Solubility enhancement of paclitaxel using a linear-dendritic block copolymer

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Solubility enhancement of paclitaxel using a linear-dendritic block copolymer Zhengyuan Zhou a , Antony D’Emanuele a,∗ , David Attwood b a b

School of Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston PR1 2HE, UK School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, UK

a r t i c l e

i n f o

Article history: Received 25 March 2013 Received in revised form 25 April 2013 Accepted 28 April 2013 Available online xxx Keywords: Paclitaxel Dendrimer Micellisation Drug solubilisation Block copolymer Worm-like micelles

a b s t r a c t The solubilising capacities of micelles of a linear-dendritic copolymer (BE-PAMAM), formed by conjugating the poly(butylene oxide) (B)–poly(ethylene oxide) (E) block copolymer B16 E42 (BE) with a G2 PAMAM dendrimer, have been compared with those of the diblock copolymer B16 E42 for the anti-cancer drug paclitaxel. The BE-PAMAM copolymer showed a greater solubility enhancement than BE under equivalent conditions. Drug-loading efficiency was improved using a solvent-loading method compared with the conventional solution-loading method. The solubility of paclitaxel was increased 3700-fold by micellar encapsulation in a 2% (w/v) BE–PAMAM copolymer solution at 37 ◦ C using this solubilisation technique. Dynamic light scattering and transmission electron microscopy studies indicated a transition of spherical to worm-like micelles of the BE copolymer induced by the encapsulation of drug molecules. A sustained release of encapsulated drug was observed, with approximately 80% and 60% paclitaxel being released from 2% (w/v) solutions of BE and BE-PAMAM respectively after 24 h of dialysis at 37 ◦ C. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Paclitaxel, which was first isolated from the trunk bark of the pacific yew tree, is a mitotic inhibitor used in chemotherapy (Horwitz et al., 1986; Wani et al., 1971). It can induce apoptosis by binding to microtubules and interfering with the normal growth of microtubules during cell division (Jordan et al., 1993). Paclitaxel has been approved for clinical use for treatment of various types of cancers. However, the drug has very poor aqueous solubility and bioavailability, which greatly limits its therapeutic efficacy. Numerous attempts have been made to develop drug carriers to enhance the solubility and delivery efficiency of paclitaxel. Those strategies fall into two categories: the first is to covalently conjugate paclitaxel to water-soluble macromolecules via an appropriate linker (prodrugs) (Skwarczynski et al., 2006). Poly(ethylene glycol) has been conjugated to 2 -OH of paclitaxel via a succinyl linker to improve the water solubility (Greenwald et al., 1996). Li et al. (1998) reported the synthesis of poly(l-glutamic acid)–paclitaxel conjugate. The derivatives showed remarkable antitumour efficacy but were less toxic. Kakinoki et al. (2008) attached paclitaxel to poly(vinyl alcohol) (PVA) via a succinic anhydride linker and an ethylene diamine spacer. The prodrug showed more effective

∗ Corresponding author. Tel.: +44 01772 895801; fax: +44 07092 030763. E-mail addresses: [email protected], [email protected], [email protected] (A. D’Emanuele).

delivery towards tumourous tissue due to the enhanced permeability and retention (EPR) effect. Etrych et al. (2010) prepared various paclitaxel and docetaxel prodrugs using a water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer. In an investigation of the effect of linkers on the stability of prodrugs it was found that the linker formed between the carbonyl group of the spacer and the hydrazide group of the side chains of the polymer was hydrolytically cleavable. Thus, these prodrugs more readily released the active compound under mildly acidic conditions. The second strategy is to physically incorporate paclitaxel into nanocarriers or to form a drug-complex, examples include nanoparticles (Fonseca et al., 2002; Koziara et al., 2006; Win and Feng, 2006; Xie and Wang, 2005), liposomes (Shieh et al., 1997; Wang et al., 2010; Yang et al., 2007; Zhao et al., 2011), microspheres (Liggins et al., 2000), cyclodextrin (Hamada et al., 2006), and polymeric micelles (Han et al., 2006; Yoncheva et al., 2012; Zhao et al., 2012). Polymeric micelles have attracted considerable attention for their pharmaceutical applications in drug solubilisation, drug delivery, controlled release and gene delivery (Kwon, 2003; Torchilin, 2001). Polymeric micelles are stable towards dilution in biological fluids due to their low critical micelle concentration. The micelle core provides a suitable microenvironment for the incorporation of a lipophilic drug while the hydrophilic micelle corona serves as a stabilising interface between the hydrophobic core and the surrounding medium, thus improving the stability and bioavailability of the drug. Polymeric micelles can also prolong the blood circulation time of

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drugs and reduce the nonspecific uptake by the reticuloendothelial system. Block copoly(oxyalkylene)s are nonionic polymeric surfactants comprising a hydrophilic poly(oxyethylene) block (E) and a hydrophobic poly(oxyalkylene) block. The best-known members of this polymer family are the poloxamers (EPE triblock copolymers, P = oxypropylene). A polymeric micellar formulation of paclitaxel and the poloxamer Pluronic P123 was found to enhance the solubility of paclitaxel, prolong blood circulation time and modify the biodistribution of the drug (Han et al., 2006). Solubilisation by block copoly(oxyalkylene)s with different hydrophobic blocks, composition and architecture has been extensively investigated over the past decades; early work has been reviewed by Attwood and Booth (Attwood and Booth, 2007; Attwood et al., 2007a,b), more recent investigations of the solubilisation of drugs include Crothers et al. (2008) and Ribeiro et al. (2009a,b). Drug solubilisation capacity is determined by the hydrophobicity of the core-forming blocks and the size and morphology of the micelles. Dendrimers or hyperbranched polymers have emerged as an interesting class of drug carriers due to their specific structure (see reviews by D‘Emanuele and Attwood, 2005; Esfand and Tomalia, 2001). These polymers can serve as ‘unimolecular micelles’ by incorporating guest molecules within the cavities between the core and branches. The highly-functionalised surface allows the binding of drug molecules either via covalent bonds or by electrostatic interaction. The properties of dendrimers can be easily modified by attachment of guest substrates such as drug, polymer, ligand or protein. Poly(amidoamine) (PAMAM) dendrimers, especially the higher generation dendrimers, have been shown to significantly increase the aqueous solubility of paclitaxel and the paclitaxel–dendrimer complexes had enhanced cytotoxicity towards prostate cancer (PC-3M) cells probably due to their increased solubility or their ability to bypass the P-glycoprotein (P-gp) efflux transporter (Devarakonda et al., 2007). Teow et al. (2013) reported the synthesis of G3 PAMAM dendrimer–paclitaxel prodrugs and evaluated their ability to overcome cellular barriers. These prodrugs were able to enhance drug solubility and bypass P-gp efflux transporters, thereby increasing drug bioavailability. Recently, ‘core–shell’ drug delivery systems based on a fourth-generation hyperbranched aliphatic polyester (Boltorn H40) have been developed (Chen et al., 2008; Kontoyianni et al., 2008). Hydrophilic or amphiphilic polymer chains were conjugated to the surface groups of the Boltorn H40 core to form a ‘unimolecular micelle’, which entrapped drug molecules within the branches. A folate moiety was attached to the corona of the carriers to facilitate targeting delivery (Li et al., 2011). In an earlier study we reported the synthesis and characterisation of a series of linear-dendritic block copolymers comprising a poly(oxybutylene)-b-poly(oxyethylene) (BE) copolymer conjugated to a PAMAM dendrimer (Zhou et al., 2009). BE-dendrimer conjugates with two BE chains per dendrimer molecule can be thought of as BE-dendrimer-EB triblock copolymers. In dilute solution ‘flowerlike’ micelles are formed in which the two polymer chains of each conjugate are looped such that the hydrophobic B blocks form the micelle core and the dendrimers with their attached E chains form the hydrophilic corona. The micelles provide multiple encapsulation sites: the hydrophobic core, the poly(oxyethylene) shell, and the dendrimer branches. The copolymers were demonstrated to have potential for use as carriers for poorly water soluble drugs in applications such as solubilisation, drug delivery and sustained release. In the present study the solubilisation capacity of BE and BE-PAMAM copolymers for the anti-cancer drug paclitaxel was compared under various conditions. The morphology of micelles before and after drug encapsulation was monitored using dynamic light scattering (DLS) and transmission electron microscopy (TEM). The release profiles of

paclitaxel from dilute micellar solutions of both copolymers were determined. 2. Experimental 2.1. Materials Generation 2 PAMAM dendrimers with an ethylenediamine core (20%, w/v in methanol) were purchased from Dendritech Inc. (Michigan, USA). Paclitaxel was purchased from Advance Tech. & Ind. Co., Ltd. (Kln, Hong Kong). 4-Nitrophenyl chloroformate (NPC) (97%), tetrahydrofuran (THF), triethylamine (TEA), dimethyl sulfoxide (DMSO), Sephadex® LH-20, and PEG 4000 were purchased from Sigma–Aldrich (UK). Slide-A-Lyzer Dialysis Cassette (MWCO 2000) was purchased from Thermo Scientific Inc. NMR grade chloroformd and methanol-d were from Goss Scientific Instruments Ltd. 2.2. Synthesis and characterisation of BE-PAMAM dendrimer copolymer Block copolymer B16 E42 (BE) was prepared by sequential oxyanionic copolymerisation of butylene oxide followed by ethylene oxide and characterised as described previously (Zhou et al., 2009). The B16 E42 -G2 PAMAM dendrimer copolymer (BE-PAMAM) was synthesised using a 4-nitrophenyl chloroformate coupling method as described in our earlier study (Zhou et al., 2009) with the following modifications. Briefly, BE copolymer was activated by reaction with NPC in THF in the presence of TEA to obtain a carbonate intermediate. After filtration and purification the intermediate was added (molar ratio 2.2:1) to G2 PAMAM dendrimer solution in DMSO and reacted for 5 days at room temperature. Flash chromatography (Sephadex LH-20, methanol:water 80:20) was then used to remove the by-products and solvent, a faster and more efficient method than the dialysis method used earlier. The BE-G2 copolymer was characterised by GPC, 1 H and 13 C NMR spectroscopy (Bruker Avance 400 MHz, Bruker, Coventry, UK) using the peak assignments determined previously. The NMR results indicated that the copolymer had an average of two BE chains attached per dendrimer with a calculated molecular weight of 9400 g mol−1 . 2.3. CMC measurement The critical micelle concentrations (CMC) of the BE copolymer and dendrimer conjugate at room temperature were determined by surface tension measurement using the pendant drop method. An FTA1000 video system (First Ten Ångstroms, Inc.) was used to visualise drops formed on the tip of a 20-gauge stainless-steel needle. The tip width of the needle was measured to perform a calibration of the video camera’s magnification. Surface tension was obtained via drop-shape analysis; measurements were repeated ten times and the results averaged. The standard deviations of the drop-shape analysis were approximately ±0.4 mN m−1 and the measurement error was less than 5%. 2.4. Drug solubilisation 2.4.1. Solution-loading Saturated drug-loaded solutions were prepared by suspending excess drug in 2 ml of 1 or 2% (w/v) polymer solutions (phosphate buffer, pH 7.4) and stirring at 25 or 37 ◦ C for 3 days. The unsolubilised drug was then removed by filtration (Millipore, 0.45 ␮m). The solubility of paclitaxel in phosphate buffer at 25 ◦ C and in 5% (w/v) PEG 4000 (phosphate buffer, pH 7.4) at 37 ◦ C was also measured for comparison.

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2.4.2. Solvent-loading Excess drug and polymer were dissolved in methanol and left at room temperature for 30 min. The solvent was removed under vacuum before adding sufficient phosphate buffer (pH 7.4) at 37 ◦ C to prepare a 2% (w/v) solution. The mixture was maintained at 37 ◦ C for 3 days and then filtered to remove any undissolved solid. The amount of drug solubilised was analysed by HPLC, using an Agilent 1100 Series HPLC system (UK) equipped with a Luna 5 ␮m, C18 column (250 mm × 4.6 mm) (Phenomenex, Cheshire, UK) at 40 ◦ C. The mobile phase was MeOH:TFA (0.05%, w/v) (80:20), with a flow rate of 1.0 ml min−1 , and UV detection was at  = 230 nm. All measurements were carried out in triplicate and the results averaged. 2.5. Micellar size and shape Analysis of micelle size distribution of the polymer solutions before and after drug loading was conducted using dynamic light scattering (Zetasizer Nano, Malvern Instruments, UK). The polymer solutions were prepared in phosphate buffer (pH 7.4) and clarified by filtering through a PVDF filter (0.45 ␮m pore size) into a clean scattering cell. The morphology of the micelles was examined using Cryo-TEM. The grids of sample were prepared in a Vitrobot using 3 ml of sample absorbed to freshly glow discharged 2-2 Quantifoil grids. Grids were continuously blotted for 4–5 s in a 90% humidity chamber at 37 ◦ C before plunge-freezing into liquid ethane. Data were then recorded on a Polara FEG operating at 200 kV on a 4 K Gatan Ultrascan CCD in low dose mode; CCD images were recorded between ˚ and had a maximum electron 2.0 and 5.0 mm defocus at 3 A/pixel dose of 20–40 e/Å2 . 2.6. Drug release study Release of paclitaxel from the saturated micellar solutions was evaluated using a dialysis technique. 2 ml of 2% (w/v) drug-loaded copolymer solutions at 37 ◦ C was placed into a pre-swollen dialysis cassette and immersed in a glass bottle containing 200 ml of phosphate buffer (pH 7.4) with 5% PEG 4000. The dialysis was performed under sink conditions in a shaking water bath at 37 ◦ C for 24 h. Samples (5 ml) from the outer phase were taken at specific time intervals followed by replenishment with the same amount of fresh buffer. The amount of paclitaxel in the samples was assayed by HPLC as described above. 3. Results and discussion The micellar properties and solubilisation characteristics of the BE copolymers and the BE-PAMAM dendrimer conjugates were measured in aqueous phosphate buffer (0.067 M, pH 7.4) to maintain the solutions at a constant (physiological) pH. The amino groups of the PAMAM dendrimer (pKa ∼10) were completely protonated in micellar solution at this pH and hence the corona surface of the dendrimer conjugates was positively charged. The effect of the buffer on the micellar properties is negligible because of its low ionic strength. 3.1. Critical micelle concentration The BE-PAMAM dendrimer copolymer is amphiphilic and can self-associate to form micelles in aqueous solution. The drop-shape analysis method used to determine the CMCs of the copolymers is more sensitive in the concentration region around the CMC and requires smaller quantities of material than the pyrene solubilisation method used in our previous study (Zhou et al., 2009); it

Fig. 1. Surface tension versus logarithm concentration (g dm−3 ) for BE and BEPAMAM dendrimer copolymer at room temperature.

does, however, lack temperature control and measurements were performed at room temperature (approx. 20 ◦ C). The CMCs of the BE and BE-PAMAM copolymers determined from inflection points in plots of surface tension versus logarithm concentration (see Fig. 1) were 0.41 and 0.59 g dm−3 , respectively, in reasonable agreement with previously reported values at 25 ◦ C (0.31 g dm−3 for BE and 0.37 g dm−3 for BE-PAMAM). As a consequence of their low CMC values, and considering the general insensitivity of the CMCs of BE copolymers to temperature between 20 and 50 ◦ C (Booth and Attwood, 2000), it may be assumed that micellisation is complete at the concentrations and temperatures used for the solubilisation determinations. 3.2. Drug solubilisation Table 1 shows a considerable increase of the solubility of paclitaxel in the micellar solutions (S) compared to that in phosphate buffer (So = 0.29 ␮g ml−1 in phosphate buffer at 25 ◦ C). For example, the solubility of paclitaxel in 2% (w/v) solutions of BE-PAMAM at 37 ◦ C using the solvent-loading method is 1.1 mg ml−1 , more than 3700 times higher than its solubility in buffer at 25 ◦ C. BEPAMAM copolymer shows a greater solubility enhancement than BE under equivalent conditions, attributable to the incorporation of drug molecules between the dendrimer branches and the E block arms within the micelles, although the hydrophobic cores are still the favoured domain for drug incorporation. The influence of the solubilisation technique on efficiency of drug loading is seen by comparing the results obtained using the solvent-loading method with those from the more conventional solution-loading method; comparing results at 37 ◦ C for 2% (w/v) solutions of BE and BE-PAMAM shows solubilisation capacities (Sbp , expressed as milligramme drug per gram of copolymer) some 8–10-fold higher using the solvent-loading method. Drug-loading efficiency of hydrophobic drugs by the solution-loading method, in which excess drug is added to micellar solutions of the polymer, is limited by the low concentration of free drug molecules in solution; in addition, their diffusion into the micelle core is a passive process hindered by the hydrophilic corona of the polymer micelles. In the solvent-loading method the drug and polymer are brought into more intimate contact by dissolving in an organic solvent, and micelles are formed by subsequent removal of the solvent and addition of phosphate buffer. In this process the drug molecules can bind

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Table 1 Solubility of paclitaxel in copolymer solutions and solvents under various conditions.a

Solution loading

Solvent loading a

Conc. (%, w/v)

Temp. (◦ C)

S (␮g ml−1 )

Sbp (mg g−1 )

BE BE-PAMAM Phos. buffer BE BE-PAMAM BE BE-PAMAM PEG 4000

1 1

23.3 56.4 0.29 29.5 79.0 63.5 123.4 8.0

2.33 5.64

1 1 2 2 5

25 25 25 37 37 37 37 37

BE BE-PAMAM

2 2

37 37

629 1080

31.5 54.0

2.95 7.90 3.18 6.17 0.16

Estimated error ±10%.

with the hydrophobic blocks of the copolymers through hydrophobic interaction during dissolution and solvent evaporation and thus are more readily encapsulated. Although solubilisation in BE micelles is lower than in micelles of BE-PAMAM, it nevertheless represents an appreciable enhancement of solubility of paclitaxel, particularly when the solvent-loading technique is used. Solubilisation in spherical micelles formed by block copoly(oxyalkylene)s will reach its maximum when the chains of core-forming blocks are fully stretched by incorporation of drug molecules (Attwood et al., 2007a,b). Any further increase is either limited by the core size or related to a transformation of micelle shape, e.g. to cylindrical or worm-like micelles. Hence the high solubilisation capacities of the BE copolymer of Table 1 suggest that the binding of drug molecules with the copolymers in the solvent-loading method may have a profound impact on the micellisation of the copolymers, probably leading to the formation of elongated micelles, a suggestion that is discussed in the following section. It is interesting to note that an increase of solution concentration from 1 to 2% (w/v) results in an approximate doubling of solubilisation of paclitaxel in the solutions of BE copolymer; because of the minimal effect of temperature on micelle formation and the low CMC, a doubling of concentration would be expected to double the number of micelles and hence the solubilisation capacity. A similar increase of solution concentration causes only a 60% increase of solubilisation in solutions of BE-PAMAM; the amino groups of the PAMAM dendrimers that form the periphery of the BE-PAMAM micelles are ionised at pH 7.4, which may affect the penetration of drug molecules resulting in the observed nonlinear influence of concentration.

the radius of the micelle core approaches the stretched length of the hydrophobic block. It has been shown that copolymers with short E blocks, (which leads to high association numbers), and with short hydrophobic blocks, (which places a low ceiling on the radius of a spherical micelle), are more likely to form elongated micelles (Booth and Attwood, 2000; Zhou et al., 2008). Several B and S (oxystyrene) block copolymers, e.g. E11 B8 (Chaibundit et al., 2002), E17 B12 (Chaibundit et al., 2005) and E17 S8 (Yang et al., 2003), have been reported to exhibit a sphere-to-cylinder transition with temperature increase; worm-like micelles have been found to form in dilute micellar solutions of copolymers E17 B12 and E17 S8 at ambient temperatures. The formation of elongated micelles by Pluronic P85 (E27 P39 E27 ) has been investigated by several groups. Evidence of large micelles was obtained either from small-angle neutron scattering (King et al., 1997; Mortensen and Pedersen, 1993) or from dynamic light scattering (Schillen et al., 1994). A DLS study of the micelle properties of copolymer B16 E42 (Zhou et al., 2009) showed one single peak attributed to spherical micelles and only a marginal increase of aggregation number over the temperature range 15–35 ◦ C; the lack of evidence of a sphere-tocylinder transition is expected considering the long block lengths of this copolymer. Hence, the formation of elongated micelles in the saturated drug-loaded micellar solution of B16 E42 from the solventloading method is attributed to the strong interaction between paclitaxel and hydrophobic blocks. The hydrophobic core-forming blocks are unable to form a compact structure when binding with the large and bulky drug molecules, which induces a transition of

3.3. Micellar size and shape 3.3.1. Dynamic light scattering Size distribution curves (not shown) for micelles of the B16 E42 copolymer in 1% (w/v) solutions at 25 ◦ C before and after solubilisation using the solution-loading method showed only a single peak taken as evidence of closed association to form spherical micelles in both unloaded and drug-loaded solutions; a 20% increase of micelle size (from ca. 10.3 nm to ca. 12.3 nm) occurred as a result of drug encapsulation. In contrast, solubilisation by micelles of the BE copolymer in 2% solutions at 37 ◦ C (Fig. 2) by the solventloading method resulted in the appearance of a broad peak at approx 125 nm, indicative of elongated (worm-like) micelles, in addition to the single peak at ca. 13 nm arising from spherical micelles which showed only a small size increase following solubilisation, The main driving forces determining the shape of micelles are the unfavourable entropy change when the conformation of a hydrophobic chain in the core is extended and the need to maintain surface coverage by the hydrophilic block. A transition from spherical to cylindrical micelles will occur in dilute solution when

Fig. 2. Intensity fraction distributions of logarithm hydrodynamic radius of micelles in a 2% (w/v) micellar solution of B16 E42 copolymer at 37 ◦ C (䊉) without drug encapsulation, and () with drug encapsulation by the solvent-loading method.

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Fig. 3. Intensity fraction distributions of logarithm hydrodynamic radius of micelles in a 2% (w/v) micellar solution of BE-PAMAM copolymer at 37 ◦ C (䊉) without drug encapsulation, and () with drug encapsulation by the solvent-loading method.

micelle shape. However, the penetration and encapsulation of drug molecules is limited when the solution-loading method is used and insufficient to alter the micelle shape. Nagarajan (1999) has discussed, from a theoretical standpoint, the effect of hydrophobic additives on micelle geometry for aqueous solutions of EPE copolymers. In an investigation of the mixing of micellar solutions of Pluronic P105 (E37 P56 E37 ) with a C14 diol using rheology and smallangle neutron scattering, Guo et al. (2001) observed a spherical to worm-like micelle transition and a concomitant change of viscosity by a factor of more than 104 , arising from the incorporation of the hydrophobic diol into the copolymer micelle cores. Similarly, Parekh et al. (2012) reported from SANS and rheological studies a sphere-to-rod shape transition of the micelles of Pluronic P85 in the presence of salicylic acid particularly when in the nonionised form. Size distribution curves of the BE-PAMAM copolymer in drugfree 2% (w/v) solutions at 37 ◦ C (Fig. 3) show more complicated micellisation behaviour. The peak at ca. 12 nm is assigned to spherical micelles while that at ca. 100 nm is thought to arise from aggregates of spherical micelles, which coexist with the micelles. Drug encapsulation causes a pronounced broadening of the size distribution of these aggregates and an increase of their mean size; more modest increases of size and size distribution of the spherical micelles were noted following solubilisation. During micellisation in dilute solution, the BE chains in the BE-PAMAM copolymers are able to fold back into the micellar cores while the PAMAM dendrimers form the periphery. Those copolymers which have more than the average of two BE chains per dendrimer molecule have a tendency to form clusters held together by BE bridges between adjacent micelles (Zhou et al., 2009). The changes in the size distribution curves caused by solubilisation suggest that the interaction and binding of drug molecules with the core-forming hydrophobic blocks has an impact on the packing of micelles and promotes the association between them. 3.3.2. Cryo-TEM This technique is able to provide a direct image of micelles in solutions, particularly when, as in the present study, no staining is used which might disturb the micellar structure. The TEM images of 2% (w/v) copolymer B16 E42 micellar solutions before and after drug encapsulation at 37 ◦ C by solvent-loading are shown in Fig. 4.

Fig. 4. TEM images of a 2% (w/v) micellar solution of B16 E42 copolymer at 37 ◦ C without (top) and with (bottom) drug encapsulation by the solvent-loading method.

Only spherical micelles were observable in the drug-free micellar solutions, whereas after drug encapsulation, although spherical micelles were still dominant, worm-like micelles could also be clearly seen. Although supporting the conclusions from analysis of the DLS data, there is an apparent discrepancy between the sizes of the spherical micelles from the two techniques; that measured by TEM (r ≈ 4.4 nm) is much smaller than that obtained from the DLS study (r ≈ 13 nm). This is probably because the poly(ethylene oxide) blocks (the corona of micelles) only scatter electrons slightly more than background (water) and, without any staining, cannot be seen in the images due to a poor signal-to-noise ratio. Hence, the particles shown in the images are considered to be the micelle cores. Assuming a length per B unit of 0.363 nm (Flory, 1969), the extended length of a B16 block will be ca. 5.8 nm and consequently the core dimension from TEM is within the size limit of a sphere. 3.4. Drug release profiles The release of paclitaxel from 2% (w/v) micellar solutions of BE and BE-PAMAM copolymers at 37 ◦ C is shown in Fig. 5. Dialysis was against phosphate buffer with added 5% (w/v) PEG 4000 to ensure sink conditions. PEG 4000 is too large to pass through the membrane and will have no effect on the micellar solutions. The release profiles exhibited an initial rapid release with approximately 60% and 40% drug releasing within 5 h from BE and BE-PAMAM solutions, respectively. Release of solubilised drug from the BE-PAMAM copolymer was more sustained than from the BE copolymer, probably due to the branching structure of the dendrimer corona which prevents the escape of drug molecules (‘cage’ effect). The release curves reached a plateau with approximately 80% and 60% paclitaxel releasing from BE and BE-PAMAM, respectively, after 24 h of dialysis. The incomplete release may be due to the strong interaction between the drugs and hydrophobic B blocks.

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Fig. 5. Drug release profiles from 2% (w/v) micellar solutions at 37 ◦ C: (䊉) BE, and () BE-PAMAM.

4. Conclusions Micelles formed in aqueous solution by B16 E42 and B16 E42 PAMAM dendrimer copolymers are good solubilisers of the hydrophobic drug paclitaxel achieving considerable enhancement of solubility compared to that in phosphate buffer. The solubilisation capacity was influenced by the solubilisation technique; a solvent-loading method, in which drug and copolymer were brought into direct contact before the formation of micelles, achieved a significantly higher drug encapsulation than the more conventional method in which drug was added to a solution containing preformed micelles. Encapsulation of drug by this technique caused a transition from spherical to worm-like micelles for BE copolymer as the chains of the core-forming hydrophobic blocks were stretched beyond the limit for maintaining micelle sphericity, by incorporation of drug into the core. The solubilisation capacity of BE-PAMAM copolymer was greater than BE under equivalent conditions, because of the possibility for incorporation of drug molecules between the dendrimer branches in addition to the hydrophobic cores. The release profiles from the micellar solutions showed that sustained release of paclitaxel was achieved for both BE and BE-PAMAM copolymers. The findings of this study suggest that these copolymers may be useful as drug carriers for very hydrophobic drugs such as paclitaxel. Acknowledgements The authors would like to thank Dr. Colin Booth, Dr. Zhuo Yang, and Dr. Frank Heatley for assistance with copolymer synthesis and characterisation. We thank Dr. Richard Collins for cryo-TEM measurement. This work was financially supported by the University of Central Lancashire under a science research programme. References Attwood, D., Booth, C., 2007. Solubilisation of a poorly aromatic drug by micellar solutions of amphiphilic block copoly(oxyalkylene)s. In: Tadros, Th.F. (Ed.), Colloid Stability and Application in Pharmacy, Colloid and Interface Science Series, 3, pp. 61–68. Attwood, D., Booth, C., Yeates, S.G., Chaibundit, C., Ricardo, N.M.P.S., 2007a. Block copolymers for drug solubilisation: relative hydrophobicities of polyether and polyester micelle-core-forming blocks. Int. J. Pharm. 345, 35–41. Attwood, D., Zhou, Z., Booth, C., 2007b. Poly(ethylene oxide) based copolymers: solubilisation capacity and gelation. Expert Opin. Drug Deliv. 4, 533–546.

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Please cite this article in press as: Zhou, Z., et al., Solubility enhancement of paclitaxel using a linear-dendritic block copolymer. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.04.075