Journal of the Chilean Chemical Society
versión On-line ISSN 0717-9707
J. Chil. Chem. Soc. v.53 n.4 Concepción dic. 2008
J. Chil. Chem. Soc, 53, N° 4 (2008) págs: 1677-1681
ENCAPSULATION OF LEU-ENKEPHALIN IN CORE-SHELL ISOBUTYLCYANOACRYLATE - THIOLATED CHITOSAN NANOPARTICLES FOR ORAL ADMINISTRATION
VÍCTOR H. CAMPOS REQUENAAB* KAWTHAR BOUCHEMALA, CHRISTINE VAUTHIERA, GILLES PONCHELA
A.UniversityParis-South XI, Faculty of Pharmacy, UMR CNRS 8612. 5, rue J.B. Clément. 92296 Chátenay Malabry, France.
B. Master in Pharmaceutical Sciences Candidate, University of Concepción, Faculty of Pharmacy, Barrio Universitario s/n, Concepción, Chile.
The present study shows the evaluation of Leu-Enkephalin (Leu-Enk) encapsulation on core-shell isobutylcyanoacrylate/thiolated chitosan (IBCA/ChitoTBA) nanoparticles as a tool of combined system of polymers and colloidal particles for oral administration of poor absorption drugs. Leu-Enk encapsulation using anionic emulsión polymerization technique was carried out previous depolymerization of MW 400,000 gmol"1 commercial chitosan, followed by thiolation of this depolymerized chitosan with Traut's reagent (2-iminothiolane), and final characterization. Later, direct quantification of the encapsulated Leu-Enk into the nanoparticles was carried out evaluating two methodologies for nanoparticles rupture solubilization with: NaOH 1 N and DMSO. Physico-chemical characterization of the obtained nanoparticles showed diameters in the nano-range between 28 and 46 nm, and yields of encapsulation of Leu-Enk between 25 and 46%, and recovery yields over 96% for co-polymer nanoparticles solubilization method with DMSO.
Keywords: nanoparticles, leu-enkephalin, isobutylcyanoacrylate, thiolated chitosan, thiomers, anionic emulsion polymerization.
Nanotechnology and polymers had captivated atremendous interest in many scientific grounds such as pharmaceutical industry and therapeutic innovation among others. Biological and synthetic polymers had been used as a powerful tool for nanoscale systems, and modified polysaccharide-coated nanoparticles are a promising tool for drug carrier systems in oral administration of poor absorption drugs in therapeutic1. Through the recent years, great development have been reached in the field of mucoadhesive polymer systems as a new tool in formulations that increase the residence time of drugs on mucosal membranes and subsequently enhance the bioavailability of poor oral absorption drugs2"3.
Leu-Enk is a low molecular peptide (fig. 1) that behaves as a potent morphinic analgesic that is currently administered by parenteral route causing both disconfort and pain in its administration. As a peptide, Leu-Enk has a poor absorption by oral route due to their susceptibility to enzymatic metabolism by aminopeptidases" and for his physicochemical and size properties which limit their membrane permeation5.
For its properties, such as enzymatic biodegradability, biocompatibility and non-toxicity, chitosan is recognized as a novel excipient for drug delivery systems6. Its natural mucoadhesive properties allows design bioadhesive drug carrier systems that can bind to the intestinal muccosa, so it can improve the residence time of drugs in the intestinal lumen and so its bioavailability7. In the last years, thiolated chitosan derivated has been developted. This thiolated polymer - so called "thiomers" - has the advantage of increase mucoadhesion based on covalent disulfide bond between both sulphydryl groups from the thiomer and from cysteine-rich subdomains of mucus glycoproteins in intestine8. In addition, thiolated chitosan has enhancing permeation properties, and antiprotease activity due to presence of thiol groups improving the paracellular absorption of macromolecules like peptides and proteins9.
Poly(alkylcyanoacrylates) had been used widely in colloidal carrier systems for their low toxicity and biodegradability, and react spontaneously with different polysaccharides like dextran and chitosan forming stables nanoparticle systems that can be used for drug encapsulation10. Isobutylcyanoacrylate (IBCA) has been extensively used in nanoparticles systems with many polysaccharides using anionic emulsión polymerization, a simple one-step reaction in aqueous media, avoidingthe use of organic solvents11"12.
For succesful drug encapsulation, several parameters must be set up including the optimal point for addition of the drug into the system, the filled-up capacity of the nanoparticles system for encapsulate the drug, among others. A critical point is the separation of loaded nanoparticles from the system, for a later rupture of the amphiphilic co-polymer nanoparticles to reléase the encapsulated drug, and then quantify directly this encapsulated fraction without loss of drug along the process.
Isobutylcyanoacrylate was kindly provided as a gift by Loctite (Dublin, Ireland). Chitosan MW 400,000 gmol"1, Leu-Enkephalin acétate and L-cysteine HC1 were purchased from Fluka (Saint-Quentin Fallavier, France). 2-Iminothiolane HC1 (Traut's reagent) was synthesized in the Department of Organic Chemistry (Biocis UMR CNRS 8076), Faculty of Pharmacy, University Paris-XI, Chátenay-Malabry, France. Acetonitrile, trifluoracetic acid and dimethylsulfoxide were HPLC grade and purchased from Fluka. Sodium hydroxide and glaciar acetic acid were reagent grade from Merck.
HPLC was made with Interchim N5C18-25QK column; Waters 1525 Binary HPLC Peristaltic Pump; Waters In-Line Degasser AF; UV Waters 2487 Dual Lambda Absorbance Detector; and Waters 717 Plus Autosampler.
Viscometer AVS400, Schott Gerate was used for chitosan molecular weight measure. Bruker MSL-400 spectrometer (Bruker Instrument Inc. Wissembourg, France) was used for :H-NMR analysis. Christ Alpha 1-4 freeze-dryer (Bioblock Scientific, Illkirch, France) were used for samples lyophilization. Sulphur elemental analysis were made in a Analyzer LECO SC144 (Service central d'analyse du CNRS, Vernaison, France). Iodine titration was assayed with a Spectrophotometer UV/VIS Lambda 11 Perkin-Elmer (Norwalk, USA). ¡¡ potential was measured by quasi-elastic light scattering with a Nanosizer* N4 PLUS (Beckman-Couter, Villepinte, France). OptimaTM Ultracentrifuge (Bekman-Coulter Instruments, USA) was used in the ultracentrifugation of nanoparticles. Scanning Electronic Microscopy (SEM) observations were performed using a LEO 1530 (LEO Electrón Microscopy Inc, Thronwood, NY) operating at 3 kV with a filament current of about 0.5 mA. Transmission Electrón Microscopy (TEM) observations were assessed using a Philips EM 208 apparatus operating at 80 kV.
Chitosan modifications and characterization: Commercial chitosan (MW 400,000 gmol"1) was selectively depolymerized following the method developed by Huang et al. using sodium nitrite to obtain three average molecular weights: 30,000, 85,000 and 145,000 gmol1 (called Chito30, Chito85 and Chito 145) which were measured by capillary viscosity13. 'H-NMR analysis were carried out for chitosan structural change and deacetylation level measures.
The inclusión of thiol groups in the different depolymerized chitosan was carried out following the method developed by Bernkop-Schnürch et al.1" reacting one gram of chitosan with Traut' s reagent (2-iminothiolane) in a weight ratio of 5:2. After an incubation period of 24 h at room temperature under continuous stirring, the resulting thiolated polymer was dialysed (Spectra/Por* 3 membrane MWCO:3500) against different aqueous media: 8 h against 5 L of 5 mmol L"1 HC1, 8 h against 5 L of 5 mmol L"1 HC1 containing 1% NaCl two times, 8 h against 5 L of 5 mmol L"1 HC1, and finally, 8 h against 5 L of 1 mmol L"1 HC1 (40 h in total). Dialysed producís were freeze-dried and stored at -20°C until use. The resulting polymers obtained were chitosan-4-thiol-butylamidine derivated, named Chito30-TBA, Chito85-TBA and Chito 145-TBA according to the original molecular weight of the corresponding unmodified polymers.
Sulphur elemental analysis and iodine titration were performed in order to determine total sulphur and reduced thiol groups contented in modified chitosan, using the same method for nanoparticles assays.
Nanoparticles preparation: Nanoparticles elaboration following anionic emulsión polymerization was carried out according to previous works15"17. Briefly 0.069 g of depolymerized chitosan, at proportions of chitosamchitosan-TBA 75:25 (w/w), were dissolved in 4 mL of 0.2 molL1 nitric acid in MilliQ® water, in a glass tube at 40°C under gentle stirring and argón bubbling. After 10 min, 1 mL of a 0.2 molL"1 nitric acid solution, and 0.25 mL of IBCA were added under vigorous magnetic stirring. Argón bubbling was kept for additional 10 min and stopped. The reaction was allowed to continué at 40°C under gentle stirring for 40 min. After cooling to room temperature, NaOH 1 N was added to raise the pH from 1.5 to 4.5 to store.
Inclusión of Leu-Enkephalin in the nanoparticles: The preparation of nanoparticles is conduced at pH 1.5 during 40 min and then pH was raised to 4.5 to store. Stability study of Leu-Enk in acidic médium at pH 1.5 and 4.5 is required. After one week of aqueous Leu-Enk solution incubation at pH 1.5 (previously at 40°C for 40 min) and at pH 4.5 for one week, samples were analysed using HPLC analysis for pH stability evaluation. The samples where assayed by reversed-phase HPLC system with UV detection at 220 nm and 10 uL loop. The mobile phase was a two-phases mixture of (A) acetonitrile/ trifuoracetic acid 0.1% in water MilliQ (10:90) and (B) acetonitrile/trifuoracetic acid 0.1% in water MilliQ (90:10). The gradient program used was 100% (A) from 0 to 1 min; from 1 to 20 min a increasing gradient from 0 to 100% (B) with a constant flow rate of 1 mL/min.
An assay for the mínimum quantity of Leu-Enk added to nanoparticles leads to determine the amount of Leu-Enk which is sufficient to fill up the nanoparticles and satúrate the system. Leu-Enk in increasing concentrations were added to the médium of polymerization, and later Leu-Enk in the supernatant was determined by HPLC, previous separation of the nanoparticles from the supernatant by ultracentrifugation at 45xl03 rpm during 30 min.
The methodology for Leu-Enk inclusión into the nanoparticles was carried out with two variations and is described in the fig. 2. The first one consisted on the addition of Leu-Enk into the chitosan acidic solution before start of the polymerization with IBCA, called "time = 0 hour" (f = Oh). The second variation consisted on the addition of Leu-Enk one hour after the end of polymerization reaction, called "time = 1 hour" (t = 1 h). This is in order to evalúate the quantity of Leu-Enk strictly encapsulated on the core of the nanoparticles and the quantity adsorpted at the surface of nanoparticles as well.
Solubilization of the co-polymer nanoparticles for encapsulation yield determination: Bertholon et al. describe methodologies for IBCA/Dextran non-loaded nanoparticles solubilization using NaOH 1N and dimethyl sulfoxide (DMSO)18. This two agents were evaluated to solubilize IBCA/ChitoTBA nanoparticles loaded with Leu-Enk (75 ugg"1) for later direct determination of the encapsulated drug released. The method of solubilization is described in fig. 3.
Yield of encapsulation and recovery assay: Leu-Enk recovery yield of the solubilization method it is necessary for assure the peptide integrity after the entire process of nanoparticle rupture for direct determination of the drug encapsulated. The yield of encapsulation (Y) was carried out adding a known Leu-Enk concentration (75 |ig g"1) in the nanoparticles formulation and was calculated according to the equation 1:
Where NP is the Leu-Enk concentration in loaded nanoparticles and T is the total concentration of Leu-Enk in the complete nanoparticles suspensión (encapsulated and non-encapsulated). The recovery yield of the solubilization method is gived by T . Loaded nanoparticles were isolated from the supernatant by using an ultracentrifugation at 45xl03 rpm during 30 min. NPEnt and T were obtained after total solubilization of nanoparticles with the NaOH 1 N or DMSO as shows in the fig. 3 and measured by HPLC in conditions previously described.
Physico-chemical characterization of the nanoparticles:
Determination of the ¡¡ potential: The electric surface charge of the co-polymer particles was deduced from the electrophoretic mobility of the particles measured by Láser Doppler Electrophoresis in a NaCl 1 mmol L"1 solution after suitable dilutions (1/200 v/v) for different nanoparticles suspensions inorderto maintain a constant ionic strength19.
Nanoparticles morphology and size: The nanoparticle morphology was analysed by Scanning Electronic Microscopy (SEM) with nanoparticle suspensions diluted in MilliQ® water from 1/1 to 1/10000. Nanoparticles size measurements were analysed by Transmission Electrón Microscopy (TEM) by direct observation after staining with phosphotunguestic acid 1 % (pH 7.4) and the size measure is ensured using Adobe Photoshop* Software.
Determination of thiol content in the nanoparticles:
Iodine titration: The degree of modification of thiolated polymer and nanoparticles was analysed by iodine titration using soluble starch, measured by spectrophotometry at 560 nm against a calibration curve of L-cystein (0.04 0.124 umolmL"1), as described by Bravo-Osuna et al20.
Elemental analysis: The total amount of sulphur in both thiolated polymers and nanoparticles was determined by elemental analysis. Samples of 10 mg were burned at 1350°C over oxygen flux and the detection of S02 was performed by infrared measurements
RESULTS AND DISCUSSION
1. Chitosan modifications and characterization:
From commercial chitosan, three average molecular weights were obtained after depolymerization: 30,000 gmol"1, 85,000 gmol"1 and 145,000 gmol"1 (called Chito30, Chito85 and Chitol45 respectively) measured by capillary viscosity. 'NMR showed no structural change and acceptable decrease in the percentage of deacetylation, with valúes about 60, 71 and 89 % for Chito30, Chito85 and Chito 145 respectively. The total sulphur in depolymerized modified chitosan (Table 1) showed no significant variation among them. The reduced thiol groups content in modified chitosan decreased according the chitosan molecular weight probably due to differences in deacetylation degree, because the thiol group from the Traut's reagent should be included on the free amino groups of the chitosan chain (fig. 4).
2. Physico-chemical characterization of nanoparticles:
The resulting dispersión made by anionic emulsión polymerization is composed by a isobutylcyanoacrylate/thiolated chitosan copolymer (fig. 4) which spontaneously auto-associates to form nanoparticles (fig. 5). The polymer particles are likely stabilized by the hydrophilic polysaccharide moiety as already suggested Passirani et al21.
The morphologic characteristics of the different nanoparticles obtained are gathered in table 2. Results obtained from the study of the electrophoretic mobility of the nanoparticles offered positive valúes of ¡¡ potential for all formulations, indicating that cationic polysaccharide was located at the surface ofthe nanoparticles (fig. 5). The polysaccharide coating completely maskedthe negative surface charge valúes characteristic of non-coated IBCA nanoparticles as well described by previous works22.
Small nanoparticles about 28 to 46 nm diameter were obtained. The size of the nanoparticles mainly depend on the molecular weight ofthe chitosan used considering the series of experiments performed with thiolated-chitosan. It can be observed that the lower the molecular weight, the smaller the nanoparticles size. Nanoparticles with small size have great importance for the stability ofthe polymer particles and leads to a high exposition of thiols groups at the surface of nanoparticles, and better mucoadhesion is expected when oral administration is perfomed.
TEM technique allowed the isolation and easy observation of spherical and very well individualized particles (fig. 6). As can be noticed, in all cases spherical particles were obtained in the nano-size range.
Total sulphur and thiol groups determination: The valúes of total amount of sulphur contained in the nanoparticles formulations showed no relationships between chitosan molecular weight. The thiol groups is reduced along chitosan molecular weight is incresed, probably due to thiol groups amount in its respective modified chitosan (Table 1).
3. Inclusión of Leu-Enkephalin into the nanoparticles:
Stability of Leu-Enk at different pH: The Leu-Enk stability at different pH and temperatures were studied. First, pH 1.5 at 40°C because the anionic emulsión polymerization occurs at this pH, and pH 4.5 because after the end of the polymerization reaction, the pH of the solution was rised to 4.5 for store. Results indícate that recovery of Leu-Enk was 101.7% ± 0.7 (n=3) for pH 1.5 and 101.8 ± 0.9 (n=3) for pH 4.5, indicating a satisfactory stability for Leu-Enk at these polymerization and store conditions.
Filled-up limit of Leu-Enkephalin encapsulation into nanoparticles: Fig. 7 shows that there is a direct response of Leu-Enk found in the supematant when peptide concentration is increased in the nanoparticles formulation. The inflection point is approximately 10 ugg"1 of Leu-Enk added, and after that point, Leu-Enk concentration start to increased directly in the supematant, while the Leu-Enk encapsulated into the nanoparticles remains constant, that can be due to a saturation of the capacity of nanoparticles.
4. Solubilization method for amphiphilic co-polymer nanoparticles and recovery yields for total Leu-Enk in the formulation:
In most of the cases, indirect methods of load drug in nanoparticles systems are developted, this is, the loaded drug is measured by the difference between the total drug added to the system, and the drug in the supematant, been this last one, directly measured. In this study, we formúlate a direct load drug determination method. First, it is necessary to develop a solubilization method for nanoparticles, so the loaded drug can be released without destruction, and then measured directly. In addition, drug in the supematant and total drug added to the system must be measured directly as it shows the fig. 3 for recovery yield determination of the method.
Co-polymer solubilization methods with NaOH 1 N and DMSO were evaluated for drug load determination. The table 3 shows that NaOH presents low total Leu-Enk recovery yields reached by direct determination of TEnt in the range of 64%. But in the case of DMSO, yields of recovery higher than 96% are reached in TEnt, and a logical correlation between the Leu-Enk in the supematant and the Leu-Enk released from nanoparticles is obtained for DMSO, that give us a more reliable co-polymer solubilization method, and no Leu-Enk destruction along the procedure is deduced.
5. Determlnation of encapsulationyield:
Once the optimal co-polymer solubilization method and filled-up limit of Leu-Enk in nanoparticles are determinated, yield of encapsulation was carried out for both methods of Leu-Enk inclusión: addingthe drugbefore polymerization (f = Oh), and adding one hour after the start ofthe polymerization (t=lh) as seen on fig. 2.
Table 4 shows that yield of encapsulation of 42-46% (0,427-1,770 ng Leu-Enk/nanoparticle) was achieved with Leu-Enk inclusión at t = 0 h, that results encapsulation into the core of nanoparticles and surface adsorption of Leu-Enk outside nanoparticles. Yield of encapsulation of 25-38% (0,388-1.290 ng Leu-Enk/nanoparticle) was obtained when Leu-Enk is added at t = 1 h, probably due only to adsorpted Leu-Enk in the surface of nanoparticles. Kinetics of formation in previous studies of IBC A/dextran nanoparticles show that after the first 15 minutes no longer nanoparticles are formated for anionic polymerization23, so most of the Leu-Enk is encapsulated fast in the first minutes of polymerization, so adding the Leu-Enk one hour after start the polymerization, a great porcentage of Leu-Enk is only adsorpted in the surface at t = 1 h, resulting theoretically 0.039-0.480 ng Leu-Enk/nanoparticle strictly encapsulated inside the nanoparticles.
Isobutylcyanoacrylate nanoparticles coated with thiolated chitosan were prepared using anionic emulsión polymerization. Nanoparticles in the order of 28-46 nm were obtained, and expected thiolation degree was found in the formulations. For direct encapsulation yield determination, nanoparticle co-polymer solubilization method with NaOH 1 N described in previous works for non-loaded nanoparticles, can not be used when formulation of loaded nanoparticles with Leu-Enk is performed. Several loss of peptide is found when nanoparticles hydrolysis with NaOH 1 N is used for solubilization of IBCA/ ChitosanTBA co-polymer, but no loss of peptide is found when DMSO is used for co-polymer solubilization, being a usefull tool for direct quantification of encapsulated Leu-Enk in IBCA/ChitosanTBA nanoparticle systems, resulting Leu-Enk recovery yields higher than 96% for this method. The encapsulation yields of Leu-Enk were satisfactory by anionic emulsión polymerization, achieving 42-47% when peptid is added before the start ofthe polymerization (direct core encapsulation and surface adsorption), and 26-38% when is added after the polymerization (only adsorption mechanism).
Authors want to thank Dr. K. Broadley from Loctite (Dublin, Ireland) for his kindness in providing the isobutylcyanoacrylate monomer. Authors want also to thank the Department of Organic Chemistry (Biocis UMR CNRS 8076), Faculty of Pharmacy, University Paris-XI (Chatenay-Malabry, France) for their help in the synthesis of 2- iminothiolane and the "Service central d'analyse du CNRS" (Vernaison, France) for the elemental analysis of thiolated polymers. Authors want also to thank Irene Bravo-Osuna, Madeleine Besnard and Alexia Aspe. Thanks to Audrey Valette (CNRS CECM Vitry-sur-Seine, France) and Jeril Degrouard (UMR CNRS 8080, Orsay, France), for their help in the characterization of nanoparticles by SEM and TEM. Finally, Víctor Campos wants to thank to CONICYT-Chile for supporting his Magister in Pharmaceutical Sciences with a two-years grant.
1. I. Bravo-Osuna, C. Vauthier, H. Chacun, G. Ponchel, Eur. J. Pharm. Biopharm. 69 (2), 436-444 (2008). [ Links ]
2. A. Bernkop-Schnürch, D. Guggi, Y. Pinter, J. Control. Reí. 94, 177-186 (2004). [ Links ]
3. M. Aboubakar, P. Couvreur, H. Pinto-Alphandary, B. Gouriton, B. Lacour, R. Farinotti, F. Puisieux, C. Vauthier, Drug Dev. Res. 49, 109-117 (2000). [ Links ]
4. Ying-Shu Quan, Takuya Fujita, Daichi Tohara, Miwako Tsuji, Makoto Kohyama, Akira Yamamoto, Life Sciences, Vol 64, N°14, 1243-1252 (1999). [ Links ]
5. G. Pauletti, F. Okumu, R. Borchardt, Pharm. Res. Vol 14, No.2, 164-168 (1997). [ Links ]
6. A. Bernkop-Schnürch, M. Hornof, D. Guggi. Eur. J. Pharm. Biopharm. 57,9-17(2004). [ Links ]
7. A. Bernkop-Schnürch, Y. Pinter, D. Guggi, H. Kahlbacher, G. Schóffmann, M. Schuh, I. Schmerold, M. Dorly, M. D'Antonio, P. Esposito, C. Huck. J. Control. Reí. 106, 26-33 (2005). [ Links ]
8. A. Bernkop-Schnürch. Adv. Drug Del. Rev. 57, 1569-1582 (2005). [ Links ]
9. A. Bernkop-Schnürch, A. Krauland, V. Leitner, T. Palmberger. Eur. J. Pharm. Biopharm. 58, 253-263 (2004). [ Links ]
10. I. Bertholon, D. Labarre, C. Vauthier. Polymer 46, 1407-1415 (2005). [ Links ]
11. I. Bertholon, C. Vauthier, D. Labarre. Pharm. Res. 23 (6), 1313-1323 (2006). [ Links ]
12. C. Chauvierre, M. Marden, C. Vauthier, D. Labarre, P. Couvreur, L. Leclerc, Biomaterials 25, 3081-3086 (2004). [ Links ]
13. M. Huang, E. Khor, L.Y. Lim, Pharm Res. 21(2), 344-353 (2004). [ Links ]
14. A. Bernkop-Schnürch, M. Hornof, T. Zoidl. Int. J. Pharm. 260, 229-237 (2003). [ Links ]
15. C. Chauvierre, D. Labarre, P. Couvreur, C. Vauthier, Macromol. 36, 6018-6027 (2003). [ Links ]
16. I. Bravo-Osuna, G. Ponchel, C. Vauthier, Eur. J. Pharm. Sci. 30 (2), 143-154(2007). [ Links ]
17. C. Chauvierre, D. Labarre, P. Couvreur, C. Vauthier, Pharm Res 20 (11) 1786-1793 (2003). [ Links ]
18. I. Bertholon, S. Lesieur, D. Labarre, M. Besnard, C. Vauthier. Macromol. 39, 3559-3567 (2006). [ Links ]
19. M.A. Arangoa, G. Ponchel, A. M. Orecchioni, M. J. Renedo, D. Duchene, J. M. Irache, Eur. J. Pharm. Sci. 11 (4), 333-341 (2000). [ Links ]
20. I. Bravo-Osuna, T. Schmitz, A. Bernkop-Schnürch, C. Vauthier, G. Ponchel, Int. J. Pharm. 316, 170-175 (2006) [ Links ]
21. C. Passirani, G. Barratt, J.P. Devissaguet, D. Labarre, Life Sci. 62 (8), 775-785 (1998). [ Links ]
22. M. T. Peracchia, C. Vauthier, C. Passirani, P. Couvreur, D. Labarre, Life Sci. 61 (7), 749-761 (1997). [ Links ]
23. C. Chauvierre, D. Labarre, P. Couvreur, C. Vauthier, J. Nano. Res. 5, 365-371 (2003) [ Links ]
(Received: November 28, 2007 - Accepted: December 22, 2008)