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Magnetoresponsive Squalenoyl Gemcitabine Composite Nanoparticles for Cancer Active Targeting Jose´ L. Arias,† L. Harivardhan Reddy,‡ and Patrick Couvreur*,‡ Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, UniVersity of Granada, 18071 Granada, Spain, and Faculte´ de Pharmacie, UMR CNRS 8612, IFR 141, UniVersite´ Paris-Sud XI, 92296 Chaˆtenay-Malabry Cedex, France ReceiVed February 20, 2008. ReVised Manuscript ReceiVed April 4, 2008 Gemcitabine is widely used against a variety of solid tumors; however, it possesses some important drawbacks such as rapid deamination leading to short biological half-life and induction of tumor resistance. We have shown previously that the covalent coupling of squalene (a precursor of cholesterol in sterol biosynthesis) to gemcitabine resulted in a potent nanomedicine, squalenoyl gemcitabine (SQdFdC), which displayed appreciable anticancer activity. Now, the present study describes the concept of magnetic responsiveness of SQdFdC nanoparticles obtained by the nanoprecipitation of SQdFdC around magnetite nanoparticles. To investigate these new core/shell nanoparticles, we have compared their structure, chemical composition and surface properties with those of either the magnetic core alone or of the SQdFdC coating material. X-ray diffraction and infrared spectroscopy studies have shown that the composite core/shell particles displayed an intermediate behavior between that of pure magnetite and of pure SQdFdC nanoparticles, whereas dark-field, high-resolution transmission electron microscopy allowed clear demonstration of the core/shell structure. Electrophoresis measurements as a function of both pH and ionic strength, as well as thermodynamic consideration, showed similar behavior of core/shell and pure SQdFdC nanoparticles, suggesting again the coating of the magnetite core by the SQdFdC prodrug. The two important parameters to be controlled in the efficient adsorption of SQdFdC onto magnetite nanocores were the magnetite/SQdFdC weight ratio and the pluronic F-68 concentration. Pluronic F-68 was found to play a key role as a surfactant in the generation of stable composite core/shell nanoparticle suspensions. Finally, the characterization of the magnetic properties of these core/shell nanoparticles revealed that if the squalenoyl shell reduced the magnetic responsiveness of the particles, it kept unchanged their soft ferrimagnetic character. Thus, the heterogeneous structure of these nanoparticles could confer them both magnetic field responsiveness and potential applicability as a drug carrier for active targeting to solid tumors.
Introduction Regular systemic chemotherapy involves the administration of important doses of anticancer drugs to obtain an acceptable therapeutic concentration at the desired site of action. However, high-dose regimens can also cause toxicity to the nontarget organs. Thus, employment of targeted delivery systems would facilitate drug transport to the desired site, simultaneously decreasing the unwanted side effects.1 Drug targeting using magnetic carriers has been the area of significant interest in the recent years.2–6 Practically, the drug is bound to a magnetic compound/carrier, and following administration into the body, it is directed to and concentrated in the target area using a magnetic field. Depending on the application, particles then release the active agent and/or provide a local effect (irradiation from radioactive particles or hyperthermia with magnetic particles). The drug release process can occur by simple diffusion or through mechanisms requiring enzymatic activity or changes in physiological conditions such as pH, osmolality, temperature, or, even, magnetically triggered from the nanoparticles.7–10 * Corresponding author. Phone: + 33 1 46 83 53 96. Fax: + 33 1 46 61 93 34. E-mail:
[email protected]. † University of Granada. ‡ Universite´ Paris-Sud XI.
(1) Couvreur, P.; Vauthier, C. Pharm. Res. 2006, 23, 1417. (2) Li, X. S.; Li, W. Q.; Wang, W. B. Cancer Biother. Radiopharm. 2007, 22, 772. (3) Tang, M.; Russell, P. J.; Khatri, A. DiscoVery Med. 2007, 7, 68. (4) Chertok, B.; Moffat, B. A.; David, A. E.; Yu, F.; Bergemann, C.; Rodd, B. D.; Yang, V. C. Biomaterials 2008, 29, 487. (5) Alexiou, C.; Schmid, R. J.; Jurgons, R.; Kremor, M.; Wanner, G.; Bergemann, C.; Huenges, E.; Nawroth, T.; Arnold, W.; Parak, F. G. Eur. Biophys. J. 2006, 35, 446. (6) Zhang, J. L.; Srivastava, R. S.; Misra, R. D. K. Langmuir 2007, 23, 6342.
Thus, the foremost advantages of magnetic nanoparticles are that they can be (i) visualized (superparamagnetic nanoparticles are used in MRI); (ii) guided or held in place by means of a magnetic field; and (iii) heated in a magnetic field to trigger drug release or to produce hyperthermia/ablation of tissues. Since the magnetic gradient decreases with the distance to the target, the major limitation of these magnetic systems relates to the strength of the external magnetic field necessary to control the residence time of the nanoparticles in the desired area or which triggers the drug desorption. The final size and the hydrophobic/hydrophilic nature of the magnetic nanoparticles are important parameters which determine their biological fate following administration. Particles under 10 nm size are rapidly removed after an extensive extravasation and renal clearance, whereas carriers over 200 nm undergo macrophage uptake and mechanical filtration by the spleen. In addition, systems with diameters greater than 5 µm induce capillary blockade.11 However, if the particles are magnetically retained in the target tissue capillaries, drug diffusion through the capillary wall induces the therapeutic action.12 With respect to the hydrophobic/hydrophilic nature of the particles, immediately upon contact with the blood, the particles interact with the plasma (7) Brigger, I.; Dubernet, C.; Couvreur, P. AdV. Drug DeliVery ReV. 2002, 54, 631. (8) Ne´mati, F.; Dubernet, C.; Fessi, H.; de Verdie`re, A. C.; Poupon, M. F.; Puisieux, F.; Couvreur, P. Int. J. Pharm. 1996, 138, 237. (9) Neuberger, T.; Scho¨pf, B.; Hoffmann, H.; Hoffmann, M.; von Rechenberg, B. J. Magn. Magn. Mater. 2005, 293, 483. (10) Ha¨feli, U. O. Int. J. Pharm. 2004, 277, 19. (11) Gupta, A. K.; Gupta, M. Biomaterials 2005, 26, 3995. (12) Goodwin, S.; Peterson, C.; Hoh, C.; Bittner, C. J. Magn. Magn. Mater. 1999, 194, 132.
10.1021/la800547s CCC: $40.75 2008 American Chemical Society Published on Web 06/10/2008
SQdFdC Nanoparticles for Cancer ActiVe Targeting
proteins; i.e., they undergo opsonization. The fact is that the protein adsorption is increased if the surface charge, the size, or the hydrophobicity increases. This is essential, as some kinds of such adsorbed proteins (opsonins) affect the ability of the cells of the reticuloendothelial system, which is part of the immune system, to phagocyte the particles and retire them from the bloodstream toward the RES organs, liver, spleen, and bone marrow. Moreover, the ease with which the particles will contact the aqueous medium retaining their individuality and decreasing their tendency to aggregate will be also controlled by their surface thermodynamics. Thus, hydrophobic particles will tend to attract each other, whereas hydrophilicity will lead to a better stability. These interactions of interfacial origin can be quantified, using proper models.13 Magnetic drug delivery systems for biomedical applications must fulfill some requirements:10,14 (i) biocompatibility, (ii) nontoxicity, (iii) nonimmunogenicity, and (iv) ease of injectability. Thus, the magnetic carriers can be designed exclusively using magnetic materials such as magnetite, maghemite, cobalt ferrite, or carbonyl iron.15 Among them, magnetite is a well-known magnetic material, whose toxicity has been demonstrated to be quite low (LD50 in rats, 400 mg/kg).16 However, the main limitations of these magnetic materials are low drug loading capacity and difficult drug release control. To resolve these problems, matrices of a diverse nature have been used, including biodegradable [liposomes, chitosan, poly(lactide), poly(D,Llactide-co-glycolide), poly(alkylcyanoacrylate), or poly(ε-caprolactone), to mention just a few] and nonbiodegradable (e.g., ethylcellulose, polystyrene, and poly(methyl methacrylate)) shells.10,12,16–21 In the present study, we have employed a magnetic core to synthesize core/shell nanoparticles of squalenoyl gemcitabine (SQdFdC), a squalenoyl prodrug of gemcitabine. This prodrug was previously obtained by covalent coupling of gemcitabine with 1,1′,2-trisnorqualenic acid.22 Due to its amphiphilic nature, SQdFdC conjugate self-assembled spontaneously in water as nanoparticles of 130 nm which displayed unique hexagonal supramolecular architecture.22–24 Unlike gemcitabine, SQdFdC nanoparticles resisted rapid deamination and overcame the drug resistance by several folds when tested in vitro.23 In addition, SQdFdC nanoparticles displayed superior anticancer activity over its parent drug gemcitabine against experimental leukemia which also resulted in long-term survivors.25 On the basis of the (13) Dura´n, J. D. G.; Arias, J. L.; Gallardo, V.; Delgado, A. V. J. Pharm. Sci. 2007, DOI: 10.1002/jps.21249. (14) Ito, A.; Shinkai, M.; Honda, H.; Kobayashi, T. J. Biosci. Bioeng. 2005, 100, 1. (15) Okon, E.; Pouliquen, D.; Okon, P.; Kovaleva, Z. V.; Stepanova, T. P.; Lavit, S. G.; Kudryavtsev, B. N.; Jallet, P. Lab. InVest. 1994, 91, 895. (16) Chatterjee, J.; Haik, Y.; Chen, C.-J. J. Magn. Magn. Mater. 2001, 225, 21. (17) Arias, J. L.; Gallardo, V.; Go´mez-Lopera, S. A.; Plaza, R. C.; Delgado, A. V. J. Controlled Release 2001, 77, 309. (18) Arias, J. L.; Gallardo, V.; Linares-Molinero, F.; Delgado, A. V. J. Colloid Interface Sci. 2006, 299, 599. (19) Arias, J. L.; Lo´pez-Viota, M.; Ruiz, M. A.; Lo´pez-Viota, J.; Delgado, A. V. Int. J. Pharm. 2007, 339, 237. (20) Jia, Z.; Yujun, W.; Yangcheng, L.; Jingyu, M.; Guangsheng, L. React. Funct. Polym. 2006, 66, 1552. (21) Ibrahim, A.; Couvreur, P.; Roland, M.; Speiser, P. J. Pharm. Pharmacol. 1983, 35, 59. (22) Couvreur, P.; Stella, B.; Cattel, L.; Rocco, F.; Renoir, J.-M.; Rosilio, V. French Patent No. WO/2006/090029, 2006. (23) Couvreur, P.; Stella, B.; Reddy, L. H.; Hillaireau, H.; Dubernet, C.; Desmae¨le, D.; Lepeˆtre-Mouelhi, S.; Rocco, F.; Dereuddre-Bosquet, N.; Clayette, P.; Rosilio, V.; Marsaud, V.; Renoir, J.-M.; Cattel, L. Nano Lett. 2006, 6, 2544. (24) Couvreur, P.; Reddy, L. H.; Mangenot, S.; Poupaert, J. H.; Desmae¨le, D.; Lepeˆtre-Mouelhi, S.; Pili, B.; Bourgaux, C.; Amenitsch, H.; Ollivon, M. Small 2008, 4, 247. (25) Reddy, L. H.; Dubernet, C.; Lepetre Mouelhi, S.; Marque, P. E.; Desmaele, D.; Couvreur, P. J. Controlled Release 2007, 124, 20.
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consideration that efficient delivery of SQdFdC nanoparticles to solid tumors could probably be achieved by employing a magnetic targeting strategy, we describe here the preparation and characterization of core/shell nanoparticles consisting of a magnetite nucleus and a SQdFdC shell. The coating efficiency of SQdFdC around the magnetic core has been qualitatively and quantitatively analyzed using electron microscopy, X-ray diffractometry, infrared absorption spectroscopy, and electrical, thermodynamic, and chemical surface characterization. We also identified the key factors for the squalenoyl adsorption to the magnetic cores to be efficient. Finally, the magnetic properties of the core/shell nanoparticle suspensions were evaluated in order to analyze the magnetic responsiveness of this new drug delivery system.
Experimental Section Materials. All chemicals used were of analytical quality from Prolabo, France, except for gemcitabine hydrochloride (Sequoia Research Products Ltd., U.K.), and pluronic F-68, squalene, formamide, and dextrose (Sigma-Aldrich Chemical Co., France). Water used in the experiments was deionized and filtered (Milli-Q Academic, Millipore, France). Synthesis of Magnetite and SQdFdC. Colloidal magnetite was prepared following the chemical co-precipitation method proposed by Massart.26,27 Briefly, it consists of adding at room temperature under mechanical stirring (≈700 rpm), 40 mL of an aqueous solution of 1 mol/L FeCl3, and 10 mL of an aqueous solution of 2 mol/L FeCl2, both in 2 mol/L HCl, to 500 mL of a 0.7 mol/L aqueous ammonia solution. To obtain stable acidic sols, the magnetite particles produced were magnetically decanted and re-dispersed in a 2 mol/L perchloric acid solution. After 12 h, the particles were again decanted with the help of a permanent magnet and re-dispersed in ethanol, as they easily undergo oxidation to maghemite if kept in water.28 Cleaning was achieved by repeated magnetic separation and redispersion in ethanol, until the supernatant was transparent and its conductivity indicated that the suspensions were clean of both unreacted chemicals and nonmagnetic particles (
