximately spherical with smooth surface and narrow dispersion as illustrated in histogram of size distribution (fig 4c). The mean diameter was 384.9 nm and the standard deviation was ±26.3 nm. In contrast to blank PLGA nanoparticles, Quercetin loaded PLGA nanoparticles were showed to have an increase in the mean size due to the presence of the drug.
a.
b.
c.
d. Fig.3:AFM images of blank PLGA nanoparticles. Height(a), Phase(b), 3D depiction(c) and size distribution(d)
a. b. c. Fig.4: AFM(a), SEM(b) images of Quercetin loaded PLGA nanoparticles and size distribution of them(c) 3.2 In vitro degradation study of blank and Quercetin loaded PLGA nanoparticles. Blank PLGA nanoparticles had almost fully degraded within a period of 20 days in PBS as a degradation medium. Morphological properties of degraded nanoparticles during the study, are shown at AFM images (figure 5). After the first day of the study, nanoparticles had lost their spherical shape (figure 5b) and they had been agglomerated (fig 5b, c). From the seventh day they had started creating films (figure 5d) and after the eleventh up to the twentieth day blank nanoparticles had degraded (figure 5e, f). From the figure 6 it can be seen that during the time of degradation the p H of PBS decreases because of the presence of acidic degradation products of PLGA. Specifically, the initial pH value of degradation medium (PBS) was 7.4 and during the time of degradation was reduced until the value 6.1
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(the fifteenth day) due to accumulation of glycolic acid and lactic acid. The pH reduction confirms the PLGA nanoparticles degradation. In the case of Quercetin loaded nanoparticles duration of degradation in PBS was fifty days. Morphological properties of degraded nanoparticles during the study, are shown at AFM images (figure 7). Analogous to the blank nanoparticles and the Quercetin loaded nanoparticles after 5 days had lost their spherical shape and they agglomerated (figure 7b,7c). After the twenty-fifth day of the degradation study were created films and as from the thirtieth fifth day up to the fiftieth had degraded. The longer period of degradation of quercetin-loaded nanoparticles is due to the existence of the drug. Results of the degradation study of drug loaded PLGA nanoparticles accordance with the results of the in vitro drug release study presented in section 3.3
Fig.5: AFM images of blank PLGA nanoparticles after 0 day (a), 1 day (b), 4 days (c), 7 days (d), 11 days (e) and 15 days(f) of in vitro degradation study.
Fig.6: Changes in the pH of the degradation medium at specific times during the degradation study
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Fig.7: AFM images of Quercetin loaded PLGA nanoparticles after 0 days (a), 5 days (b), 15 days (c), 25 days (d), 35 days (e) and 50 days(f) of degradation study 3.3 In vitro drug release study The in vitro release behavior of Quercetin from PLGA nanoparticles is shown in figure 8. As can be seen, Quercetin release from nanoparticles followed a biphasic release model with a primary release conducted in the first 24hrs followed by a sustained release for 59 days. The burst drug release of PLGA nanoparticles was climbed at 27% due to the fact of the existence of quercetin molecules on or near the surface of polymeric nanoparticles. Simultaneously, the sustained release was shown to reach a 96.1% caused by the degradation of the polymeric matrix carried out in more than 50 days as shown in session 3.2.
Fig.8: In vitro drug release profile of quercetin loaded PLGA nanoparticles 3.4 Physical status of Quercetin in PLGA NPs In order to determine the physical status of drug molecule, polymer and polymeric nanoparticles we used the X-ray diffraction. The XRD patterns for pure Quercetin powder (black diagram), blank PLGA nanoparticles (red diagram) and quercetin-loaded PLGA nanoparticles (green diagram) are shown in figure 9. For pure drug, the XRD pattern showed characteristic peaks because of its crystalline structure. For blank PLGA nanoparticles, the XRD pattern
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showed no peak, suggesting that polymeric blank nanoparticles are amorphous. For quercetin-loaded PLGA nanoparticles the XRD pattern also showed no peak claim that Quercetin exists dispersed in a non-crystalline state within the PLGA polymeric matrix.
Fig.9: XRD patterns of Quercetin powder, blank PLGA nanoparticles and Quercetin loaded PLGA nanoparticles. 4. CONCLUSIONS Electrohydrodynamic Atomization process is a suitable technique for fabrication of polymeric nanoparticles that used in drug delivery systems. PLGA nanoparticles were prepared successfully and used to encapsulate Quercetin to protect the drug and control pharmacokinetics properties. Morphology analysis (AFM and SEM measurements) illustrated that PLGA nanoparticles are spherical with an average size of 343 nm for PLGA nanoparticles and 384 nm for Quercetin loaded PLGA nanoparticles. Quercetin encapsulated within the polymeric matrix in an noncrystalline state. Quercetin loaded nanoparticles exhibited a biphasic release model of the drug with a rapid release over a few hours and a sustained slow release over 59 days. Therefore, this kind of drug delivery system of the antioxidant drug Quercetin, has great potential to be used in prevention of atherosclerosis and other relative cardiovascular diseases. 5. REFERENCES [1] World Heart Organization global atlas on cardiovascular disease prevention and control, Geneva 2011 [2] Parul Lakhanpal et al., Role of quercetin in cardiovascular diseases”, Internet Journal of Medical Update, Vol. 3, No. 1, Jan-Jun 2008 [3] Alexander RW,”Hypertension and the pathogenesis of atherosclerosis: oxidative stress and the mediation of arterial inflammatory response: a new perspective.”Hypertension , pp:155-61,1995 [4] Avnesh Kumari et al., Colloids and Surfaces B: Biointerfaces, 80 (2010) 184-192 2010 [5] Aneela Maalik et al., Tropical Journal of Pharmaceutical Research, 13 (2014) 1561-1566 [6] Hector Pool et al., “Antioxidant Effects of Quercetin and Catechin Encapsulated into PLGA Nanoparticles”, Hindawi Publishing Corporation Journal of Nanomaterials Vol 2012 [7] Lucia Ruxandra Tefas et. al., Clujul Medical 88 (2015) no. 2 [8] B. T. Midhun et al., Journal of Biomaterials Science, 22 (2011) 2431–2444 [9] Alejandro Sosnik, J. Biomed. Nanotechnol, 10 (2014) 9 [10] Maedeh Zamani et. al., International Journal of Nanomedicine 8 (2013) 2997–3017 [11] Leilei Zhang et al., HHS Author Manuscripts, pp 595–612,2012 [12] Varvara Karagkiozaki et al., J. Biomed. Nanotechnol. 11 (2015) 191–210 [13] Keyvan Karimi Galougahi et al., European Heart Journal 36 (2015) 1576–1582 [14] Varvara Karagkiozaki, “Horizons in clinical nanomedicine” [15] Fatemeh Sadat et al., Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 [16] S.L. Nuttall et al., Q J Med 92 (1999) 239–244 [17] FredricJ.Pashkow et al., , International Journal of Inflammation Vol. 2011, 2011 [18] Magdalena Stevanović and Dragan Uskoković, “Influence of Different Degradation Medium on Release of Ascorbic Acid from PLGA Nano ans Microspheres”, in proc. of “Physical Chemistry 2008. 9th International Conference on Fundamental and Applied Aspects of Physical Chemistry, Belgrade, September 24-26, 200”