1. INTRODUCTION:

Polymer nanotechnologies have the pivotal role to achieve drug delivery challenges such as reduction of side effects, drug targeting, and controlled drug delivery (Vauthier and Bouchemali 2009). Nanoparticle consists of macromolecular materials (polymer) in which the drugs are entrapped, encapsulated, dissolved, adsorbed or attached (Soppimath et al. 2001). Biodegradable polyesters have been used widely in various fields like implants, tissue engineering and drug delivery (Lee et al. 2009). Poly (ε caprolactone; PCL), an FDA (Food and drug administration) approved polyester is widely used as a drug carrier. Additionally, PCL is non-toxic, non-mutagenic and comparatively more economic than other polyesters (Pitt, 1990; Woodruff and Hutmatcher 2010).

PCL based nanoparticles have been mostly prepared by the solvent displacement method, solvent evaporation method, emulsification diffusion method, interfacial polymer disposition method and dialysis method (Sinha et al. 2004).

Received on 14.05.2015 Modified on 05.06.2015

Research J. Pharm. and Tech. 8(7): July, 2015; Page 836-840

DOI: 10.5958/0974-360X.2015.00136.5

Solvent displacement and emulsification-diffusion methods used most often for preparing biodegradable submicron particles (Mora-Huertas et al. 2011).

Quercetin is a polyphenolic compound found in fruits, vegetables and beverages that are regularly consumed by humans. Quercetin (3, 5, 7, 3′, 4′- pentahydroxylflavone) possesses a wide range of pharmacological properties like anti-oxidative (Bischoff 2008), anti-leishmanial (Silva et al. 2012; Kumar et al. 2014), antiviral (Ohnishi and Bannai 1993) anti-inflammatory (Boots et al. 2008), and anti-proliferative (Hirpara et al. 2009). Though quercetin have various pharmacological properties, the problem related to low aqueous solubility, short half-life and low bioavailability, limit the therapeutic utility of quercetin. Recent research has focused on delivering quercetin in various carrier systems like liposomes (Gang et al. 2012), nanoparticles (Pool et al. 2012), nanocrystals (Sahoo et al. 2011), phospholipid complexes (Singh et al. 2012) and solid dispersion techniques (Costa et al. 2011), to overcome above mentioned problems without affecting its efficacy.

In this present work, we have encapsulate quercetin in Poly (ε caprolactone) based biodegradable nanoparticles by the solvent displacement method (nano-precipitation). We hypothesized that quercetin loaded nanoparticles would be capable of improving the bioavailability owing to submicron size of nanoparticles and controlled drug release. To achieve the above objective, investigate the formulation variables (polymer and surfactant concentrations) on the characteristics of nanoparticles such as particle size in different media, drug loading, dissolution efficiency and morphological analysis. Present study would assist to layout effective delivery system for protection, encapsulation and controlled release of bioactive compounds, which have potential to improve clinical utility.

2. MATERIALS AND METHODS:

Quercetin (95%) was purchased from Sigma (Sigma-Aldrich, India), PCL (Mw, 14, 000,) was purchased from Aldrich (Sigma-Aldrich, India), Pluronic F127 was obtained from Ranbaxy Laboratory, India. Milli-Q water was used in all experiments.

2.1. Preparation of PCL based quercetin nanoparticles:

The PCL based nanoparticles were prepared by modified-solvent displacement method (Fessi et al. 1988; Fessi et al. 1989). The solvent phases (organic phase) consist of drug (quercetin) and PCL, non-solvent phase (aqueous phase) is supplemented with pluronic F-127 (surfactant). The organic phase was slowly added to the aqueous phase under magnetic stirring. Nanoparticles are formed instantaneously and the solvent is removed by using evaporation under reduced pressure. The nano-suspension was then centrifuged at 20,000 RPM using ultracentrifuge (Optima MAX-XP, Beckman Coulter, USA) for 20 min at 4^oC.The resulting pellets were re-suspended in distilled water and frozen at -20^oC for 12 h and, subsequently, frozen nanoparticle dispersion was freeze dried at −70 °C for 48 h using freeze dryer (Heto Power Dry LL3000, Thermo Corporation, Czech Republic). The freeze-dried nanoparticles were stored at 4 ⁰C until further use.

Table 1 Formulation compositions used for constituting quercetin loaded nanoparticles

Formulation code	Compositions
	PCL concentration (mg)	Pluronic F-127 (%)
F1	50	0.5
F2	50	0.6
F3	50	0.7
F4	100	0.5
F5	100	0.6
F6	100	0.7
F7	150	0.5
F8	150	0.6
F9	150	0.7

2.2. Drug loading (%):

Drug loading was determined using a solvent extraction method (Natarajan et al. 2011). Dried nanoparticles (equivalent to 2 mg of drug) were treated with dichloromethane: methanol (1:9) to extract quercetin by vortex for 1 min and centrifugation at 5000 rpm for 15 min. The supernatant containing quercetin was suitably diluted using methanol and the absorbance was measured at 371nm by UV–Vis spectro-photometer (UV 1800, Shimadzu, Japan). Drug loading (%) was calculated using the ratio of the weight of quercetin determined by the weight of total nanoparticles, as shown in equation 1. Experiments were carried out in triplicates for each formulation and the results were expressed as mean ± SD.

2.3. Particle size and polydispersity index:

The particle size analysis and polydispersity Index (PDI) of drug loaded nanoparticles was determined by photon correlation spectroscopy using a Zetasizer (Nano ZS, Malvern Instruments, Worcestershire, UK). Each sample was diluted (10 mL) with different media viz. 0.1N HCl and Phosphate buffer (pH 7.4) until the appropriate concentration of particle dispersion to avoid multi-scattering events. All measurements were done in triplicate. The measurement conditions were: He–Ne Red laser, 4.0 mW, 633 nm; temperature, 25^oC; refractive index, 1.333; or with adjustment if needed. The polydispersity index (PI) is a measure of dispersion homogeneity and ranges from 0 to 1. Values close to 0 indicate a homogeneous dispersion while those greater than 0.3 indicate high heterogeneity.

2.4 Dissolution studies:

The in vitro dissolution study of nanoparticles formulations (F1-F9) of quercetin was performed by the method described by Dinesh Kumar et al. 2015. Quercetin loaded nanoparticles equivalent to 2 mg of drug were placed into dialysis bags. The dialysis bags were placed into a USP dissolution apparatus 2 (TDT-08L, Electrolab, Mumbai, India) containing 250 ml of dissolution medium, maintained at 37±0.5°C and paddle speed set at 100 rpm. The withdrawn sample was replenished with 2 ml of fresh media. The concentration of quercetin was determined at 371 nm using UV Visible spectrometer (UV- 1800, Shimadzu). The in vitro dissolution profile of dissolution efficiency was calculated from the release data obtained from our previous study (Dinesh Kumar et al. 2015) using OriginPro 8 (Origin Lab Corporation, Massachusetts, USA) software.

2.4. Zeta potential:

Zeta potential was measured by electrophoretic mobility which is determined by laser Doppler anemometry in a micro-eletrophoresis cell using the Zetasizer (Nano ZS, Malvern Instruments, Worcestershire, UK) equipped with 4.0 mW He–Ne red laser (633 nm) which measures the potential ranged from -120 to 120 V. Nanoparticles are diluted with de-ionized water (10mL), for the measurement of zeta potential. Each sample was measured at 25^oC, in triplicate.

2.5. High Resolution Transmission Electron Microscopy (HR-TEM):

The morphology of nanoparticles was investigated by HR-TEM (High-resolution transmission electron microscopy (HR-TEM); JEM-2100, JEOL Ltd., Akishima-shi, Japan). Samples for the TEM studies were prepared by placing a drop of the aqueous suspension of particles spread over carbon-coated copper grids followed by solvent evaporation under vacuum. Add 1% (w/v) phosphotungstic acid to give a negative stain. The grid was allowed to stand for 20s being removed excess stain as described above. The grid was air-dried at ambient temperature before loading in the microscope.

2.6. Thermogravimetric Analysis:

Thermal analysis of quercetin, PCL, Pluronic F-127 and Physical Mixture were performed by thermogravimetric analysis using DTG-60 (Shimadzu, Japan). Samples of 5.0 ± 0.5 mg were placed into platinum crucible and heated, at a constant rate of 10 °C per min, from ambient to 600°C under nitrogen atmosphere (nitrogen flow rate 50 ml/min).

3. RESULTS AND DISCUSSION:

In this study, we utilized modified nanoprecipitation method for the preparation of quercetin loaded nanoparticles as shown in Fig.1.

3.1. Particle size and polydispersity index:

Polymer concentration is an important tool with respect to particle size in different media. In the present study, three different polymer concentrations (0.5 %, 0.6 % and

0.7 %) were selected based on preliminary test. The mean particle size of nanoparticles ranged from 189.10 ± 0.28 to 252.14 ± 2.32 in 0.1 N HCl and 184.06 ± 3.84 to 288.54 ± 2.86 in phosphate buffer pH 7.4 (Table 2). Particle size was increased by increasing PCL concentration from 0.5 to 0.7 %. This might be due to the increased viscosity of organic phase by increasing polymer concentration. Similar findings were also described by (Song et al. 2008; Thioune et al. 1997). Surfactant concentration is one of the key factors for stable formulation. In this study, a marginal increase in particle size was observed with an increasing amount of surfactant concentration. It may due to surface coating of surfactant over nanoparticles. Similar findings were also reported by (Redhead et al. 2001). The Polydispersity of the studied formulations (F1-F9) varied from 0.091 ± 0.12 to 0.184 ± 1.22. From the results, it confirms that all formulations (F1-F9) are homogenous nature (Table 2).

3.2. Zeta potential:

The zeta potential is a key factor in the stability of colloidal dispersions. The magnitude of the zeta potential indicates the degree of electrostatic repulsion between adjacent, similarly charged particles in a dispersion. In this study, zeta potential of the quercetin loaded nanoparticles was less negative with an increase in PCL concentration and particle size of the nanoparticles (Table 2). The zeta potential of the nanoparticles was negative owing to the presence of terminal carboxylic groups. The zeta potential of the studied formulations (F1-F9) varied from -10.1 ± 0.35 to -15.1 ± 0.28.

3.3. Drug loading:

Drug loading (%) of formulation F1-F9 was in the range of 7.18 ± 0.25 to 9.27 ± 0.50 (Table 2). Quercetin loading (%) was decreased by increasing polymer concentration. Increase in PCL amount led to an increase total mass of polymer concentration, which is owing to lower drug to polymer ratio. Similar findings were also reported by Natarajan et al. 2011. The increase in polymer concentration might have increased the viscosity of primary emulsion, thereby reducing the partition of drugs into external phase and subsequently increasing the drug entrapment efficiency.

3.4. Dissolution efficiency:

The dissolution efficiency of formulations (F1-F9) was in the range of 46.42 ± 5.42 to 72.20 ± 0.68 (Table 2). Results showed a decrease in dissolution efficiency with an increase amount of polymer. Conversely an increase in surfactant concentration from 0.5-0.7 % led to an increase in drug dissolution efficiency.

3.5. Morphological analysis by HR-TEM:

The HR-TEM image of the quercetin loaded nanoparticles and their surface morphology are shown in Fig. 2. It could be observed that most of the quercetin loaded nanoparticles revealed a uniform and spherical shape with the size 200-250 nm in diameter, which was in coincidence with the data from photon correlation spectroscopy.

Table 2 Characterization of quercetin loaded nanoparticles

Formulation code	Particle size (nm)		Drug loading (%)	Zeta potential (mV)	Polydispersity (PI)	Dissolution efficiency (%)
Formulation code	0.1 HCl	Phosphate buffer (pH 7.4)	Drug loading (%)	Zeta potential (mV)	Polydispersity (PI)	Dissolution efficiency (%)
F1	189.10 ± 0.28	184.06 ± 3.84	9.27 ± 0.50	-15.1 ± 0.28	0.091 ± 0.12	60.74 ± 1.24
F2	194.90 ± 1.90	196.22 ± 4.56	8.51 ± 0.35	-14.5 ± 0.99	0.108 ± 0.58	65.62 ± 2.76
F3	200.68 ± 7.24	214.78 ± 7.78	8.34 ± 0.08	-14.9 ± 0.21	0.128 ± 0.82	72.20 ± 0.68
F4	196.72 ± 2.66	206.70 ± 2.34	8.23 ± 0.02	-13.6 ± 0.42	0.149 ± 1.08	56.50 ± 4.76
F5	204.56 ± 0.54	219.34 ± 8.28	8.00 ± 0.24	-13.4 ± 0.28	0.156 ± 0.94	57.58 ± 3.56
F6	212.44 ± 0.14	227.52 ± 3.12	7.82 ± 0.21	-13.1 ± 0.63	0.172 ± 0.32	61.61 ± 2.24
F7	221.56 ± 0.99	228.78 ± 2.75	7.25 ± 0.10	-12.9 ± 0.14	0.150 ± 0.62	46.42 ± 5.42
F8	236.60 ± 0.42	246.80 ± 5.26	7.53 ± 0.24	-12.3 ± 0.14	0.168 ± 0.78	49.63 ± 8.86
F9	252.14 ± 2.32	288.54 ± 2.86	7.18 ± 0.25	-10.1 ± 0.35	0.184 ± 1.22	50.37 ± 4.18

Values indicate Mean ± SD, n=3

Figure 2: HR-TEM images of quercetin loaded nanoparticles (F8)

3.6. Drug-excipient compatibility study (TGA)

The TGA curve of quercetin, PCL, Pluronic F127 and physical mixture are illustrated in figure 3. The TG cure exhibited 89.55% mass loss between 308 to 570 °C, due to decomposition of quercetin. PCL showed 91.23% of mass loss between 340 to 450 °C. Pluronic F-127 showed 99.03% of mass loss between 230 to 500 °C (Figure 3). All the TGA curves of physical mixtures can be considered as a superposition of TG curve of pure quercetin and excipients, as a proof of compatibility between quercetin with the used excipients.

Figure 3 TGA curve of (a) quercetin (b) PCL (c) pluronic F127 (d) Physical mixture 1(quercetin+PCL) and (e) Physical mixture 2 (quercetin+Pluronic F127)

4. CONCLUSION:

Quercetin seems to possess minimal therapeutic window through oral delivery, which is demanding to produce required therapeutic concentration at the site of action. This emphasizes for the development of polymeric nanoparticles to have controlled release through oral route which may be beneficial by increasing the duration of quercetin release to the site of action and consequently reducing the dosing intervals. Quercetin loaded nanoparticles were prepared by the nano- precipitation method. Quercetin loaded nanoparticles showed the release of quercetin for more than 48 hours in a controlled manner. Results have been shown that the polymer concentration has significantly affected the particle size, zeta potential and drug loading (%). In contrast, surfactant concentration did not significantly affect drug loading and zeta potential. These polymeric nanoparticles may therefore be suitable for encapsulating bioactive components within food or pharmaceutical products, can lead to improvements in both the stability and bioavailability of the bioactive compounds.

Abbreviations used:

PCL poly (ε caprolactone)

PDI Poly dispersibility index

TGA Thermogravimetric analysis

HR-TEM High Resolution-Transmission Electron Microscopy

5. ACKNOWLEDGEMENT:

This article does not contain any studies with human and animal subjects performed by any of the authors. All authors Dinesh Kumar V. and Priya Ranjan Prasad Verma declare that they have no conflict of interest. Dinesh Kumar V. is thankful to UGC for the financial support as UGC-BSR (Grant No. F.4-1/2006 (BSR)/7-36/2007(BSR)). The authors are thankful to CIF, BIT Mesra (India) for SEM, AFM, DLS and STIC, Cochin University (India) for XRD analysis. The authors are also thankful to BIT Mesra (India), for providing necessary facilities for this project.

6. REFERENCES:

1 Bischoff SC. Quercetin: potentials in the prevention and therapy of disease. Curr Opin Clin Nutr Metab Care. 11; 2008: 733-740.

2 Boots AW, Haenen GR, and Bast A. Health effects of quercetin: from antioxidant to nutraceutical. Eur J Pharmacol. 585; 2008: 325-337.

3 Costa ARM, Marquiafavel FS, Vaz Mmoll, Rocha BA, Bueno PCP, Amaral PLM, Barud HS, Berreta-Silva AA. Quercetin-PVP K25 solid dispersions. J Therm Anal Calorim. 104; 2011: 273–278.

4 Fessi H, Puisieux F, and Devissaguet JP. Nanocapsule formation by interfacial polymer deposition following solvent displacement. Int J Pharm, 55; 1988: R1-R4.

5 Fessi H, Puisieux F, Devissaguet JP, Ammoury N, and Benita S. Nanocapsule formation by interfacial polymer deposition following solvent displacement. Int J Pharm. 55; 1989: 25-28.

6 Galindo-Rodriguez S, Allemann E, Fessi H, and Doelker E. Physiochemical parameters associated with nanoparticles formation in the salting out, emulsification-diffusion, and nanoprecipitation method. Pharm Res. 21; 2004: 1428-1439.

7 Gang W, Jie WJ, Ping ZL, Ming DS, Ying LJ, Lei W, and Fang Y. Liposomal quercetin: evaluating drug delivery in vitro and bio-distribution in vivo. Expert Opin Drug Deliv. 9; 2012: 599-613.

8 Hirpara KV, Aggarwal P, Mukherjee AJ, Joshi N, and Burman AC. Quercetin and its derivatives: synthesis, pharmacological uses with special emphasis on anti-tumor properties and prodrug with enhanced bio-availability. Anticancer Agents Med Chem. 9; 2009: 138-161.

9 Kumar VD Verma PRP, and Singh SK. New insights into the diagnosis and chemotherapy for visceral leishmaniasis. Curr Drug Delivery. 11; 2014: 200-213.

10 Lee JS, Hwang SJ, Lee DS, Chulkim S, and Kim DJ. Formation of poly (ethylene glycol)- Poly (ε caprolactone) nanoparticles via nanoprecipitation. Macromol Res. 17 (2); 2009: 72-78.

11 Mora-Huertas CE, Fessi H, and Elaissari A. Influence of process and formulation parameters on the formation of submicron particles by solvent displacement and emulsification-diffusion methods critical comparison. Adv Colloid Interface Sci. 163; 2011: 90-122.

12 Natarajan V, Krithica N, Madhan B, and Sehgal PK. Formulation and evaluation of quercetin poly (ε caprolactone) microspheres for the treatment of rheumatoid arthritis. J Pharm Sci. 100 (1); 2011: 195-205.

13 Ohnishi E, and Bannai H. Quercetin potentiates TNF-induced antiviral activity. Antiviral Res 22 (4); 1999: 327-331.

14 Pitt CG. Poly (ε caprolactone) and its copolymer. In M. Chasin, and R. lange (Eds.), Biodegradable polymers as drug delivery systems (pp. 71-120). New York: Marcel Dekker. 1990.

15 Pool H, and Quintanar D, Figueroa JD, Bechara JEH, Clements DJ, Mendoza S. Polymeric Nanoparticles as Oral Delivery Systems for Encapsulation and Release of Polyphenolic Compounds: Impact on Quercetin Antioxidant Activity and Bioaccessibility. Food Biophys 7; 2012: 276–288.

16 Sahoo NG, Kakran M, Shaal LA, Li L, Muller RH, Pal M, Tan LP. Preparation and characterization of quercetin nanocrystals. J Pharm Sci. 100; 2011: 2379–2390.

17 Silva ER, Maquiaveli CC, and Magalhaes PP. The leishmanicidal flavonols quercetin and quercitrin target Leishmania (Leishmania) Amazonensis arginase. Exp Parasitol. 130; 2012: 183-188.

18 Singh D, Rawat MSM, Semalty A, and Semalty M. Quercetin-phospholipid complex: An amorphous pharmaceutical system in herbal drug delivery. Curr Drug Discovery Technol. 9; 2012: 17-24.

19 Sinha VR, Bansal K, Kaushik R, Kumria R, Trehan A. Poly (εcaprolactone) microspheres and nanospheres: an overview. Inter J Pharm. 278; 2004: 1-23.

20 Song X, Zhao Y, Wu W, Bi Y, Cai Z, Chen, Q, Li Y, Hou S. PLGA nanoparticles loaded with vincristine sulfate and verapamil hydrochloride: systematic study of particle size and drug entrapment efficiency. Int J Pharm. 350; 2008: 320-329.

21 Soppimath KS, Aminabhavi TM, Kulkarni AR, Rudzinski WE. Biodegradable polymeric nanoparticles as drug delivery devices. J Control Release. 70; 2001: 1–20.

22 Thioune O, Fessi H, Devissaguet JP, and Puisieux F. Preparation of pseudolatex by nanoprecipitation: influence of the solvent nature on intrinsic viscosity and interaction constant. Int J Pharm. 146; 1997: 233-248.

23 Vauthier C, Bouchemali K. Methods for the Preparation and Manufacture of Polymeric Nanoparticles. Pharma Res. 26 (5); 2009: 1025-58.

24 Woodruff MA, and Hutmacher DW. The return of a forgotten polymer-poly ε caprolactone in the 21st century. Prog Polym Sci. 35; 2010: 1217-1256.