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Influence of the preparation method on the physicochemical properties of econazole-loaded poly(butyl cyanoacrylate) colloidal nanoparticles
Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 260–265
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Influence of the preparation method on the physicochemical properties of econazole-loaded poly(butyl cyanoacrylate) colloidal nanoparticles Georgi Yordanov ∗ Faculty of Chemistry, Sofia University St. Kliment Ohridski, 1 James Bourchier Blvd., 1164 Sofia, Bulgaria
a r t i c l e
i n f o
Article history: Received 31 October 2011 Received in revised form 8 December 2011 Accepted 21 December 2011 Available online 27 December 2011 Keywords: Econazole Poly(butyl cyanoacrylate) Nanoparticles Nanoprecipitation Emulsion polymerization Poloxamer 188 Polysorbate 80 Dextran 40
a b s t r a c t This article describes the influence of the preparation method on the physicochemical properties of econazole-loaded poly(butyl cyanoacrylate) nanoparticles. The nanoparticles were prepared by using two different methods – nanoprecipitation and emulsion polymerization. Three different non-ionic colloidal stabilizers (poloxamer 188, polysorbate 80 and dextran 40) were used to obtain nanoparticles with various surface coatings. The econazole-loaded nanoparticles were characterized for morphology, size distribution, chemical composition, zeta-potential, drug loading efficiency and drug content. It was found that the average nanoparticle size, yield and drug content depend mainly on the preparation method, while the utilization of different colloidal stabilizers resulted in nanoparticles with different -potentials. The colloidal stability of the formulations was found to depend on the method of preparation, as well as on the type of colloidal stabilizer. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Econazole (ECN) is an antifungal medicine of the imidazole class (Fig. 1a) with a broad antimycotic activity with some action against Gram-positive bacteria [1–3]. It is used topically in dermatomycoses also orally and parenterally. Econazole prevents fungal organisms from producing vital substances required for growth and function. Recent advancements in pharmaceutical nanotechnology have provided many proofs for therapeutic benefits from classical antifungal agents formulated in novel colloidal drug delivery systems [4–7]. There are studies that have reported the preparation of various colloidal formulations of econazole, including entrapment of the drug in alginate nanoparticles [4], PLG nanoparticles [4,8], micelles [9], submicron emulsions [10], liposomes [11], nanosized vesicles [12], and lipid nanoparticles [13]. Although, the nanoparticles of poly(butyl cyanoacrylate) (PBCA; Fig. 1b) are considered as one of the promising polymer carriers for drug delivery [14–23], to the best of the author’s knowledge there is no systematic study on the influence of the preparation method on the properties of ECN-loaded PBCA nanoparticles that can be found in the available literature.
∗ Tel.: +359 2 8161 331; fax: +359 2 9625 438. E-mail address: [email protected] 0927-7757/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2011.12.060
There are two main strategies for the preparation of polymeric nanoparticles: (i) dispersion of preformed polymers, and (ii) polymerization of monomers. There are many different methods based on these two general approaches [24]. PACA-based colloidal systems are classically prepared by emulsion polymerization of the respective monomers in aqueous medium [25,26]. The mechanism of polymerization can be anionic, radical or zwitterionic. Previous studies on the preparation of poly(alkyl cyanoacrylate) nanoparticles by polymerization-based methods demonstrated that many physicochemical characteristics of the polymer and the nanoparticles strongly depend on the conditions of the reaction (pH, time of polymerization, type of surfactant, concentration of monomer, etc.) [27–29]. The nanoprecipitation approach has also been adapted for the preparation of PBCA nanoparticles, starting from presynthesized PBCA polymer [23]. In comparison with the polymerization-based methods, the nanoprecipitation has the advantage that the used polymer is well characterized and its characteristics do not depend on the conditions of the colloid preparation. The nanoprecipitation has also two additional advantages: (i) it does not require acidic medium for the formation of nanoparticles (which is usually the case in most of the classic preparations of PBCA nanoparticles by polymerization-based methods) and is therefore suitable for the entrapment of acid-sensitive drugs; and (ii) avoids utilization of the highly reactive alkyl cyanoacrylate monomers, which can react with some drugs. Drugs, which are stable in acidic medium, can be entrapped in PBCA nanoparticles by
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volume of the dispersion was set to 10 ml by addition of distilled water. 2.3. Preparation of ECN-PBCA nanoparticles by nanoprecipitation
Fig. 1. Chemical structures of (a) econazole, and (b) poly(butyl cyanoacrylate).
both methods, leading to the formation of nanoparticles of different characteristics [23,30]. This article reports the successful preparation of ECN-loaded PBCA colloidal nanoparticles by using two different production methods: emulsion polymerization and nanoprecipitation. It is demonstrated that the utilization of the two different approaches for the preparation of ECN-PBCA nanoparticles allows the formation of particles with different physicochemical characteristics. Three different colloidal stabilizers, approved for biomedical applications, were used – dextran 40, poloxamer 188 and polysorbate 80. The study is focused on the physicochemical aspects of the preparation methods and characterization of the obtained drug-loaded nanoparticles for morphology, yield, size distribution, chemical composition, drug content, drug loading efficiency, -potential and colloidal stability. It is expected that the obtained data could be useful for scientists from biomedical fields for further development of novel colloidal formulations of ECN and similar antifungal drugs. 2. Experimental 2.1. Chemicals Butylcyanoacrylate (BCA) monomer was from Special Polymers Ltd. (Bulgaria). Econazole nitrate (ECN), dextran 40 (D40; Mw ∼40,000 Da), poloxamer 188 (P188; PEO-PPO-PEO triblock copolymer), polysorbate 80 (P80; PEGylated sorbitan monooleate), acetone and phosphate-buffered saline (PBS; pH 7.4) were from Sigma (Germany). Glucose (10%) was from Actavis (Bulgaria). Poly(butyl cyanoacrylate) (PBCA; Mw ∼2000) was prepared as described previously [23]. 2.2. Preparation of ECN-PBCA nanoparticles by emulsion polymerization The preparation of ECN-PBCA nanoparticles by emulsion polymerization is based on the controlled anionic polymerization of the monomer, which is added as acetone solution together with the drug to the polymerization medium at room temperature. The polymerization medium contained 5% glucose and 5 mg/ml of the respective colloidal stabilizer (P188, P80 or D40) dissolved in distilled water. BCA (100 l) and ECN (10 mg) were dissolved in dry acetone (5 ml) and added dropwise to the polymerization medium (10 ml) upon intensive stirring (∼400 rpm). The obtained dispersion was stirred on open air for 3 h at room temperature and the residual acetone was removed by rotary evaporation. Then, the final
The protocol for the nanoprecipitation approach is similar to the emulsion polymerization protocol, but in this case presynthesized polymer (PBCA) is used instead of the monomer (BCA). For the preparation of ECN-PBCA nanoparticles, presynthesized PBCA (100 mg) and ECN (10 mg) were dissolved in dry acetone (5 ml) and the obtained solution was dropwise added to the nanoprecipitation medium (10 ml). The nanoprecipitation medium had the same composition as the polymerization medium described in the previous paragraph, that is glucose (5%) and the respective colloidal stabilizer (5 mg/ml) dissolved in distilled water. The obtained milky dispersion was stirred on open air for 3 hours and the residual acetone was removed by rotary evaporation. The volume of the final dispersion was set to 10 ml by addition of distilled water. 2.4. Structural characterization The nanoparticles were observed by using scanning electron microscope JSM-5510 (JEOL). The nanoparticles for Fourier transform infrared (FTIR) and nuclear magnetic resonance (1 H NMR) spectroscopy were purified by centrifugation and washing with distilled water and then dried under vacuum to obtain dry white powdered material. The sample for FTIR analysis was prepared using the KBr tablet technique. The FTIR spectrum was taken in the interval 400–3400 cm−1 (Bruker Tensor 27 spectrometer). The 1 H NMR spectra were taken with Bruker Avance II+ 600 spectrometer (spectrometer frequency 600.13 MHz). The samples were prepared by dissolving dried nanoparticles (ca. 5 mg) in acetone-d6 (0.6 ml). The spectra were taken at room temperature in the interval 0–15 ppm. 2.5. Light scattering experiments The size distribution and -potential of the different ECNPBCA nanoparticles were determined in 0.1× PBS (pH 7.4; total ionic strength 16.5 mM; conductivity 2.0 mS/cm) by dynamic light scattering (DLS) system Zetasizer Nano ZS (Malvern Instruments, UK). The average nanoparticle sizes, diffusion coefficients and potentials were calculated based on the average values of five measurements. All light scattering experiments have been carried out at scattering angle 90◦ , using dilute nanoparticle dispersions in PBS buffer at 25 ◦ C. 2.6. Determination of the drug content and the drug loading efficiency The drug content (DC) is defined as the mass fraction (given in %) of ECN in the ECN-PBCA nanoparticles. It was determined by the following way. An aliquot of the as-prepared nanoparticle dispersion was centrifuged (14,000 rpm, 60 min) in a pre-weighted tube, and then the nanoparticles were washed with distilled water and dried in vacuum. The dried nanoparticles were weighted to determine the nanoparticle yield (the mass of drug-loaded nanoparticles per milliliter of dispersion). Each sample of accurately weighted and dried particles was dissolved in a fixed volume (5.00 ml) of tetrahydrofuran (THF). Calibration curve was used, the extinction coefficient for ECN was found to be 485 ± 15 L/mol cm at 271 nm. The THF was freshly distilled before use to remove any impurities that absorb light at this wavelength. The pure PBCA and pure THF do not absorb light at 271 nm (using a double-beam spectrophotometer Shimadzu UV-190), so only the ECN can be determined. The drug content was determined by assuming that ECN is loaded in the
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particles in its neutral form (which is expected taking into account that the interior of the particles is hydrophobic). All measurements were made at least triplicate. Quartz cuvettes with Teflon stoppers were used (to avoid evaporation of the volatile THF during measurements). The drug content could be also determined directly by using data from the NMR spectra (see Section 3.3). The drug loading efficiency (Le) is defined as the fraction of loaded ECN with respect to the total amount of drug. The loaded ECN was determined as described above and the total amount of ECN in all experiments was the same, ca. 10 mg per preparation. 3. Results and discussion
Table 1 Physicochemical characteristics of different ECN-PBCA nanoparticles determined by dynamic light scattering (DLS) at 25 ◦ C. The -potential was measured by electrophoretic light scattering in PBS (with ionic strength 16.5 mM; conductivity 2.0 mS/cm; pH 7.4). Stabilizer
Average size (nm)
PDI
Diff. coeff. (2 /s)
-potential (mV)
D40a P188a P80a D40b P188b P80b
121 ± 3 124 ± 3 111 ± 3 235 ± 4 230 ± 3 217 ± 4
0.12 ± 0.01 0.10 ± 0.01 0.12 ± 0.02 0.15 ± 0.01 0.15 ± 0.01 0.15 ± 0.02
4.1 ± 0.2 3.9 ± 0.2 4.4 ± 0.2 2.1 ± 0.1 2.2 ± 0.1 2.3 ± 0.1
−3.3 ± 0.5 −0.2 ± 0.2 −4.4 ± 0.6 −3.2 ± 0.4 −0.1 ± 0.7 −4.5 ± 0.7
a b
3.1. Particle morphology and size distribution All of the obtained ECN-PBCA nanoparticles are of spherical shape with smooth surface, as seen from the SEM images in Fig. 2. The respective size distributions obtained by DLS analysis are shown in Fig. 3. The average sizes (hydrodynamic diameters), polydispersity indexes (PDI) and diffusion coefficients are given in Table 1. The larger in size particles have lower value of the diffusion coefficient (as expected from the Stokes–Einstein equation). In all cases the size distribution is monomodal and the average sizes
Nanoparticles prepared by polymerization. Nanoparticles prepared by nanoprecipitation.
obtained by DLS well correspond to the respective sizes, seen from the SEM images. Fig. 3 represents the size distributions by volume, which is generally accepted to provide quite accurate data. The nanoparticles obtained by polymerization are smaller in size and with smaller polydispersity index (PDI) than the nanoparticles, obtained by nanoprecipitation. The average size of ECN-PBCA nanoparticles prepared by polymerization in the presence of D40 and P188 are similar, around 120 nm. The nanoparticles, prepared
Fig. 2. Representative SEM images of ECN-PBCA nanoparticles prepared by (a) nanoprecipitation using dextran 40; (b) nanoprecipitation, using poloxamer 188; (c) nanoprecipitation, using polysorbate 80; (d) polymerization, using dextran 40; (e) polymerization, using poloxamer 188; (f) polymerization, using polysorbate 80.
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Fig. 4. FTIR spectrum of ECN-PBCA nanoparticles, prepared by polymerization in the presence of D40 as a colloidal stabilizer.
its cationic form in aqueous solutions, one can expect that it could influence the -potential of PBCA colloids. Despite the low value of the -potential in the case of ECN-PBCA nanoparticles, the respective colloidal dispersion is rather stable, which indicates that the steric repulsion between the chains of the non-ionic surfactant on the nanoparticle surface seems to be a more important factor for the stability of the colloid then the electrostatic repulsion between the particles. The -potentials do not seem to depend significantly on the type of the preparation method, but seem to depend on the type of the colloidal stabilizer. It is worth noting that the D40coated nanoparticles are less stable in the PBS buffer, which may result in difficulties during measurements. The colloidal stability of the formulations is further discussed in Section 3.6. Fig. 3. Representative size distributions of ECN-PBCA nanoparticles prepared by (a) polymerization; (b) nanoprecipitation. The abbreviations of the different colloidal stabilizers used are given in the legend. The size distributions were obtained from DLS measurements.
in the presence of P80 are slightly smaller, ∼111 nm. The nanoprecipitation approach resulted in ECN-PBCA nanoparticles of larger sizes, around 230 nm in the cases of using D40 and P188. Previously reported preparations of pure and chlorambucil-loaded PBCA colloids resulted in particles of similar size [23]. In this case, the utilization of P80 as a colloidal stabilizer also leads to the formation of slightly smaller particles, of average size ∼217 nm. Taking into account the data shown in Table 1 and Figs. 2 and 3, one can conclude that the preparation method is the main factor that determines the nanoparticle size, while the utilization of different colloidal stabilizers does not have a significant effect on the average size of the obtained particles. 3.2. Measurements of -potential The data from the measurements of the -potential are summarized in Table 1. As seen, the type of colloidal stabilizer has some effect on the value of the -potential, which is negative for the D40- and P80-coated nanoparticles, and is close to zero for the P188-coated ones. Previous studies on the -potential of pure PACA colloids have showed negative values and it has been suggested that this could be a result from ionized free carboxylic groups [19]. These studies also showed that the absolute value of the -potential depends on the ionic strength of the dispersing medium and lower values have been measured at higher ionic strength [19]. There are also previous reports, which have shown that loaded drugs can influence the value of the -potential (for example, adsorption of cationic drugs on particles with negative -potential can decrease its absolute value) [20]. Taking into account that ECN is in
3.3. Chemical structure characterization A representative FTIR spectrum of ECN-PBCA nanoparticles prepared by polymerization in the presence of D40 is shown in Fig. 4. The characteristic absorbance bands of PBCA are observed as follows: for the carbonyl C O ester (1752 cm−1 ), C N groups (2248 cm−1 ), C H (2876–2964 cm−1 ), and C O C (absorbance at 1256 and 1016 cm−1 correspond to the asymmetric and symmetric stretches, respectively). The absorbance bands of PBCA are similar to previously reported data [31,32]. The absorbance peak at 1591 cm−1 seems to be from the entrapped econazole, although it appears at 1585 cm−1 in the spectrum of pure ECN-nitrate. The other absorbance bands of ECN overlap with those of PBCA and cannot be clearly distinguished. It should be noted that FTIR is not powerful enough to analyze the ECN bands, thus NMR was also used to complement the chemical structure characterization, while it also confirmed data for drug content obtained by UV–Vis (see also Section 3.4). The 1 H NMR-spectrum in Fig. 5a corresponds to pure PBCA, and the spectrum in Fig. 5b corresponds to ECN-PBCA nanoparticles prepared by polymerization in the presence of D40 as a colloidal stabilizer. In the spectrum of pure PBCA, the signals at 1.50 ppm (2H), 1.73 ppm (2H), 2.90 ppm (2H), and 4.26 ppm (2H) correspond all to methylene protons. The signal at 0.97 ppm (3H) corresponds to protons from the methyl groups. The signal at 2.05 ppm is from the solvent (acetone-d6) and is used for calibration. The spectrum of pure PBCA is similar to previously reported studies [23,30,33]. Additional signals from the entrapped ECN molecules appear in the spectrum of ECN-PBCA nanoparticles, of which those from the aromatic protons are well distinguishable between 6.9 and 7.8 ppm (10H). The integral intensity of signals from aromatic protons (IAr ), when compared with the integral intensity of the signals from methylene protons (IMe ) in PBCA, can be used for calculation of the drug content (Eqs. (1) and (2)).
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G. Yordanov / Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 260–265 Table 2 Drug content (DC), drug loading efficiency (Le) and yield of ECN-PBCA nanoparticles. Stabilizer
DC (%)
D40a P188a P80a D40b P188b P80b
9.5 8.8 9.5 5.6 5.4 5.5
a b
± ± ± ± ± ±
Le (%) 0.2 0.2 0.1 0.1 0.1 0.3
61 64 66 52 49 50
± ± ± ± ± ±
2 2 4 2 2 3
Yield (mg/ml) 6.5 7.3 7.1 9.3 9.2 9.2
± ± ± ± ± ±
0.1 0.3 0.3 0.3 0.1 0.1
Nanoparticles prepared by polymerization. Nanoparticles prepared by nanoprecipitation.
is more likely to be entrapped within the nanoparticles in the coarse of the anionic polymerization of butyl cyanoacrylate. The drug content in nanoparticles can be directly determined also from the NMR spectrum (see Section 3.3), but UV–vis spectroscopy seems to be more convenient and easier method. 3.5. Yield of nanoparticles
Fig. 5. 1 H NMR spectra of (a) pure PBCA; (b) ECN-PBCA nanoparticles prepared by polymerization in the presence of D40 as a colloidal stabilizer. The signals from aromatic H-atoms from ECN appear at 7–8 ppm. Data from the 1 H NMR spectrum of ECN-PBCA can be used to determine the drug content (see the text for details).
MBCA nBCA MBCA IMe /3 mBCA = = mECN MECN nECN MECN IAr /10 DC (%) =
mECN × 100 mBCA + mECN
(1) (2)
Here mECN and mBCA are the masses of ECN and BCA monomer units, respectively; nECN and nECN are the number of ECN molecules and BCA monomer units, respectively; MECN and MBCA are the respective molar masses. From the spectrum in Fig. 5b, the ratio of the integral intensity of the methylene protons (0.90–1.04 ppm) to that of the aromatic protons (6.9–7.8 ppm) is 1.000/0.141. The molar mass of the butyl cyanoacrylate (BCA) unit is 153.18 g/mol, and that of ECN is 381.68 g/mol. Then, by using Eqs. (1) and (2), it can be calculated that the drug content is 9.5%, which exactly corresponds to the drug content of the same sample determined by UV-vis spectroscopy (see Section 3.4). 3.4. Drug content and loading efficiency The data obtained from determination of the drug content and the drug loading efficiencies by means of UV-vis spectroscopy (as described in Section 2) are summarized in Table 2. The drug content for the nanoparticles prepared by polymerization is higher (8.8–9.5%) then for the nanoparticles prepared by nanoprecipitation (5.4–5.6%). The respective loading efficiencies are also higher. It seems that the type of colloidal stabilizer does not have a significant effect on the drug loading. However, the type of preparation method has such an effect, leading to higher drug content and loading efficiencies in the case of polymerization. It seems that the ECN
The yield of nanoparticles is defined as the mass of nanoparticles per millilitre of the obtained dispersion. Since, in all experiments the amount of monomer/polymer, ECN and dispersing medium are the same, then one can compare the values given in Table 2. As seen, the yield of nanoparticles does not depend significantly on the type of the colloidal stabilizer, but seems to depend mainly on the preparation method. The yield is lower for ECN-PBCA nanoparticles prepared by the polymerization-based method. This could be a result from deposition of some monomer and polymerization on the stirring bar, as well as formation of small oligomers that are solubilized in the dispersing medium by the colloidal stabilizer thus decreasing the overall yield of nanoparticles. 3.6. Colloidal stability Preliminary evaluations showed that both, the preparation method and the type of colloidal stabilizer affect the colloidal stability of the obtained ECN-PBCA colloids. Unstable formulations are found to be those, prepared by using D40 as a stabilizer. The D40-coated particles tend to aggregate in few days of storage at room temperature or at 4 ◦ C. In general, the particles prepared by polymerization are found to be less stable than those, prepared by nanoprecipitation. The nanoparticles prepared by polymerization tend to coagulate in few months of storage at 4 ◦ C. The particles prepared by nanoprecipitation could sediment upon storage, but can be redispersed again upon sonication. The most stable formulations are those prepared by nanoprecipitation in the presence of P188 and P80. These colloids could be stored at 4 ◦ C for at least 6 months without any observable change in colloidal stability. The concentration of stabilizers in this study was chosen to be in the ranges, which are usually used for the preparations of PACA colloids. It may be expected that higher concentrations of stabilizers may be beneficial for the colloidal stability, but higher concentrations of stabilizers is not recommended and should be avoided for colloidal systems that are intended for biomedical use (because of risks to increase the toxicity of the formulations). The observed differences in the colloidal stability of the ECN-PBCA dispersions could be due to the difference in particle size, drug content and/or probably differences in the properties of the polymer, although the main factors that determine the colloidal stability should be studied further in more details. 4. Conclusions Econazole-loaded PBCA nanoparticles are prepared by using two different methods: nanoprecipitation and polymerization, and
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three different colloidal stabilizers (dextran 40, polysorbate 80 and poloxamer 188). The obtained nanoparticles are of spherical shape and possess monomodal size distribution. The nanoparticles, prepared by polymerization-based method were smaller in size and had a higher drug content than those, prepared by nanoprecipitation. The type of colloidal stabilizer did not have a significant effect on the average nanoparticle size and drug content at all other constant conditions. The utilization of polysorbate 80 and dextran 40 as colloidal stabilizers resulted in negative -potentials, while using poloxamer 188 resulted in -potentials close to zero. Preliminary studies of the colloidal stability indicated that the nanoparticles, coated with poloxamer 188 and polysorbate 80, are relatively more stable than the dextran-coated nanoparticles. The nanoparticles obtained by nanoprecipitation were more stable than those prepared by polymerization. Acknowledgements GY is thankful for the support from COST Action D43. The technical support from Mr. Nikola Dimitrov and Dr. Mariana Boneva with SEM observation and DLS measurements, respectively, is greatly acknowledged. NMR and FTIR spectra were measured at the Institute of Organic Chemistry (BAS). References [1] D. Thienpont, J. Van Cutsem, J.M. Van Nueten, C.J. Niemegeers, R. Marsboom, Biological and toxicological properties of econazole, a broad-spectrum antimycotic, Arzneimittel-Forsch. 25 (1975) 224–230. [2] R.C. Heel, R.N. Brogden, T.M. Speight, G.S. Avery, Econazole: a review of its antifungal activity and therapeutic efficacy, Drugs 16 (1978) 177–201. [3] M. Borgers, Mechanism of action of antifungal drugs, with special reference to the imidazole derivatives, Rev. Infect. Dis. 2 (1980) 520–534. [4] R. Pandey, Z. Ahmad, S. Sharma, G.K. Khuller, Nano-encapsulation of azole antifungals: potential applications to improve oral drug delivery, Int. J. Pharm. 301 (2005) 268–276. [5] T. Prow, J. Grice, L. Lin, R. Faye, M. Butler, W. Becker, E. Wurm, C. Yoong, T. Robertson, H. Soyer, M. Roberts, Nanoparticles and microparticles for skin drug delivery, Adv. Drug Deliv. Rev. 63 (2011) 470–491. [6] D.Q. Luo, J.H. Guo, F.J. Wang, Z.X. Jin, X.L. Cheng, J.C. Zhu, C.Q. Peng, C. Zhang, Anti-fungal efficacy of polybutylcyanoacrylate nanoparticles of allicin and comparison with pure allicin, J. Biomater. Sci. Polym. Ed. 20 (2009) 21–31. [7] N. Xu, J. Gu, Y. Zhu, H. Wen, Q. Ren, J. Chen, Efficacy of intravenous amphotericin B–polybutylcyanoacrylate nanoparticles against cryptococcal meningitis in mice, Int. J. Nanomed. 6 (2011) 905–913. [8] Z. Ahmad, R. Pandey, S. Sharma, G.K. Khuller, Novel chemotherapy for tuberculosis: chemotherapeutic potential of econazole- and moxifloxacin-loaded PLG nanoparticles, Int. J. Antimicrob. Agents 31 (2008) 142–146. [9] Y.G. Bachhav, K. Mondon, Y.N. Kalia, R. Gurny, M. Möller, Novel micelle formulations to increase cutaneous bioavailability of azole antifungals, J. Control. Release 153 (2011) 126–132. [10] M.P.Y. Piemi, D. Korner, S. Benita, J.-P. Marty, Positively and negatively charged submicron emulsions for enhanced topical delivery of antifungal drugs, J. Control. Release 58 (1999) 177–187. [11] S. Cogswell, S. Berger, D. Waterhouse, M.B. Bally, E.K. Wasan, A parenteral econazole formulation using a novel micelle-to-liposome transfer method: in vitro characterization and tumor growth delay in a Breast Cancer Xenograft Model, Pharm. Res. 23 (2006) 2575–2585.
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Influence of the preparation method on the physicochemical properties of econazole-loaded poly(butyl cyanoacrylate) colloidal nanoparticles Georgi Yordanov ∗ Faculty of Chemistry, Sofia University St. Kliment Ohridski, 1 James Bourchier Blvd., 1164 Sofia, Bulgaria
a r t i c l e
i n f o
Article history: Received 31 October 2011 Received in revised form 8 December 2011 Accepted 21 December 2011 Available online 27 December 2011 Keywords: Econazole Poly(butyl cyanoacrylate) Nanoparticles Nanoprecipitation Emulsion polymerization Poloxamer 188 Polysorbate 80 Dextran 40
a b s t r a c t This article describes the influence of the preparation method on the physicochemical properties of econazole-loaded poly(butyl cyanoacrylate) nanoparticles. The nanoparticles were prepared by using two different methods – nanoprecipitation and emulsion polymerization. Three different non-ionic colloidal stabilizers (poloxamer 188, polysorbate 80 and dextran 40) were used to obtain nanoparticles with various surface coatings. The econazole-loaded nanoparticles were characterized for morphology, size distribution, chemical composition, zeta-potential, drug loading efficiency and drug content. It was found that the average nanoparticle size, yield and drug content depend mainly on the preparation method, while the utilization of different colloidal stabilizers resulted in nanoparticles with different -potentials. The colloidal stability of the formulations was found to depend on the method of preparation, as well as on the type of colloidal stabilizer. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Econazole (ECN) is an antifungal medicine of the imidazole class (Fig. 1a) with a broad antimycotic activity with some action against Gram-positive bacteria [1–3]. It is used topically in dermatomycoses also orally and parenterally. Econazole prevents fungal organisms from producing vital substances required for growth and function. Recent advancements in pharmaceutical nanotechnology have provided many proofs for therapeutic benefits from classical antifungal agents formulated in novel colloidal drug delivery systems [4–7]. There are studies that have reported the preparation of various colloidal formulations of econazole, including entrapment of the drug in alginate nanoparticles [4], PLG nanoparticles [4,8], micelles [9], submicron emulsions [10], liposomes [11], nanosized vesicles [12], and lipid nanoparticles [13]. Although, the nanoparticles of poly(butyl cyanoacrylate) (PBCA; Fig. 1b) are considered as one of the promising polymer carriers for drug delivery [14–23], to the best of the author’s knowledge there is no systematic study on the influence of the preparation method on the properties of ECN-loaded PBCA nanoparticles that can be found in the available literature.
∗ Tel.: +359 2 8161 331; fax: +359 2 9625 438. E-mail address: [email protected] 0927-7757/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2011.12.060
There are two main strategies for the preparation of polymeric nanoparticles: (i) dispersion of preformed polymers, and (ii) polymerization of monomers. There are many different methods based on these two general approaches [24]. PACA-based colloidal systems are classically prepared by emulsion polymerization of the respective monomers in aqueous medium [25,26]. The mechanism of polymerization can be anionic, radical or zwitterionic. Previous studies on the preparation of poly(alkyl cyanoacrylate) nanoparticles by polymerization-based methods demonstrated that many physicochemical characteristics of the polymer and the nanoparticles strongly depend on the conditions of the reaction (pH, time of polymerization, type of surfactant, concentration of monomer, etc.) [27–29]. The nanoprecipitation approach has also been adapted for the preparation of PBCA nanoparticles, starting from presynthesized PBCA polymer [23]. In comparison with the polymerization-based methods, the nanoprecipitation has the advantage that the used polymer is well characterized and its characteristics do not depend on the conditions of the colloid preparation. The nanoprecipitation has also two additional advantages: (i) it does not require acidic medium for the formation of nanoparticles (which is usually the case in most of the classic preparations of PBCA nanoparticles by polymerization-based methods) and is therefore suitable for the entrapment of acid-sensitive drugs; and (ii) avoids utilization of the highly reactive alkyl cyanoacrylate monomers, which can react with some drugs. Drugs, which are stable in acidic medium, can be entrapped in PBCA nanoparticles by
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volume of the dispersion was set to 10 ml by addition of distilled water. 2.3. Preparation of ECN-PBCA nanoparticles by nanoprecipitation
Fig. 1. Chemical structures of (a) econazole, and (b) poly(butyl cyanoacrylate).
both methods, leading to the formation of nanoparticles of different characteristics [23,30]. This article reports the successful preparation of ECN-loaded PBCA colloidal nanoparticles by using two different production methods: emulsion polymerization and nanoprecipitation. It is demonstrated that the utilization of the two different approaches for the preparation of ECN-PBCA nanoparticles allows the formation of particles with different physicochemical characteristics. Three different colloidal stabilizers, approved for biomedical applications, were used – dextran 40, poloxamer 188 and polysorbate 80. The study is focused on the physicochemical aspects of the preparation methods and characterization of the obtained drug-loaded nanoparticles for morphology, yield, size distribution, chemical composition, drug content, drug loading efficiency, -potential and colloidal stability. It is expected that the obtained data could be useful for scientists from biomedical fields for further development of novel colloidal formulations of ECN and similar antifungal drugs. 2. Experimental 2.1. Chemicals Butylcyanoacrylate (BCA) monomer was from Special Polymers Ltd. (Bulgaria). Econazole nitrate (ECN), dextran 40 (D40; Mw ∼40,000 Da), poloxamer 188 (P188; PEO-PPO-PEO triblock copolymer), polysorbate 80 (P80; PEGylated sorbitan monooleate), acetone and phosphate-buffered saline (PBS; pH 7.4) were from Sigma (Germany). Glucose (10%) was from Actavis (Bulgaria). Poly(butyl cyanoacrylate) (PBCA; Mw ∼2000) was prepared as described previously [23]. 2.2. Preparation of ECN-PBCA nanoparticles by emulsion polymerization The preparation of ECN-PBCA nanoparticles by emulsion polymerization is based on the controlled anionic polymerization of the monomer, which is added as acetone solution together with the drug to the polymerization medium at room temperature. The polymerization medium contained 5% glucose and 5 mg/ml of the respective colloidal stabilizer (P188, P80 or D40) dissolved in distilled water. BCA (100 l) and ECN (10 mg) were dissolved in dry acetone (5 ml) and added dropwise to the polymerization medium (10 ml) upon intensive stirring (∼400 rpm). The obtained dispersion was stirred on open air for 3 h at room temperature and the residual acetone was removed by rotary evaporation. Then, the final
The protocol for the nanoprecipitation approach is similar to the emulsion polymerization protocol, but in this case presynthesized polymer (PBCA) is used instead of the monomer (BCA). For the preparation of ECN-PBCA nanoparticles, presynthesized PBCA (100 mg) and ECN (10 mg) were dissolved in dry acetone (5 ml) and the obtained solution was dropwise added to the nanoprecipitation medium (10 ml). The nanoprecipitation medium had the same composition as the polymerization medium described in the previous paragraph, that is glucose (5%) and the respective colloidal stabilizer (5 mg/ml) dissolved in distilled water. The obtained milky dispersion was stirred on open air for 3 hours and the residual acetone was removed by rotary evaporation. The volume of the final dispersion was set to 10 ml by addition of distilled water. 2.4. Structural characterization The nanoparticles were observed by using scanning electron microscope JSM-5510 (JEOL). The nanoparticles for Fourier transform infrared (FTIR) and nuclear magnetic resonance (1 H NMR) spectroscopy were purified by centrifugation and washing with distilled water and then dried under vacuum to obtain dry white powdered material. The sample for FTIR analysis was prepared using the KBr tablet technique. The FTIR spectrum was taken in the interval 400–3400 cm−1 (Bruker Tensor 27 spectrometer). The 1 H NMR spectra were taken with Bruker Avance II+ 600 spectrometer (spectrometer frequency 600.13 MHz). The samples were prepared by dissolving dried nanoparticles (ca. 5 mg) in acetone-d6 (0.6 ml). The spectra were taken at room temperature in the interval 0–15 ppm. 2.5. Light scattering experiments The size distribution and -potential of the different ECNPBCA nanoparticles were determined in 0.1× PBS (pH 7.4; total ionic strength 16.5 mM; conductivity 2.0 mS/cm) by dynamic light scattering (DLS) system Zetasizer Nano ZS (Malvern Instruments, UK). The average nanoparticle sizes, diffusion coefficients and potentials were calculated based on the average values of five measurements. All light scattering experiments have been carried out at scattering angle 90◦ , using dilute nanoparticle dispersions in PBS buffer at 25 ◦ C. 2.6. Determination of the drug content and the drug loading efficiency The drug content (DC) is defined as the mass fraction (given in %) of ECN in the ECN-PBCA nanoparticles. It was determined by the following way. An aliquot of the as-prepared nanoparticle dispersion was centrifuged (14,000 rpm, 60 min) in a pre-weighted tube, and then the nanoparticles were washed with distilled water and dried in vacuum. The dried nanoparticles were weighted to determine the nanoparticle yield (the mass of drug-loaded nanoparticles per milliliter of dispersion). Each sample of accurately weighted and dried particles was dissolved in a fixed volume (5.00 ml) of tetrahydrofuran (THF). Calibration curve was used, the extinction coefficient for ECN was found to be 485 ± 15 L/mol cm at 271 nm. The THF was freshly distilled before use to remove any impurities that absorb light at this wavelength. The pure PBCA and pure THF do not absorb light at 271 nm (using a double-beam spectrophotometer Shimadzu UV-190), so only the ECN can be determined. The drug content was determined by assuming that ECN is loaded in the
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particles in its neutral form (which is expected taking into account that the interior of the particles is hydrophobic). All measurements were made at least triplicate. Quartz cuvettes with Teflon stoppers were used (to avoid evaporation of the volatile THF during measurements). The drug content could be also determined directly by using data from the NMR spectra (see Section 3.3). The drug loading efficiency (Le) is defined as the fraction of loaded ECN with respect to the total amount of drug. The loaded ECN was determined as described above and the total amount of ECN in all experiments was the same, ca. 10 mg per preparation. 3. Results and discussion
Table 1 Physicochemical characteristics of different ECN-PBCA nanoparticles determined by dynamic light scattering (DLS) at 25 ◦ C. The -potential was measured by electrophoretic light scattering in PBS (with ionic strength 16.5 mM; conductivity 2.0 mS/cm; pH 7.4). Stabilizer
Average size (nm)
PDI
Diff. coeff. (2 /s)
-potential (mV)
D40a P188a P80a D40b P188b P80b
121 ± 3 124 ± 3 111 ± 3 235 ± 4 230 ± 3 217 ± 4
0.12 ± 0.01 0.10 ± 0.01 0.12 ± 0.02 0.15 ± 0.01 0.15 ± 0.01 0.15 ± 0.02
4.1 ± 0.2 3.9 ± 0.2 4.4 ± 0.2 2.1 ± 0.1 2.2 ± 0.1 2.3 ± 0.1
−3.3 ± 0.5 −0.2 ± 0.2 −4.4 ± 0.6 −3.2 ± 0.4 −0.1 ± 0.7 −4.5 ± 0.7
a b
3.1. Particle morphology and size distribution All of the obtained ECN-PBCA nanoparticles are of spherical shape with smooth surface, as seen from the SEM images in Fig. 2. The respective size distributions obtained by DLS analysis are shown in Fig. 3. The average sizes (hydrodynamic diameters), polydispersity indexes (PDI) and diffusion coefficients are given in Table 1. The larger in size particles have lower value of the diffusion coefficient (as expected from the Stokes–Einstein equation). In all cases the size distribution is monomodal and the average sizes
Nanoparticles prepared by polymerization. Nanoparticles prepared by nanoprecipitation.
obtained by DLS well correspond to the respective sizes, seen from the SEM images. Fig. 3 represents the size distributions by volume, which is generally accepted to provide quite accurate data. The nanoparticles obtained by polymerization are smaller in size and with smaller polydispersity index (PDI) than the nanoparticles, obtained by nanoprecipitation. The average size of ECN-PBCA nanoparticles prepared by polymerization in the presence of D40 and P188 are similar, around 120 nm. The nanoparticles, prepared
Fig. 2. Representative SEM images of ECN-PBCA nanoparticles prepared by (a) nanoprecipitation using dextran 40; (b) nanoprecipitation, using poloxamer 188; (c) nanoprecipitation, using polysorbate 80; (d) polymerization, using dextran 40; (e) polymerization, using poloxamer 188; (f) polymerization, using polysorbate 80.
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Fig. 4. FTIR spectrum of ECN-PBCA nanoparticles, prepared by polymerization in the presence of D40 as a colloidal stabilizer.
its cationic form in aqueous solutions, one can expect that it could influence the -potential of PBCA colloids. Despite the low value of the -potential in the case of ECN-PBCA nanoparticles, the respective colloidal dispersion is rather stable, which indicates that the steric repulsion between the chains of the non-ionic surfactant on the nanoparticle surface seems to be a more important factor for the stability of the colloid then the electrostatic repulsion between the particles. The -potentials do not seem to depend significantly on the type of the preparation method, but seem to depend on the type of the colloidal stabilizer. It is worth noting that the D40coated nanoparticles are less stable in the PBS buffer, which may result in difficulties during measurements. The colloidal stability of the formulations is further discussed in Section 3.6. Fig. 3. Representative size distributions of ECN-PBCA nanoparticles prepared by (a) polymerization; (b) nanoprecipitation. The abbreviations of the different colloidal stabilizers used are given in the legend. The size distributions were obtained from DLS measurements.
in the presence of P80 are slightly smaller, ∼111 nm. The nanoprecipitation approach resulted in ECN-PBCA nanoparticles of larger sizes, around 230 nm in the cases of using D40 and P188. Previously reported preparations of pure and chlorambucil-loaded PBCA colloids resulted in particles of similar size [23]. In this case, the utilization of P80 as a colloidal stabilizer also leads to the formation of slightly smaller particles, of average size ∼217 nm. Taking into account the data shown in Table 1 and Figs. 2 and 3, one can conclude that the preparation method is the main factor that determines the nanoparticle size, while the utilization of different colloidal stabilizers does not have a significant effect on the average size of the obtained particles. 3.2. Measurements of -potential The data from the measurements of the -potential are summarized in Table 1. As seen, the type of colloidal stabilizer has some effect on the value of the -potential, which is negative for the D40- and P80-coated nanoparticles, and is close to zero for the P188-coated ones. Previous studies on the -potential of pure PACA colloids have showed negative values and it has been suggested that this could be a result from ionized free carboxylic groups [19]. These studies also showed that the absolute value of the -potential depends on the ionic strength of the dispersing medium and lower values have been measured at higher ionic strength [19]. There are also previous reports, which have shown that loaded drugs can influence the value of the -potential (for example, adsorption of cationic drugs on particles with negative -potential can decrease its absolute value) [20]. Taking into account that ECN is in
3.3. Chemical structure characterization A representative FTIR spectrum of ECN-PBCA nanoparticles prepared by polymerization in the presence of D40 is shown in Fig. 4. The characteristic absorbance bands of PBCA are observed as follows: for the carbonyl C O ester (1752 cm−1 ), C N groups (2248 cm−1 ), C H (2876–2964 cm−1 ), and C O C (absorbance at 1256 and 1016 cm−1 correspond to the asymmetric and symmetric stretches, respectively). The absorbance bands of PBCA are similar to previously reported data [31,32]. The absorbance peak at 1591 cm−1 seems to be from the entrapped econazole, although it appears at 1585 cm−1 in the spectrum of pure ECN-nitrate. The other absorbance bands of ECN overlap with those of PBCA and cannot be clearly distinguished. It should be noted that FTIR is not powerful enough to analyze the ECN bands, thus NMR was also used to complement the chemical structure characterization, while it also confirmed data for drug content obtained by UV–Vis (see also Section 3.4). The 1 H NMR-spectrum in Fig. 5a corresponds to pure PBCA, and the spectrum in Fig. 5b corresponds to ECN-PBCA nanoparticles prepared by polymerization in the presence of D40 as a colloidal stabilizer. In the spectrum of pure PBCA, the signals at 1.50 ppm (2H), 1.73 ppm (2H), 2.90 ppm (2H), and 4.26 ppm (2H) correspond all to methylene protons. The signal at 0.97 ppm (3H) corresponds to protons from the methyl groups. The signal at 2.05 ppm is from the solvent (acetone-d6) and is used for calibration. The spectrum of pure PBCA is similar to previously reported studies [23,30,33]. Additional signals from the entrapped ECN molecules appear in the spectrum of ECN-PBCA nanoparticles, of which those from the aromatic protons are well distinguishable between 6.9 and 7.8 ppm (10H). The integral intensity of signals from aromatic protons (IAr ), when compared with the integral intensity of the signals from methylene protons (IMe ) in PBCA, can be used for calculation of the drug content (Eqs. (1) and (2)).
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G. Yordanov / Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 260–265 Table 2 Drug content (DC), drug loading efficiency (Le) and yield of ECN-PBCA nanoparticles. Stabilizer
DC (%)
D40a P188a P80a D40b P188b P80b
9.5 8.8 9.5 5.6 5.4 5.5
a b
± ± ± ± ± ±
Le (%) 0.2 0.2 0.1 0.1 0.1 0.3
61 64 66 52 49 50
± ± ± ± ± ±
2 2 4 2 2 3
Yield (mg/ml) 6.5 7.3 7.1 9.3 9.2 9.2
± ± ± ± ± ±
0.1 0.3 0.3 0.3 0.1 0.1
Nanoparticles prepared by polymerization. Nanoparticles prepared by nanoprecipitation.
is more likely to be entrapped within the nanoparticles in the coarse of the anionic polymerization of butyl cyanoacrylate. The drug content in nanoparticles can be directly determined also from the NMR spectrum (see Section 3.3), but UV–vis spectroscopy seems to be more convenient and easier method. 3.5. Yield of nanoparticles
Fig. 5. 1 H NMR spectra of (a) pure PBCA; (b) ECN-PBCA nanoparticles prepared by polymerization in the presence of D40 as a colloidal stabilizer. The signals from aromatic H-atoms from ECN appear at 7–8 ppm. Data from the 1 H NMR spectrum of ECN-PBCA can be used to determine the drug content (see the text for details).
MBCA nBCA MBCA IMe /3 mBCA = = mECN MECN nECN MECN IAr /10 DC (%) =
mECN × 100 mBCA + mECN
(1) (2)
Here mECN and mBCA are the masses of ECN and BCA monomer units, respectively; nECN and nECN are the number of ECN molecules and BCA monomer units, respectively; MECN and MBCA are the respective molar masses. From the spectrum in Fig. 5b, the ratio of the integral intensity of the methylene protons (0.90–1.04 ppm) to that of the aromatic protons (6.9–7.8 ppm) is 1.000/0.141. The molar mass of the butyl cyanoacrylate (BCA) unit is 153.18 g/mol, and that of ECN is 381.68 g/mol. Then, by using Eqs. (1) and (2), it can be calculated that the drug content is 9.5%, which exactly corresponds to the drug content of the same sample determined by UV-vis spectroscopy (see Section 3.4). 3.4. Drug content and loading efficiency The data obtained from determination of the drug content and the drug loading efficiencies by means of UV-vis spectroscopy (as described in Section 2) are summarized in Table 2. The drug content for the nanoparticles prepared by polymerization is higher (8.8–9.5%) then for the nanoparticles prepared by nanoprecipitation (5.4–5.6%). The respective loading efficiencies are also higher. It seems that the type of colloidal stabilizer does not have a significant effect on the drug loading. However, the type of preparation method has such an effect, leading to higher drug content and loading efficiencies in the case of polymerization. It seems that the ECN
The yield of nanoparticles is defined as the mass of nanoparticles per millilitre of the obtained dispersion. Since, in all experiments the amount of monomer/polymer, ECN and dispersing medium are the same, then one can compare the values given in Table 2. As seen, the yield of nanoparticles does not depend significantly on the type of the colloidal stabilizer, but seems to depend mainly on the preparation method. The yield is lower for ECN-PBCA nanoparticles prepared by the polymerization-based method. This could be a result from deposition of some monomer and polymerization on the stirring bar, as well as formation of small oligomers that are solubilized in the dispersing medium by the colloidal stabilizer thus decreasing the overall yield of nanoparticles. 3.6. Colloidal stability Preliminary evaluations showed that both, the preparation method and the type of colloidal stabilizer affect the colloidal stability of the obtained ECN-PBCA colloids. Unstable formulations are found to be those, prepared by using D40 as a stabilizer. The D40-coated particles tend to aggregate in few days of storage at room temperature or at 4 ◦ C. In general, the particles prepared by polymerization are found to be less stable than those, prepared by nanoprecipitation. The nanoparticles prepared by polymerization tend to coagulate in few months of storage at 4 ◦ C. The particles prepared by nanoprecipitation could sediment upon storage, but can be redispersed again upon sonication. The most stable formulations are those prepared by nanoprecipitation in the presence of P188 and P80. These colloids could be stored at 4 ◦ C for at least 6 months without any observable change in colloidal stability. The concentration of stabilizers in this study was chosen to be in the ranges, which are usually used for the preparations of PACA colloids. It may be expected that higher concentrations of stabilizers may be beneficial for the colloidal stability, but higher concentrations of stabilizers is not recommended and should be avoided for colloidal systems that are intended for biomedical use (because of risks to increase the toxicity of the formulations). The observed differences in the colloidal stability of the ECN-PBCA dispersions could be due to the difference in particle size, drug content and/or probably differences in the properties of the polymer, although the main factors that determine the colloidal stability should be studied further in more details. 4. Conclusions Econazole-loaded PBCA nanoparticles are prepared by using two different methods: nanoprecipitation and polymerization, and
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three different colloidal stabilizers (dextran 40, polysorbate 80 and poloxamer 188). The obtained nanoparticles are of spherical shape and possess monomodal size distribution. The nanoparticles, prepared by polymerization-based method were smaller in size and had a higher drug content than those, prepared by nanoprecipitation. The type of colloidal stabilizer did not have a significant effect on the average nanoparticle size and drug content at all other constant conditions. The utilization of polysorbate 80 and dextran 40 as colloidal stabilizers resulted in negative -potentials, while using poloxamer 188 resulted in -potentials close to zero. Preliminary studies of the colloidal stability indicated that the nanoparticles, coated with poloxamer 188 and polysorbate 80, are relatively more stable than the dextran-coated nanoparticles. The nanoparticles obtained by nanoprecipitation were more stable than those prepared by polymerization. Acknowledgements GY is thankful for the support from COST Action D43. The technical support from Mr. Nikola Dimitrov and Dr. Mariana Boneva with SEM observation and DLS measurements, respectively, is greatly acknowledged. NMR and FTIR spectra were measured at the Institute of Organic Chemistry (BAS). References [1] D. Thienpont, J. Van Cutsem, J.M. Van Nueten, C.J. Niemegeers, R. Marsboom, Biological and toxicological properties of econazole, a broad-spectrum antimycotic, Arzneimittel-Forsch. 25 (1975) 224–230. [2] R.C. Heel, R.N. Brogden, T.M. Speight, G.S. Avery, Econazole: a review of its antifungal activity and therapeutic efficacy, Drugs 16 (1978) 177–201. [3] M. Borgers, Mechanism of action of antifungal drugs, with special reference to the imidazole derivatives, Rev. Infect. Dis. 2 (1980) 520–534. [4] R. Pandey, Z. Ahmad, S. Sharma, G.K. Khuller, Nano-encapsulation of azole antifungals: potential applications to improve oral drug delivery, Int. J. Pharm. 301 (2005) 268–276. [5] T. Prow, J. Grice, L. Lin, R. Faye, M. Butler, W. Becker, E. Wurm, C. Yoong, T. Robertson, H. Soyer, M. Roberts, Nanoparticles and microparticles for skin drug delivery, Adv. Drug Deliv. Rev. 63 (2011) 470–491. [6] D.Q. Luo, J.H. Guo, F.J. Wang, Z.X. Jin, X.L. Cheng, J.C. Zhu, C.Q. Peng, C. Zhang, Anti-fungal efficacy of polybutylcyanoacrylate nanoparticles of allicin and comparison with pure allicin, J. Biomater. Sci. Polym. Ed. 20 (2009) 21–31. [7] N. Xu, J. Gu, Y. Zhu, H. Wen, Q. Ren, J. Chen, Efficacy of intravenous amphotericin B–polybutylcyanoacrylate nanoparticles against cryptococcal meningitis in mice, Int. J. Nanomed. 6 (2011) 905–913. [8] Z. Ahmad, R. Pandey, S. Sharma, G.K. Khuller, Novel chemotherapy for tuberculosis: chemotherapeutic potential of econazole- and moxifloxacin-loaded PLG nanoparticles, Int. J. Antimicrob. Agents 31 (2008) 142–146. [9] Y.G. Bachhav, K. Mondon, Y.N. Kalia, R. Gurny, M. Möller, Novel micelle formulations to increase cutaneous bioavailability of azole antifungals, J. Control. Release 153 (2011) 126–132. [10] M.P.Y. Piemi, D. Korner, S. Benita, J.-P. Marty, Positively and negatively charged submicron emulsions for enhanced topical delivery of antifungal drugs, J. Control. Release 58 (1999) 177–187. [11] S. Cogswell, S. Berger, D. Waterhouse, M.B. Bally, E.K. Wasan, A parenteral econazole formulation using a novel micelle-to-liposome transfer method: in vitro characterization and tumor growth delay in a Breast Cancer Xenograft Model, Pharm. Res. 23 (2006) 2575–2585.
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