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Development of Nonsurfactant Cyclodextrin Nanoparticles Loaded With Anticancer Drug Paclitaxel
Development of Nonsurfactant Cyclodextrin Nanoparticles Loaded With Anticancer Drug Paclitaxel ¨ RKAYNAK,1 MEVLUT ERTAN,2 MURAT S¸EN,3 A. ATILLA HINCAL1 EREM BILENSOY,1 OYA GU 1
Faculty of Pharmacy, Department of Pharmaceutical Technology, Hacettepe University, 06100 Sihhiye, Ankara, Turkey
2
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Hacettepe University, 06100 Sihhiye, Ankara, Turkey
3
Department of Polymer Chemistry, Division of Chemistry, Hacettepe University, 06532 Beytepe, Ankara, Turkey
Received 5 December 2006; revised 2 April 2007; accepted 23 May 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21111
ABSTRACT: In the current formulation of clinical use paclitaxel (PCX) is associated with solubilizers that may produce severe side effects. In this study, PCX was complexed to an amphiphilic cyclodextrin (CD), 6-O-CAPRO-b-CD, capable of forming nanoparticles spontaneously in order to mask its physicochemical properties via the formation of inclusion complexes of the drug with amphiphilic CD before the nanoparticle is formed. Complexes have been characterized with various techniques such as 1H NMR, Fourier Transform Infrared (FTIR), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM) confirming the formation of inclusion complex between PCX and 6-O-CAPRO-b-CD. Nanospheres and nanocapsules were prepared directly from the preformed PCX/6-O-CAPRO-b-CD inclusion complex by the nanoprecipitation technique, showing a size from 150 to 250 nm for nanospheres and from 500 to 500 nm for nanocapsules. Zeta potentials of the nanospheres and nanocapsules indicate stable colloidal dispersions within the range of 18 to 39 mV. A 12-month physical stability was demonstrated for blank nanoparticles. PCX encapsulation was high with three-fold increase in loading when nanoparticles are prepared directly from preformed inclusion complexes of the drug with 6-O-CAPRO-b-CD. In vitro liberation profiles of PCX from CD nanoparticles show a prolonged release profile for this drug up to 12 h for nanospheres and 24 h for nanocapsules. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:1519–1529, 2008
Keywords: cyclodextrins; nanospheres; nanocapsules; complexation; NMR; thermal analysis; controlled release; physical stability; FTIR; particle sizing
INTRODUCTION Paclitaxel (PCX) is an antitumor agent with a unique mechanism of action promoting the assembly of microtubules from tubule dimers and prevents them from depolarizing.1–3 This leads to the loss of normal microtubule dynamics
Correspondence to: Erem Bilensoy (Telephone: 90-312-31015-24; Fax: 90-312-305-43-69; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 1519–1529 (2008) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
necessary for cell division and other vital processes and consequently causes cell death. The ability of this drug to stabilize microtubules makes it significantly effective against various types of solid tumors including breast cancer, advanced ovarian carcinoma, lung cancer, head and neck carcinomas, and acute leukemias.4,5 However, the clinical application and the bioavailability of PCX is mainly limited by its narrow therapeutic index and very poor solubility in water (0.5 mg/L) and other pharmaceutically acceptable solvents. The current formulation of injectable PCX (Taxol1) consists of Cremophor EL
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and dehydrated alcohol at 50:50 v/v ratio. This vehicle has been demonstrated to cause severe hypersensitivity reactions upon administration and has shown incompatibility with common PVC intravenous infusion sets.6–10 To eliminate Cremophor EL from PCX injectable formulations, alternative delivery systems have been suggested including parenteral emulsions,11,12 liposomes,13,14 nanoparticles of different polymeric structures,15–20 water-soluble drugs and conjugates,21–23 and inclusion complexes with CDs.24–28 Recently Abraxane1, a Cremophor-free, albuminbound nanoparticle PCX of mean diameter of 130 nm has been approved by the FDA for the therapy of recurrent metastatic breast cancer that does not respond to combination therapy. Albumin has been used to formulate PCX in nanoparticulate form for cancer therapy.29,30 In fact, administration of poorly soluble anticancer drugs in nanoparticulate carrier systems seems to be advantageous due to reduced size of these delivery systems (<400 nm) which help escape from reticuloendothelial system RES uptake and facilitate the extravasation through the leaky vasculature typical of the tumor site as well as the avoidance of Cremophor as the solubilizer. This is believed to reduce side effects and increase therapeutic efficacy while altering the tissue distribution and the pharmacokinetics of the anticancer drug.31,32 Furthermore it has been demonstrated that nanoparticles may overcome MDR type P-gp increasing the drug content inside the neoplastic cells.33 This may also help increase the therapeutic efficacy of PCX since an acquired resistance to the drug has already been reported.34 Amphiphilic cyclodextrins (CDs) are obtained by the chemical per-modification of natural CDs (b-CD or g-CD) by the selective substitution of aliphatic chains of varying length (2C to 18C), structure (linear or branched) linked with varying bonds (ester, ether, amide, thio, fluoro) of high purity.35–37 These CD derivatives were demonstrated to yield nanospheres or nanocapsules spontaneously using the nanoprecipitation technique with or without the presence of surfactants.36 Some amphiphilic b-CD derivatives such as b-CDC6 modified on the secondary face with 6C aliphatic esters and 6-N-CAPRO-b-CD modified on the primary face with a 6C aliphatic amide were demonstrated to give stable nanoparticles of high drug loading capacity and reduction of burst effect during the drug release process when nanoparticles are prepared directly JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008
from preformed drug/amphiphilic CD inclusion complex.38–40 The objective of this study was to combine for the first time two different alternative approaches to PCX formulation which are complexation to CDs and incorporation of PCX into nanoparticulate carrier systems. For this reason, a new amphiphilic CD derivative, 6-O-CAPRO-b-CD, modified on the primary face with 6C aliphatic esters suggesting biodegradability, was synthesized to form inclusion complexes with PCX. Nanoparticles were prepared directly from the preformed inclusion complex in order to enhance the therapeutic concentration of the drug while avoiding the use of Cremophor, to improve the physical stability of PCX, to achieve high encapsulation efficiency, and controlled release profile.
EXPERIMENTAL SECTION Materials Reagents and chemicals were used as received in the synthesis of 6-O-CAPRO-b-CD. b-CD 100% pure according to nuclear magnetic resonance spectroscopy was extensively dried under vacuum over phosphorous pentoxide before use. PCX seen in Figure 1a was a kind gift of Zhejiang Pharmaceuticals, Hangzhou, China (EA648838320CN). Cremophor1EL used to solubilize PCX was purchased from BioChemika/Fluka, Buchs, Belgium. Miglyol 812 used as the oil phase in nanocapsule preparation was purchased from Condea Chimie, Witten, Germany. All other reagents were of HPLC grade and were used without further purification. Ultrapure water MilliQ (Millipore Simplicity 185, Molsheim, France) was used in the preparation of nanoparticles.
Synthesis and Analytical Characterization of 6-O-CAPRO-b-CD Amphiphilic CD derivative, 6-O-CAPRO-b-CD (Fig. 1b) was synthesized according to the previously described technique.35 Briefly, 6-O-CAPRO-bCD was obtained by iodination of the primary hydroxyl groups and reaction with cesium hexanoate which proved to be a good nucleophile35 in DMF resulting in the per-modification of primary hydroxyls with 6C aliphatic esters. Final product of 1.9 g (92%) white powder was characterized by DOI 10.1002/jps
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molar ratios of drug: CD were studied. In this technique, PCX and 6-O-CAPRO-b-CD were dissolved in 20 mL of absolute alcohol in fixed amounts and this solution was poured into 40 mL of distilled water in a round-bottomed flask. The suspension was stirred for equilibration at room temperature for 7 days. Organic solvent was then evaporated under vacuum and the resulting suspension was lyophilized to obtain the inclusion complex in dry powder form.
Characterization of PCX/6-O-CAPRO-b-CD Inclusion Complexes
Figure 1. Schematic representation of (a) PCX and (b) 6-O-CAPRO-b-CD.
elemental analysis, 1H NMR spectrometry, Fourier Transform Infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC) analysis, and melting point determination to confirm the product’s purity, thermal characteristics, and structure to be in accordance with the previously obtained results.35
Preparation of PCX/6-O-CAPRO-b-CD Inclusion Complexes Colyophilization technique was used to prepare inclusion complexes of the drug with amphiphilic CD derivative 6-O-CAPRO-b-CD. 1:1 and 1:2 DOI 10.1002/jps
DSC was used to determine the thermal behavior of PCX, 6-O-CAPRO-b-CD, and the 1:1 and 1:2 M complexes. DSC analysis was performed with a Dupont DSC912 (Wilmington, DE) instrument within the temperature range of 25-2508C under nitrogen atmosphere. Samples weighing approximately 3 mg were heated in hermetically sealed aluminum pan at a rate of 108C/min. Fourier Transform Infrared (FTIR) Spectroscopy was performed with a Nicolet 520 FTIR spectrophotometer (Waltham, MA), using discs of each sample and previously prepared KBr containing 0.01 g of sample and 0.1 g KBr between wavelengths 40 and 4000 cm1. 1H NMR spectra at 400 MHz were taken in CDCl3 and DMSO with a Varian Mercury 400 MHz. Chemical shifts are given to external tetramethylsilane at 0 ppm with calibration using solvent signals. Scanning electron microscopy (SEM) was used to determine the crystalline structure of PCX, 6-O-CAPRO-b-CD, and their complexes with a JEOL SEM ASID-10 instrument, Tokyo, Japan. Samples were fixed on metal plates and sputtered with gold–palladium mixture at a thickness of ˚ and observed at an accelerated voltage of 100 A 80 kV.
Preparation of Blank and PCX-Loaded Nanoparticles Nanospheres were prepared according to the nanoprecipitation technique introduced by Fessi et al.41 According to this technique, an organic phase consisting of 6-O-CAPRO-b-CD (1 mg) dissolved in absolute alcohol (1 mL) was added with an Eppendorf injector at room temperature under constant stirring to an aqueous phase consisting of only Ultrapure Water (MilliQ) JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008
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(2 mL). Nanospheres formed spontaneously. This dispersion was stirred for equilibrium for 30 min followed by evaporation of organic solvent under vacuum to the desired volume (2 mL). To obtain nanocapsules, the same technique is used with the exception of an oil phase of Miglyol 812 (50 mL) added to the organic phase. Drug-loaded nanoparticles were prepared with either the conventional loading (CL) technique and the previously reported high-loading technique (HL) based on the preparation of nanoparticles from preformed inclusion complexes.38,39 The loading techniques can be described briefly as follows: Conventional loading (CL): Nanoparticles are loaded by adding fixed amount of ethanolic PCX solution (200 mg) to the organic phase during preparation. Preloading technique (PL): Nanoparticles are prepared by dissolving only a fixed amount (1 mg) of PCX/6-O-CAPRO-b-CD complex in the organic phase for the preparation. High-loading technique (HL): Nanoparticles are prepared from fixed amount of PCX/6-OCAPRO-b-CD complex (1 mg) with an overloading of ethanolic PCX solution (200 mg) in the organic phase during preparation.
long-term physical stability of the nanoparticles. Entrapment efficiency of PCX to amphiphilic CD nanoparticles was determined by the following technique; free undissolved drug in the nanoparticle dispersion was centrifuged at 5000 rpm and the resulting supernatant was separated and lyophilized (HETO Lyolab Freeze Dryer, Allerod, Denmark). The dry powder consisting of nanoparticles and nanoparticle-bound PCX was then dissolved in acetonitrile: water (70:30 v/v) and PCX quantity was assayed with an HPLC technique using HP 1100 Agilent Series instrument with a mobile phase of acetonitrile:water (70:30), flow rate of 1 mL/min, injection volume of 50 mL at 227 nm with a diode array detector using a reverse phase column C-18 octadecylsilane (ODS) (Hichrom, Berkshire 5 C18 column, 250 4.6 mm, UK) giving a retention time of 4.2 min for PCX. The HPLC technique was analytically validated (linearity r2: 0.9999, repeatability CV%: 0.4%, reproducibility CV%: 1.1%, LOD: 0.028 mg/ mL, LOQ: 0.095 mg/mL). Drug loading data were expressed in terms of entrapped drug quantity, entrapment efficiency, and associated drug %. Entrapped drug quantity is the drug quantity determined in nanoparticle sample (mg/mL). Entrapment efficiency and associated drug % values were calculated according to the following formula: Entrapment efficiency
Characterization of Blank and PCX-Loaded Nanoparticles Particle size distributions (mean diameter (nm), polydispersity index) of nanoparticles were determined by quasi-elastic light scattering using Malvern Zetasizer, Malvern Instruments, Worcestershire, UK. Measurements were realized in triplicate at a 908 angle at 258C. Zeta potential values were taken using the Malvern Zetasizer, Malvern Instruments, also in triplicate at 1208 angle and 258C with suitable dilution of samples with deionized water. Atomic force microscopy (AFM) was used to image the PCX-loaded nanoparticle dispersions with a Q-Scope1350 Multimode Atomic force Microscope, Ambios & Quasant, Santa Cruz, CA. Physical stability of blank CD nanoparticles was determined through a 12-month followup period during which the samples were stored as aqueous nanoparticle dispersions at 88C. Repeated measurements of particle size and zeta potential were performed in order to evaluate the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008
¼
Entrapped drug quantity ðmmolÞ 100 Initial CD amount ðmmolÞ
Associated drug% ¼
Entrapped drug quantity ðmgÞ 100 Initial drug amount ðmgÞ
In vitro PCX release profiles from 6-O-CAPROb-CD nanospheres and nanocapsules were determined in capped Eppendorf tubes in a 20 mL of medium of isotonic phosphate buffer saline pH 7.4 containing 0.1% polysorbate 80 for sink conditions at 378C in a thermostated shaker bath (Memmert, Schwabach, Germany) at 80 rpm. At given time intervals 0.5 mL of sample was taken from the release medium and replaced with fresh medium at 378C. Samples were then centrifuged at 13500 rpm for 15 min to precipitate the nanoparticles and the clear supernatant was freeze dried. Resulting dry powder was dissolved in 0.5 mL of mobile phase and assayed for PCX content. DOI 10.1002/jps
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RESULTS AND DISCUSSION Characterization of 6-O-CAPRO-b-CD 6-O-CAPRO-b-CD, amphiphilic CD derivative modified on the primary face with 6C aliphatic ester was characterized after synthesis by multiple techniques including 1H NMR spectroscopy, FTIR spectroscopy, DSC, and elemental analysis. 1 H NMR data for 6-O-CAPRO-b-CD are as follows; 1 H NMR (dH, 400 MHz, DMSO-d6): 7.99 (DMF, H–C¼O), 6.04 (dd, –OH), 5.86 (d, –OH), 4.92 (d, H1), 4.32 (d, -H-6), 4.21 (dd, H-60 ), 3.86 (t, H-5), 3.66 (m, H-3), 3.37 (m, H-4), 3.4 (DMSO-H2O), 2.7, 2.9 (DMF, -N-(CH3)2), 2.54 (DMSO), 2.31 (m, CH2–CO–), 1.52 (m, CH2–CH2–CO–), 1.28 (m, –(CH2)-2), 0.88 (t, CH3). FTIR spectrum (data not shown) of 6-OCAPRO-b-CD gave the following typical bands confirming the ester structure of this derivative; O–H stretching vibrations at 3700–3100 cm1, aliphatic C–H stretching vibrations at 3000– 2850 cm1, C – O stretching bands characteristic of the ester group at 1800–1700 cm1. DSC thermogram taken between 25 and 2508C shows a distinct and sharp melting endotherm for 6-O-CAPRO-b-CD at 2298C (data not shown). Table 1 shows the elemental analysis data for the newly synthesized 6-O-CAPRO-b-CD proving the purity of the final product.
Characterization of PCX/6-O-CAPRO-b-CD Inclusion Complexes The interaction at the solid state and in solution between PCX and CD cavity was evaluated by different techniques such as DSC analysis, FTIR spectroscopy, 1H NMR spectrometry, SEM imaging. Figure 2 displays the DSC thermograms of lyophilized PCX, lyophilized 6-O-CAPRO-b-CD, 1:1 and 1:2 molar ratio complexes of the drug, and CD. As seen from the DSC thermograms, melting endotherm of 6-O-CAPRO-b-CD at 2298C and melting and decomposition endotherms and dehydration at 1008C of PCX at 224 and 2328C have disappeared or broadened suggesting
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the absence of free crystalline drug within the colyophilizate. FTIR spectra for PCX, 6-O-CAPROb-CD, and their 1:1 and 1:2 complexes are seen in Figure 3 indicating a reduction in the O–H stretching vibration band at 3300 cm1. The peaks about 1500 and 1200 cm1 relative to aromatic ring and C–O–C stretching respectively show significant changes. The peak about 1700 cm1 corresponding to aryl and saturated ketone groups also indicates a possible interaction through the aromatic rings of the drug molecule seen in Figure 1a. These data indicate that there exists a chemical interaction between PCX and 6-OCAPRO-b-CD which is most probably indicative of a partial inclusion of the drug within the amphiphilic CD cavity. This interaction is more pronounced for the 1:2 molar ratio complex as seen in Figure 3. 1 H NMR spectra of PCX, 6-O-CAPRO-b-CD, and 1:1 and 1.2 molar ratio complexes were taken at 400 MHz and shifts in the signals of H-3 and H-5 along with other protons of amphiphilic CD were evaluated in terms of a possible inclusion in CD cavity which is expected to cause negative shifts42 in the internal protons (H-3 and H-5) of the CD cavity. Table 2 represents the chemical shift values of 6-O-CAPRO-b-CD protons before and after complexation to PCX. It can be observed that internal protons H-3 and H-5 display shifts of 0.02 to 0.03 ppm for 1:1 and 1:2 molar ratio complexes. More interestingly, H-6 and H-60 which are the protons of the aliphatic ester grafted to the primary hydroxyl neighboring the CD glucopyranose unit has also shifted significantly as seen from Table 2. This suggests that PCX molecule has entered the CD cavity and also interacted with the aliphatic chains grafted to the primary face. Thompson et al.43 has proposed that leaving the secondary face which is the wide side of the cavity unsubstituted may facilitate the entrance of hydrophobic drug molecule to the cavity. Modification of the primary face with long aliphatic chains contributes to the complexation ability of the CD derivative by providing a second hydrophobic zone to interact with after the CD cavity. SEM photomicrographs taken for PCX, 6-OCAPRO-b-CD, 1:1 and 1:2 molar ratio complexes
Table 1. Elemental Analysis Data of Amphiphilic CD, 6-O-CAPRO-b-CD
Amphiphilic CD 6-O-CAPRO-b-CD
DOI 10.1002/jps
Ampirical Formula
Molecular Weight (g/mol)
Theoretical Percentage
Practical Percentage
C84H140O42
1820
(C%) 55.3 (H%) 7.7
(C%) 50.2 (H%) 7.3
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Table 2. Chemical Shift Values (ppm) of 6-O-CAPROb-CD Protons before and after Complexation to PCX in 1:1 and 1:2 Molar Ratio Protons
dCDa
d1:1b
d1:2b
~d1:1c
~d1:2d
H-1 H-3 H-4 H-5 H-6 H-60
4.92 3.66 3.37 3.86 4.32 4.21
(—)e 3.64 3.36 3.83 4.31 4.16
(—) 3.64 3.37 3.84 4.31 4.16
(–) 0.02 0.01 0.03 0.01 0.05
(—) 0.02 0 0.02 0.01 0.05
a
Free state. Complexed state. ~d1:1 ¼ dCD d1:1. d ~d1:1 ¼ dCD d1:2. e Peaks could not be separated since H-1 proton of 6-OCAPRO-b-CD and H-5 proton at the taxane core of PCX have given resonance at the same ppm value. b c
Figure 2. DSC thermograms of PCX, 6-O-CAPRO-bCD, 1:1 and 1:2 molar ratio complexes of PCX/6-OCAPRO-b-CD.
Figure 3. FTIR spectra of PCX, 6-O-CAPRO-b-CD, and 1:1 and 1:2 molar ratio complexes. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008
DOI 10.1002/jps
Figure 4. SEM photomicrographs of (a) PCX (b) 6-O-CAPRO-b-CD, (c) 1:1 complex, and (d) 1:2 complex. Table 3. Particle Size and Zeta Potential Values for Blank and PCX-Loaded Amphiphilic CD Nanoparticles Formulation Blank nanosphere Blank nanocapsule CL nanosphere PL nanosphere HL nanosphere CL nanocapsule PL nanocapsule HL nanocapsule DOI 10.1002/jps
Particle Size (Mean Diameter, nm, SD)
Zeta Potential (mV)
157 5 587 36 219 32 232 5 263 24 606 37 450 31 639 37
13.8 2 14.7 4 19.6 1 18.8 2 34.6 5 32.5 3 37.1 2 33.0 5
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indicate that the needle-like crystalline structure typical of PCX does not exist in the complexes which both show an amorphous state. Figure 4a–d represents the SEM photomicrographs of the drug, CD, and the different complexes. This change in the crystalline form could be a result of the colyophilization process which is very similar to the nanoprecipitation process used to prepare the nanoparticles.44 Characterization of Blank and PCX-Loaded Nanoparticles Particle size and zeta potential values of blank and PCX-loaded 6-O-CAPRO-b-CD nanoparticles prepared with different loading techniques are seen in Table 3. The data suggest that loaded nanoparticles are slightly larger than blank nanoparticles probably due to the adsorption of the drug on the particle surface. Nanocapsules are limited to the oil droplet size45 that form during the preparation so they are considerably larger than nanospheres but still in the appropriate nm range for injectable use. Nanospheres in particular are around 150–250 nm which suggests favorable properties for injectable systems, possibility to escape from RES uptake, and leaking from the vasculature surrounding the tumor site resulting in an eventual passive targeting of the system.46 The sizes of nanospheres were believed to be slightly affected by the preparation technique being preloading, CL or high-loading due to the different amounts of drug entrapment. Zeta potentials of the blank nanoparticles are around 19 mV with a slight increase up to 37 mV for loaded formulation. It is believed that this increase in zeta potential is due to the charge of the adsorbed PCX on the particle surface. In fact, zeta potential of PCX dispersion in buffer PBS was determined to be 30 mV confirming this theory. Long-term physical stability of the blank nanoparticles was measured by repeated particle size and zeta potential measurements. The data are presented in Figures 5 and 6 for particle size and zeta potential measurements, respectively. The data indicate the 12-month physical stability of 6-O-CAPRO-b-CD nanospheres and nanocapsules in aqueous dispersion form upon storage at 88C. Changes in the particle size and zeta potential during the 12-month follow-up period are not statistically significant ( p < 0.05). Drug loading values of PCX are seen in Table 4 in terms of entrapped drug quantity, entrapment JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008
Figure 5. Time-dependent particle size of 6-OCAPRO-b-CD nanospheres and nanocapsules in aqueous dispersion.
efficiency, and associated drug percentage. It is seen that loading techniques increase drug loading values significantly up to threefold for highloaded nanoparticles. It is also observed that nanocapsules entrap almost twofold PCX when compared to nanospheres. This may be a result of the affinity of this very poorly soluble drug to the oily core of the nanocapsules. It has been reported that drug physicochemical properties including octanol: water partition coefficient, k1:1 affinity constant for CD, and aqueous solubility affect loading characteristics significantly.47 In accordance with this fact, PCX with its very poor water solubility, high partition coefficient, and demonstrated affinity to CDs show a high encapsulation efficiency to CD nanoparticles. In vitro release of PCX was assessed from nanospheres and nanocapsules into isotonic phosphate buffer pH 7.4 containing 0.1%
Figure 6. Time-dependent zeta potential values of 6-O-CAPRO-b-CD nanospheres and nanocapsules in aqueous dispersion. DOI 10.1002/jps
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Table 4. Drug Loading Values for PCX for 6-O-CAPRO-b-CD Nanoparticles Prepared With Different Loading Techniques
Dosage Form
Entrapped Drug Quantity (mg) SD
Associated Drug Percentage (%, a/a)
Entrapment Efficiency (%) (mmol Drug/mmol CD) 100
Nanosphere Nanocapsule Nanosphere Nanocapsule Nanosphere Nanocapsule
67.4 0.7 120.7 0.3 65.4 0.6 117.2 0.5 143.5 0.9 186.4 0.9
33.7 60.4 32.7 58.6 36.8 47.8
24 43 24.6 44.2 53 68
polysorbate 80. Figure 7 displays the in vitro release profiles of conventionally loaded and highloaded nanospheres and nanocapsules. It can be seen that nanocapsules liberate the drug in a considerably slower release profile. Another factor affecting release seems to be the loading technique with CL resulting in a burst effect. PCX is released in a period of 12 h with complete release achieved in 24 h for nanocapsules. PCX’s burst effect is observed in the first 15 min for conventionally loaded formulations. However, high-loaded formulations displayed biphasic release profile with complete PCX release achieved around 12 h.
CONCLUSION
Loading Technique CL PL HL
In the light of the data obtained in this study, it can be concluded that amphiphilic CD nanoparticles may provide a promising alternative for nonsurfactant, Cremophor-free, nanoscale carrier for injectable use of PCX. Further studies should focus on the safety and efficacy of this delivery system to ensure an improvement in the therapeutic efficacy and clinical application of this potent anticancer agent.
ACKNOWLEDGMENTS Authors thank the TUBITAK SBAG CNRS-3 project and Hacettepe University Research Fund Project NO. 0202301005 for their financial support to this study.
REFERENCES
Figure 7. In vitro release profiles for PCX from amphiphilic CD nanospheres and nanocapsules (n ¼ 3, SD) (PCX HL NS, High-loaded nanospheres; PCX CL NS, conventionally loaded nanospheres; PCX HL NC, high-loaded nanocapsules; PCX CL NC, conventionally loaded nanocapsules). DOI 10.1002/jps
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7. Dorr RT. 1994. Pharmacology and toxicology of Cremophor EL diluent. Ann Pharmacother 28: S11–S14. 8. Weiss RB, Donehower RC, Wiernik PH. 1990. Hypersensitivity reactions from taxol. J Clin Oncol 8:1263–1268. 9. Waugh W, Trissel LA, Stella VJ. 1991. Stability, compatibility and plasticizer extraction of taxol (NSC-125973) injection diluted in infusion solutions and stored in various containers. Am J Hosp Pharm 48:1520–1524. 10. Gelderblom H, Verweij J, Nooter K, Sparreboom A. 2001. Cremophor EL: The drawbacks and advantages of vehicle selection for drug formulation. Eur J Cancer 37:1590–1598. 11. Lundberg B. 1997. A submicron lipid emulsion coated with amphipathic polyethylene glycol for parenteral administration of paclitaxel (taxol). J Pharm Pharmacol 49:16–21. 12. Kan P, Chen ZB, Lee CJ, Chu LM. 1999. Development of nonionic surfactant/phospholipids o/w emulsion as a paclitaxel delivery system. J Control Release 58:271–278. 13. Sharma A, Straubinger RM. 1994. Novel taxol formulations: Preparation and characterization of taxol containing liposomes. Pharm Res 11:889–896. 14. Campbel RB, Balasubramanian SV, Straubinger RM. 2001. Influence of cationic lipids on the stability and membrane properties of paclitaxel-containing liposomes. J Pharm Sci 90:1091–1105. 15. Fonseca C, Simoes S, Gaspar R. 2002. Paclitaxelloaded PLGA nanoparticles, preparation, physicochemical characterization and in vitro antitumoral activity. J Control Release 83:273–286. 16. Kim SY, Lee YM, Baik DJ, Kang JS. 2003. Toxic characteristics of methoxy polyethyleneglycol/poly epsilon caprolactone nanospheres: In vitro and in vivo studies in the normal mice. Biomaterials 24: 55–63. 17. Mitra A, Lin S. 2003. Effect of surfactant on fabrication and characterization of paclitaxelloaded polybutylcyanoacrylate nanoparticulate delivery systems. J Pharm Pharmacol 55:895– 902. 18. Mu L, Feng SS. 2003. A novel controlled release formulation for the anticancer drug paclitaxel (taxol): PLGA nanoparticles containing Vitamin A TPGS. J Control Release 86:33–48. 19. Potineni A, Lynn DM, Langer R, Amiji M. 2003. Polyethylene oxide modified poly beta-amino ester nanoparticles as a pH-sensitive biodegradable system for paclitaxel delivery. J Control Release 86: 223–234. 20. Suh H, Jeong B, Rathi R, Kim SW. 1998. Regulation of smooth muscle cell proliferation using paclitaxel-loaded poly(ethylene oxide)-poly(lactide/glycolide) nanospheres. J Biomed Mater Res 42:331–338.
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21. Pendri P, Conover CD, Greenwald RB. 1998. Antitumor activity of paclitaxel-2(-glycinate conjugated to polyethylene glycol: A water soluble drug. Anticancer Drug Des 13:387–395. 22. Rodrigues M, Carter P, Wirth C, Mullins S, Lee A, Blackburn BK. 1995. Synthesis and b-lactamase mediated activation of cephalosporin-taxol prodrug. Chem Biol 2:223–227. 23. Safovy A, Bonner JA, Waksal HW, Buchsbaum DJ, Gillespie GY, Khazaeli MB, Raisch KP. 2003. Synthesis and biological evaluation of paclitaxelC225 conjugate as a model for targeted drug drug delivery. Bioconjug Chem 14:302–310. 24. Sharma US, Balasubramanian SV, Straubinger RM. 1995. Pharmaceutical and physical properties of paclitaxel (taxol) complexes with cyclodextrins. J Pharm Sci 84:1223–1230. 25. Cserhati T, Hollo J. 1994. Interaction of taxol and other anticancer drugs with hydroxypropyl-b-cyclodextrin. Int J Pharm 108:69–75. 26. Cserhati T, Forgacs E, Hollo J. 1995. Interaciton of taxol and other anticancer drugs with a-cyclodextrin. J Pharm Biomed Anal 13:533–541. 27. Dordunoo SK, Burt HM. 1996. Solubility and stability of taxol: Effects of buffers and cyclodextrins. Int J Pharm 133:191–201. 28. Alcaro S, Ventura CA, Paolino D, Battaglia D, Ortuso F, Cattel L, Puglisi G, Fresta M. 2002. Preparation, characterization, molecular modeling and in vitro activity of paclitaxel-cyclodextrin complexes. Bioorg Med Chem Lett 12:1637–1641. 29. Sparreboom A, Scripture CD, Trieu V, Williams PJ, De T, Yang A, Beals B, Figg WD, Hawkins M, Desai N. 2005. Comparative, preclinical and clinical pharmacokinetics of a cremophor-free, nanoparticle albumin-bound paclitacel (ABI-007) and paclitaxel formulated in cremophor (taxol). Clin Cancer Res 11:4136–4143. 30. Micha JP, Goldstein BH, Birk L, Rottenmeier MA, Brown JV, III. 2006. Abraxane in the treatment of ovarian cancer: The absence of hypersensivity reactions. Gynecol Oncol 100:437–438. 31. Couvreur P, Kante B, Lenaerts V, Scailteur V, Roland M, Speiser P. 1980. Tissue distribution of antitumor drugs associated with polyalkylcyanoacrylate nanoparticles. J Pharm Sci 69:199–202. 32. Rolland A. 1989. Clinical pharmacokinetics of doxorubicin in hepatoma patients after a single intravenous injection of free and nanoparticle-bound anthracyclin. Int J Pharm 54:113–121. 33. Bennis S, Chapey C, Couvreur P, Robert J. 1994. Enhanced cytotoxicity of doxorubicin encapsulated in polyisohexylcyanoacrylate nanospheres against multidrug resistant cell culture Eur J Cancer 30A: 89–93. 34. Rowinsky E, Donehower RD. 1995. Drug therapy: Paclitaxel-review article. N Engl J Med 332:1004– 1014.
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35. Memisoglu E, Bochot A, S¸en M, Charon D, Duchene D, Hıncal AA. 2002. Amphiphilic b-cyclodextrins modified on the primary face: Synthesis, characterization and evaluation of their potential as novel excipients in the preparation of nanocapsules. J Pharm Sci 91:1214–1224. 36. Memisoglu-Bilensoy E, Trichard L, Bochot A, Duchene D, Hıncal AA. 2006. Amphiphilic cyclodextrins and microencapsulation. In: Benita S, editor. Microencapsulation—methods and industrial applications, 2nd edition, New York: Taylor & Francis Ltd. 37. Duchene D, Wouessidjewe D, Ponchel G. 1999. Cyclodextrins and carrier systems. J Control Release 62:263–268. 38. Memisoglu E, Bochot A, S¸en M, Duchene D, Hıncal AA. 2003. Non-surfactant nanospheres of progesterone inclusion complexes with amphiphilic b-cyclodextrins. Int J Pharm 251:143–153. 39. Memisoglu E, Bochot A, S¸en M, Duchene D, Hıncal AA. 2003. Direct formation of nanospheres from amphiphilic b-cyclodextrin inclusion complexes. Pharm Res 20:117–125. 40. Memisoglu-Bilensoy E, Vural I, Bochot A, Renoir JM, Duchene D, Hıncal AA. 2005. Tamoxifen citrate loaded amphiphilic cyclodextrin nanoparticles: In vitro characterization and cytotoxicity. J Control Release 104:489–496.
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41. Fessi H, Devissaguet JP, Puisieux F, Thies C. 1992. Process for the preparation of dispersible colloidal systems of a substance in the form of nanoparticles. US Patent No 5,118,528. 42. Hedges A, Shieh J. 1991. Complexation in modified cyclodextrins compared with unmodified cyclodextrins. In: Duchene D, editor. New trends in cyclodextrins and derivatives. Paris: Editions de Sant. 43. Thompson DO. 1997. Cyclodextrins as enabling excipients: Their present and future use in pharmaceuticals. CRC Crit Ther Drug Carrier Syst 14: 1–104. 44. Kawashima Y, Okumura M, Takenaka H. 1982. Spherical crystallization: Direct spherical agglomerization of salicylic acid crystals during crystallization. Science 216:1127–1128. 45. Legrand P, Barratt G, Mosqueira V, Fessi H, Devissaguet JP. 1999. Polymeric nanocapsules as drug delivery systems: A review. STP Pharma Sci 9: 411–418. 46. Brannon-Peppas L, Blanchette JO. 2004. Nanoparticles and targeted systems for cancer therapy. Adv Drug Deliv Rev 56:1649–1659. 47. Memisoglu-Bilensoy E, S¸en M, Hıncal AA. 2006. Effect of drug physicochemical properties on in vitro characteristics of amphiphilic cyclodextrin nanospheres and nanocapsules. J Microencapsul 23:59–68.
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Faculty of Pharmacy, Department of Pharmaceutical Technology, Hacettepe University, 06100 Sihhiye, Ankara, Turkey
2
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Hacettepe University, 06100 Sihhiye, Ankara, Turkey
3
Department of Polymer Chemistry, Division of Chemistry, Hacettepe University, 06532 Beytepe, Ankara, Turkey
Received 5 December 2006; revised 2 April 2007; accepted 23 May 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21111
ABSTRACT: In the current formulation of clinical use paclitaxel (PCX) is associated with solubilizers that may produce severe side effects. In this study, PCX was complexed to an amphiphilic cyclodextrin (CD), 6-O-CAPRO-b-CD, capable of forming nanoparticles spontaneously in order to mask its physicochemical properties via the formation of inclusion complexes of the drug with amphiphilic CD before the nanoparticle is formed. Complexes have been characterized with various techniques such as 1H NMR, Fourier Transform Infrared (FTIR), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM) confirming the formation of inclusion complex between PCX and 6-O-CAPRO-b-CD. Nanospheres and nanocapsules were prepared directly from the preformed PCX/6-O-CAPRO-b-CD inclusion complex by the nanoprecipitation technique, showing a size from 150 to 250 nm for nanospheres and from 500 to 500 nm for nanocapsules. Zeta potentials of the nanospheres and nanocapsules indicate stable colloidal dispersions within the range of 18 to 39 mV. A 12-month physical stability was demonstrated for blank nanoparticles. PCX encapsulation was high with three-fold increase in loading when nanoparticles are prepared directly from preformed inclusion complexes of the drug with 6-O-CAPRO-b-CD. In vitro liberation profiles of PCX from CD nanoparticles show a prolonged release profile for this drug up to 12 h for nanospheres and 24 h for nanocapsules. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:1519–1529, 2008
Keywords: cyclodextrins; nanospheres; nanocapsules; complexation; NMR; thermal analysis; controlled release; physical stability; FTIR; particle sizing
INTRODUCTION Paclitaxel (PCX) is an antitumor agent with a unique mechanism of action promoting the assembly of microtubules from tubule dimers and prevents them from depolarizing.1–3 This leads to the loss of normal microtubule dynamics
Correspondence to: Erem Bilensoy (Telephone: 90-312-31015-24; Fax: 90-312-305-43-69; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 1519–1529 (2008) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
necessary for cell division and other vital processes and consequently causes cell death. The ability of this drug to stabilize microtubules makes it significantly effective against various types of solid tumors including breast cancer, advanced ovarian carcinoma, lung cancer, head and neck carcinomas, and acute leukemias.4,5 However, the clinical application and the bioavailability of PCX is mainly limited by its narrow therapeutic index and very poor solubility in water (0.5 mg/L) and other pharmaceutically acceptable solvents. The current formulation of injectable PCX (Taxol1) consists of Cremophor EL
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and dehydrated alcohol at 50:50 v/v ratio. This vehicle has been demonstrated to cause severe hypersensitivity reactions upon administration and has shown incompatibility with common PVC intravenous infusion sets.6–10 To eliminate Cremophor EL from PCX injectable formulations, alternative delivery systems have been suggested including parenteral emulsions,11,12 liposomes,13,14 nanoparticles of different polymeric structures,15–20 water-soluble drugs and conjugates,21–23 and inclusion complexes with CDs.24–28 Recently Abraxane1, a Cremophor-free, albuminbound nanoparticle PCX of mean diameter of 130 nm has been approved by the FDA for the therapy of recurrent metastatic breast cancer that does not respond to combination therapy. Albumin has been used to formulate PCX in nanoparticulate form for cancer therapy.29,30 In fact, administration of poorly soluble anticancer drugs in nanoparticulate carrier systems seems to be advantageous due to reduced size of these delivery systems (<400 nm) which help escape from reticuloendothelial system RES uptake and facilitate the extravasation through the leaky vasculature typical of the tumor site as well as the avoidance of Cremophor as the solubilizer. This is believed to reduce side effects and increase therapeutic efficacy while altering the tissue distribution and the pharmacokinetics of the anticancer drug.31,32 Furthermore it has been demonstrated that nanoparticles may overcome MDR type P-gp increasing the drug content inside the neoplastic cells.33 This may also help increase the therapeutic efficacy of PCX since an acquired resistance to the drug has already been reported.34 Amphiphilic cyclodextrins (CDs) are obtained by the chemical per-modification of natural CDs (b-CD or g-CD) by the selective substitution of aliphatic chains of varying length (2C to 18C), structure (linear or branched) linked with varying bonds (ester, ether, amide, thio, fluoro) of high purity.35–37 These CD derivatives were demonstrated to yield nanospheres or nanocapsules spontaneously using the nanoprecipitation technique with or without the presence of surfactants.36 Some amphiphilic b-CD derivatives such as b-CDC6 modified on the secondary face with 6C aliphatic esters and 6-N-CAPRO-b-CD modified on the primary face with a 6C aliphatic amide were demonstrated to give stable nanoparticles of high drug loading capacity and reduction of burst effect during the drug release process when nanoparticles are prepared directly JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008
from preformed drug/amphiphilic CD inclusion complex.38–40 The objective of this study was to combine for the first time two different alternative approaches to PCX formulation which are complexation to CDs and incorporation of PCX into nanoparticulate carrier systems. For this reason, a new amphiphilic CD derivative, 6-O-CAPRO-b-CD, modified on the primary face with 6C aliphatic esters suggesting biodegradability, was synthesized to form inclusion complexes with PCX. Nanoparticles were prepared directly from the preformed inclusion complex in order to enhance the therapeutic concentration of the drug while avoiding the use of Cremophor, to improve the physical stability of PCX, to achieve high encapsulation efficiency, and controlled release profile.
EXPERIMENTAL SECTION Materials Reagents and chemicals were used as received in the synthesis of 6-O-CAPRO-b-CD. b-CD 100% pure according to nuclear magnetic resonance spectroscopy was extensively dried under vacuum over phosphorous pentoxide before use. PCX seen in Figure 1a was a kind gift of Zhejiang Pharmaceuticals, Hangzhou, China (EA648838320CN). Cremophor1EL used to solubilize PCX was purchased from BioChemika/Fluka, Buchs, Belgium. Miglyol 812 used as the oil phase in nanocapsule preparation was purchased from Condea Chimie, Witten, Germany. All other reagents were of HPLC grade and were used without further purification. Ultrapure water MilliQ (Millipore Simplicity 185, Molsheim, France) was used in the preparation of nanoparticles.
Synthesis and Analytical Characterization of 6-O-CAPRO-b-CD Amphiphilic CD derivative, 6-O-CAPRO-b-CD (Fig. 1b) was synthesized according to the previously described technique.35 Briefly, 6-O-CAPRO-bCD was obtained by iodination of the primary hydroxyl groups and reaction with cesium hexanoate which proved to be a good nucleophile35 in DMF resulting in the per-modification of primary hydroxyls with 6C aliphatic esters. Final product of 1.9 g (92%) white powder was characterized by DOI 10.1002/jps
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molar ratios of drug: CD were studied. In this technique, PCX and 6-O-CAPRO-b-CD were dissolved in 20 mL of absolute alcohol in fixed amounts and this solution was poured into 40 mL of distilled water in a round-bottomed flask. The suspension was stirred for equilibration at room temperature for 7 days. Organic solvent was then evaporated under vacuum and the resulting suspension was lyophilized to obtain the inclusion complex in dry powder form.
Characterization of PCX/6-O-CAPRO-b-CD Inclusion Complexes
Figure 1. Schematic representation of (a) PCX and (b) 6-O-CAPRO-b-CD.
elemental analysis, 1H NMR spectrometry, Fourier Transform Infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC) analysis, and melting point determination to confirm the product’s purity, thermal characteristics, and structure to be in accordance with the previously obtained results.35
Preparation of PCX/6-O-CAPRO-b-CD Inclusion Complexes Colyophilization technique was used to prepare inclusion complexes of the drug with amphiphilic CD derivative 6-O-CAPRO-b-CD. 1:1 and 1:2 DOI 10.1002/jps
DSC was used to determine the thermal behavior of PCX, 6-O-CAPRO-b-CD, and the 1:1 and 1:2 M complexes. DSC analysis was performed with a Dupont DSC912 (Wilmington, DE) instrument within the temperature range of 25-2508C under nitrogen atmosphere. Samples weighing approximately 3 mg were heated in hermetically sealed aluminum pan at a rate of 108C/min. Fourier Transform Infrared (FTIR) Spectroscopy was performed with a Nicolet 520 FTIR spectrophotometer (Waltham, MA), using discs of each sample and previously prepared KBr containing 0.01 g of sample and 0.1 g KBr between wavelengths 40 and 4000 cm1. 1H NMR spectra at 400 MHz were taken in CDCl3 and DMSO with a Varian Mercury 400 MHz. Chemical shifts are given to external tetramethylsilane at 0 ppm with calibration using solvent signals. Scanning electron microscopy (SEM) was used to determine the crystalline structure of PCX, 6-O-CAPRO-b-CD, and their complexes with a JEOL SEM ASID-10 instrument, Tokyo, Japan. Samples were fixed on metal plates and sputtered with gold–palladium mixture at a thickness of ˚ and observed at an accelerated voltage of 100 A 80 kV.
Preparation of Blank and PCX-Loaded Nanoparticles Nanospheres were prepared according to the nanoprecipitation technique introduced by Fessi et al.41 According to this technique, an organic phase consisting of 6-O-CAPRO-b-CD (1 mg) dissolved in absolute alcohol (1 mL) was added with an Eppendorf injector at room temperature under constant stirring to an aqueous phase consisting of only Ultrapure Water (MilliQ) JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008
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(2 mL). Nanospheres formed spontaneously. This dispersion was stirred for equilibrium for 30 min followed by evaporation of organic solvent under vacuum to the desired volume (2 mL). To obtain nanocapsules, the same technique is used with the exception of an oil phase of Miglyol 812 (50 mL) added to the organic phase. Drug-loaded nanoparticles were prepared with either the conventional loading (CL) technique and the previously reported high-loading technique (HL) based on the preparation of nanoparticles from preformed inclusion complexes.38,39 The loading techniques can be described briefly as follows: Conventional loading (CL): Nanoparticles are loaded by adding fixed amount of ethanolic PCX solution (200 mg) to the organic phase during preparation. Preloading technique (PL): Nanoparticles are prepared by dissolving only a fixed amount (1 mg) of PCX/6-O-CAPRO-b-CD complex in the organic phase for the preparation. High-loading technique (HL): Nanoparticles are prepared from fixed amount of PCX/6-OCAPRO-b-CD complex (1 mg) with an overloading of ethanolic PCX solution (200 mg) in the organic phase during preparation.
long-term physical stability of the nanoparticles. Entrapment efficiency of PCX to amphiphilic CD nanoparticles was determined by the following technique; free undissolved drug in the nanoparticle dispersion was centrifuged at 5000 rpm and the resulting supernatant was separated and lyophilized (HETO Lyolab Freeze Dryer, Allerod, Denmark). The dry powder consisting of nanoparticles and nanoparticle-bound PCX was then dissolved in acetonitrile: water (70:30 v/v) and PCX quantity was assayed with an HPLC technique using HP 1100 Agilent Series instrument with a mobile phase of acetonitrile:water (70:30), flow rate of 1 mL/min, injection volume of 50 mL at 227 nm with a diode array detector using a reverse phase column C-18 octadecylsilane (ODS) (Hichrom, Berkshire 5 C18 column, 250 4.6 mm, UK) giving a retention time of 4.2 min for PCX. The HPLC technique was analytically validated (linearity r2: 0.9999, repeatability CV%: 0.4%, reproducibility CV%: 1.1%, LOD: 0.028 mg/ mL, LOQ: 0.095 mg/mL). Drug loading data were expressed in terms of entrapped drug quantity, entrapment efficiency, and associated drug %. Entrapped drug quantity is the drug quantity determined in nanoparticle sample (mg/mL). Entrapment efficiency and associated drug % values were calculated according to the following formula: Entrapment efficiency
Characterization of Blank and PCX-Loaded Nanoparticles Particle size distributions (mean diameter (nm), polydispersity index) of nanoparticles were determined by quasi-elastic light scattering using Malvern Zetasizer, Malvern Instruments, Worcestershire, UK. Measurements were realized in triplicate at a 908 angle at 258C. Zeta potential values were taken using the Malvern Zetasizer, Malvern Instruments, also in triplicate at 1208 angle and 258C with suitable dilution of samples with deionized water. Atomic force microscopy (AFM) was used to image the PCX-loaded nanoparticle dispersions with a Q-Scope1350 Multimode Atomic force Microscope, Ambios & Quasant, Santa Cruz, CA. Physical stability of blank CD nanoparticles was determined through a 12-month followup period during which the samples were stored as aqueous nanoparticle dispersions at 88C. Repeated measurements of particle size and zeta potential were performed in order to evaluate the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008
¼
Entrapped drug quantity ðmmolÞ 100 Initial CD amount ðmmolÞ
Associated drug% ¼
Entrapped drug quantity ðmgÞ 100 Initial drug amount ðmgÞ
In vitro PCX release profiles from 6-O-CAPROb-CD nanospheres and nanocapsules were determined in capped Eppendorf tubes in a 20 mL of medium of isotonic phosphate buffer saline pH 7.4 containing 0.1% polysorbate 80 for sink conditions at 378C in a thermostated shaker bath (Memmert, Schwabach, Germany) at 80 rpm. At given time intervals 0.5 mL of sample was taken from the release medium and replaced with fresh medium at 378C. Samples were then centrifuged at 13500 rpm for 15 min to precipitate the nanoparticles and the clear supernatant was freeze dried. Resulting dry powder was dissolved in 0.5 mL of mobile phase and assayed for PCX content. DOI 10.1002/jps
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RESULTS AND DISCUSSION Characterization of 6-O-CAPRO-b-CD 6-O-CAPRO-b-CD, amphiphilic CD derivative modified on the primary face with 6C aliphatic ester was characterized after synthesis by multiple techniques including 1H NMR spectroscopy, FTIR spectroscopy, DSC, and elemental analysis. 1 H NMR data for 6-O-CAPRO-b-CD are as follows; 1 H NMR (dH, 400 MHz, DMSO-d6): 7.99 (DMF, H–C¼O), 6.04 (dd, –OH), 5.86 (d, –OH), 4.92 (d, H1), 4.32 (d, -H-6), 4.21 (dd, H-60 ), 3.86 (t, H-5), 3.66 (m, H-3), 3.37 (m, H-4), 3.4 (DMSO-H2O), 2.7, 2.9 (DMF, -N-(CH3)2), 2.54 (DMSO), 2.31 (m, CH2–CO–), 1.52 (m, CH2–CH2–CO–), 1.28 (m, –(CH2)-2), 0.88 (t, CH3). FTIR spectrum (data not shown) of 6-OCAPRO-b-CD gave the following typical bands confirming the ester structure of this derivative; O–H stretching vibrations at 3700–3100 cm1, aliphatic C–H stretching vibrations at 3000– 2850 cm1, C – O stretching bands characteristic of the ester group at 1800–1700 cm1. DSC thermogram taken between 25 and 2508C shows a distinct and sharp melting endotherm for 6-O-CAPRO-b-CD at 2298C (data not shown). Table 1 shows the elemental analysis data for the newly synthesized 6-O-CAPRO-b-CD proving the purity of the final product.
Characterization of PCX/6-O-CAPRO-b-CD Inclusion Complexes The interaction at the solid state and in solution between PCX and CD cavity was evaluated by different techniques such as DSC analysis, FTIR spectroscopy, 1H NMR spectrometry, SEM imaging. Figure 2 displays the DSC thermograms of lyophilized PCX, lyophilized 6-O-CAPRO-b-CD, 1:1 and 1:2 molar ratio complexes of the drug, and CD. As seen from the DSC thermograms, melting endotherm of 6-O-CAPRO-b-CD at 2298C and melting and decomposition endotherms and dehydration at 1008C of PCX at 224 and 2328C have disappeared or broadened suggesting
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the absence of free crystalline drug within the colyophilizate. FTIR spectra for PCX, 6-O-CAPROb-CD, and their 1:1 and 1:2 complexes are seen in Figure 3 indicating a reduction in the O–H stretching vibration band at 3300 cm1. The peaks about 1500 and 1200 cm1 relative to aromatic ring and C–O–C stretching respectively show significant changes. The peak about 1700 cm1 corresponding to aryl and saturated ketone groups also indicates a possible interaction through the aromatic rings of the drug molecule seen in Figure 1a. These data indicate that there exists a chemical interaction between PCX and 6-OCAPRO-b-CD which is most probably indicative of a partial inclusion of the drug within the amphiphilic CD cavity. This interaction is more pronounced for the 1:2 molar ratio complex as seen in Figure 3. 1 H NMR spectra of PCX, 6-O-CAPRO-b-CD, and 1:1 and 1.2 molar ratio complexes were taken at 400 MHz and shifts in the signals of H-3 and H-5 along with other protons of amphiphilic CD were evaluated in terms of a possible inclusion in CD cavity which is expected to cause negative shifts42 in the internal protons (H-3 and H-5) of the CD cavity. Table 2 represents the chemical shift values of 6-O-CAPRO-b-CD protons before and after complexation to PCX. It can be observed that internal protons H-3 and H-5 display shifts of 0.02 to 0.03 ppm for 1:1 and 1:2 molar ratio complexes. More interestingly, H-6 and H-60 which are the protons of the aliphatic ester grafted to the primary hydroxyl neighboring the CD glucopyranose unit has also shifted significantly as seen from Table 2. This suggests that PCX molecule has entered the CD cavity and also interacted with the aliphatic chains grafted to the primary face. Thompson et al.43 has proposed that leaving the secondary face which is the wide side of the cavity unsubstituted may facilitate the entrance of hydrophobic drug molecule to the cavity. Modification of the primary face with long aliphatic chains contributes to the complexation ability of the CD derivative by providing a second hydrophobic zone to interact with after the CD cavity. SEM photomicrographs taken for PCX, 6-OCAPRO-b-CD, 1:1 and 1:2 molar ratio complexes
Table 1. Elemental Analysis Data of Amphiphilic CD, 6-O-CAPRO-b-CD
Amphiphilic CD 6-O-CAPRO-b-CD
DOI 10.1002/jps
Ampirical Formula
Molecular Weight (g/mol)
Theoretical Percentage
Practical Percentage
C84H140O42
1820
(C%) 55.3 (H%) 7.7
(C%) 50.2 (H%) 7.3
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Table 2. Chemical Shift Values (ppm) of 6-O-CAPROb-CD Protons before and after Complexation to PCX in 1:1 and 1:2 Molar Ratio Protons
dCDa
d1:1b
d1:2b
~d1:1c
~d1:2d
H-1 H-3 H-4 H-5 H-6 H-60
4.92 3.66 3.37 3.86 4.32 4.21
(—)e 3.64 3.36 3.83 4.31 4.16
(—) 3.64 3.37 3.84 4.31 4.16
(–) 0.02 0.01 0.03 0.01 0.05
(—) 0.02 0 0.02 0.01 0.05
a
Free state. Complexed state. ~d1:1 ¼ dCD d1:1. d ~d1:1 ¼ dCD d1:2. e Peaks could not be separated since H-1 proton of 6-OCAPRO-b-CD and H-5 proton at the taxane core of PCX have given resonance at the same ppm value. b c
Figure 2. DSC thermograms of PCX, 6-O-CAPRO-bCD, 1:1 and 1:2 molar ratio complexes of PCX/6-OCAPRO-b-CD.
Figure 3. FTIR spectra of PCX, 6-O-CAPRO-b-CD, and 1:1 and 1:2 molar ratio complexes. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008
DOI 10.1002/jps
Figure 4. SEM photomicrographs of (a) PCX (b) 6-O-CAPRO-b-CD, (c) 1:1 complex, and (d) 1:2 complex. Table 3. Particle Size and Zeta Potential Values for Blank and PCX-Loaded Amphiphilic CD Nanoparticles Formulation Blank nanosphere Blank nanocapsule CL nanosphere PL nanosphere HL nanosphere CL nanocapsule PL nanocapsule HL nanocapsule DOI 10.1002/jps
Particle Size (Mean Diameter, nm, SD)
Zeta Potential (mV)
157 5 587 36 219 32 232 5 263 24 606 37 450 31 639 37
13.8 2 14.7 4 19.6 1 18.8 2 34.6 5 32.5 3 37.1 2 33.0 5
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indicate that the needle-like crystalline structure typical of PCX does not exist in the complexes which both show an amorphous state. Figure 4a–d represents the SEM photomicrographs of the drug, CD, and the different complexes. This change in the crystalline form could be a result of the colyophilization process which is very similar to the nanoprecipitation process used to prepare the nanoparticles.44 Characterization of Blank and PCX-Loaded Nanoparticles Particle size and zeta potential values of blank and PCX-loaded 6-O-CAPRO-b-CD nanoparticles prepared with different loading techniques are seen in Table 3. The data suggest that loaded nanoparticles are slightly larger than blank nanoparticles probably due to the adsorption of the drug on the particle surface. Nanocapsules are limited to the oil droplet size45 that form during the preparation so they are considerably larger than nanospheres but still in the appropriate nm range for injectable use. Nanospheres in particular are around 150–250 nm which suggests favorable properties for injectable systems, possibility to escape from RES uptake, and leaking from the vasculature surrounding the tumor site resulting in an eventual passive targeting of the system.46 The sizes of nanospheres were believed to be slightly affected by the preparation technique being preloading, CL or high-loading due to the different amounts of drug entrapment. Zeta potentials of the blank nanoparticles are around 19 mV with a slight increase up to 37 mV for loaded formulation. It is believed that this increase in zeta potential is due to the charge of the adsorbed PCX on the particle surface. In fact, zeta potential of PCX dispersion in buffer PBS was determined to be 30 mV confirming this theory. Long-term physical stability of the blank nanoparticles was measured by repeated particle size and zeta potential measurements. The data are presented in Figures 5 and 6 for particle size and zeta potential measurements, respectively. The data indicate the 12-month physical stability of 6-O-CAPRO-b-CD nanospheres and nanocapsules in aqueous dispersion form upon storage at 88C. Changes in the particle size and zeta potential during the 12-month follow-up period are not statistically significant ( p < 0.05). Drug loading values of PCX are seen in Table 4 in terms of entrapped drug quantity, entrapment JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008
Figure 5. Time-dependent particle size of 6-OCAPRO-b-CD nanospheres and nanocapsules in aqueous dispersion.
efficiency, and associated drug percentage. It is seen that loading techniques increase drug loading values significantly up to threefold for highloaded nanoparticles. It is also observed that nanocapsules entrap almost twofold PCX when compared to nanospheres. This may be a result of the affinity of this very poorly soluble drug to the oily core of the nanocapsules. It has been reported that drug physicochemical properties including octanol: water partition coefficient, k1:1 affinity constant for CD, and aqueous solubility affect loading characteristics significantly.47 In accordance with this fact, PCX with its very poor water solubility, high partition coefficient, and demonstrated affinity to CDs show a high encapsulation efficiency to CD nanoparticles. In vitro release of PCX was assessed from nanospheres and nanocapsules into isotonic phosphate buffer pH 7.4 containing 0.1%
Figure 6. Time-dependent zeta potential values of 6-O-CAPRO-b-CD nanospheres and nanocapsules in aqueous dispersion. DOI 10.1002/jps
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Table 4. Drug Loading Values for PCX for 6-O-CAPRO-b-CD Nanoparticles Prepared With Different Loading Techniques
Dosage Form
Entrapped Drug Quantity (mg) SD
Associated Drug Percentage (%, a/a)
Entrapment Efficiency (%) (mmol Drug/mmol CD) 100
Nanosphere Nanocapsule Nanosphere Nanocapsule Nanosphere Nanocapsule
67.4 0.7 120.7 0.3 65.4 0.6 117.2 0.5 143.5 0.9 186.4 0.9
33.7 60.4 32.7 58.6 36.8 47.8
24 43 24.6 44.2 53 68
polysorbate 80. Figure 7 displays the in vitro release profiles of conventionally loaded and highloaded nanospheres and nanocapsules. It can be seen that nanocapsules liberate the drug in a considerably slower release profile. Another factor affecting release seems to be the loading technique with CL resulting in a burst effect. PCX is released in a period of 12 h with complete release achieved in 24 h for nanocapsules. PCX’s burst effect is observed in the first 15 min for conventionally loaded formulations. However, high-loaded formulations displayed biphasic release profile with complete PCX release achieved around 12 h.
CONCLUSION
Loading Technique CL PL HL
In the light of the data obtained in this study, it can be concluded that amphiphilic CD nanoparticles may provide a promising alternative for nonsurfactant, Cremophor-free, nanoscale carrier for injectable use of PCX. Further studies should focus on the safety and efficacy of this delivery system to ensure an improvement in the therapeutic efficacy and clinical application of this potent anticancer agent.
ACKNOWLEDGMENTS Authors thank the TUBITAK SBAG CNRS-3 project and Hacettepe University Research Fund Project NO. 0202301005 for their financial support to this study.
REFERENCES
Figure 7. In vitro release profiles for PCX from amphiphilic CD nanospheres and nanocapsules (n ¼ 3, SD) (PCX HL NS, High-loaded nanospheres; PCX CL NS, conventionally loaded nanospheres; PCX HL NC, high-loaded nanocapsules; PCX CL NC, conventionally loaded nanocapsules). DOI 10.1002/jps
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