International Journal of Pharmaceutics 485 (2015) 357–364
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Self-assembled filomicelles prepared from polylactide/poly(ethylene glycol) block copolymers for anticancer drug delivery Katarzyna Jelonek a, * , Suming Li b, ** , Xiaohan Wu c, Janusz Kasperczyk a,d, Andrzej Marcinkowski a a
Centre of Polymer and Carbon Materials, Polish Academy of Sciences, Curie-Sklodowska 34 Street, 41-819 Zabrze, Poland European Institute of Membranes, UMR CNRS 5635, University Montpellier 2, Place Eugene Bataillon, 34095 Montpellier Cedex 5, France Max Mousseron Institute on Biomolecules, UMR CNRS 5247, University Montpellier 1, 34090 Montpellier Cedex 5, France d School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Katowice, Poland, Department of Biopharmacy, Jednosci 8, Sosnowiec, Poland b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 January 2015 Received in revised form 13 March 2015 Accepted 16 March 2015 Available online 18 March 2015
Bioresorbable filomicelles present many advantageous as drug delivery systems e.g., long circulation time and high loading efficiency. The aim of this study was to develop polylactide/poly(ethylene glycol) (PLA/PEG) filomicelles for drug delivery applications. A series of PLA/PEG diblock copolymers were synthesized using non-toxic initiator, and characterized by means of NMR and GPC. Analysis of morphology of micelles determined by TEM revealed that apart from the weight fraction also the molar mass of PEG and the stereochemistry of PLA block must be considered for tailoring micellar structures. The CMC was found to be dependent on the length and structure of the hydrophobic block. It was observed that the drug loading properties could be improved by selection of appropriate copolymer and encapsulation method. Slower release of paclitaxel was observed for mPEG5000 initiated copolymers than mPEG2000 initiated copolymers. Moreover, the influence of the length of hydrophobic block and its stereoisomeric form on drug release rate was evidenced. Therefore, PLA/PEG filomicelles with good stability, high drug loading capacity and sustained drug release appear most attractive for drug delivery applications. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Polymeric micelles Filomicelles Paclitaxel PLA/PEG Drug delivery
1. Introduction Amphiphilic block copolymers are able to self-assemble in water environment to form various aggregates, including spherical micelles, rod-like micelles, filomicelles (also called worm-like micelles) and polymerosomes (Rajagopal et al., 2010). The evolution of distinct morphologies has been reported to depend on different factors such as copolymer chain structure, polymer concentration, solvent, salt concentration and presence of small molecule stabilizers. The physical properties of these aggregates such as size, size distribution and morphology influence their stability, drug loading and release properties, as well as in vivo pharmacokinetics and biodistribution (Fairley et al., 2008). Compared to surfactant micelles, block copolymer-based micelles are characterized by higher thermodynamic stability,
* Corresponding author. Tel.: +48 32 2716077; fax: +48 32 2712969. ** Corresponding author. Tel.: +33 467 149 121. E-mail addresses: [email protected] (K. Jelonek), [email protected] (S. Li). http://dx.doi.org/10.1016/j.ijpharm.2015.03.032 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
and larger versatility for controlling micellar structure and functionality by choices of polymer composition, architecture, molecular weight and monomer chemistry (Loverde et al., 2010; Nishiyama and Kataoka, 2006a; Zhu and Hayward, 2008). This makes them most attractive for novel applications in drug delivery. Among the various aggregates, flexible worm-like “filomicelles” – in analogy to filoviruses – possess a long circulation time up to a week in the bloodstream because their unique visco-elastic properties and hydrodynamics could reduce interactions with the blood vessel walls (Loverde et al., 2011; Venkataraman et al., 2011). Moreover, filomicelles display almost twice higher drug loading capacity as compared to spherical micelles due to larger core volume per carrier (Cai et al., 2007). Amphiphilic block copolymers composed of a hydrophilic block such as poly(ethylene glycol) (PEG) and a degradable and hydrophobic polyester block such as polylactide (PLA) or poly (e-caprolactone) (PCL) have been extensively investigated as potential drug carrier (Branco and Schneider, 2009; Geng and Discher 2006; Yang et al., 2010). PEG is commonly used in biomedical applications due to its outstanding physicochemical and biological properties including solubility in water and in
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organic solvents, non-toxicity and resistance to protein adsorption and cellular adhesion, which minimizes the detection by the immune system and consequently increases the plasma half-life of micelles (Batrakova et al., 2006 Heald et al., 2002; Hu et al., 2003; Yang et al., 2009). It has been demonstrated that long-circulating drug carriers can preferentially and efficiently accumulate in solid tumors due to the enhanced permeability and retention (EPR) effect (Nishiyama and Kataoka, 2006b). Paclitaxel is one of the most efficient cytostatic presenting activity against a wide variety of tumors, including ovarian, metastatic breast cancer, non-small cell lung cancer, head and neck malignancies and others (Kim et al., 2001; Singla et al., 2002; Yang et al., 2009). The current challenge is to formulate paclitaxel in a nontoxic vehicle so as to eliminate the toxicity of Cremophor EL, the currently used formulation vehicle. The usefulness of PLA/PEG spherical micelles as a paclitaxel carrier has already been confirmed (Yang et al., 2009). Significant improvement would be obtained by development of PLA/PEG filomicelles with this drug. Therefore, understanding how the structure of micelles obtained from amphiphilic copolymers may be controlled by specific parameters is still a major challenge (Loverde et al., 2011). Dalhaimer et al. (2003) observed that assembly of PCL–PEG diblock copolymers into particular microstructures depends primarily on the weight fraction of PEG block relative to the total polymer weight. Formation of worm-like micelles was observed for copolymers with PEG weight fraction in the range from 45% to 55% (Dalhaimer et al., 2003). Nevertheless, other factors such as copolymer concentration and preparation method could also affect the morphology of aggregates. Fairley et al. (2008) found that the amphiphilic copolymer PCL(4100)/PEG(5000) formed different morphologies in aqueous solution, including long rod-like, short rod-like, or spherical aggregates with increasing copolymer concentration. Giacomelli and Borsali (2006) reported that PCL–PEG copolymers copolymers with PCL volume fractions ranging from 0.35 to 0.58 could form spherical or worm-like micelles when a solvent was used before micellization. However, PLA–PEG diblock filomicelles have been rarely considered, so far. In the present study, the influence of copolymer composition (weight ratio of EO moieties), molar mass of mPEG and preparation method on the morphology of micelles was analyzed. The potential of micelles for encapsulation and release of paclitaxel was also evaluated. 2. Materials and methods 2.1. Materials L-, D-, and DL-lactides were obtained from Purac Biochem (Goerinchem, The Netherlands) and purified by crystallization from ethyl acetate. Monomethoxy poly(ethylene glycol) (mPEG) with molar masses of 2000 or 5000, and zinc lactate were purchased from Sigma–Aldrich (St-Quentin Fallavier, France). All other organic solvents were of analytic grade from Sigma–Aldrich and used without further purification. Paclitaxel was purchased from LC Laboratories (Woburn, MA). 2.2. Synthesis and characterization of PLA/PEG copolymers A series of PLA/PEG diblock copolymers were synthesized by ring opening polymerization of lactide with mPEG using zinc lactate as non-toxic initiator as previously described (Yang et al., 2010). Briefly, predetermined amounts of mPEG and lactide were introduced into a polymerization tube, and sealed under vacuum. Polymerization then proceeded for 3 days at 140 C. The product was recovered by dissolution in dichloromethane and precipitation in diethyl ether, and dried under vacuum to constant weight.
The composition of copolymers was determined by means of proton nuclear magnetic resonance (1H NMR) recorded at Bruker spectrometer (250 MHz) using CDCl3 as a solvent. Chemical shifts (d) were given in ppm using tetramethylsilane as an internal reference. The molar mass and molar mass distribution of block copolymers were measured by gel permeation chromatography (Waters 410) equipped with an RI detector. Tetrahydrofuran (THF) was used as the mobile phase at a flow rate of 1.0 mL/min. 20 mL of each polymer solution at a concentration of 10 g/L was injected for analysis. 2.3. Preparation of PLA/PEG micelles Micelles were prepared by co-solvent evaporation method according to the procedure described by Cai et al. (2007). Briefly, 5 mg of polymer was dissolved in 100 mL of chloroform. 5 mL of distilled water was then added. In case of micelles obtained from mixture of D-PLA/PEG and L-PLA/PEG, equal amounts of both polymers were used. The solution was stirred vigorously at room temperature for 3.5 h and left for solvent evaporation for 24 h. The resulting micellar solution was finally filtered through 0.85 mm filter and stored at 4 C. Micelles were also prepared using dialysis method. 10 mg of polymer was dissolved in 1 mL of THF and the solution was transferred into a dialysis bag. The polymer solution was dialyzed for 48 h under stirring against 1 L of water which was frequently renewed. The samples were diluted with distilled water to a concentration of 1 g/L, filtered through 0.85 mm filter and stored at 4 C. 2.4. Characterization of PLA/PEG micelles The morphology of micelles was observed by using transmission electron microscopy (TEM) performed on Hitachi H-600 microscope, operating at an accelerating voltage of 75 kV. 5 mL of micellar solution was placed on a copper grid covered with nitrocellulose membrane, stained negatively with 2% phosphotungstic acid (PTA) and air dried at room temperature before measurements. The morphology of selected micelles was also analyzed using multimode atomic force microscopy (AFM) instrument (MultiMode, di-Veeco, USA, CA) with NanoScope 3D operating in the tapping mode in air with standard 125 mm single-crystal silicon cantilevers (Model TESP; Bruker; USA, CA). The 1.0 g/L micellar solution was 20 times diluted. A drop of the diluted solution was placed on a silica matrix and air dried overnight before measurement at room temperature. The software package WSxM (Nanotec Electronica) was used for image processing. Dynamic light scattering (DLS) measurements were conducted at 25 C with 90 scattering angle using Malvern Zetasizer Nano spectrometer (Malvern Instrument, Worcs, UK). The critical micelle concentration (CMC) of copolymers was measured by fluorescence spectroscopy method (Hitachi F-2500) using pyrene as fluorescence probe (Dominguez et al., 1997). Pyrene was excited at 334 nm and its emission was recorded at 375 nm and 395 nm at polymer concentrations ranging from 9.7 104 to 0.5 mg/mL and constant concentration of pyrene (2.0 106 M). The CMC values were estimated from the cross-over point of the plots of the intensity ratio (I375/I395) from pyrene excitation spectra vs. log C (where C is concentration in mg/mL). 2.5. In vitro drug release Two methods were applied for paclitaxel encapsulation to micelles obtained by co-solvent evaporation method. In method 1, predetermined amount of paclitaxel in methanol was added to drug
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free micelles and stirred for 4.5 h. In method 2, drug incorporation and micelles formation were realized simultaneously: paclitaxel in chloroform was added to a copolymer solution in chloroform, mixed with water and stirred for 4.5 h. In both methods, PLA/PEG micelles were prepared with a theoretical loading of 10% or 20%. Paclitaxel-loaded micelles were centrifuged at 3000 rpm for 5 min. The supernatants were recovered and the sediments were used to determine the amount of paclitaxel which was not encapsulated in micelles (free paclitaxel). The drug loading content (LC) and drug encapsulation efficiency (EE) were calculated according to the following equations (Yang et al., 2009): total paclitaxel amount free paclitaxel amount LC ¼ 100 amount of paclitaxel loaded micelle
EE ¼
total paclitaxel amount free paclitaxel amount 100 total paclitaxel amount
Drug loaded micellar solutions were introduced in a dialysis membrane (MWCO = 3500). Drug release was realized under in vitro conditions at 37 C in phosphate buffered saline (PBS) at pH 7.4. At the predetermined time intervals, the release medium was sampled and fresh PBS was added to maintain the initial volume. The amount of paclitaxel was measured by using high performance liquid chromatography (HPLC). A SunFireTM C18 column (4.6 150 mm, particle size 5 mm, Waters) was used and UV detector set at 227 nm. The mobile phase was 55:45 (v/v) HPLC grade water–acetonitrile. The flow rate was set at 1.0 mL/min and the total analysis time was 8 min. A linear calibration curve was previously obtained using 10 standard solutions in the range of 0.25–1000 mg/mL (R2 = 0.996). 3. Results and discussion 3.1. Characterization of PLA/PEG copolymers A series of PLA/PEG diblock copolymers were synthesized using zinc lactate as non-toxic initiator. Polymers are named as LxEOy, DxEOy or DLxEOy where L, D and DL represent L-PLA, D-PLA or DL-PLA blocks, respectively, x and y designate the number-average degree of polymerization (DP) of corresponding blocks. The composition of copolymers, including the EO/LA molar ratio and DP values were obtained from the integrations of NMR resonances
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belonging to the methylene protons of PEG at 3.6 ppm and to the methine proton of PLA at 5.2 ppm, as previously described (Li and Vert, 2003). The molar mass of copolymers was determined by means of GPC and NMR. Six mPEG2000-initiated (LAxEO45) and four mPEG5000-initiated (LAxEO114) diblock copolymers were synthesized. The characteristics of the various copolymers are presented in Table 1. The copolymers were obtained in a wide range of EO/LA ratio from 0.62 to 3.22. The weight fraction of EO (fEO) in mPEG2000 and mPEG5000initiated copolymers ranges from 0.28 to 0.60, and from 0.45 to 0.56, respectively. According to the literature, copolymers with fEO around 0.45 are susceptible to form wormlike micelles (Dalhaimer et al., 2003). 3.2. Characteristics of self-assembling structures 3.2.1. Micelle morphology Polymeric micelles were prepared by means of co-solvent evaporation method and characterized in terms of morphology and stability. A wide range of copolymers were used so as to determine the influence of polymer structure, Mw of PEG and fEO on micelles morphology. Micelles were prepared from both LAxEO45 and LAxEO114 copolymers with various fEO values (Table 1). L22EO45 and D21EO45 were also used as a mixture for micelles preparation. According to the literature, mixed micelles containing two PLA stereoisomers exhibit higher stability due to stronger interactions between L-PLA and D-PLA blocks (Yang et al., 2007). Thus, higher drug-loading efficiency was expected for mixed micelles than for single micelles. L85EO114 and DL80EO114 were compared to determine the effect of chirality of the hydrophobic blocks on micellar morphology. The morphology of micelles was observed using TEM. Filomicelles were obtained from mPEG2000-initiated copolymers in a wide range of fEO (0.28–0.55) as shown in Fig. 1. Two copolymers were not considered for further study: the most hydrophobic L73EO45 (Fig.1A) which precipitated during preparation making this process not effective, and the most hydrophilic L18EO45 which yielded spherical micelles only (Fig. 1F). Coexistence of filomicelles and spherical micelles was observed for L41EO45 (Fig. 1B) and L28EO45 (Fig. 1C). Studies of Rajagopal et al. (2010) on PCL–PEG micelles revealed that coexistence of several morphologies is a common phenomenon in transition regions. In fact, with decrease
Table 1 Characteristics of PLA/PEG diblock copolymers. Copolymer
MnPEG
EO/LAa
DPPEGb
DPPLAc
Mn(NMR)d
Mn(GPC)e
fEO
CMCf (mg/mL)
L73EO45 L41EO45 L28EO45 L22EO45 D21EO45 L22EO45 + D21EO45 L18EO45 L85EO114 DL80EO114 L63EO114 L54EO114
2000 2000 2000 2000 2000 – 2000 5000 5000 5000 5000
0.62 1.10 1.58 2.00 2.05 – 3.22 1.33 1.42 1.80 2.09
45 45 45 45 45 – 45 114 114 114 114
73 41 28 22 22 – 18 85 80 63 54
7200 4900 4048 3600 3500 – 3300 11 100 10 770 9500 8900
5900 4950 4100 3650 4700 – 3800 11 300 11 300 12500 12000
0.28 0.40 0.49 0.55 0.56 – 0.60 0.45 0.46 0.52 0.56
0.004 0.004 0.004 0.006 – 0.006 0.006 0.003 0.004 0.005 0.005
a b c d e f
Calculated from the integrations of NMR bands belonging to PEG blocks at 3.6 ppm and to PLA blocks at 5.2 ppm. DPPEG = MnPEG/44. DPPLA = DPPEG/(EO/LA). Mn = MnPEG + DPPLA 72. Determined by GPC. Determined by fluorometry at 25 C.
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Fig. 1. TEM images of LAxEO45 micelles: L73EO45 (A), L41EO45 (B), L28EO45 (C), L22EO45 (D), L22EO45 + D21EO45 (E) and L18EO45 (F).
of the fEO, the morphology turns from spherical micelles to filomicelles, and then to bilayered polymersomes (Wu et al., 2013). It is of interest to note that the coexistence region is much larger than the region of exclusive filomicelle formation (fEO 0.5–0.55), as observed for strongly segregating poly(ethylene oxide)-b-poly (1,2-butadiene (PEO-b-PBD) copolymers (Rajagopal et al., 2010). L22EO45 copolymer formed exclusively filomicelles (Fig. 1D). Interestingly, mixture of L22EO45 and D21EO45 also yielded filomicelles, but the micelle size was not uniform (Fig. 1E). In the group of mPEG5000-initiated copolymers (LAxEO114) (Fig. 2), formation of filomicelles was observed for L85EO114 (Fig. 2A). More spherical micelles coexisted with filomicelles in the case of L63EO114 and L54EO114 with shorter L-PLA blocks (Fig. 2B and C). Surprisingly, filomicelles and spherical micelles were almost exclusively observed in different regions of L63EO114 (Fig. 2B). It is also of interest to compare the micellar morphologies of copolymers with similar PLA block lengths but different lactide stereoisomers (Fig. 2A and Fig. 3). Only spherical forms were observed for DL80EO114 (Fig. 3), in contrast to L85EO114 which mainly formed filomicelles. These findings were confirmed by AFM analysis (Fig. 4). Therefore, polymer chain stereoregularity seems to strongly affect the micelle’s morphology. In fact, stereoregular
L-PLA blocks are more rigid than DL-PLA ones with randomly distributed L- and D-lactidyl units, and rigid blocks seem to be more inclined to form filomicelles. Dialysis was also used to prepare micelles in comparison with co-solvent evaporation method. Fig. 5 shows that similar filomicelles are obtained by dialysis method, and spherical micelles are also present. In the case of DL80EO114 (Fig. 5D), only spherical micelles are obtained as with co-solvent evaporation method. Moreover, mPEG2000 derived LAxEO45 copolymers present the same behaviors (data not shown). 3.2.2. Critical micelle concentration The critical micelle concentration (CMC) is an important parameter that influences the in vivo performance of micelles, in particular the micellar stability after intravenous injection. The CMC of copolymers was determined using fluorescence spectroscopy with pyrene as a hydrophobic probe. Fig. 6 shows the I375/I395 vs. log C plots of L85EO114 copolymer. A CMC value of 0.003 mg/mL was obtained from the cross-over point of the plots. Dependence of the CMC on the length of hydrophobic block was observed for both mPEG2000 and mPEG5000 initiated copolymers (Table 1). Thus, copolymers with longer PLA blocks exhibit lower CMC values. This
Fig. 2. TEM images of LAxEO114 micelles by co-solvent evaporation method: L85EO114 (A), L63EO114 (B1, B2) and L54EO114 (C).
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Fig. 3. TEM image (A) and DLS graph (B) of DL80EO114 micelles.
finding is consistent with literature data, assigning longer hydrophobic blocks with better self-assembling properties and lower CMC values (Liggins and Burt, 2002; Yasugi et al., 1999). In fact, entanglement of longer hydrophobic blocks in the inner core may provide higher structural stability of micelles. No difference in CMC was found for single micelles of L22EO45 and mixed micelles of L22EO45 + D21EO45, in contrast to reports of Yang et al. (2007). This might be caused by the different preparation methods of micelles. In Yang’s work, micelles were prepared by direct dissolution of copolymers in water, in contrast to co-solvent evaporation used in this work. It must be emphasized that the CMC values of all copolymers are very low (3–6 mg/mL), which ensures the micellar stability after administration in the body and dilution in bloodstream. These values are comparable to those of other copolymers that generally show very low CMC values ranging from 1 mg/mL to 10 mg/mL (Yokoyama, 2011), and are much lower than typical CMC values of low molar mass surfactants (Zhu and Hayward, 2008). 3.3. Drug encapsulation properties of PLA/PEG micelles The hydrophobic core of polymeric micelles allows to incorporate drugs of poor water solubility and serves as nano-reservoir, while the outer hydrophilic corona provides biocompatibility and prolonged circulation in blood by avoiding rapid clearance by the liver and spleen after intravenous administration (Geng and Discher, 2006; Nishiyama and Kataoka, 2006a). The encapsulation properties of different PLA/PEG copolymers were evaluated, using paclitaxel as a hydrophobic model drug.
3.3.1. Optimization of drug encapsulation method Preliminary studies were conducted with L85EO114 in order to select an appropriate method for effective incorporation of paclitaxel in the core of micelles. Two encapsulation methods based on co-solvent evaporation method were compared. Method 1 consists in addition of drug solution in previously prepared micelles, while method 2 involves simultaneous micellar formation and drug incorporation. 10% or 20% of theoretical drug content was applied. As shown in Table 2, the EE and LC values were higher for micelles obtained by method 1, in contrast to Cai's work (Cai et al., 2007) which showed no difference of encapsulation efficiency between the two methods for PCL–PEG micelles. Moreover, 10% theoretical drug load yielded higher EE and LC values as compared to 20%. Therefore, method 1 with 10% theoretical drug loading was selected for paclitaxel encapsulation in further studies. 3.3.2. Comparison of drug loading properties of different PLA/PEG micelles In the next step, drug loading properties were evaluated for all the PLA/PEG micelles as presented in Table 3. In the case of mPEG2000 initiated copolymers, the EE and LC depended on the length of PLA blocks. Micelles with longer PLA block length led to higher EE and LC. On the other hand, mixed micelles of L22EO45 and D21EO45 showed much better drug loading properties than single L22EO45 micelles. This finding is consistent with literature data (Yang et al., 2009). In fact, mixed micelles containing both L-PLA/PEG and D-PLA/PEG copolymers with more compact core structure could favor drug encapsulation.
Fig. 4. AFM images of micelles obtained from L85EO114 (A) and DL80EO114 (B).
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Fig. 5. TEM images of LAxEO114 micelles prepared by using dialysis method: L85EO114 (A), L63EO114 (B), L54EO114 (C) and DL80EO114 (D).
Micelles of mPEG5000 initiated copolymers present better drug loading properties than those of mPEG2000 initiated copolymers. Similar findings were observed for PCL/PEG worm-like micelles (Cai et al., 2007). Interestingly, no dependence of drug loading on the PLA block length is observed (Table 3). On the other hand, much lower EE and LC values were obtained for spherical micelles obtained from DL80EO114 micelles than for L85EO114 filomicelles. This difference could be assigned to the fact that filomicelles present higher drug encapsulation potential than spherical micelles (Cai et al., 2007). 3.3.3. In vitro drug release Paclitaxel release from the various micellar systems was studied under in vitro conditions. Figs. 7 and 8 present the release
profiles of paclitaxel from mPEG2000 and mPEG5000 initiated copolymer micelles, respectively. Less than 35% of drug was released during 71 days in all cases. However, differences in drug release rates were noticed. Paclitaxel release was faster from mPEG2000 initiated copolymers than from mPEG5000 initiated copolymers and was faster for copolymers with shorter PLA blocks in both groups of micelles. The slowest release was observed for L44EO45 and L85EO114 with the longest PLA blocks in both groups. On the other hand, paclitaxel release was slower from mixed micelles of L22EO45 and D21EO45 than from L22EO45 single micelles, 12.6% and 21.6% of drug being released in 71 days, respectively. This finding might be assigned to the more compact core structure of mixed micelles which disfavors drug diffusion. 30.6% release was observed for spherical micelles of DL80EO114 in 71 days, in contrast to 9.8% for L85EO114 filomicelles. The release profile was similar for all the micellar systems, which is characterized by a gradual initial release and an increase of release rate after 40 days. The acceleration of drug release was probably caused by progress of degradation of the PLA core.
Table 2 Encapsulation efficiency and loading content data of paclitaxel in L85EO114 micelles.
Fig. 6. Plots of I375/I395 vs. log C of L85EO114.
Method
Initial drug loading (wt.%)
EE (%)a
LC (%)a
1 1 2 2
10 20 10 20
69.4 6.0 64.3 13.7 51.8 6.2 42.5 4.2
6.5 0.5 11.3 2.1 4.9 0.6 7.8 0.7
a
Data represent mean value S.D., n = 3
K. Jelonek et al. / International Journal of Pharmaceutics 485 (2015) 357–364 Table 3 Encapsulation efficiency and loading content data of paclitaxel in different PLA/PEG micelles. Copolymer
EE (%)a
LC (%)a
L41EO45 L28EO45 L22EO45 L22EO45 + D21EO45 L85EO114 DL81EO114 L63EO114 L54EO114
57.8 4.3 49.9 6.4 48.3 11.6 65.0 4.6 69.4 6.0 52.3 6.0 68.2 8.7 69.0 3.5
5.5 0.4 4.7 0.6 4.7 0.6 6.1 0.4 6.5 0.5 5.0 0.6 6.4 0.8 6.4 0.3
a
Data represent mean value S.D., n = 3.
Generally, the process of drug release proceeded very slowly for all copolymers. This finding could be assigned to the morphology of the micelles. In fact, it was reported that filomicelles provide longer drug release than spherical micelles (Cai et al., 2007). As shown by Yang et al. (2007), even spherical PLA/PEG micelles may provide sustained drug release for over 30 days. The chemical structures and properties of the micellar core-forming blocks significantly affect the drug loading efficiency and drug release rate (Branco and Schneider, 2009). In this study, L-, D- or DL-PLA was used as the hydrophobic block. A solid-like inner core was formed inside micelles (Heald et al., 2002). Copolymers with relatively long PLA blocks provided effective entrapment of drug molecules that remained in the core of micelles for longer time. The fact that mixed micelles exhibit slower release than single micelles, and
Fig. 7. In vitro release of paclitaxel from micelles obtained from mPEG2000 initiated copolymers (S.D. shown as error bars, n = 3).
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that L85EO114 filomicelles exhibit slower release than DL80EO114 spherical micelles could be assigned to the difference of core structure or core stability. 4. Conclusion This work aimed to elucidate the influence of PLA/PEG diblock copolymer composition, molar mass of mPEG and preparation method on the morphology, CMC, drug encapsulation and drug release properties of micelles. Analysis of micellar morphology determined by TEM revealed that apart from the weight fraction of PEG block (fEO), also the molar mass of PEG was an important factor that affects selfassembling structures. Formation of filomicelles was observed at fEO 0.55 for mPEG2000-initiated copolymers, and at fEO 0.45 for mPEG5000-initiated copolymers. However, differences in morphology of L85EO114 and DL81EO114 micelles indicated that the stereochemistry of PLA block must be considered for tailoring micellar structures. In contrast, preparation method (co-solvent evaporation and dialysis) did not affect the structure of micelles. The low CMC values of all copolymers (3–6 mg/mL) ensure the stability of micelles after administration in the body and dilution in bloodstream. Dependence of the CMC on the PLA block length was observed. The lowest CMC values were obtained for copolymers with the longest PLA blocks. The copolymer composition, PLA block length, mixed or single micelles, and PLA stereochemistry are identified as factors influencing drug encapsulation and drug release properties of PLA/PEG micelles. Micelles with longer PLA blocks lead to higher encapsulation efficiency and load content for mPEG2000 initiated copolymers. In contrast, drug loading properties did not depend on the PLA block length for mPEG5000 initiated copolymers. The latter presents better drug loading properties than the former. On the other hand, spherical micelles of DL81EO114 micelles present lower encapsulation efficiency and load content as compared to L85EO114 filomicelles. Paclitaxel release was faster from mPEG2000 initiated copolymers than from mPEG5000 initiated copolymers, and was faster for copolymers with shorter PLA blocks in both groups of micelles. Mixed micelles exhibit slower release rate than single micelles due to the more compact core structure of mixed micelles which disfavors drug diffusion. Faster release was observed for DL80EO114 than for L85EO114 micelles. Therefore, filomicelles prepared from PLA/PEG dilbock copolymers are promising for prolonged delivery of hydrophobic antitumor drugs. Acknowledgements This study was conducted in the frame of postdoc fellowship supported by Polish Ministry of Higher Education “Mobility Plus” and realized at the Institut des Biomolecules Max Mousseron, UMR CNRS 5247, University of Montpellier I, France. The authors thank Dr. Josephine Laikeehim of Centre de Biochimie Structurale (Montpellier, France) for TEM measurements. References
Fig. 8. In vitro release of paclitaxel from micelles obtained from mPEG5000 initiated copolymers (S.D. shown as error bars, n = 3).
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Nishiyama, N., Kataoka, K., 2006b. Current state achievements, and future prospects of polymeric micelles as nanocarriers for drug and gene delivery. Pharmacol. Ther. 112, 630–648. Rajagopal, K., Mahmud, A., Christian, D.A., Pajerowski, J.D., Brown, A.E.X., Loverde, S. M., Discher, D.E., 2010. Curvature-coupled hydration of semicrystalline polymer amphiphilies yields flexible worm micelles but favors rigid vesicles: polycaprolactone-based block copolymers. Macromolecules 43, 9736–9746. Singla, A.K., Garg, A., Aggarwal, D., 2002. Paclitaxel and its formulations. Int. J. Pharm. 235, 179–192. Venkataraman, S., Hedric, J.L., Ong, Z.Y., Yang, C., Ee, P.L.R., Hammond, P.T., Yang, Y.Y., 2011. The effects of polymeric nanostructure shape on drug delivery. Adv. Drug Delivery Rev. 63, 1228–1246. Wu, X., Li, S., Coumes, F., Darcos, V., Lai Kee Him, J., Bron, P., 2013. Modeling and selfassembly behavior of PEG–PLA–PEG triblock copolymers in aqueous solution. Nanoscale 5, 9010–9017. Yang, L., Qi, X., El Ghazoui, A., Li, S., 2010. Aggregation behavior of self-assembling polylactide/poly(ethylene glycol) micelles for sustained drug delivery. Int. J. Pharm. 394, 43–49. Yang, L., Wu, X., Liu, F., Duan, Y., Li, S., 2009. Novel biodegradable polylactide/poly (ethylene glycol) micelles prepared by direct dissolution method for controlled delivery of anticancer drugs. Pharm. Res. 26 (10), 2332–2342. Yang, L., Zhao, Z., El Ghazoui, A., Li, S., 2007. Micelles formed by self-assembling of polylactide/poly(ethylene glycol) block copolymers in aqueous solutions. J. Colloid Interface Sci. 314, 470–477. Yasugi, K., Nagasaki, Y., Kato, M., Kataoka, K., 1999. Preparation and characterization of polymer micelles from poly(ethylene glycol)–poly(D,L-lactide) block copolymers as potential drug carrier. J. Controlled Release 62, 89–100. Yokoyama, M., 2011. Clinical applications of polymeric micelle carrier systems in chemotherapy and image diagnosis of solid tumors. J. Exp. Clin. Med. 3 (4), 151–158. Zhu, J., Hayward, R.C., 2008. Spontaneous generation of amphiphilic block copolymer micelles with multiple morphologies through interfacial instabilities. J. Am. Chem. Soc. 130, 7496–7502.
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Self-assembled filomicelles prepared from polylactide/poly(ethylene glycol) block copolymers for anticancer drug delivery Katarzyna Jelonek a, * , Suming Li b, ** , Xiaohan Wu c, Janusz Kasperczyk a,d, Andrzej Marcinkowski a a
Centre of Polymer and Carbon Materials, Polish Academy of Sciences, Curie-Sklodowska 34 Street, 41-819 Zabrze, Poland European Institute of Membranes, UMR CNRS 5635, University Montpellier 2, Place Eugene Bataillon, 34095 Montpellier Cedex 5, France Max Mousseron Institute on Biomolecules, UMR CNRS 5247, University Montpellier 1, 34090 Montpellier Cedex 5, France d School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Katowice, Poland, Department of Biopharmacy, Jednosci 8, Sosnowiec, Poland b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 January 2015 Received in revised form 13 March 2015 Accepted 16 March 2015 Available online 18 March 2015
Bioresorbable filomicelles present many advantageous as drug delivery systems e.g., long circulation time and high loading efficiency. The aim of this study was to develop polylactide/poly(ethylene glycol) (PLA/PEG) filomicelles for drug delivery applications. A series of PLA/PEG diblock copolymers were synthesized using non-toxic initiator, and characterized by means of NMR and GPC. Analysis of morphology of micelles determined by TEM revealed that apart from the weight fraction also the molar mass of PEG and the stereochemistry of PLA block must be considered for tailoring micellar structures. The CMC was found to be dependent on the length and structure of the hydrophobic block. It was observed that the drug loading properties could be improved by selection of appropriate copolymer and encapsulation method. Slower release of paclitaxel was observed for mPEG5000 initiated copolymers than mPEG2000 initiated copolymers. Moreover, the influence of the length of hydrophobic block and its stereoisomeric form on drug release rate was evidenced. Therefore, PLA/PEG filomicelles with good stability, high drug loading capacity and sustained drug release appear most attractive for drug delivery applications. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Polymeric micelles Filomicelles Paclitaxel PLA/PEG Drug delivery
1. Introduction Amphiphilic block copolymers are able to self-assemble in water environment to form various aggregates, including spherical micelles, rod-like micelles, filomicelles (also called worm-like micelles) and polymerosomes (Rajagopal et al., 2010). The evolution of distinct morphologies has been reported to depend on different factors such as copolymer chain structure, polymer concentration, solvent, salt concentration and presence of small molecule stabilizers. The physical properties of these aggregates such as size, size distribution and morphology influence their stability, drug loading and release properties, as well as in vivo pharmacokinetics and biodistribution (Fairley et al., 2008). Compared to surfactant micelles, block copolymer-based micelles are characterized by higher thermodynamic stability,
* Corresponding author. Tel.: +48 32 2716077; fax: +48 32 2712969. ** Corresponding author. Tel.: +33 467 149 121. E-mail addresses: [email protected] (K. Jelonek), [email protected] (S. Li). http://dx.doi.org/10.1016/j.ijpharm.2015.03.032 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
and larger versatility for controlling micellar structure and functionality by choices of polymer composition, architecture, molecular weight and monomer chemistry (Loverde et al., 2010; Nishiyama and Kataoka, 2006a; Zhu and Hayward, 2008). This makes them most attractive for novel applications in drug delivery. Among the various aggregates, flexible worm-like “filomicelles” – in analogy to filoviruses – possess a long circulation time up to a week in the bloodstream because their unique visco-elastic properties and hydrodynamics could reduce interactions with the blood vessel walls (Loverde et al., 2011; Venkataraman et al., 2011). Moreover, filomicelles display almost twice higher drug loading capacity as compared to spherical micelles due to larger core volume per carrier (Cai et al., 2007). Amphiphilic block copolymers composed of a hydrophilic block such as poly(ethylene glycol) (PEG) and a degradable and hydrophobic polyester block such as polylactide (PLA) or poly (e-caprolactone) (PCL) have been extensively investigated as potential drug carrier (Branco and Schneider, 2009; Geng and Discher 2006; Yang et al., 2010). PEG is commonly used in biomedical applications due to its outstanding physicochemical and biological properties including solubility in water and in
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organic solvents, non-toxicity and resistance to protein adsorption and cellular adhesion, which minimizes the detection by the immune system and consequently increases the plasma half-life of micelles (Batrakova et al., 2006 Heald et al., 2002; Hu et al., 2003; Yang et al., 2009). It has been demonstrated that long-circulating drug carriers can preferentially and efficiently accumulate in solid tumors due to the enhanced permeability and retention (EPR) effect (Nishiyama and Kataoka, 2006b). Paclitaxel is one of the most efficient cytostatic presenting activity against a wide variety of tumors, including ovarian, metastatic breast cancer, non-small cell lung cancer, head and neck malignancies and others (Kim et al., 2001; Singla et al., 2002; Yang et al., 2009). The current challenge is to formulate paclitaxel in a nontoxic vehicle so as to eliminate the toxicity of Cremophor EL, the currently used formulation vehicle. The usefulness of PLA/PEG spherical micelles as a paclitaxel carrier has already been confirmed (Yang et al., 2009). Significant improvement would be obtained by development of PLA/PEG filomicelles with this drug. Therefore, understanding how the structure of micelles obtained from amphiphilic copolymers may be controlled by specific parameters is still a major challenge (Loverde et al., 2011). Dalhaimer et al. (2003) observed that assembly of PCL–PEG diblock copolymers into particular microstructures depends primarily on the weight fraction of PEG block relative to the total polymer weight. Formation of worm-like micelles was observed for copolymers with PEG weight fraction in the range from 45% to 55% (Dalhaimer et al., 2003). Nevertheless, other factors such as copolymer concentration and preparation method could also affect the morphology of aggregates. Fairley et al. (2008) found that the amphiphilic copolymer PCL(4100)/PEG(5000) formed different morphologies in aqueous solution, including long rod-like, short rod-like, or spherical aggregates with increasing copolymer concentration. Giacomelli and Borsali (2006) reported that PCL–PEG copolymers copolymers with PCL volume fractions ranging from 0.35 to 0.58 could form spherical or worm-like micelles when a solvent was used before micellization. However, PLA–PEG diblock filomicelles have been rarely considered, so far. In the present study, the influence of copolymer composition (weight ratio of EO moieties), molar mass of mPEG and preparation method on the morphology of micelles was analyzed. The potential of micelles for encapsulation and release of paclitaxel was also evaluated. 2. Materials and methods 2.1. Materials L-, D-, and DL-lactides were obtained from Purac Biochem (Goerinchem, The Netherlands) and purified by crystallization from ethyl acetate. Monomethoxy poly(ethylene glycol) (mPEG) with molar masses of 2000 or 5000, and zinc lactate were purchased from Sigma–Aldrich (St-Quentin Fallavier, France). All other organic solvents were of analytic grade from Sigma–Aldrich and used without further purification. Paclitaxel was purchased from LC Laboratories (Woburn, MA). 2.2. Synthesis and characterization of PLA/PEG copolymers A series of PLA/PEG diblock copolymers were synthesized by ring opening polymerization of lactide with mPEG using zinc lactate as non-toxic initiator as previously described (Yang et al., 2010). Briefly, predetermined amounts of mPEG and lactide were introduced into a polymerization tube, and sealed under vacuum. Polymerization then proceeded for 3 days at 140 C. The product was recovered by dissolution in dichloromethane and precipitation in diethyl ether, and dried under vacuum to constant weight.
The composition of copolymers was determined by means of proton nuclear magnetic resonance (1H NMR) recorded at Bruker spectrometer (250 MHz) using CDCl3 as a solvent. Chemical shifts (d) were given in ppm using tetramethylsilane as an internal reference. The molar mass and molar mass distribution of block copolymers were measured by gel permeation chromatography (Waters 410) equipped with an RI detector. Tetrahydrofuran (THF) was used as the mobile phase at a flow rate of 1.0 mL/min. 20 mL of each polymer solution at a concentration of 10 g/L was injected for analysis. 2.3. Preparation of PLA/PEG micelles Micelles were prepared by co-solvent evaporation method according to the procedure described by Cai et al. (2007). Briefly, 5 mg of polymer was dissolved in 100 mL of chloroform. 5 mL of distilled water was then added. In case of micelles obtained from mixture of D-PLA/PEG and L-PLA/PEG, equal amounts of both polymers were used. The solution was stirred vigorously at room temperature for 3.5 h and left for solvent evaporation for 24 h. The resulting micellar solution was finally filtered through 0.85 mm filter and stored at 4 C. Micelles were also prepared using dialysis method. 10 mg of polymer was dissolved in 1 mL of THF and the solution was transferred into a dialysis bag. The polymer solution was dialyzed for 48 h under stirring against 1 L of water which was frequently renewed. The samples were diluted with distilled water to a concentration of 1 g/L, filtered through 0.85 mm filter and stored at 4 C. 2.4. Characterization of PLA/PEG micelles The morphology of micelles was observed by using transmission electron microscopy (TEM) performed on Hitachi H-600 microscope, operating at an accelerating voltage of 75 kV. 5 mL of micellar solution was placed on a copper grid covered with nitrocellulose membrane, stained negatively with 2% phosphotungstic acid (PTA) and air dried at room temperature before measurements. The morphology of selected micelles was also analyzed using multimode atomic force microscopy (AFM) instrument (MultiMode, di-Veeco, USA, CA) with NanoScope 3D operating in the tapping mode in air with standard 125 mm single-crystal silicon cantilevers (Model TESP; Bruker; USA, CA). The 1.0 g/L micellar solution was 20 times diluted. A drop of the diluted solution was placed on a silica matrix and air dried overnight before measurement at room temperature. The software package WSxM (Nanotec Electronica) was used for image processing. Dynamic light scattering (DLS) measurements were conducted at 25 C with 90 scattering angle using Malvern Zetasizer Nano spectrometer (Malvern Instrument, Worcs, UK). The critical micelle concentration (CMC) of copolymers was measured by fluorescence spectroscopy method (Hitachi F-2500) using pyrene as fluorescence probe (Dominguez et al., 1997). Pyrene was excited at 334 nm and its emission was recorded at 375 nm and 395 nm at polymer concentrations ranging from 9.7 104 to 0.5 mg/mL and constant concentration of pyrene (2.0 106 M). The CMC values were estimated from the cross-over point of the plots of the intensity ratio (I375/I395) from pyrene excitation spectra vs. log C (where C is concentration in mg/mL). 2.5. In vitro drug release Two methods were applied for paclitaxel encapsulation to micelles obtained by co-solvent evaporation method. In method 1, predetermined amount of paclitaxel in methanol was added to drug
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free micelles and stirred for 4.5 h. In method 2, drug incorporation and micelles formation were realized simultaneously: paclitaxel in chloroform was added to a copolymer solution in chloroform, mixed with water and stirred for 4.5 h. In both methods, PLA/PEG micelles were prepared with a theoretical loading of 10% or 20%. Paclitaxel-loaded micelles were centrifuged at 3000 rpm for 5 min. The supernatants were recovered and the sediments were used to determine the amount of paclitaxel which was not encapsulated in micelles (free paclitaxel). The drug loading content (LC) and drug encapsulation efficiency (EE) were calculated according to the following equations (Yang et al., 2009): total paclitaxel amount free paclitaxel amount LC ¼ 100 amount of paclitaxel loaded micelle
EE ¼
total paclitaxel amount free paclitaxel amount 100 total paclitaxel amount
Drug loaded micellar solutions were introduced in a dialysis membrane (MWCO = 3500). Drug release was realized under in vitro conditions at 37 C in phosphate buffered saline (PBS) at pH 7.4. At the predetermined time intervals, the release medium was sampled and fresh PBS was added to maintain the initial volume. The amount of paclitaxel was measured by using high performance liquid chromatography (HPLC). A SunFireTM C18 column (4.6 150 mm, particle size 5 mm, Waters) was used and UV detector set at 227 nm. The mobile phase was 55:45 (v/v) HPLC grade water–acetonitrile. The flow rate was set at 1.0 mL/min and the total analysis time was 8 min. A linear calibration curve was previously obtained using 10 standard solutions in the range of 0.25–1000 mg/mL (R2 = 0.996). 3. Results and discussion 3.1. Characterization of PLA/PEG copolymers A series of PLA/PEG diblock copolymers were synthesized using zinc lactate as non-toxic initiator. Polymers are named as LxEOy, DxEOy or DLxEOy where L, D and DL represent L-PLA, D-PLA or DL-PLA blocks, respectively, x and y designate the number-average degree of polymerization (DP) of corresponding blocks. The composition of copolymers, including the EO/LA molar ratio and DP values were obtained from the integrations of NMR resonances
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belonging to the methylene protons of PEG at 3.6 ppm and to the methine proton of PLA at 5.2 ppm, as previously described (Li and Vert, 2003). The molar mass of copolymers was determined by means of GPC and NMR. Six mPEG2000-initiated (LAxEO45) and four mPEG5000-initiated (LAxEO114) diblock copolymers were synthesized. The characteristics of the various copolymers are presented in Table 1. The copolymers were obtained in a wide range of EO/LA ratio from 0.62 to 3.22. The weight fraction of EO (fEO) in mPEG2000 and mPEG5000initiated copolymers ranges from 0.28 to 0.60, and from 0.45 to 0.56, respectively. According to the literature, copolymers with fEO around 0.45 are susceptible to form wormlike micelles (Dalhaimer et al., 2003). 3.2. Characteristics of self-assembling structures 3.2.1. Micelle morphology Polymeric micelles were prepared by means of co-solvent evaporation method and characterized in terms of morphology and stability. A wide range of copolymers were used so as to determine the influence of polymer structure, Mw of PEG and fEO on micelles morphology. Micelles were prepared from both LAxEO45 and LAxEO114 copolymers with various fEO values (Table 1). L22EO45 and D21EO45 were also used as a mixture for micelles preparation. According to the literature, mixed micelles containing two PLA stereoisomers exhibit higher stability due to stronger interactions between L-PLA and D-PLA blocks (Yang et al., 2007). Thus, higher drug-loading efficiency was expected for mixed micelles than for single micelles. L85EO114 and DL80EO114 were compared to determine the effect of chirality of the hydrophobic blocks on micellar morphology. The morphology of micelles was observed using TEM. Filomicelles were obtained from mPEG2000-initiated copolymers in a wide range of fEO (0.28–0.55) as shown in Fig. 1. Two copolymers were not considered for further study: the most hydrophobic L73EO45 (Fig.1A) which precipitated during preparation making this process not effective, and the most hydrophilic L18EO45 which yielded spherical micelles only (Fig. 1F). Coexistence of filomicelles and spherical micelles was observed for L41EO45 (Fig. 1B) and L28EO45 (Fig. 1C). Studies of Rajagopal et al. (2010) on PCL–PEG micelles revealed that coexistence of several morphologies is a common phenomenon in transition regions. In fact, with decrease
Table 1 Characteristics of PLA/PEG diblock copolymers. Copolymer
MnPEG
EO/LAa
DPPEGb
DPPLAc
Mn(NMR)d
Mn(GPC)e
fEO
CMCf (mg/mL)
L73EO45 L41EO45 L28EO45 L22EO45 D21EO45 L22EO45 + D21EO45 L18EO45 L85EO114 DL80EO114 L63EO114 L54EO114
2000 2000 2000 2000 2000 – 2000 5000 5000 5000 5000
0.62 1.10 1.58 2.00 2.05 – 3.22 1.33 1.42 1.80 2.09
45 45 45 45 45 – 45 114 114 114 114
73 41 28 22 22 – 18 85 80 63 54
7200 4900 4048 3600 3500 – 3300 11 100 10 770 9500 8900
5900 4950 4100 3650 4700 – 3800 11 300 11 300 12500 12000
0.28 0.40 0.49 0.55 0.56 – 0.60 0.45 0.46 0.52 0.56
0.004 0.004 0.004 0.006 – 0.006 0.006 0.003 0.004 0.005 0.005
a b c d e f
Calculated from the integrations of NMR bands belonging to PEG blocks at 3.6 ppm and to PLA blocks at 5.2 ppm. DPPEG = MnPEG/44. DPPLA = DPPEG/(EO/LA). Mn = MnPEG + DPPLA 72. Determined by GPC. Determined by fluorometry at 25 C.
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Fig. 1. TEM images of LAxEO45 micelles: L73EO45 (A), L41EO45 (B), L28EO45 (C), L22EO45 (D), L22EO45 + D21EO45 (E) and L18EO45 (F).
of the fEO, the morphology turns from spherical micelles to filomicelles, and then to bilayered polymersomes (Wu et al., 2013). It is of interest to note that the coexistence region is much larger than the region of exclusive filomicelle formation (fEO 0.5–0.55), as observed for strongly segregating poly(ethylene oxide)-b-poly (1,2-butadiene (PEO-b-PBD) copolymers (Rajagopal et al., 2010). L22EO45 copolymer formed exclusively filomicelles (Fig. 1D). Interestingly, mixture of L22EO45 and D21EO45 also yielded filomicelles, but the micelle size was not uniform (Fig. 1E). In the group of mPEG5000-initiated copolymers (LAxEO114) (Fig. 2), formation of filomicelles was observed for L85EO114 (Fig. 2A). More spherical micelles coexisted with filomicelles in the case of L63EO114 and L54EO114 with shorter L-PLA blocks (Fig. 2B and C). Surprisingly, filomicelles and spherical micelles were almost exclusively observed in different regions of L63EO114 (Fig. 2B). It is also of interest to compare the micellar morphologies of copolymers with similar PLA block lengths but different lactide stereoisomers (Fig. 2A and Fig. 3). Only spherical forms were observed for DL80EO114 (Fig. 3), in contrast to L85EO114 which mainly formed filomicelles. These findings were confirmed by AFM analysis (Fig. 4). Therefore, polymer chain stereoregularity seems to strongly affect the micelle’s morphology. In fact, stereoregular
L-PLA blocks are more rigid than DL-PLA ones with randomly distributed L- and D-lactidyl units, and rigid blocks seem to be more inclined to form filomicelles. Dialysis was also used to prepare micelles in comparison with co-solvent evaporation method. Fig. 5 shows that similar filomicelles are obtained by dialysis method, and spherical micelles are also present. In the case of DL80EO114 (Fig. 5D), only spherical micelles are obtained as with co-solvent evaporation method. Moreover, mPEG2000 derived LAxEO45 copolymers present the same behaviors (data not shown). 3.2.2. Critical micelle concentration The critical micelle concentration (CMC) is an important parameter that influences the in vivo performance of micelles, in particular the micellar stability after intravenous injection. The CMC of copolymers was determined using fluorescence spectroscopy with pyrene as a hydrophobic probe. Fig. 6 shows the I375/I395 vs. log C plots of L85EO114 copolymer. A CMC value of 0.003 mg/mL was obtained from the cross-over point of the plots. Dependence of the CMC on the length of hydrophobic block was observed for both mPEG2000 and mPEG5000 initiated copolymers (Table 1). Thus, copolymers with longer PLA blocks exhibit lower CMC values. This
Fig. 2. TEM images of LAxEO114 micelles by co-solvent evaporation method: L85EO114 (A), L63EO114 (B1, B2) and L54EO114 (C).
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Fig. 3. TEM image (A) and DLS graph (B) of DL80EO114 micelles.
finding is consistent with literature data, assigning longer hydrophobic blocks with better self-assembling properties and lower CMC values (Liggins and Burt, 2002; Yasugi et al., 1999). In fact, entanglement of longer hydrophobic blocks in the inner core may provide higher structural stability of micelles. No difference in CMC was found for single micelles of L22EO45 and mixed micelles of L22EO45 + D21EO45, in contrast to reports of Yang et al. (2007). This might be caused by the different preparation methods of micelles. In Yang’s work, micelles were prepared by direct dissolution of copolymers in water, in contrast to co-solvent evaporation used in this work. It must be emphasized that the CMC values of all copolymers are very low (3–6 mg/mL), which ensures the micellar stability after administration in the body and dilution in bloodstream. These values are comparable to those of other copolymers that generally show very low CMC values ranging from 1 mg/mL to 10 mg/mL (Yokoyama, 2011), and are much lower than typical CMC values of low molar mass surfactants (Zhu and Hayward, 2008). 3.3. Drug encapsulation properties of PLA/PEG micelles The hydrophobic core of polymeric micelles allows to incorporate drugs of poor water solubility and serves as nano-reservoir, while the outer hydrophilic corona provides biocompatibility and prolonged circulation in blood by avoiding rapid clearance by the liver and spleen after intravenous administration (Geng and Discher, 2006; Nishiyama and Kataoka, 2006a). The encapsulation properties of different PLA/PEG copolymers were evaluated, using paclitaxel as a hydrophobic model drug.
3.3.1. Optimization of drug encapsulation method Preliminary studies were conducted with L85EO114 in order to select an appropriate method for effective incorporation of paclitaxel in the core of micelles. Two encapsulation methods based on co-solvent evaporation method were compared. Method 1 consists in addition of drug solution in previously prepared micelles, while method 2 involves simultaneous micellar formation and drug incorporation. 10% or 20% of theoretical drug content was applied. As shown in Table 2, the EE and LC values were higher for micelles obtained by method 1, in contrast to Cai's work (Cai et al., 2007) which showed no difference of encapsulation efficiency between the two methods for PCL–PEG micelles. Moreover, 10% theoretical drug load yielded higher EE and LC values as compared to 20%. Therefore, method 1 with 10% theoretical drug loading was selected for paclitaxel encapsulation in further studies. 3.3.2. Comparison of drug loading properties of different PLA/PEG micelles In the next step, drug loading properties were evaluated for all the PLA/PEG micelles as presented in Table 3. In the case of mPEG2000 initiated copolymers, the EE and LC depended on the length of PLA blocks. Micelles with longer PLA block length led to higher EE and LC. On the other hand, mixed micelles of L22EO45 and D21EO45 showed much better drug loading properties than single L22EO45 micelles. This finding is consistent with literature data (Yang et al., 2009). In fact, mixed micelles containing both L-PLA/PEG and D-PLA/PEG copolymers with more compact core structure could favor drug encapsulation.
Fig. 4. AFM images of micelles obtained from L85EO114 (A) and DL80EO114 (B).
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Fig. 5. TEM images of LAxEO114 micelles prepared by using dialysis method: L85EO114 (A), L63EO114 (B), L54EO114 (C) and DL80EO114 (D).
Micelles of mPEG5000 initiated copolymers present better drug loading properties than those of mPEG2000 initiated copolymers. Similar findings were observed for PCL/PEG worm-like micelles (Cai et al., 2007). Interestingly, no dependence of drug loading on the PLA block length is observed (Table 3). On the other hand, much lower EE and LC values were obtained for spherical micelles obtained from DL80EO114 micelles than for L85EO114 filomicelles. This difference could be assigned to the fact that filomicelles present higher drug encapsulation potential than spherical micelles (Cai et al., 2007). 3.3.3. In vitro drug release Paclitaxel release from the various micellar systems was studied under in vitro conditions. Figs. 7 and 8 present the release
profiles of paclitaxel from mPEG2000 and mPEG5000 initiated copolymer micelles, respectively. Less than 35% of drug was released during 71 days in all cases. However, differences in drug release rates were noticed. Paclitaxel release was faster from mPEG2000 initiated copolymers than from mPEG5000 initiated copolymers and was faster for copolymers with shorter PLA blocks in both groups of micelles. The slowest release was observed for L44EO45 and L85EO114 with the longest PLA blocks in both groups. On the other hand, paclitaxel release was slower from mixed micelles of L22EO45 and D21EO45 than from L22EO45 single micelles, 12.6% and 21.6% of drug being released in 71 days, respectively. This finding might be assigned to the more compact core structure of mixed micelles which disfavors drug diffusion. 30.6% release was observed for spherical micelles of DL80EO114 in 71 days, in contrast to 9.8% for L85EO114 filomicelles. The release profile was similar for all the micellar systems, which is characterized by a gradual initial release and an increase of release rate after 40 days. The acceleration of drug release was probably caused by progress of degradation of the PLA core.
Table 2 Encapsulation efficiency and loading content data of paclitaxel in L85EO114 micelles.
Fig. 6. Plots of I375/I395 vs. log C of L85EO114.
Method
Initial drug loading (wt.%)
EE (%)a
LC (%)a
1 1 2 2
10 20 10 20
69.4 6.0 64.3 13.7 51.8 6.2 42.5 4.2
6.5 0.5 11.3 2.1 4.9 0.6 7.8 0.7
a
Data represent mean value S.D., n = 3
K. Jelonek et al. / International Journal of Pharmaceutics 485 (2015) 357–364 Table 3 Encapsulation efficiency and loading content data of paclitaxel in different PLA/PEG micelles. Copolymer
EE (%)a
LC (%)a
L41EO45 L28EO45 L22EO45 L22EO45 + D21EO45 L85EO114 DL81EO114 L63EO114 L54EO114
57.8 4.3 49.9 6.4 48.3 11.6 65.0 4.6 69.4 6.0 52.3 6.0 68.2 8.7 69.0 3.5
5.5 0.4 4.7 0.6 4.7 0.6 6.1 0.4 6.5 0.5 5.0 0.6 6.4 0.8 6.4 0.3
a
Data represent mean value S.D., n = 3.
Generally, the process of drug release proceeded very slowly for all copolymers. This finding could be assigned to the morphology of the micelles. In fact, it was reported that filomicelles provide longer drug release than spherical micelles (Cai et al., 2007). As shown by Yang et al. (2007), even spherical PLA/PEG micelles may provide sustained drug release for over 30 days. The chemical structures and properties of the micellar core-forming blocks significantly affect the drug loading efficiency and drug release rate (Branco and Schneider, 2009). In this study, L-, D- or DL-PLA was used as the hydrophobic block. A solid-like inner core was formed inside micelles (Heald et al., 2002). Copolymers with relatively long PLA blocks provided effective entrapment of drug molecules that remained in the core of micelles for longer time. The fact that mixed micelles exhibit slower release than single micelles, and
Fig. 7. In vitro release of paclitaxel from micelles obtained from mPEG2000 initiated copolymers (S.D. shown as error bars, n = 3).
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that L85EO114 filomicelles exhibit slower release than DL80EO114 spherical micelles could be assigned to the difference of core structure or core stability. 4. Conclusion This work aimed to elucidate the influence of PLA/PEG diblock copolymer composition, molar mass of mPEG and preparation method on the morphology, CMC, drug encapsulation and drug release properties of micelles. Analysis of micellar morphology determined by TEM revealed that apart from the weight fraction of PEG block (fEO), also the molar mass of PEG was an important factor that affects selfassembling structures. Formation of filomicelles was observed at fEO 0.55 for mPEG2000-initiated copolymers, and at fEO 0.45 for mPEG5000-initiated copolymers. However, differences in morphology of L85EO114 and DL81EO114 micelles indicated that the stereochemistry of PLA block must be considered for tailoring micellar structures. In contrast, preparation method (co-solvent evaporation and dialysis) did not affect the structure of micelles. The low CMC values of all copolymers (3–6 mg/mL) ensure the stability of micelles after administration in the body and dilution in bloodstream. Dependence of the CMC on the PLA block length was observed. The lowest CMC values were obtained for copolymers with the longest PLA blocks. The copolymer composition, PLA block length, mixed or single micelles, and PLA stereochemistry are identified as factors influencing drug encapsulation and drug release properties of PLA/PEG micelles. Micelles with longer PLA blocks lead to higher encapsulation efficiency and load content for mPEG2000 initiated copolymers. In contrast, drug loading properties did not depend on the PLA block length for mPEG5000 initiated copolymers. The latter presents better drug loading properties than the former. On the other hand, spherical micelles of DL81EO114 micelles present lower encapsulation efficiency and load content as compared to L85EO114 filomicelles. Paclitaxel release was faster from mPEG2000 initiated copolymers than from mPEG5000 initiated copolymers, and was faster for copolymers with shorter PLA blocks in both groups of micelles. Mixed micelles exhibit slower release rate than single micelles due to the more compact core structure of mixed micelles which disfavors drug diffusion. Faster release was observed for DL80EO114 than for L85EO114 micelles. Therefore, filomicelles prepared from PLA/PEG dilbock copolymers are promising for prolonged delivery of hydrophobic antitumor drugs. Acknowledgements This study was conducted in the frame of postdoc fellowship supported by Polish Ministry of Higher Education “Mobility Plus” and realized at the Institut des Biomolecules Max Mousseron, UMR CNRS 5247, University of Montpellier I, France. The authors thank Dr. Josephine Laikeehim of Centre de Biochimie Structurale (Montpellier, France) for TEM measurements. References
Fig. 8. In vitro release of paclitaxel from micelles obtained from mPEG5000 initiated copolymers (S.D. shown as error bars, n = 3).
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