Journal of Controlled Release 111 (2006) 271 – 280 www.elsevier.com/locate/jconrel
Novel composite core-shell nanoparticles as busulfan carriers A. Layre a , P. Couvreur a , H. Chacun a , J. Richard b,1 , C. Passirani c , D. Requier b , J.P. Benoit c , R. Gref a,⁎ a
UMR CNRS 8612, Faculty of Pharmacy, Paris-Sud University, 5, rue Jean Baptiste Clément, 92296 Châtenay-Malabry, France b Ethypharm, 194 Bureau de la colline, 92213 Saint-Cloud, France c INSERM U646, Faculty of Pharmacy, 10 rue A. Boquel, 49100 Angers, France Received 23 August 2005; accepted 10 January 2006 Available online 20 February 2006
Abstract This study presents a method for the design of novel composite core-shell nanoparticles able to encapsulate busulfan, a crystalline drug. They were obtained by co-precipitation of mixtures of poly(isobutylcyanoacrylate) (PIBCA) and of a diblock copolymer, poly(ε-caprolactone)–poly (ethylene glycol) (PCL–PEG), in different mass ratios. The nanoparticle size, morphology and surface charge were assessed. The chemical composition of the top layers was determined by X-ray photo-electron spectroscopy (XPS). 3H-labelled busulfan was used in order to determine the drug loading efficiency and the in vitro drug release by liquid scintillation counting. Physico-chemical techniques such as Zeta potential determination and XPS analysis provided evidence about a preferential surface distribution of the PCL–PEG polymer. Therefore, composite nanoparticles have a ‘core-shell’-type structure, where the “core” is essentially formed by the PIBCA polymer and the “shell” by the PCL–PEG copolymer. The use of PIBCA to form the core of the nanoparticles leads to a 2–4 fold drug loading increase, in comparison to the single PCL– PEG nanoparticles. In addition, the complement activation results showed a significant difference between the composite nanoparticles and the single PIBCA nanoparticles, thus demonstrating that PEG at the surface of the nanoparticles reduced the complement consumption. The PIBCA: PCL–PEG composite nanoparticles prepared using the new co-precipitation method here described represent an original approach for busulfan administration. © 2006 Elsevier B.V. All rights reserved. Keywords: Nanoparticle; Poly(ethylene glycol); Drug delivery; XPS; Complement
1. Introduction Busulfan is a bifunctional alkylating agent [1], which is widely used at high dose as a part of myeloablative regimen before both allogenic and autologous bone marrow transplantation for the treatment of haematological malignancies [2] and non-malignant disorders such as immunodeficiency [3]. For a long time, busulfan has been available only in oral form and a wide intra-patient and inter-patient bioavailability variability in both adult and children has been reported [4]. Moreover, severe
⁎ Corresponding author. Tel.: +33 1 46 83 59 09; fax: +33 1 46 61 93 34. E-mail address:
[email protected] (R. Gref). 1 New permanent address: Serono, Via di Valle Caia, 22, 00040 Ardea (Roma), Italia. 0168-3659/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2006.01.002
side effects were reported such as the veino-occlusive disease (VOD). This pathology has been correlated with a high systemic exposure to busulfan expressed as the area under the plasma concentration–time curve [5]. In order to overcome these problems, intravenous formulations of busulfan were developed, using cosolvent mixtures [6,7]. However, these organic solvents have their own welldocumented toxicity [8,9]. Therefore, to avoid the massive use of organic solvents, injectable colloidal carriers, such as conventional liposomes [10] and biodegradable polymer nanoparticles have been elaborated [11,12] . However, these carriers had encapsulation efficiencies lower than 1% (w/w). Indeed, successful encapsulation of busulfan into nanoparticles has never been described, yet. In previous studies, we have established the ability of poly (isobutylcyanoacrylate) (PIBCA) nanoparticles to encapsulate larger amount of busulfan than other polymers [13].
272
A. Layre et al. / Journal of Controlled Release 111 (2006) 271–280
Unfortunately, such nanoparticles displaying large hydrophobic surface areas were rapidly recognised by the mononuclear phagocyte system (MPS), eliminated from the blood stream within minutes and ended up mainly in liver and spleen [14]. The liver uptake of busulfan-loaded nanoparticles might run the risk to increase the occurrence of the VOD. It is our hypothesis that by using long circulating nanoparticles, able to dramatically reduce liver accumulation, the probability for the VOD to occur would be reduced. Long-circulating nanoparticles can be obtained by coating or grafting flexible and non-ionic polymers, such as poly(ethylene glycol) (PEG) onto the hydrophobic nanoparticles' surface [15,16]. Several methods have been investigated to prepare PEG-coated poly(alkylcyanoacrylate) nanoparticles, such as the adsorption of amphiphilic Poloxamer or Poloxamine copolymers onto nanoparticles' surface [17]. However, no significant biodistribution changes were observed in comparison with uncoated nanoparticles. Indeed, it was supposed that in vivo, these copolymers might be competitively displaced from the surface by plasma proteins having a higher affinity for PIBCA [17] or that they could be released as a consequence of polymer's bioerosion [18]. Another approach to design PEG-coated PIBCA nanoparticles consisted in grafting PEG onto the nanoparticles during the emulsion polymerization process of the isobutylcyanoacrylate monomer [19]. However, it has been shown that some drugs were able to react with the cyanoacrylate monomers too, which dramatically affected both nanoparticle formation and the biological activity of the drug [20,21]. Since busulfan is also a very strongly reactive drug, this method is likely not adapted for the preparation of busulfan-loaded nanoparticles. More recently, the synthesis of block copolymers containing PEG and polyalkylcyanoacrylate blocks has been reported [22]. These copolymers could be nanoprecipitated under the form of nanoparticles with increased blood circulation times compared with the uncoated poly(alkylcyanoacrylate) nanoparticles [23]. Nevertheless, it was observed that the blood half life in rats of these “PEGylated” polyalkylcyanoacrylate nanoparticles was significantly lower than that of the PEGylated polyester nanoparticles [23,24]. Thus, the aim of this study was to design novel composite core-shell nanoparticles able to combine the ability of the PIBCA to efficiently encapsulate busulfan, with the excellent stealth properties of the diblock copolymer, poly(ε-caprolactone)–poly(ethylene glycol) (PCL–PEG), which is expected to provide increased blood half lives to the nanoparticles [16,24]. This paper describes the feasibility of these composite nanoparticles with special emphasis on their surface characterization. Loading efficiency of busulfan and in vitro drug release were investigated, too. 2. Materials and methods 2.1. Materials Poly(isobutylcyanoacrylate) (PIBCA) was synthesized by an anionic polymerization of isobutylcyanoacrylate monomers in water. The monomer (1 ml) was added in one shot to water
(15 ml). The polymerization carried on during 1 h 30 at 40 °C under magnetic stirring (1200 rpm). After this time, a milky suspension was obtained together with a polymer aggregate around the magnetic stirrer. This polymer was collected in two fractions: the milky suspension was freeze dried (fraction 1), and the aggregated polymer was dissolved in acetone and dried under vacuum at room temperature (fraction 2). The polymer thus obtained in fraction 2 (representing more than 85% of the polymer synthesized) was further used in the nanoprecipitation process. The poly(ε-caprolactone)–poly(ethylene glycol) diblock copolymer was synthesized as previously described [25]. Briefly, given amounts of the monomer (ε-caprolactone) freshly distilled and monomethoxy polyethylene glycol (MPEG) (weight average molar mass 2000 g/mol, SigmaAldrich, Germany) were dissolved in xylene. The weight ratio ε-caprolactone : MPEG was 9 : 1. Stannous octanoate (SigmaAldrich, Germany) purified by distillation was used as a catalyst in equimolar quantity with regard to MPEG. The reaction was carried on at 110 °C for 6 h. Average molar masses (Mw) were determined using gel permeation chromatography equipped with a refractometric and a multiangle light scattering detector (Wyatt Dawn Model F, Milton Roy, Wyatt Technology). The diblock copolymer symbolized PCL10k– PEG2K had a PCL block with a Mw of 10,000 g/mol and a PEG block with a Mw of 2000 g/mol. Busulfan was purchased from Sigma-Aldrich (Germany) and Tritium-labelled busulfan from RC TRITEC (Switzerland). Acetone was obtained from Carlo-Erba (France). Poloxamer 188 (Synperonic PE/F68) and D-(+)trehalose were purchased from Fluka (Switzerland). Rat plasma was obtained from Charles River (USA). 2.2. Nanoparticle preparation To prepare the composite nanoparticles, firstly two PIBCA and PCL10k–PEG2k solutions at 20 mg/ml were mixed in different volume ratios (80 / 20, 70 / 30, 60 / 40, 50 / 50). Secondly, busulfan was dissolved in acetone at 4 mg/ml. After, 500 μl of the polymer solution and 500 μl of the busulfan solution were mixed. The resulting solution (1 ml) was then injected into water (2 ml) under magnetic stirring (1200 rpm) at room temperature leading to spontaneous formation of composite nanoparticles. Acetone was eliminated using a rotative evaporator (rotavapor®) at room temperature. The suspensions were purified by centrifugation (MR22i, Jouan, France) (5 min at 630 ×g), prefiltration (Acrodisc, Glass fiber membrane, 1 μm, Pall, USA), and finally filtration (Millex®-HV, 0.45 μm, Millipore, USA) in order to eliminate drug crystals which might form during acetone evaporation step [26]. The composite nanoparticles are further named CNP(PIBCA:PCL10k–PEG2k). For example, CNP(80 : 20) was obtained from a PIBCA:PCL10k–PEG2k mixture in 80:20 volume ratio. Single PIBCA and single PCL10K–PEG2K nanoparticles were prepared by the nanoprecipitation process, as previously described by Fessi and Devissaguet [27]. Briefly, an organic solution of polymer (PIBCA or PCL10K–PEG2K) (10 mg) and
A. Layre et al. / Journal of Controlled Release 111 (2006) 271–280
busulfan (2 mg) in acetone (1 ml) was injected into 2 ml water under magnetic stirring (1200 rpm) at room temperature. Nanoparticle suspensions were purified as described previously. Drug free nanoparticles were prepared according to the same procedure, without the purification steps. The drug content in nanoparticles was assessed using tritiumlabelled busulfan. 3H-Busulfan loaded CNP(PIBCA:PCL10k–PEG2k) nanoparticles and single PIBCA or PCL10K–PEG2K nanoparticles were prepared as described above, with a theoretical activity of 1.6 μCi/mg drug. The nanoparticles were collected by centrifugation (30,000 ×g for 30 min) (MR22i, Jouan, France) and dried in desiccators under vacuum at room temperature over 24 h. After weight determination, the dried nanoparticles were dissolved in 1 ml acetone. The drug loading was determined by liquid scintillation. It was expressed as the weight of drug in the nanoparticles divided by the weight of the dried nanoparticles collected. 2.3. Nanoparticle characterization 2.3.1. Nanoparticle size measurement The mean particle diameters were measured by laser light scattering using a nanosizer (Coulter® N4MD, Coulter Electronics Inc, Hialeath, USA). Each sample was properly diluted in water, in order to maintain the number of counts per second between 5 · 104 and 1 · 106. Water was filtered with a 0.22 μm filter to remove any impurities that could affect scattering of the light. Each sample was measured three times for at least three minutes at 20 °C and at an angle of 90°. Both unimodal and size distribution processor (SDP) analysis were performed. 2.3.2. Nanoparticle morphology Nanoparticles were observed using transmission electron microscopy after freeze fracture. A small drop of an aqueous nanoparticle suspension was deposited into a 100 μm deep symmetric cup. Then, the sample was frozen using a high pressure cooling device HPM 010 (Bal-Tec). Fracturing, etching and shadowing, using Pt–C, were performed in a Bal-Tec Model BAF 400T apparatus. The replicas of the surface were then floated off by specimen submerging in successive baths of water/acetone, water, NaOH (1 M), water and acetone. Finally, the replicas were collected onto naked 400 mesh grids which were subsequently mounted in a transmission electron microscopy (TEM) for inspection. TEM observations were performed on a LEO 912 Omega high resolution microscope working at 120 kV. 2.4. Surface characterization 2.4.1. Zeta potential determination The nanoparticle zeta potential measurements were carried out using a Zeta Sizer 4 (Malvern Instruments Ltd. UK). Each sample was properly diluted in NaCl (1 mM), in order to maintain the number of counts per second around 600. Three measurements were carried out for each sample and the mean values and standard deviations were calculated.
273
2.4.2. X-ray photoelectron spectroscopy analysis In order to investigate the chemical composition of the nanoparticle top layers and to gain evidence about the preferential localization of the PCL10k–PEG2k copolymers, XPS analysis was carried out on the dry PIBCA, PCL10k– PEG2k and CNP(50 : 50) nanoparticles. Indeed, XPS allows to determine the elemental and average chemical composition of a material at its very surface (about 10 nm depth), by measuring the binding energy of the 1s electrons emitted from the atoms in these top layers. The spectra were recorded with a thermo VG Scientific ESCALAB 250 spectrometer (VG Instrument, UK) equipped with a monochromatic Al Kα X-ray source (1486.6 eV) at a spot size of 650 μm. The take-off angle relative to the sample holder surface was 90°. The pressure in the analysis chamber was ca. 2–3 · 10− 8 mbar. The pass energy was set at 150 and 40 eV for the survey and the narrow scans, respectively. The step size was 1.0 eV for the survey spectra and 0.1 for the narrow regions, respectively. A 4 eV flood gun combined with an argon gun was used to neutralize the surface charge. The spectra were calibrated against the N1s peak PIBCA centred at 399.7 eV. Identification of chemical functional groups was obtained from the high-resolution peak analysis of carbon 1s (C1s), oxygen 1s (O1s), and nitrogen 1s (N1s) envelopes. Data acquisition and processing software was achieved using Aventage software, version 2. The surface composition was determined using the manufacturer's sensitivity factors. The fractional concentration of a particular element A (% A) was computed using: %A¼
ðIA =SA Þ 100 n X ðIi=SiÞ i¼1
Where Ii and Si are the integrated peak areas of each of the n detected elements and the sensitivity factors, respectively. The comparison between the composition in atomic ratios obtained from XPS analysis and from the theoretical calculations should provide evidence about a preferential surface distribution or a uniform distribution of chemical elements. The theoretical atomic ratios were calculated from the chemical formulas of PIBCA ((C8NO2)n) and PCL10k–PEG2k ((C6O2)88– (C2O)45), supposing that all the different chemical elements were uniformly distributed within the analysed samples. Since hydrogen is not detected in XPS, this element was not taken into account in the theoretical formulas. 2.5. Complement activation The method used to assess the interactions of the nanoparticles with complement was the haemolytic CH50 test. The principle of the procedure is based on the fact that, when sensitized sheep erythrocytes are in contact with the complement proteins in human serum, the classical complement pathway is activated, resulting in the lysis of erythrocytes and the release of haemoglobin. This technique consisted in the determination of the amount of units of serum able to lyse 50% of a fixed number of sensitized sheep erythrocytes (CH50 units). When the human serum is put in contact with activating
274
A. Layre et al. / Journal of Controlled Release 111 (2006) 271–280
nanoparticles, opsonization occurs and less complement proteins remain in the serum to lyse the sheep erythrocytes, thus the CH50 units decrease. The CH50 test was described previously [28]. Briefly, Veronal-buffered saline containing 0.15 mM Ca2+ and 0.5 mM Mg2+ (VBS+) were prepared as described elsewhere [29]. Human serum was a pool of forty donor blood samples provided by the ‘Etablissement Français du Sang’ (Angers, France), aliquoted and stored at − 80 °C until used. Sheep erythrocytes (Eurobio, France) were sensitized by rabbit anti-sheep erythrocyte antibodies (Haemolytic Serum, Eurobio, France) and suspended at a final concentration of 1 · 108 cells/ml in VBS+. First, a little amount of Poloxamer 188 (2.5 · 10− 1 μg per cm2 of nanoparticle surface) was incubated with the nanoparticles, in order to avoid the nanoparticle aggregation that occurred in presence of VBS+. Increasing amount of nanoparticle suspensions were added to human serum in VBS+, so that the final dilution of the human serum in the reaction mixture was 1 / 4 (v/v) in a final volume of 1 ml. After a 60 min incubation at 37 °C with gentle agitation, the suspension was diluted 1 / 25 (v/v) with VBS+. Aliquots at different dilutions were added to a given volume of sensitized sheep erythrocytes. After a 45 min incubation at 37 °C, the suspension was centrifuged (2000 rpm, 10 min). Then, the optical density at 415 nm of the supernatant, related to the lytic capacity of the serum, was recorded. The amount of CH50 units remaining in the serum was determined and compared to the result obtained with control serum. The results were expressed as consumption of CH50 units. In order to compare the effect of nanoparticles with different mean diameters, the amount of nanoparticles in contact with serum was expressed in terms of surface area. Nanoparticle surface area was calculated as described elsewhere [30]. The distance between two terminally attached PEG chains at the surface was calculated, assuming that all the PEG chains migrate at the nanoparticles' surface during their preparation [15] and that all the prepared nanoparticles are homogeneous in terms of composition and size. Thus, it is possible to calculate the surface SPEG that would occupy each PEG chain on the surface of the nanoparticles, taking into account the nanoparticle mean radius (r).
Assuming that each PEG chain would occupy the centre of a square at the nanoparticles' pffiffiffiffiffiffiffiffiffisurface, the distance between two PEG chain is dPEG ¼ SPEG . It results that in the cases studied: ° CNPð80 : 20Þ : r ¼ 70d10−7 cm; f ¼ 0:03 and dPEG ¼ 20 A ° CNPð50 : 50Þ : r ¼ 60d10−7 cm; f ¼ 0:08 and dPEG ¼ 13 A −7 ° PCL10k PEG2k : r ¼ 40d10 cm; f ¼ 0:16 and dPEG ¼ 11 A
2.6. Freeze-drying process Aliquots of composite nanoparticles (400 μl) at a concentration of 5 mg/ml were added to sugar and surfactant solution (400 μl) at various concentrations before freeze-drying. The mass ratio polymer : cryoprotectant in the nanoparticle suspension ranged from 1 : 2 to 1 : 10. Freezing was performed in a conventional freezer (− 20 °C). The frozen nanoparticles were then lyophilized using a freeze-drying system (Christ-Alpha 1-4, Bioblock Scientific, France) over 24 h. Temperature cycle was −30 °C: +30 °C and the vacuum was 8 · 10− 3 mbar. 2.7. In vitro release study The drug release experiments were carried out for the CNP(50 : 50) suspension. These experiments were performed at 37 °C in water and in rat plasma. Freshly prepared tritium labelled busulfan-loaded nanoparticle suspensions (5 μCi/mg busulfan) were diluted with the release medium studied. Then, the nanoparticle suspension was separated in 1 ml aliquots and placed on a shaker (Titramax 101, Heidolph, Germany) at 37 °C. At each given time-point, one of those aliquots was centrifuged (10 min at 30,000 ×g). Busulfan in the supernatant was assessed by liquid scintillation. Nanoparticles were collected and dried in a desiccator during 24 h under vacuum at room temperature. After weight measurement, the nanoparticles were dissolved in 1 ml acetone. The busulfan quantity in the nanoparticle fraction was determined by liquid scintillation. 3. Results and discussion
SPEG ¼ 4pr2 =NPEG
Feasibility and characterization of drug-free composite nanoparticles.
where NPEG is the total number of PEG chains in one nanoparticle. Or,
3.1. Size determination
NPEG ¼ mNP f N =MPEG where MPEG is the molar mass of the block PEG (2000 g/mol), N is the Avogadro number, f is the weight fraction of PEG in the nanoparticles and mNP is the mass of one nanoparticle. mNP is given by: mNP ¼ 4pr3 dNP =3 where the density of the nanoparticle (dNP) was equal to 1.2 g/ cm3 [31]. We finally obtain: SPEG ¼ ð3MPEG Þ=ðr dNP f N Þ
The mean diameter of single PIBCA, single PCL10k–PEG2k nanoparticles and composite nanoparticles (weight ratios 80 : 20 to 50 : 50) are reported in Table 1. The co-precipitation of PIBCA and PCL10k–PEG2k provided nanoparticles with a size lower than 200 nm, whatever the polymers weight ratio. However, the nanoparticle mean diameter was affected by the PIBCA : PCL10k–PEG2k ratio, the mean diameter of pure PIBCA nanoparticles was 143 (± 37 nm) and it decreased until 106 (± 33 nm) for the CNP(50 : 50) nanoparticles. Thus, an increase in the amount of PCL10k–PEG2k in the PIBCA: PCL10k–PEG2k mixtures lead to a decrease of the resulting nanoparticle mean diameter. These data might be explained by
A. Layre et al. / Journal of Controlled Release 111 (2006) 271–280 Table 1 Z-average mean diameter and zeta potential values of single PIBCA, single PCL10k–PEG2k and CNP composite nanoparticles (weight ratios from 80 : 20 to 50 : 50) Particle type
Mean diameter (nm)
Zeta potential (mV)
PIBCA CNP(80 : 20) CNP(70 : 30) CNP(60 : 40) CNP(50 : 50) PCL10k–PEG2k
143 ± 37 139 ± 43 124 ± 34 123 ± 38 106 ± 33 67 ± 22
− 39 ± 0.1 − 42 ± 0.6 − 26 ± 0.3 − 28 ± 0.3 − 22 ± 1.1 − 15 ± 0.9
275
PIBCA (Fig. 1A) and PCL10k–PEG2k (Fig. 1B) nanoparticles presented a spherical shape and the latter ones were smaller. The CNP(50 : 50) typically showed a core-shell structure (Fig. 1C. and 1D). No second population of smaller diameter could be detected in these samples, as in the PCL10k–PEG2k ones. These observations suggest that both PIBCA and PCL10k–PEG2k associate to form mixed nanoparticles. We presume that the amphiphilic PCL10k–PEG2k would organize at the surface during particle formation, and finally form a shell around the hydrophobic PIBCA core.
Each value was the average of three different experiments ± SD.
3.3. Drug-free nanoparticle surface characterization the amphiphilic intrinsic properties of the PCL10k–PEG2k copolymer, reducing the interfacial tension between the aqueous and the organic phase. 3.2. Nanoparticle morphology In order to investigate the morphology of the nanoparticles, their cross section was observed by transmission electron microscopy after freeze fracture. Photomicrographs of the single PIBCA, and single PCL10k–PEG2k nanoparticles as well as the CNP(50 : 50) composite nanoparticles are presented in Fig. 1. The
3.3.1. Surface charge of nanoparticles The Zeta potential of both single PIBCA and PCL10k–PEG2k nanoparticles, as well as that of composite nanoparticles (weight ratios from 80/ 20 to 50 /50) are reported in Table 1. The single PIBCA nanoparticles displayed a Zeta potential of around −40 mV, whereas the PCL10k–PEG2k nanoparticles had a Zeta potential of − 15 mV. The CNP had intermediate Zeta potential values and the higher the PCL10k–PEG2k content in the polymer mixtures, the higher the Zeta potential values. For example, the CNP(50 : 50) nanoparticles displayed Zeta potential values of − 22 mV, close to the one of the PCL10k–PEG2k nanoparticles. These findings
Fig. 1. Transmission electronic microscopy after freeze-fracture images of single PIBCA (A); single PCL10k–PEG2k (B) and CNP(50 : 50) (C and D) nanoparticles. Bar represents 200 nm.
276
A. Layre et al. / Journal of Controlled Release 111 (2006) 271–280
also suggested the preferential localization of the PCL10k– PEG2k copolymers at the surface of the composite nanoparticles. 3.4. XPS analysis
Sample
The XPS scans of the different dried nanoparticles are presented in Fig. 2. In the XPS spectra of PCL10k–PEG2k nanoparticles (Fig. 2B), the main peaks were C1s and O1s centred at 285 and 533 eV, respectively. In PIBCA and CNP(50 : 50) XPS spectra (Fig. 2A and C, respectively), the main peaks were C1s, N1s, and O1s centred at 286, 400 and 533 eV, respectively. XPS is of particular interest in our case, as only PIBCA displays a N peak. The relative intensity of the N1s peak of the CNP(50 : 50) was lower than that of PIBCA nanoparticles. This is an indication that PIBCA core was covered with
A
CN HO CH2 C H n O C O CH2
O1s
CH CH3CH3
1000
800
C1s
N1s
600
400
200
Binding energy (eV)
B HO
1000
O1s
O O x
OH y
800
C1s
600
400
200
Binding energy (eV)
C
O1s C1s
800
600
400
XPS elemental ratios (%) C
N
O
Composition in atomic ratios C/N
C/O
CO / C = O
Theor XPS Theor XPS Theor XPS PIBCA 75.0 7.8 17.2 8 PCL10k–PEG2k 75.1 0.0 25.0 0 CNP(50 : 50) 74.8 2.2 23.0 15.9
9.6 4 0 2.8 34 3.3
4.4 3.0 3.2
– 2 –
– 1.7 –
PCL10k–PEG2k. To have a quantitative estimation about this coating, Table 2 presents the atomic composition (C, N, O) and the composition in atomic ratios (C / N, C / O and CO / C = O) calculated from the data obtained using XPS analysis as well as the theoretical ones (see Materials and methods section). The comparison between the ratios obtained from XPS analysis and from the theoretical calculations should provide evidence about a preferential surface distribution or a uniform distribution of chemical elements. The experimental and theoretical atomic ratios (C / N and C / O) determined with PIBCA nanoparticles were equal (Table 2), which proves there is a uniform distribution of each chemical element in the nanoparticles. In the case of the PCL10k–PEG2k nanoparticles, the experimental and the theoretical CO / C=O ratios were equal too, corresponding to a uniform distribution of the poly(ethylene glycol) block and of the poly(ε-caprolactone) block. Therefore, XPS analysis could not prove a core-shell organisation of PCL10k–PEG2k polymer. This finding could be due to the XPS analysis depth (about 10 nm). In fact, the PEG layer formed by PEG 2000 g/mol has a lower thickness than 10 nm [32]. Therefore, the analysis depth (10 nm) is higher than the ‘PEGylated shell’ layer thickness and the elemental ratio (CO / C = O) obtained by XPS takes into account the shell (PEG2k) and part of the core depth (PCL10k) of the nanoparticles. Indeed, only with PEG blocks with molar masses higher than 5000 g/mol, it was possible using XPS to detect the preferred localization of PEG at the surface [16,33]. The theoretical C / N ratio of the CNP(50 : 50) nanoparticles was 2-fold lower than the experimental ratio (Table 2). In other words, the experimental nitrogen content in the nanoparticles was lower than the nitrogen content in the polymer blends. These data provide evidence about a preferential surface distribution of the PCL10k–PEG2k polymer. Therefore, CNP(50 : 50) nanoparticles have a ‘core-shell’-type structure, where the “core” is essentially formed by the PIBCA polymer and the “shell” by the PCL10k–PEG2k polymer. 3.5. Complement activation
N1s
1000
Table 2 XPS elemental ratios (C, N, O) and composition in atomic ratios C / N, C / O, and CO / C = O of single PIBCA, single PCL 10k –PEG 2k and CNP (50 : 50) nanoparticles
200
Binding energy (eV) Fig. 2. XPS scans of pure PIBCA (A), pure PCL10k–PEG2k (B), and CNP(50 : 50) nanoparticles (C).
It is now well established that nanoparticle phagocytosis is mediated by opsonization, and that serum complement is a major component of the opsonin system [34]. The evaluation of the nanoparticles–complement interactions represents a good preliminary experiment predictive of the in vivo fate of the colloidal drug carriers after intravenous administration [35].
CH50 units consumption (%)
A. Layre et al. / Journal of Controlled Release 111 (2006) 271–280
277
In Jeon's model, the minimum average distance between two PEG chains needed for repelling proteins was estimated around 20 Å. So, our data are in the same range as these theoretical predictions. Similar distances were also found in the case of nanoparticles prepared using diblock PLA–PEG copolymers, to reduce complement consumption [28], plasma protein adsorption and uptake by polymorphonuclear cells [25].
100 80 60 40 20
3.6. Freeze-drying of nanoparticles
0 0
200
400
600
800
1000
1200
Surface area (cm2/ml) Fig. 3. Consumption of CH50 units in the presence of single PIBCA nanoparticles (mean diameter 150 nm) (♦), CNP(80 : 20) nanoparticles (mean diameter 140 nm) (▵), CNP(50 : 50) nanoparticles (mean diameter 120 nm) (●) and single PCL10k–PEG2k nanoparticles (mean diameter 80 nm) (□).
Complement consumption, as a function of the PIBCA, PCL10k–PEG2k, CNP(80 : 20) and CNP(50 : 50) nanoparticle surface area, is reported in Fig. 3. A dose-dependent consumption of CH50 units was obtained in all cases. It is well established that a larger amount of nanoparticles increased the contact between the nanoparticles and serum opsonins, leading to higher amounts of adsorbed opsonins [36]. The PIBCA nanoparticles were clearly the best activating suspensions. Indeed, the complement consumption was faster and stronger than with the other nanoparticle suspensions. The complement consumption decreased when the PCL10k–PEG2k content in the nanoparticles increased. Therefore, the novel “core-shell” composite nanoparticles were able to diminish the complement consumption as compared with the control PIBCA nanoparticles. For example, for a surface area of 900 cm2/ml, 100% complement consumption occurred for CNP(80 : 20), while only 60% and 20% complement consumption were obtained in the case of CNP(50 : 50) and PCL10k–PEG2k, respectively. In conclusion, the complement consumption with the “PEGylated” nanoparticles was dependant on their average PEG surface density, as already described by Jeon and Andrade and Jeon et al. [37,38]. Assuming that all the PEG chains migrate at the nanoparticles' surface, the calculated distance between two terminally attached PEG chains at the surface of CNP(80 : 20), CNP(50 : 50) and PCL10k–PEG2k conventional nanoparticles were 20, 13 and 11 Å, respectively (see Materials and methods).
The characteristics of the freeze-dried composite nanoparticles (weight ratios 80 : 20 to 50 : 50) are presented in Table 3. The CNP preserved the Tyndall effect upon redispersion whatever the freeze-drying conditions. The ratio between the nanoparticles' diameter after and before freeze-drying (Sf / Si) in the presence of a mixture of Poloxamer 188 (weight ratio 1 : 2) and trehalose (weight ratios 1 : 5 and 1 : 10) increased when the PCL10k–PEG2k proportion increased. However, the use of a mixture of Poloxamer 188 (weight ratio 1 : 10) and trehalose (weight ratios 1 : 10 and 1 : 5), whatever the nanoparticle composite suspension, resulted in a good redispersion with a Sf / Si ratio close to one. Thus, freeze-drying could be successfully used to dry composite nanoparticle suspensions (weight ratios 80 : 20 to 50 : 50) after the addition of a mixture of Poloxamer 188 (weight ratio 1 : 10) and trehalose (weight ratio 1 : 10 or 1 : 5). 3.7. Busulfan loaded nanoparticle characterization Table 4 shows the mean diameter of the nanoparticles before and after purification (see materials and methods), and the values of busulfan loading into PIBCA and PCL10k–PEG2k pure nanoparticles as well as into composite nanoparticles (weight ratios from 80 : 20 to 50 : 50). In all cases, the nanoparticle size slightly decreased after purification. As specified in the Materials and methods section, the nanoparticle suspensions were filtrated on the 0.45 μm filter. Indeed, this filter enables to remove the busulfan crystals in the suspension medium, but also retains some of the largest nanoparticles, which accounts for the observed size reduction after purification. The nanoparticle size was dependent on the PIBCA: PCL10k–PEG2k ratio. It increased when the PCL10k–PEG2k decreased, except for the CNP(50 : 50). These data could be
Table 3 Characteristics of freeze-dried composite nanoparticles (weight ratios from 80 : 20 to 50 : 50) in the presence of poloxamer 188 (P188) and trehalose (treh) for different polymer : cryoprotectant mass ratios CNP(70 : 30)
CNP(60 : 40)
CNP(50 : 50)
Polymer : cryoprotectant mass ratio
CNP(80 : 20)
P188
Treh
AS
TE
Sf / Si
AS
TE
Sf / Si
AS
TE
Sf / Si
AS
TE
Sf / Si
1:2 1:2 1 : 10 1 : 10
1:5 1 : 10 1:5 1 : 10
− − − −
+ + + +
1.5 1.7 1.2 1.1
− − − −
+ + + +
1.4 2.3 1.2 1.3
− − − −
+ + + +
1.5 1.6 1.3 1.2
− − − −
+ + + +
2.3 1.9 1.1 1.2
(AS) Aggregation scale: (−) absent; (+) scarce. (TE) Tyndall effect: (−) absent, (+) present. Sf / Si: ratio between the mean diameters, after and before lyophilization.
A. Layre et al. / Journal of Controlled Release 111 (2006) 271–280
Table 4 PIBCA, PCL10k–PEG2k pure nanoparticles and composite nanoparticles (weight ratios from 80 : 20 to 50 : 50) characteristics: Z-average mean diameter before and after purification, and drug loading efficiency Particle type
Particle diameter (nm)
PIBCA CNP(80 : 20) CNP(70 : 30) CNP(60 : 40) CNP(50 : 50) PCL10k–PEG2k
Before purification
After purification
176 ± 40 168 ± 37 142 ± 37 135 ± 32 151 ± 39 85 ± 28
169 ± 39 152 ± 36 136 ± 31 129 ± 37 147 ± 34 87 ± 23
Drug loading (% (w/w)) 5.9 ± 0.2 3.4 ± 0.3 2.3 ± 0.1 1.6 ± 0.2 1.7 ± 0.2 0.8 ± 0.2
100%
released busulfan (%)
278
90% 80% 70% 60% 50% 40% 0
200
300
400
time (min)
Each value is the average value from six different experiments ± SD.
explained by the amphiphilic characteristics of the PCL10k– PEG2k copolymer, reducing the interfacial tension between the aqueous and organic phases. The drug loading of the CNP was dependent on the PIBCA : PCL10k–PEG2k ratio (Table 4). For example, the drug loading of PCL10k–PEG2k nanoparticles was only 0.8 ± 0.2 (% (w/w)) whereas it increased to 3.4 ± 0.2 (% (w/w)) in the case of the CNP(80 : 20). The higher the amount of PIBCA in the PIBCA: PCL10k–PEG2k mixtures, the higher the drug loading. Thus, the busulfan loading of the composite nanoparticles was increased up to 4 folds in comparison to the drug loading of the PCL10k–PEG2k nanoparticles. Therefore, the use of PIBCA as “core” of the composite nanoparticles provided higher encapsulation efficiency. For example, the busulfan encapsulation efficiency of PCL10k–PEG2k nanoparticles was only 4.0 ± 0.2% whereas it was increased to 17.0 ± 0.2% (w/w) in the case of CNP(80 : 20). 3.8. In vitro release The in vitro release studies were performed in water or rat plasma (Fig. 4), under “sink” conditions with the CNP(50 : 50) suspensions containing 1.7 ± 0.2% (% (w/w)) busulfan. After a fast release of 70% (water) and 78% (plasma) of the entrapped drug during the first ten minutes, the remaining drug was
100%
released busulfan (%)
100
90%
Fig. 5. Busulfan release profiles from CNP(50 : 50) at various total busulfan concentration in the medium (busulfan both in nanoparticles and release medium). The experiments were performed in water at 37 °C. The busulfan concentration in the medium was: ♦ 7 μg/ml; 13 μg/ml; ▴ 27 μg/ml; 36 μg/ml.
▪
•
released slower over six hours. This profile may be explained by the fact that busulfan is a semi-polar drug (log P = −0.59) [39], which rapidly partitions in favour of the dispersion medium, accounting for the immediate release. To confirm this point, four additional experiments were performed in water at 37 °C with CNP(50 : 50) suspensions by varying the total busulfan content in the medium (nanoparticles and supernatant) (Fig. 5). The CNP(50 : 50) suspension containing 1.7 ± 0.2% (w/w) busulfan were appropriately diluted with water to achieve busulfan concentration in the medium from 7 to 36 μg/ml. All these concentrations are below the solubility value of busulfan in water (260 μg/ml). Under these conditions, when the busulfan concentration in the medium increased, the drug was released more slowly. Indeed, after a 2 h incubation, all the busulfan was released when the busulfan concentration in the release medium was 7 μg/ml, whereas only 75% of busulfan came out of the nanoparticles when the concentration in the release medium was 36 μg/ml (Fig. 5). It has been reported in the literature that busulfan is mainly taken up by liver in the first 10 to 30 min after intravenous or oral administration [40–42]. We therefore expect that despite the rapid busulfan release from the CNP, these carriers, potentially able to reduce liver uptake, would improve busulfan pharmacokinetics and allow the distribution of this drug towards other organs than the liver.
80%
4. Conclusion
70% 60% 50% 40% 0
50
100
150
200
time (min) Fig. 4. Busulfan release profiles from CNP(50 : 50) containing initially 1.7 ± 0.2% (w/w) drug were performed at 37 °C under “sink condition” in water (open square) or rat plasma (close diamond). Each value is derived from three different experiments ± SD.
The encapsulation of busulfan represents a challenge due to the physico-chemical characteristics of this molecule which crystallises spontaneously in water solution. In this paper, we have shown that novel core-shell composite nanoparticles can be prepared, making it possible to get significant drug loading ratio values. In addition, they may combine the advantages of the poly(isobutylcyanoacrylate) core for its encapsulation efficiency with the steric repulsive effect of the poly(ε-caprolactone)–poly(ethylene glycol)-surface layer, preventing rapid elimination from the blood stream.
A. Layre et al. / Journal of Controlled Release 111 (2006) 271–280
Acknowledgements The authors would like to acknowledge the French national research center (CNRS) and Ethypharm for their financial support, G. Frebourg and J.P. Lechaire from the Service Microscopie Electronique de l'IFR de Biologie Interactive (PARIS V) for the freeze-fracture observations, Dr. M. Chehimi from Interfaces, Traitements, Organisation et Dynamique des Systèmes (PARIS VII) for the XPS analysis and Dr Alain Chevailler from the Laboratoire d'Immunologie et Allergologie, Centre Hospitalo–Universitaire d'Angers for the supply of human serum.
References [1] C.D.R. Dunn, The chemical and biological properties of busulfan (‘Myleran’), Hemat 2 (1974) 101–117. [2] D.A.G. Galton, Myleran in chronic myeloid leukemia: results of treatment, Lancet 1 (1953) 208–213. [3] B.R. Blazar, N.K.C. Ramsay, J.H. Kersey, W. Krivit, D.C. Arthur, A.H. Filipovich, Pretransplant conditioning with busulfan (Myleran) and cyclophosphamide for nonmalignant diseases, Transplantation 39 (1985) 597–603. [4] M. Hassan, G. Oberg, A.N. Bekassy, J. Aschan, H. Ehrsson, P. Ljungman, G. Lonnerholm, B. Smedmyr, A. Taube, I. Wallin, et al., Pharmacokinetics of high-dose busulphan in relation to age and chronopharmacology, Cancer Chemother. Pharmacol. 28 (2) (1991) 130–134. [5] L.B. Grochow, R.J. Jones, R.B. Brundrett, H.G. Braine, T.L. Chen, R. Saral, G.W. Santos, O.M. Colvin, Pharmacokinetics of busulfan: correlation with veino-occlusive disease in patients undergoing bone marrow transplantation, Cancer Chemother. Pharmacol. 25 (1) (1989) 55–61. [6] H.P. Bhagwatwar, S. Phadungpojna, D.S.L. Chow, B.S. Andersson, Formulation and stability of busulfan for intravenous administration in high-dose chemotherapy, Cancer Chemother. Pharmacol. 37 (1996) 401–408. [7] U.S. Schuler, U.D. Renner, F. Kroschinsky, C. Johne, A. Jenke, R. Naumann, M. Bornhaüser, H.J. Deeg, G. Ehninger, Intravenous busulfan for conditioning before autologous or allogenic human blood stem cell transplantation, Br. J. Haematol. 114 (2001) 944–950. [8] G.L.J. Kennedy, H. Sherman, Acute and subchronic toxicity of dimethylformamide and dimethylacetamide following various routes of administration, Drug Chem. Toxicol. 9 (2) (1986) 147–170. [9] P. Yellowlees, C. Greenfield, N. McIntyre, Dimethylsulphoxide-induced toxicity, Lancet 2 (8202) (1980) 1004–1006. [10] Z. Hassan, C. Nilsson, M. Hassan, Liposomal busulfan: bioavailability and effect on bone marrow in mice, Bone Marrow Transplant. 22 (1998) 913–918. [11] J. Bouligand, Développement de Nanosphères Furtives de Busulfan, Université Paris V, Paris, 2001 (master report). [12] A.M. Layre, R. Gref, J. Richard, D. Requier, H. Chacun, M. Appel, A.J. Domb, P. Couvreur, Nanoencapsulation of a crystalline drug, Int. J. Pharm. 298 (2) (2005) 323–327. [13] A.M. Layre, P. Couvreur, H. Chacun, C. Aymes-Chodur, N.-E. Ghermani, J. Poupaert, J. Richard, D. Requier, R. Gref, Busulfan loading into poly (alkyl cyanoacrylate) nanoparticles: physico-chemistry and molecular modelling, J Biomed Mater Res Part B (Accepted for publication). [14] V. Lenaerts, J.F. Nagelkerke, T.J. Van Berkel, P. Couvreur, L. Grislain, M. Roland, P. Speiser, In vivo uptake of polyisobutyl cyanoacrylate nanoparticles by rat liver Kupffer, endothelial, and parenchymal cells, J. Pharm. Sci. 73 (7) (1984) 980–982. [15] M.T. Peracchia, C. Vauthier, C. Passirani, P. Couvreur, D. Labarre, Complement consumption by poly(ethylene glycol) in different conformations chemically coupled to poly(isobutyl 2-cyanoacrylate) nanoparticles, Life Sci. 61 (7) (1997) 749–761.
279
[16] R. Gref, Y. Minamitake, M.T. Peracchia, V. Trubetskoy, V. Torchilin, R. Langer, Biodegradable long-circulating polymeric nanospheres, Science 263 (1994) 1600–1603. [17] S.J. Douglas, S.S. Davis, L. Illum, Biodistribution of poly(butyl 2-cyanoarcrylate) nanoparticles in rabbits, Int. J. Pharm. 34 (1–2) (1986) 145–152. [18] V. Lenaerts, P. Couvreur, D. Christiaens-Leyh, E. Joiris, M. Roland, B. Rollman, P. Speiser, Degradation of poly(isobutyl cyanoacrylate) nanoparticles, Biomaterials 5 (2) (1984) 65–68. [19] M.T. Peracchia, C. Vauthier, F. Puisieux, P. Couvreur, Development of sterically stabilized poly(isobutyl 2-cyanoacrylate) nanoparticles by chemical coupling of poly(ethylene glycol), J. Biomed. Mater. Res. 34 (3) (1997) 317–326. [20] J.L. Grangier, M. Puygrenier, J.C. Gautier, P. Couvreur, Nanoparticles as carriers for growth hormone releasing factor, J. Control. Release 15 (1) (1991) 3–13. [21] M. Simeonova, R. Velichkova, G. Ivanova, V. Enchev, I. Abrahams, Poly (butylcyanoacrylate) nanoparticles for topical delivery of 5-fluorouracil, Int. J. Pharm. 263 (1–2) (2003) 133–140. [22] M.T. Peracchia, D. Desmaële, P. Couvreur, J. d'Angelo, Synthesis of novel poly(MePEG cyanoacrylate–co-alkyl cyanoacrylate) amphiphilic copolymer for nanoparticle technology, Macromolecules 30 (4) (1997) 846–851. [23] P. Calvo, B. Gouritin, H. Chacun, D. Desmaele, J. D'Angelo, J.P. Noel, D. Georgin, E. Fattal, J.P. Andreux, P. Couvreur, Long-circulating PEGylated polycyanoacrylate nanoparticles as new drug carrier for brain delivery, Pharm. Res. 18 (8) (2001) 1157–1166. [24] D. Bazile, C. Prud'homme, M.-T. Bassoullet, M. Marlard, G. Spenlehauer, M. Veillard, Stealth Me.PEG–PLA nanoparticles avoid uptake by the mononuclear phagocytes system, J. Pharm. Sci. 84 (4) (1995) 493–498. [25] R. Gref, M. Luck, P. Quellec, M. Marchand, E. Dellacherie, S. Harnisch, T. Blunk, R.H. Muller, ‘Stealth’ corona-core nanoparticles surface modified by polyethylene glycol (PEG): influences of the corona (PEG chain length and surface density) and of the core composition on phagocytic uptake and plasma protein adsorption, Colloids Surf., B. Biointerfaces 18 (3–4) (2000) 301–313. [26] A. Layre, R. Gref, P. Couvreur, J. Richard, Nanoparticules polymériques composites, FR 2872703, July 7, 2004. [27] H.C. Fessi, J.P. Devissaguet, Process for the preparation of dispersible colloidal systems of a substance in the form of nanoparticles, US Patent 5,118,528, June 2, 1992. [28] M. Vittaz, D. Bazile, G. Spenlehauer, T. Verrecchia, M. Veillard, F. Puisieux, D. Labarre, Effect of PEO surface density on long-circulating PLA–PEO nanoparticles which are very low complement activators, Biomaterials 17 (16) (1996) 1575–1581. [29] M.D. Kazatchkine, G. Hauptmann, U.E. Nydegger, Technique du complement, INSERM (1986) 22–23. [30] C. Passirani, G. Barratt, J.-P. Devissaguet, D. Labarre, Interactions of nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate) with the complement system, Life Sci. 62 (8) (1998) 775–785. [31] C. Vauthier, C. Schimdt, P. Couvreur, Measurement of the density of polymeric nanoparticulate drug carriers by isopycnic centrifugation, J. Nanopart. Res. 1 (3) (1999) 411–418. [32] J. Groll, Z. Ademovic, T. Ameringer, D. Klee, M. Moeller, Comparison of coatings from reactive star shaped PEG-stat-PPG prepolymers and grafted linear PEG for biological and medical applications, Biomacromolecules 6 (2) (2005) 956–962. [33] P. Quellec, R. Gref, L. Perrin, E. Dellacherie, F. Sommer, J.M. Verbavatz, M.J. Alonso, Protein encapsulation within polyethylene glycol-coated nanospheres. I. Physicochemical characterization, J. Biomed. Mater. Res. 42 (1) (1998) 45–54. [34] E. Allemann, P. Gravel, J.C. Leroux, L. Balant, R. Gurny, Kinetics of blood component adsorption on poly(D,L-lactic acid) nanoparticles: evidence of complement C3 component involvement, J. Biomed. Mater. Res. 37 (2) (1997) 229–234. [35] C. Passirani, J.P. Benoit, in: R.I. Mahato (Ed.), Biomaterials for Delivery and Targeting of Proteins and Nucleic Acids, 2005, pp. 187–229.
280
A. Layre et al. / Journal of Controlled Release 111 (2006) 271–280
[36] M.F. Zambaux, F. Bonneaux, R. Gref, E. Dellacherie, C. Vigneron, MPEO–PLA nanoparticles: effect of MPEO content on some of their surface properties, J. Biomed. Mater. Res. 44 (1) (1999) 109–115. [37] S.I. Jeon, J.D. Andrade, Protein–surface interactions in the presence of polyethylene oxide: II. Effect of protein size, J. Colloid Interface Sci. 142 (1) (1991) 159–166. [38] S.I. Jeon, J.H. Lee, J.D. Andrade, P.G. De Gennes, Protein–surface interactions in the presence of polyethylene oxide: I. Simplified theory, J. Colloid Interface Sci. 142 (1) (1991) 149–158. [39] G.R. Westerhof, R.E. Ploemacher, A. Boudewijn, I. Blokland, J.H. Dillingh, A.T. McGown, J.A. Hadfield, M.J. Dawson, J.D. Down, Comparison of different busulfan analogues for depletion of hematopoietic stem cells and promotion of donor-type chimerism in murine bone marrow transplant recipients, Cancer Res. 60 (19) (2000) 5470–5478.
[40] M. Hassan, G. Oberg, K. Ericson, H. Ehrsson, L. Eriksson, M. Ingvar, S. Stone-Elander, J.O. Thorell, B. Smedmyr, N. Warne, et al., In vivo distribution of [11C]-busulfan in cynomolgus monkey and in the brain of a human patient, Cancer Chemother. Pharmacol. 30 (2) (1992) 81–85. [41] J.T. Slattery, Letter, Biol. Blood Marrow Transplant. 9 (4) (2003) 282–284. [42] A. Kashyap, J. Wingard, P. Cagnoni, J. Roy, S. Tarantolo, W. Hu, K. Blume, J. Niland, J.M. Palmer, W. Vaughan, H. Fernandez, R. Champlin, S. Forman, B.S. Andersson, Intravenous versus oral busulfan as part of a busulfan/cyclophosphamide preparative regimen for allogeneic hematopoietic stem cell transplantation: decreased incidence of hepatic venoocclusive disease (HVOD), HVOD-related mortality, and overall 100-day mortality, Biol. Blood Marrow Transplant. 8 (9) (2002) 493–500.
