http://informahealthcare.com/phd ISSN: 1083-7450 (print), 1097-9867 (electronic) Pharm Dev Technol, Early Online: 1–5 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2013.860551
ORIGINAL RESEARCH
Formulation and evaluation of cholesterol-rich nanoemulsion (LDE) for drug delivery potential of cholesteryl-maleoyl-5-fluorouracil Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Purdue University on 03/13/15 For personal use only.
Fars K. Alanazi1, Nazrul Haq1,2, Awwad A. Radwan1, Ibrahim A. Alsarra1,2, and Faiyaz Shakeel1,2 1
Kayyali Chair for Pharmaceutical Industry, Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia and Center of Excellence in Biotechnology Research (CEBR), Riyadh, Saudi Arabia
2
Abstract
Keywords
It has been reported that cholesterol-rich nanoemulsions (LDE) can bind to low density lipoprotein (LDL) receptors which can concentrate anticancer drugs in the tissues via LDL receptor overexpression and reduced the adverse effects of the treatment. Therefore, in this study, LDE nanoemulsions of cholesteryl-maleoyl-5-fluorouracil (5-FU conjugate) were developed and evaluated in vitro. LDE nanoemulsions were prepared by high-energy emulsification technique. Developed formulations were characterized in terms of droplet size, polydispersity index, zeta potential, viscosity and refractive index. Optimized formulation (L5) was also evaluated for surface morphology using transmission electron microscopy (TEM). Developed formulations were subjected to in vitro drug release studies through dialysis membrane. The droplet size (50 nm), polydispersity index (0.109) and viscosity (32.16 cp) were found to be lowest for optimized formulation L5. The results of zeta potential indicated the stable formation of developed LDE nanoemulsions. TEM images of optimized formulation indicated non-spherical shape of droplets. About 97% of conjugate was found to be released from L5 after 24 h of study. Overall, these results indicated that developed LDE nanoemulsions could be successfully used for oral delivery of 5-FU conjugate.
Cholesteryl-maleoyl-5-fluorouracil, droplet size, drug release, nanoemulsion, zeta potential
Introduction The 5-fluorouracil (5-FU), the most popular cytotoxic drug which belongs to an antimetabolite class of anticancer drugs has been recommended clinically for the treatment of various types of cancers and solid tumors such as colorectal, breast and ovarian tumor1,2. It has been reported as hydrophilic drug with aqueous solubility of 12.2 mg/ml2. Oral administration of 5-FU showed incomplete and erratic pharmacokinetic profile, therefore it is administered via intravenous injection in which maintenance of blood concentration of drug is still short due to its rapid metabolism3,4. Chemical modification of 5-FU via prodrug/ conjugation approach has been successfully used to enhance its lipophilicity in order to enhance its therapeutic efficacy and to reduce adverse effects as evidenced by its various derivatives such as tegafur, capecitabine and carmofur5–7. Therefore, ester conjugate of 5-FU (cholesteryl-maleoyl-5-FU with molecular weight of 596.77; Figure 1) was synthesized in order to improve its therapeutic efficacy and to reduce adverse effects. Various drug delivery approaches namely niosomes, liposomes, ethosomes, microparticles, nanoparticles, nanogels and microemulsions have also been tried to enhance its therapeutic efficacy and to reduce adverse effects8–18. Cholesterol-rich nanoemulsions (LDE) are known to bind with low density lipoprotein (LDL) receptors which can concentrate
Address for correspondence: Dr. F. Shakeel, King Saud University, Riyadh, Saudi Arabia. Tel: +966-537507318. E-mail: [email protected]
History Received 17 September 2013 Revised 5 October 2013 Accepted 7 October 2013 Published online 25 November 2013
anticancer drugs in the tissues by LDL receptor overexpression and reduced the adverse effects of the treatment19,20. They are formed by monolayer of phosphatidylcholine surrounding a core of cholesteryl ester19. LDE nanoemulsions mimic the lipidic portion of LDL and acquire apolipoprotein-E from the circulating native lipoproteins when come in contact with plasma20. It has been reported that the most of the cancer cells show LDL receptor upregulation and LDE nanoemulsions target these cells and deliver the anticancer drugs into the cells via LDL-receptor mediated endocytosis19,20. The potential of LDE nanoemulsions has been proved in enhancing therapeutic efficacy and reducing adverse effects of the treatment of various anticancer drugs such as carmustine, paclitaxel, etoposide and methotrexate either preclinically or clinically19–26. LDE nanoemulsions of 5-FU or 5-FU conjugate have not been investigated either in vitro or in vivo in literature so far. Therefore, in this study, attempts were made to develop and evaluate LDE nanoemulsions of newly synthesized conjugate cholesteryl-maleoyl-5-fluorouracil (5-FU conjugate) in order to enhance its therapeutic efficacy and to reduce adverse effects.
Materials and methods Materials Chromatographic grade methanol and 5-FU (purity 99.2%) were purchased from BDH Laboratory supplies (Liverpool, UK) and Alfa Aesar (Ward Hill, MA), respectively. Cholesterylmaleoyl-5-fluorouracil conjugate (purity 98.9%) was synthesized and characterized in the Laboratory. Cholesterol (purity 98.0%), triolein, cholesteryloleate, phosphatidylcholine and Tween-80
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Table 1. Composition of different LDE formulations (L1–L5). Ingredients 5-FU conjugate (mg) Cholesterol (mg) Triolein (mg) Phosphatidylcholine (mg) Cholesteryloleate (mg) Tween-80 (ml) Labrasol (ml) Deionized water q.s. (ml)
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Figure 1. Molecular structure of cholesterol-maleoyl-5-fluorouracil (Mol. Wt. 596.77).
were purchased from Sigma Aldrich (St. Louis, MO). Labrasol was procured as a kind gift sample from Gattefosse (Cedex, France). The purity of all these chemicals was greater than 99.0%. Dialysis bag (MWCO 12 000g/mol) was purchased from Spectrum Medical Industries (Mumbai, India). Ultra-pure water (chromatographic grade) was obtained from ELGA water purification unit (Wycombe, Bucks, UK). All other chemicals and reagents were of analytical grade. Analytical methodology Ultra high performance liquid chromatographic (UHPLC) method with diode array detector (DAD) was used for quantification of 5-FU conjugate. UHPLC-DAD analysis was carried out at room temperature (22 1 C) using Thermo Scientific UHPLC system (Thermo Scientific, Waltham, MA) equipped with a 3000 LC pump, 3000 autosampler, binary pumps, a programmable DAD detector, ultimate 3000 column oven, ultimate 3000 controller with an inline vacuum degasser. UHPLC data was analyzed using Chromeleon, version 6.8 software (Waltham, MA). The chromatographic identification of 5-FU conjugate was achieved with a Thermo Hypersil GOLD 50 2.1 mm RP C18 column (Thermo Scientific, Waltham, MA) having a 1.9 mm packing as a stationary phase. The mobile phase was methanol:water (80:20% v/v) with a flow rate of 0.4 ml/min. The DAD wavelength was set at 276 nm. Samples (1 ml) were injected into UHPLC system using an ultimate 3000 series Thermo auto sampler (Thermo Scientific, Waltham, MA). The proposed analytical method was developed and validated for linearity, accuracy, precision, robustness, specificity and stability-indicating properties in the laboratory (unpublished data).
L1
L2
L3
L4
L5
6.0 0.5 1.0 20.0 40.0 0.1 0.1 2.0
6.0 0.5 1.0 20.0 40.0 0.15 0.15 2.0
6.0 0.5 1.0 20.0 40.0 0.2 0.2 2.0
6.0 0.5 1.0 20.0 40.0 0.25 0.25 2.0
6.0 0.5 1.0 20.0 40.0 0.3 0.3 2.0
q.s., Quantity sufficient to produce 2 ml of total LDE nanoemulsion; 5-FU conjugate, choesteryl-maleoyl-5-fluorouracil.
order to evaluate the impact of surfactants on physicochemical properties and in vitro drug release of LDE formulations. The composition of different LDE formulations is listed in Table 1. Thermodynamic stability test As nanoemulsions are thermodynamically/thermokinetically stable systems, different thermodynamic stability tests were performed in order to eliminate metastable or unstable formulations27. Prepared LDE nanoemulsions were subjected to different thermodynamic stability tests such as centrifugation (at 5000 rpm), heating and cooling cycles (three cycles) and freeze–pump–thaw cycles (three cycles) as reported in previous literature27–29. Measurement of droplet size, polydispersity and zeta potential The droplet size, polydispersity index (PI) and zeta potential (ZP) of LDE nanoemulsions (L1–L5) were measured using Malvern Mastersizer (Malvern Instruments Ltd., Holtsville, NY) at room temperature (25 C) with a scattering angle of 90 . Each sample of drug-loaded LDE nanoemulsion was diluted suitably with deionized water (1:200), sonicated for 10 min and filtered through 0.45 mm membrane filter. About 3 ml of each formulation was transferred to a disposable cuvette for droplet size and PI measurement. For the measurement of ZP of each LDE nanoemulsion, glass electrode cuvettes were used instead of disposable cuvettes. Measurement of viscosity and refractive index
Preparation of cholesterol-rich nanoemulsion (LDE) of 5-FU conjugate Various LDE formulations of 5-FU conjugates (L1–L5) were prepared by high energy emulsification technique as reported by Maranhao et al.20 and Moura et al.21 with slight modifications. Lipid mixture composed of 0.5 mg of cholesterol, 1 mg of triolein, 20 mg of phosphatidylcholine and 40 mg of cholesteryloleate was emulsified with the required quantity of deionized water (aqueous phase) by prolonged ultrasonic irradiation followed by a two-step ultracentrifugation of crude emulsion with density adjustment by the addition of KBr to obtain LDE nanoemulsions20. Prepared LDE nanoemulsions (L1–L5) were dialyzed against normal saline and passed through 0.22 mm membrane filters. A 6 mg of 5-FU conjugate was solubilized in the mixture of Tween-80 and Labrasol and incorporated into prepared LDE nanoemulsions. The rest of the procedure was similar to that described by Maranhao et al.20 and Moura et al.21. In order to mimic lowdensity lipoprotein (LDL) receptors, the composition of phospholipids (lipid phase) was similar in all formulations. However, the composition of surfactants (Tween-80 and Labrasol) was varied in
The viscosity of prepared LDE nanoemulsions (L1–L5) was measured without any dilution using Brookfield Viscometer (Brookfield Engineering Laboratories, Inc, Middleboro, MA) as reported previously27. However, the RI of each LDE nanoemulsion was measured using Abbes type Refractometer (Precision Standard Testing Equipment Corporation, Germany) at 25 C without any dilution as reported previously28. Transmission electron microscopy Transmission electron microscopic (TEM) analysis was performed on optimized LDE nanoemulsion L5. High resolution TEM operating at 200 KV (Tecnai TF20, Hillsboro, OR) was performed to reveal surface morphology and size of LDE nanoemulsion. Optimized formulation (L5) was diluted 200 times with deionized water and a drop of formulation was then deposited directly on the holey film carbon-coated copper grid of 300 mesh. After 2 min, the excess liquid was blotted with the help of tissue paper. Negative staining was performed with a 5 ml drop of 2% phosphotungestic acid for 10 s and blotted dry. Formulation
Cholesterol-rich nanoemulsion of cholesteryl-maleoyl-5-fluorouracil
DOI: 10.3109/10837450.2013.860551
applied grid was observed under light microscopy at 200 KV for surface morphology and size.
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In vitro drug release studies In vitro drug release studies were performed to compare the release of 5-FU conjugate from different LDE nanoemulsions. Drug release studies were carried out in 500 ml of deionized water (dissolution media) using United States Pharmacopoeia (USP) XXIV method at 100 rpm and 37 0.5 C27. Two ml of each LDE nanoemulsion (all containing 6 mg of 5-FU conjugate) was placed in ready to use dialysis bag (Spectrum Medical Industries, Mumbai, India). Samples (1 ml) were withdrawn at regular time intervals (0, 1, 2, 3, 6, 18 and 24 h) and same amount of drug-free fresh deionized water was replaced every time. The samples of in vitro drug studies were analyzed for drug content by validated UHPLC-DAD method as described in the previous section.
Results and discussion The 5-FU conjugate (cholesteryl-maleoyl-5FU) was synthesized and well characterized by thin-layer chromatography, ultraviolet and infrared spectral analysis, mass spectrometry and nuclear magnetic resonance spectra in the laboratory (unpublished data). The molecular weight of the newly synthesized conjugate was determined by mass spectrometry and its molecular structure was elucidated using the above-mentioned spectral analysis (Figure 1). Thermodynamic stability test For the development of robust LDE nanoemulsions, the prepared formulations (L1–L5) were subjected to different thermodynamic tests. These tests were performed qualitatively to eliminate metastable or unstable LDE nanoemulsions. It was observed that all the prepared LDE formulations were found to be stable at all stress conditions of thermodynamic stability tests (Table 1). Measurement of droplet size, PI and ZP The results of droplet size distribution, PI and ZP are listed in Table 2. The mean droplet size of different LDE nanoemulsions (L1–L5) was observed in the range of 50.0–95.2 nm. The droplet size of LDE nanoemulsions was found to be decreased by increasing the concentration of surfactants (Tween-80 and Labrasol) in the formulation that could be due to the enhanced surface area for the absorption of drug in the presence of higher concentration of surfactants. The droplet size was found to be lowest (50 nm) and highest (95.2 nm) in formulations L5 and L1, respectively (Table 2). The lowest and highest droplet size in formulations L5 and L1 were probably due to highest and lowest concentration of surfactants in L5 and L1, respectively28.
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The PI of LDE nanoemulsions was observed in the range of 0.109–0.343 (Table 2). The PI in all formulations was low which indicated the uniformity of droplet size distribution in all LDE nanoemulsions. The lowest (0.109) and highest (0.343) PI were also observed in formulations L5 and L1, respectively, which could be probably due to narrow and wide droplet size distribution range in L5 and L1, respectively. However, ZP of LDE nanoemulsions was observed in the range of 28.80 to 26.54 mV (Table 2). The negative value of ZP in all formulations was probably due to the presence of phosphatidylcholine in all formulations. The results of ZP indicated the stable formation of all LDE nanoemulsions because the ZP value in the magnitude of 30 mV indicated the stability potential of prepared formulations as reported in the literature28,29. ZP values of our study were very close to 30 mV. Measurement of viscosity and RI The results of viscosity and RI measurement are listed in Table 2. The viscosity of LDE nanoemulsions was observed in the range of 32.16–52.26 cp. The viscosity was observed lowest (32.16 cp) in formulation L5 and highest (52.26 cp) in formulation L1 (Table 2). The lowest and highest viscosities in formulations L5 and L1 were probably due to lowest and highest droplet sizes as well as highest and lowest concentration of surfactants in formulations L5 and L1, respectively. The RI of LDE nanoemulsions was observed in the range of 1.335–1.340. The RI values in all formulations were very close to the RI of water (1.33) which indicated oil-in-water behavior of all formulations. The lowest value of RI (1.335) was observed in formulation L5. Overall, physicochemical data of LDE nanoemulsions supported the proper development of formulations. Transmission electron microscopy Surface characterization of optimized LDE nanoemulsion (L5) was studied using high-resolution TEM in terms of surface morphology and shape of droplets. Photomicrograph of formulation L5 was taken and interpreted for droplet size and surface morphology. It was observed that all droplets were distributed within submicron range (Figure 2). The size of all droplets was found to be less than 100 nm. The droplet size distributions recorded by high-resolution TEM were in agreement with droplet size distributions recorded by light scattering technique using Malvern Mastersizer (Table 2). The shape of droplets was observed as non-spherical that could be due to the presence of phosphatidylcholine in the formulation.
Table 2. Physicochemical data of different LDE nanoemulsions (L1–L5).
Formulation L1 L2 L3 L4 L5
Ddm SD (nm)
PI
95.20 8.92 88.60 7.46 80.65 7.02 54.51 6.34 50.00 5.10
0.343 0.245 0.209 0.128 0.109
ZP (mV) 26.54 27.54 28.00 28.20 28.80
RI SD
SD (cps)
1.340 0.006 1.339 0.005 1.337 0.004 1.336 0.004 1.335 0.005
52.26 5.22 45.20 5.02 40.55 4.32 36.43 4.10 32.16 3.65
Ddm, Mean droplet diameter; PI, polydispersity index (PI); ZP, zeta potential; mV, millivolt; RI, refractive index; Z, viscosity; SD, standard deviation.
Figure 2. Transmission electron microscopic image of optimized LDE nanoemulsion L5.
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Based on highest drug release (97%), lowest droplet size (50 nm), lowest polydispersity index (0.109), lowest viscosity (32.16 cp), optimum values of zeta potential ( 28.8 mV) and refractive index (1.335) and higher concentration of surfactants, formulation L5 has been optimized as an effective formulation. From these results, it was concluded that developed LDE nanoemulsions could be successfully used for the oral delivery of 5-fluorouracil conjugate. However, further cytotoxicity and in vivo studies are required to prove complete anticancer potential of developed formulations.
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Declaration of interest This study was supported by National Plan for Science & Technology and Innovation for generous financial support (Grant No. 11 NAN 1286-02). The authors report no declaration of interest. The authors alone are responsible for the content and writing of this article.
References Figure 3. In vitro drug release profile of 5-FU conjugate from different LDE nanoemulsions (L1–L5) through dialysis membrane.
In vitro drug release studies In vitro drug release studies were performed to compare the release profile of 5-FU conjugate trough dialysis membrane from different LDE nanoemulsions (L1–L5) in order to optimize the formulation for cytotoxicity and preclinical studies. The results of 5-FU conjugate release are presented in Figure 3. It was observed that initial release of 5-FU conjugate from all formulations was higher (immediate release profile). More than 70% of 5-FU conjugate was found to be released from all formulations after 6 h of study (Figure 3). After 6 h of study period, all LDE nanoemulsions showed slower release of 5-FU conjugate (sustained release profile). The highest drug release profile was observed with formulation L5 (Figure 3). The % amount of 5-FU conjugate that was released from L5 after 24 h of study was found to be 97 as compared to other formulations. More than 80% of 5-FU conjugate was released from L5 after 6 h of study. However, the lowest drug release profile was observed with formulation L1. About 78.6% of 5-FU conjugate was released from L1 after 24 h of study and 78.8% of 5-FU conjugate was released from L1 after 6 h of study. The results of in vitro drug release studies were in agreement with the results of droplet size distribution, PI, RI and viscosity. The lowest drug release profile of formulation L1 was probably due to highest droplet size, highest viscosity and presence of lowest concentration of surfactants (Tween-80 and Labrasol). However, the highest drug release profile of L5 was probably due to lowest droplet size, lowest viscosity, presence of highest concentration of surfactants and eventually highest surface area which allowed rapid drug release. All formulations showed drug release profile in two steps. In first step, the immediate release profile (from 1 to 6 h of study) was observed (Figure 3). In final step, sustained release profile was observed. Two steps release profile of 5-FU conjugate indicated diffusion controlled dissolution rate of 5-FU conjugate from all LDE nanoemulsions28,30. Based on highest drug release (97%), lowest droplet size (50 nm), lowest PI (0.109), lowest viscosity (32.16 cp), optimum ZP ( 28.8 mV) and higher concentration of surfactants, formulation L5 was selected as an optimized formulation for further evaluation.
Conclusion In this study, cholesterol-rich nanoemulsions of cholesterylmaleoyl-5-fluorouracil conjugate were developed and evaluated.
1. Rossella P, Massimo B, Simona G, Christopher AJ. Hydrophilic interaction liquid chromatography–APCI–mass spectrometry determination of 5-fluorouracil in plasma and tissues. J Pharm Biomed Anal 2005;38:738–745. 2. Dong Z, Zheng W, Xu Z, Yin Z. Improved stability and tumor targeting of 5-fluorouracil by conjugation with hyaluronan. J Appl Polym Sci 2013;130:927–932. 3. Presant CA, Jacobson J, Wolf W, et al. Does leucovorin alter the intratumoral pharmacokinetics of 5-fluorouracil? A Southwest oncology group study. Invest New Drugs 2002;20:369–376. 4. Longley DB, Harkin DP, Johnston PG. Reviews and comments from the nature publishing group. Nat Rev Cancer 2003;3:330. 5. Koukourakis GV, Kouloulias V, Koukourakis MJ, et al. Efficacy of the oral fluorouracil prodrug capecitabine in cancer treatment: a review. Molecules 2008;13:1897–1922. 6. Kuzuhara S, Ohkoshi N, Kanemaru K, et al. Subacute leucoencephalopathy induced by carmofur, a 5f-fluorouracil derivative. Neurology 1987;234:365–370. 7. Casado E, Pfeiffer P, Feliu J, et al. UFT (tegafur uracil) in rectal cancer. Ann Oncol 2008;19:1371–1378. 8. Cosco D, Paolino D, Muzzalupo R, et al. Novel PEG-coated niosomes based on bola-surfactant as drug carriers for 5-fluorouracil. Biomed Microdevices 2009;11:1115–1125. 9. Gupta RR, Jain SK, Varshney M. AOT water-in-oil microemulsions as a potential enhancer in transdermal drug delivery of 5-fluorouracil. Colloids Surf B 2005;41:25–32. 10. Li S, Wang A, Jiang W, Guan Z. Pharmacokinetic characteristics and anticancer effects of 5-fluorouracil loaded nanoparticles. BMC Cancer 2008;8:E103. 11. Lin Y, Li Y, Ooi CP. 5-Fluorouracil encapsulated HA/PLGA composite microspheres for cancer therapy. J Mater Sci Mater Med 2012;23:2453–2460. 12. Ahmad MZ, Akhtar S, Anwar M, Ahmad FJ. Assam Bora rice starch based biocompatible mucoadhesive microsphere for targeted delivery of 5-fluorouracil in colorectal cancer. Mol Pharm 2012;9: 2986–2994. 13. Ashwanikumar N, Kumar NA, Nair SA, Kumar GV. Methacrylicbased nanogels for the pH-sensitive delivery of 5-fluorouracil in the colon. Int J Nanomed 2012;7:5769–5779. 14. Shishu K, Maheshwari M. Development and evaluation of novel microemulsion based oral formulations of 5-fluorouracil using noneverted rat intestine sac model. Drug Dev Ind Pharm 2012;38: 294–300. 15. Thomas AM, Kapanen AI, Hare JI, et al. Development of a liposomal nanoparticle formulation of 5-fluorouracil for parenteral administration: formulation design, pharmacokinetics and efficacy. J Controlled Release 2011;150:212–219. 16. Yanyu X, Fang L, Qineng P, Hao C. The influence of the structure and the composition of water/AOT-Tween 85/IPM microemulsion system on transdermal delivery of 5-fluorouracil. Drug Dev Ind Pharm 2012;38:1521–1529. 17. Yassin AEB, Anwer MK, Mowafy HA, et al. Optimization of 5-fluorouracil solid-lipid nanoparticles: a preliminary study to treat colon cancer. Int J Med Sci 2010;7:398–408.
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DOI: 10.3109/10837450.2013.860551
Cholesterol-rich nanoemulsion of cholesteryl-maleoyl-5-fluorouracil
18. Zhang Z, Wo Y, Zhang Y, et al. In vitro study of ethosome penetration in human skin and hypertrophic scar tissue. Nanomedicine 2012;8:1026–1033. 19. Valduga CJ, Fernandes DC, Lo Prete AC, et al. Use of a cholesterolrich microemulsion that binds to low-density lipoprotein receptors as vehicle for etoposide. J Pharm Pharmacol 2003;55:1615–1622. 20. Moura JA, Valduga CJ, Tavares ER, et al. Novel formulation of a methotrexate derivative with a lipid nanoemulsion. Int J Nanomed 2011;6:2285–2265. 21. Maranha˜o RC, Ce´sar TB, Pedroso-Mariani SR, et al. Metabolic behavior in rats of a non-protein microemulsion resembling low density lipoprotein. Lipids 1993;28:691–696. 22. Ades A, Carvalho JP, Graziani SR, et al. Uptake of a cholesterol rich emulsion by neoplastic ovarian tissues. Gynecol Oncol 2001;81: 84–87. 23. Graziani SR, Igreja FAF, Hegg R, et al. Uptake of a cholesterol-rich emulsion by breast cancer. Gynecol Oncol 2002;85:493–497. 24. Azevedo CH, Carvalho JP, Valduga CJ, Maranha˜o RC. Plasma kinetics and uptake by the tumor of a cholesterol-rich microemulsion (LDE) associated to etoposide oleate in patients with ovarian carcinoma. Gynecol Oncol 2005;97:178–182.
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25. Rodrigues DG, Maria DA, Fernandes DC, et al. Improvement of paclitaxel therapeutic index by derivatization and association to a cholesterol-rich microemulsion: in vitro and in vivo studies. Cancer Chemother Pharmacol 2005;55:565–576. 26. Dias ML, Carvalho JP, Rodrigues DG, et al. Pharmacokinetics and tumor uptake of a derivatized form of paclitaxel associated to a cholesterol-rich nanoemulsion (LDE) in patients with gynecologic cancers. Cancer Chemother Pharmacol 2007;59:105–111. 27. Shafiq S, Shakeel F, Talegaonkar S, et al. Development and bioavailability assessment of ramipril nanoemulsion formulation. Eur J Pharm Biopharm 2007;66:227–243. 28. Shakeel F, Haq N, Elbadry M, et al. Ultra fine super selfnanoemulsifying drug delivery system enhanced solubility and dissolution of indomethacin. J Mol Liq 2013;180:89–94. 29. Bali V, Ali M, Ali J. Nanocarrier for the enhanced bioavailability of a cardiovascular agent: in vitro, pharmacodynamics, pharmacokinetic and stability assessment. Int J Pharm 2011;403:46–56. 30. Villar AM, Naveros BC, Campmany AC, et al. Design and optimization of self-nanoemulsifying drug delivery systems (SNEDDS) for enhanced dissolution of gemfibrozil. Int J Pharm 2012;431:161–175.
ORIGINAL RESEARCH
Formulation and evaluation of cholesterol-rich nanoemulsion (LDE) for drug delivery potential of cholesteryl-maleoyl-5-fluorouracil Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Purdue University on 03/13/15 For personal use only.
Fars K. Alanazi1, Nazrul Haq1,2, Awwad A. Radwan1, Ibrahim A. Alsarra1,2, and Faiyaz Shakeel1,2 1
Kayyali Chair for Pharmaceutical Industry, Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia and Center of Excellence in Biotechnology Research (CEBR), Riyadh, Saudi Arabia
2
Abstract
Keywords
It has been reported that cholesterol-rich nanoemulsions (LDE) can bind to low density lipoprotein (LDL) receptors which can concentrate anticancer drugs in the tissues via LDL receptor overexpression and reduced the adverse effects of the treatment. Therefore, in this study, LDE nanoemulsions of cholesteryl-maleoyl-5-fluorouracil (5-FU conjugate) were developed and evaluated in vitro. LDE nanoemulsions were prepared by high-energy emulsification technique. Developed formulations were characterized in terms of droplet size, polydispersity index, zeta potential, viscosity and refractive index. Optimized formulation (L5) was also evaluated for surface morphology using transmission electron microscopy (TEM). Developed formulations were subjected to in vitro drug release studies through dialysis membrane. The droplet size (50 nm), polydispersity index (0.109) and viscosity (32.16 cp) were found to be lowest for optimized formulation L5. The results of zeta potential indicated the stable formation of developed LDE nanoemulsions. TEM images of optimized formulation indicated non-spherical shape of droplets. About 97% of conjugate was found to be released from L5 after 24 h of study. Overall, these results indicated that developed LDE nanoemulsions could be successfully used for oral delivery of 5-FU conjugate.
Cholesteryl-maleoyl-5-fluorouracil, droplet size, drug release, nanoemulsion, zeta potential
Introduction The 5-fluorouracil (5-FU), the most popular cytotoxic drug which belongs to an antimetabolite class of anticancer drugs has been recommended clinically for the treatment of various types of cancers and solid tumors such as colorectal, breast and ovarian tumor1,2. It has been reported as hydrophilic drug with aqueous solubility of 12.2 mg/ml2. Oral administration of 5-FU showed incomplete and erratic pharmacokinetic profile, therefore it is administered via intravenous injection in which maintenance of blood concentration of drug is still short due to its rapid metabolism3,4. Chemical modification of 5-FU via prodrug/ conjugation approach has been successfully used to enhance its lipophilicity in order to enhance its therapeutic efficacy and to reduce adverse effects as evidenced by its various derivatives such as tegafur, capecitabine and carmofur5–7. Therefore, ester conjugate of 5-FU (cholesteryl-maleoyl-5-FU with molecular weight of 596.77; Figure 1) was synthesized in order to improve its therapeutic efficacy and to reduce adverse effects. Various drug delivery approaches namely niosomes, liposomes, ethosomes, microparticles, nanoparticles, nanogels and microemulsions have also been tried to enhance its therapeutic efficacy and to reduce adverse effects8–18. Cholesterol-rich nanoemulsions (LDE) are known to bind with low density lipoprotein (LDL) receptors which can concentrate
Address for correspondence: Dr. F. Shakeel, King Saud University, Riyadh, Saudi Arabia. Tel: +966-537507318. E-mail: [email protected]
History Received 17 September 2013 Revised 5 October 2013 Accepted 7 October 2013 Published online 25 November 2013
anticancer drugs in the tissues by LDL receptor overexpression and reduced the adverse effects of the treatment19,20. They are formed by monolayer of phosphatidylcholine surrounding a core of cholesteryl ester19. LDE nanoemulsions mimic the lipidic portion of LDL and acquire apolipoprotein-E from the circulating native lipoproteins when come in contact with plasma20. It has been reported that the most of the cancer cells show LDL receptor upregulation and LDE nanoemulsions target these cells and deliver the anticancer drugs into the cells via LDL-receptor mediated endocytosis19,20. The potential of LDE nanoemulsions has been proved in enhancing therapeutic efficacy and reducing adverse effects of the treatment of various anticancer drugs such as carmustine, paclitaxel, etoposide and methotrexate either preclinically or clinically19–26. LDE nanoemulsions of 5-FU or 5-FU conjugate have not been investigated either in vitro or in vivo in literature so far. Therefore, in this study, attempts were made to develop and evaluate LDE nanoemulsions of newly synthesized conjugate cholesteryl-maleoyl-5-fluorouracil (5-FU conjugate) in order to enhance its therapeutic efficacy and to reduce adverse effects.
Materials and methods Materials Chromatographic grade methanol and 5-FU (purity 99.2%) were purchased from BDH Laboratory supplies (Liverpool, UK) and Alfa Aesar (Ward Hill, MA), respectively. Cholesterylmaleoyl-5-fluorouracil conjugate (purity 98.9%) was synthesized and characterized in the Laboratory. Cholesterol (purity 98.0%), triolein, cholesteryloleate, phosphatidylcholine and Tween-80
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Table 1. Composition of different LDE formulations (L1–L5). Ingredients 5-FU conjugate (mg) Cholesterol (mg) Triolein (mg) Phosphatidylcholine (mg) Cholesteryloleate (mg) Tween-80 (ml) Labrasol (ml) Deionized water q.s. (ml)
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Figure 1. Molecular structure of cholesterol-maleoyl-5-fluorouracil (Mol. Wt. 596.77).
were purchased from Sigma Aldrich (St. Louis, MO). Labrasol was procured as a kind gift sample from Gattefosse (Cedex, France). The purity of all these chemicals was greater than 99.0%. Dialysis bag (MWCO 12 000g/mol) was purchased from Spectrum Medical Industries (Mumbai, India). Ultra-pure water (chromatographic grade) was obtained from ELGA water purification unit (Wycombe, Bucks, UK). All other chemicals and reagents were of analytical grade. Analytical methodology Ultra high performance liquid chromatographic (UHPLC) method with diode array detector (DAD) was used for quantification of 5-FU conjugate. UHPLC-DAD analysis was carried out at room temperature (22 1 C) using Thermo Scientific UHPLC system (Thermo Scientific, Waltham, MA) equipped with a 3000 LC pump, 3000 autosampler, binary pumps, a programmable DAD detector, ultimate 3000 column oven, ultimate 3000 controller with an inline vacuum degasser. UHPLC data was analyzed using Chromeleon, version 6.8 software (Waltham, MA). The chromatographic identification of 5-FU conjugate was achieved with a Thermo Hypersil GOLD 50 2.1 mm RP C18 column (Thermo Scientific, Waltham, MA) having a 1.9 mm packing as a stationary phase. The mobile phase was methanol:water (80:20% v/v) with a flow rate of 0.4 ml/min. The DAD wavelength was set at 276 nm. Samples (1 ml) were injected into UHPLC system using an ultimate 3000 series Thermo auto sampler (Thermo Scientific, Waltham, MA). The proposed analytical method was developed and validated for linearity, accuracy, precision, robustness, specificity and stability-indicating properties in the laboratory (unpublished data).
L1
L2
L3
L4
L5
6.0 0.5 1.0 20.0 40.0 0.1 0.1 2.0
6.0 0.5 1.0 20.0 40.0 0.15 0.15 2.0
6.0 0.5 1.0 20.0 40.0 0.2 0.2 2.0
6.0 0.5 1.0 20.0 40.0 0.25 0.25 2.0
6.0 0.5 1.0 20.0 40.0 0.3 0.3 2.0
q.s., Quantity sufficient to produce 2 ml of total LDE nanoemulsion; 5-FU conjugate, choesteryl-maleoyl-5-fluorouracil.
order to evaluate the impact of surfactants on physicochemical properties and in vitro drug release of LDE formulations. The composition of different LDE formulations is listed in Table 1. Thermodynamic stability test As nanoemulsions are thermodynamically/thermokinetically stable systems, different thermodynamic stability tests were performed in order to eliminate metastable or unstable formulations27. Prepared LDE nanoemulsions were subjected to different thermodynamic stability tests such as centrifugation (at 5000 rpm), heating and cooling cycles (three cycles) and freeze–pump–thaw cycles (three cycles) as reported in previous literature27–29. Measurement of droplet size, polydispersity and zeta potential The droplet size, polydispersity index (PI) and zeta potential (ZP) of LDE nanoemulsions (L1–L5) were measured using Malvern Mastersizer (Malvern Instruments Ltd., Holtsville, NY) at room temperature (25 C) with a scattering angle of 90 . Each sample of drug-loaded LDE nanoemulsion was diluted suitably with deionized water (1:200), sonicated for 10 min and filtered through 0.45 mm membrane filter. About 3 ml of each formulation was transferred to a disposable cuvette for droplet size and PI measurement. For the measurement of ZP of each LDE nanoemulsion, glass electrode cuvettes were used instead of disposable cuvettes. Measurement of viscosity and refractive index
Preparation of cholesterol-rich nanoemulsion (LDE) of 5-FU conjugate Various LDE formulations of 5-FU conjugates (L1–L5) were prepared by high energy emulsification technique as reported by Maranhao et al.20 and Moura et al.21 with slight modifications. Lipid mixture composed of 0.5 mg of cholesterol, 1 mg of triolein, 20 mg of phosphatidylcholine and 40 mg of cholesteryloleate was emulsified with the required quantity of deionized water (aqueous phase) by prolonged ultrasonic irradiation followed by a two-step ultracentrifugation of crude emulsion with density adjustment by the addition of KBr to obtain LDE nanoemulsions20. Prepared LDE nanoemulsions (L1–L5) were dialyzed against normal saline and passed through 0.22 mm membrane filters. A 6 mg of 5-FU conjugate was solubilized in the mixture of Tween-80 and Labrasol and incorporated into prepared LDE nanoemulsions. The rest of the procedure was similar to that described by Maranhao et al.20 and Moura et al.21. In order to mimic lowdensity lipoprotein (LDL) receptors, the composition of phospholipids (lipid phase) was similar in all formulations. However, the composition of surfactants (Tween-80 and Labrasol) was varied in
The viscosity of prepared LDE nanoemulsions (L1–L5) was measured without any dilution using Brookfield Viscometer (Brookfield Engineering Laboratories, Inc, Middleboro, MA) as reported previously27. However, the RI of each LDE nanoemulsion was measured using Abbes type Refractometer (Precision Standard Testing Equipment Corporation, Germany) at 25 C without any dilution as reported previously28. Transmission electron microscopy Transmission electron microscopic (TEM) analysis was performed on optimized LDE nanoemulsion L5. High resolution TEM operating at 200 KV (Tecnai TF20, Hillsboro, OR) was performed to reveal surface morphology and size of LDE nanoemulsion. Optimized formulation (L5) was diluted 200 times with deionized water and a drop of formulation was then deposited directly on the holey film carbon-coated copper grid of 300 mesh. After 2 min, the excess liquid was blotted with the help of tissue paper. Negative staining was performed with a 5 ml drop of 2% phosphotungestic acid for 10 s and blotted dry. Formulation
Cholesterol-rich nanoemulsion of cholesteryl-maleoyl-5-fluorouracil
DOI: 10.3109/10837450.2013.860551
applied grid was observed under light microscopy at 200 KV for surface morphology and size.
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In vitro drug release studies In vitro drug release studies were performed to compare the release of 5-FU conjugate from different LDE nanoemulsions. Drug release studies were carried out in 500 ml of deionized water (dissolution media) using United States Pharmacopoeia (USP) XXIV method at 100 rpm and 37 0.5 C27. Two ml of each LDE nanoemulsion (all containing 6 mg of 5-FU conjugate) was placed in ready to use dialysis bag (Spectrum Medical Industries, Mumbai, India). Samples (1 ml) were withdrawn at regular time intervals (0, 1, 2, 3, 6, 18 and 24 h) and same amount of drug-free fresh deionized water was replaced every time. The samples of in vitro drug studies were analyzed for drug content by validated UHPLC-DAD method as described in the previous section.
Results and discussion The 5-FU conjugate (cholesteryl-maleoyl-5FU) was synthesized and well characterized by thin-layer chromatography, ultraviolet and infrared spectral analysis, mass spectrometry and nuclear magnetic resonance spectra in the laboratory (unpublished data). The molecular weight of the newly synthesized conjugate was determined by mass spectrometry and its molecular structure was elucidated using the above-mentioned spectral analysis (Figure 1). Thermodynamic stability test For the development of robust LDE nanoemulsions, the prepared formulations (L1–L5) were subjected to different thermodynamic tests. These tests were performed qualitatively to eliminate metastable or unstable LDE nanoemulsions. It was observed that all the prepared LDE formulations were found to be stable at all stress conditions of thermodynamic stability tests (Table 1). Measurement of droplet size, PI and ZP The results of droplet size distribution, PI and ZP are listed in Table 2. The mean droplet size of different LDE nanoemulsions (L1–L5) was observed in the range of 50.0–95.2 nm. The droplet size of LDE nanoemulsions was found to be decreased by increasing the concentration of surfactants (Tween-80 and Labrasol) in the formulation that could be due to the enhanced surface area for the absorption of drug in the presence of higher concentration of surfactants. The droplet size was found to be lowest (50 nm) and highest (95.2 nm) in formulations L5 and L1, respectively (Table 2). The lowest and highest droplet size in formulations L5 and L1 were probably due to highest and lowest concentration of surfactants in L5 and L1, respectively28.
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The PI of LDE nanoemulsions was observed in the range of 0.109–0.343 (Table 2). The PI in all formulations was low which indicated the uniformity of droplet size distribution in all LDE nanoemulsions. The lowest (0.109) and highest (0.343) PI were also observed in formulations L5 and L1, respectively, which could be probably due to narrow and wide droplet size distribution range in L5 and L1, respectively. However, ZP of LDE nanoemulsions was observed in the range of 28.80 to 26.54 mV (Table 2). The negative value of ZP in all formulations was probably due to the presence of phosphatidylcholine in all formulations. The results of ZP indicated the stable formation of all LDE nanoemulsions because the ZP value in the magnitude of 30 mV indicated the stability potential of prepared formulations as reported in the literature28,29. ZP values of our study were very close to 30 mV. Measurement of viscosity and RI The results of viscosity and RI measurement are listed in Table 2. The viscosity of LDE nanoemulsions was observed in the range of 32.16–52.26 cp. The viscosity was observed lowest (32.16 cp) in formulation L5 and highest (52.26 cp) in formulation L1 (Table 2). The lowest and highest viscosities in formulations L5 and L1 were probably due to lowest and highest droplet sizes as well as highest and lowest concentration of surfactants in formulations L5 and L1, respectively. The RI of LDE nanoemulsions was observed in the range of 1.335–1.340. The RI values in all formulations were very close to the RI of water (1.33) which indicated oil-in-water behavior of all formulations. The lowest value of RI (1.335) was observed in formulation L5. Overall, physicochemical data of LDE nanoemulsions supported the proper development of formulations. Transmission electron microscopy Surface characterization of optimized LDE nanoemulsion (L5) was studied using high-resolution TEM in terms of surface morphology and shape of droplets. Photomicrograph of formulation L5 was taken and interpreted for droplet size and surface morphology. It was observed that all droplets were distributed within submicron range (Figure 2). The size of all droplets was found to be less than 100 nm. The droplet size distributions recorded by high-resolution TEM were in agreement with droplet size distributions recorded by light scattering technique using Malvern Mastersizer (Table 2). The shape of droplets was observed as non-spherical that could be due to the presence of phosphatidylcholine in the formulation.
Table 2. Physicochemical data of different LDE nanoemulsions (L1–L5).
Formulation L1 L2 L3 L4 L5
Ddm SD (nm)
PI
95.20 8.92 88.60 7.46 80.65 7.02 54.51 6.34 50.00 5.10
0.343 0.245 0.209 0.128 0.109
ZP (mV) 26.54 27.54 28.00 28.20 28.80
RI SD
SD (cps)
1.340 0.006 1.339 0.005 1.337 0.004 1.336 0.004 1.335 0.005
52.26 5.22 45.20 5.02 40.55 4.32 36.43 4.10 32.16 3.65
Ddm, Mean droplet diameter; PI, polydispersity index (PI); ZP, zeta potential; mV, millivolt; RI, refractive index; Z, viscosity; SD, standard deviation.
Figure 2. Transmission electron microscopic image of optimized LDE nanoemulsion L5.
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F. K. Alanazi et al.
Pharm Dev Technol, Early Online: 1–5
Based on highest drug release (97%), lowest droplet size (50 nm), lowest polydispersity index (0.109), lowest viscosity (32.16 cp), optimum values of zeta potential ( 28.8 mV) and refractive index (1.335) and higher concentration of surfactants, formulation L5 has been optimized as an effective formulation. From these results, it was concluded that developed LDE nanoemulsions could be successfully used for the oral delivery of 5-fluorouracil conjugate. However, further cytotoxicity and in vivo studies are required to prove complete anticancer potential of developed formulations.
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Declaration of interest This study was supported by National Plan for Science & Technology and Innovation for generous financial support (Grant No. 11 NAN 1286-02). The authors report no declaration of interest. The authors alone are responsible for the content and writing of this article.
References Figure 3. In vitro drug release profile of 5-FU conjugate from different LDE nanoemulsions (L1–L5) through dialysis membrane.
In vitro drug release studies In vitro drug release studies were performed to compare the release profile of 5-FU conjugate trough dialysis membrane from different LDE nanoemulsions (L1–L5) in order to optimize the formulation for cytotoxicity and preclinical studies. The results of 5-FU conjugate release are presented in Figure 3. It was observed that initial release of 5-FU conjugate from all formulations was higher (immediate release profile). More than 70% of 5-FU conjugate was found to be released from all formulations after 6 h of study (Figure 3). After 6 h of study period, all LDE nanoemulsions showed slower release of 5-FU conjugate (sustained release profile). The highest drug release profile was observed with formulation L5 (Figure 3). The % amount of 5-FU conjugate that was released from L5 after 24 h of study was found to be 97 as compared to other formulations. More than 80% of 5-FU conjugate was released from L5 after 6 h of study. However, the lowest drug release profile was observed with formulation L1. About 78.6% of 5-FU conjugate was released from L1 after 24 h of study and 78.8% of 5-FU conjugate was released from L1 after 6 h of study. The results of in vitro drug release studies were in agreement with the results of droplet size distribution, PI, RI and viscosity. The lowest drug release profile of formulation L1 was probably due to highest droplet size, highest viscosity and presence of lowest concentration of surfactants (Tween-80 and Labrasol). However, the highest drug release profile of L5 was probably due to lowest droplet size, lowest viscosity, presence of highest concentration of surfactants and eventually highest surface area which allowed rapid drug release. All formulations showed drug release profile in two steps. In first step, the immediate release profile (from 1 to 6 h of study) was observed (Figure 3). In final step, sustained release profile was observed. Two steps release profile of 5-FU conjugate indicated diffusion controlled dissolution rate of 5-FU conjugate from all LDE nanoemulsions28,30. Based on highest drug release (97%), lowest droplet size (50 nm), lowest PI (0.109), lowest viscosity (32.16 cp), optimum ZP ( 28.8 mV) and higher concentration of surfactants, formulation L5 was selected as an optimized formulation for further evaluation.
Conclusion In this study, cholesterol-rich nanoemulsions of cholesterylmaleoyl-5-fluorouracil conjugate were developed and evaluated.
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DOI: 10.3109/10837450.2013.860551
Cholesterol-rich nanoemulsion of cholesteryl-maleoyl-5-fluorouracil
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