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Biomacromolecules 2008, 9, 886–895
Unimolecular Micelles Based On Hydrophobically Derivatized Hyperbranched Polyglycerols: Ligand Binding Properties Rajesh Kumar Kainthan,† Clement Mugabe,‡ Helen M. Burt,‡ and Donald E. Brooks*,†,§ Centre for Blood Research, Departments of Pathology & Laboratory Medicine and Chemistry and Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia V6T 2B5, Canada Received November 1, 2007; Revised Manuscript Received December 12, 2007
This paper discusses the binding and release properties of hydrophobically modified hyperbranched polyglycerol-polyethylene glycol copolymers that were originally developed as human serum albumin (HSA) substitutes. Their unimolecular micellar nature in aqueous solution has been proven by size measurements and other spectroscopic methods. These polymers aggregate weakly in solution, but the aggregates are broken down by low shear forces or by encapsulating a hydrophobic ligand within the polymer. The small molecule binding properties of these polymers are compared with those of HSA. The preliminary in vitro paclitaxel release studies showed very promising sustained drug release characteristics achieved by these unimolecular micelles.
Introduction Polymer micelles with sizes ranging from 20 to 100 nm are potential drug and gene delivery vehicles.1–3 Because their diameters are above the glomerular filtration limit and below the size recognized by the reticuloendothelial system, they avoid premature excretion from the body. They are also being developed as contrast agents for various imaging applications. These micelles, with a hydrophobic core and hydrophilic shell, can encapsulate hydrophobic molecules such as drugs and hormones in the core due to hydrophobic interactions while the hydrophilic shell keeps the system soluble in water and can protect it against host defense systems, leading to a longer circulation half-life. Polymeric micelles are formed when amphiphilic block copolymers are dissolved in water above the critical micelle concentration (CMC). A number of such polymer systems have been developed in the past and has been reviewed several times.1–4 One of the disadvantages of these thermodynamic aggregates is their instability upon dilution to below the CMC due to the dissociation of micelles into free polymer chains. This is often the case when such micellar systems are injected into the blood stream or after oral administration, leading to the abrupt release of encapsulated drugs, potential toxicity problems, and decreased efficacy. Other environmental changes such as in temperature, pH, and the presence of other molecules can also affect the stability of these systems. Branched polymers with a covalently linked hydrophobic core and hydrophilic shell, resembling a micellar architecture, are being developed to overcome this problem. Such dendritic polymers, including dendrimers and amphiphilic star block copolymers, have also been shown to encapsulate hydrophobic molecules. These systems, which have no CMC, are often referred to as “unimolecular micelles” due to the presence of a hydrophobic core and hydrophilic shell connected by covalent bonds in a single molecule.5 Their natural * Corresponding author. E-mail:
[email protected]. Fax: (604) 8227742. Address: Centre for Blood Research, Life Sciences Centre, 2350 Health Sciences Mall, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. † Department of Pathology & Laboratory Medicine and Centre for Blood Research. ‡ Faculty of Pharmaceutical Sciences. § Department of Chemistry.
stability to various environmental effects such as dilution, shear force, and pH combined with their binding capacity make these excellent candidates as drug delivery systems.6–9 The outer surface of these branched polymers can be derivatized with suitable functionalities for multivalent interactions in order to achieve targeted drug delivery. Moreover, the surface groups can also be decorated with cell membrane penetrating ligands (molecular transporters) such as peptides for enhanced drug delivery.10,11 Encapsulation of hydrophobic drugs and studies of their properties have been reported for suitably modified dendrimers such as poly(amidoamine) (PAMAM) and polypropylenimine diaminobutane (DAB).12,13 Much higher encapsulation and slower drug release were achieved for acidic drugs such as methotrexate due to complementary electrostatic interactions.14 Hyperbranched polymers are structurally related to dendrimers although branching is less perfect, typically ∼60% of the monomers being branched.15,16 Hyperbranched polymers are synthesized in a single synthetic procedure, whereas dendrimers are prepared in tedious multistep synthetic and purification steps, which makes the former commercially viable alternatives to symmetric and perfectly branched dendrimers. Hyperbranched polyglycerols (HPGs) are one of the few hyperbranched polymers that can be synthesized in a controlled manner with predetermined molecular weights and narrow polydispersity.17–19 This new class of water-soluble polymers is highly biocompatible, with no evident animal toxicity.20–22 Recently, we reported the development of a second-generation synthetic substitute for albumin based on derivatized hyperbranched polyglycerol (HPGC18-PEG) that closely mimics the binding and transport properties of the natural material and is considered to hold advantages over the current plasma expanders used clinically.23 Reported in this article are their binding properties in detail and their suitability as drug delivery agents.
Materials and Methods Materials. All chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON) and used without further purification. 1,2Epoxyoctadecane was synthesized by the peroxidation of octadecene with m-chloroperbenzoic acid. R-Epoxy, ω-methoxy polyethylene glycol 350 (MPEG epoxide), was synthesized from a reaction of MPEG 350,
10.1021/bm701208p CCC: $40.75 2008 American Chemical Society Published on Web 02/02/2008
Unimolecular Micelles from Hyperbranched Polyglycerols sodium hydroxide, and epichlorohydrin. 13COOH labeled palmitic acid was obtained from Cambridge Isotope Ltd. The potassium salt of palmitic acid was prepared according to a reported procedure.24 Millipore water with a conductivity of 18.2 MΩ/cm was used for making solutions. Paclitaxel was obtained from Hauser Chemical Company (Boulder, CO). Dialysis membrane tubing was purchased from Spectrum Laboratories (Rancho Dominguez, CA). All solvents were HPLC grade and were obtained from Fisher Scientific. Polymer Synthesis and Characterization. Polymerizations were carried out in a three-neck round-bottom flask equipped with a mechanical stirrer. The second neck was connected to a dual manifold Schlenk line, and the third was closed with a rubber septum though which reagents were added. A typical polymerization reaction procedure (dHPG-B) was as follows. Initially, the initiator trimethyloyl propane (TMP, 120 mg) was added to the flask under argon atmosphere followed by 0.1 mL of potassium methylate solution in methanol (20 wt %). The mixture was stirred using a magnetic stir bar for 15 min, after which excess methanol was removed in a vacuum. The flask was kept in an oil bath at 95 °C, and 5.5 mL glycidol was added dropwise over a period of 12 h using a syringe pump. After completion of monomer addition, the mixture was stirred for an additional 5 h. 1,2-Epoxyoctadecane (1.0 g) was then added and the mixture stirred for 24 h. To this mixture, 15 mL of MPEG-epoxide was added dropwise over a period of 12 h and was stirred for additional 5 h. The product was dissolved in methanol and neutralized by passing it three times through a cation exchange column (Amberlite IRC-150). The unreacted 1,2epoxyoctadecane was removed by extraction with hexane. Methanol was removed and polymer was then dialysed for three days against water using cellulose acetate dialysis tubing (MWCO 1000 g/mol, Spectrum Laboratories Inc.), with three water changes per day. The dry polymer was then obtained by freeze-drying. 1 H NMR spectra were recorded on a Bruker Avance 400 MHz NMR spectrometer using deuterated solvents (Cambridge Isotope Laboratories, 99.8% D) with the solvent peak as a reference. Molecular weights and polydispersities of the polymers were determined by gel permeation chromatography (GPC) connected to a MALLS detector (GPCMALLS); details have been reported earlier.19 Intrinsic viscosity measurements were performed on filtered aqueous polymer solutions using an Ubbelohde viscometer and a temperature controlled bath. The concentrations were verified by determination of the solid content after evaporation of the solvent. The intrinsic viscosities were determined from plots of ηsp/C against concentrations. Fluorescence and UV–visible spectra were obtained with a Varian Cary Eclipse spectroflourimeter and Varian Cary 4000 spectrophotometer, respectively. Hydrodynamic radii of the polymers in free-standing solutions were measured using a microcuvette (mc) at room temperature (22 °C) using DAWN EOS multiangle laser light scattering (MALLS) instrument from Wyatt Technology Corp. equipped with a Wyatt quasi-elastic light scattering (QELS) detector. The particle size distribution analysis was performed using Dynals software. Fatty Acid and Pyrene Binding Experiments. Proton-decoupled 13 C NMR spectra were recorded on a Bruker (75.47 MHz for carbon) Avance NMR spectrometer. Samples were dissolved in water and locking was done using CD3OD in a capillary tube inserted into the sample. The multiplet at 49.15 from CD3OD was used as an internal reference. An excess of pyrene was stirred with known concentrations of aqueous polymer solutions for 12 h, filtered twice using 0.2 µM syringe filters, and the resultant solution analyzed by 1HNMR and UV-vis spectroscopy. The literature value of 29500 for max at λ ) 338 nm was used for concentration measurements.25 ANS Binding. 1-Anilinonaphthalene-8-sulfonic acid (ANS) and fatty acid free human serum albumin (fatty acid content e0.007%) were obtained from Sigma-Aldrich. Phosphate buffered saline (PBS: 150 mmol/L NaCl, 1.9 mmol/L NaH2PO4, 8.1 mmol/L Na2HPO4, pH 7.4) was used for making solutions. Fluorescence spectra were recorded using a Varian Cary Eclipse spectroflourimeter at room temperature. The excitation wavelength was set at 370 nm, and the emission range
Biomacromolecules, Vol. 9, No. 3, 2008 887 was set between 400 and 600 nm. The binding capacity (n) and the binding constant (Kb) for the dHPG polymers were determined by a double fluorometric titration technique as described.26 In the first titration, increasing concentrations of polymers were added to a constant concentration of ANS (C1ANS) and the maximum intensity of ANS fluorescence (Fmax), representing complete binding of ANS to polymer, was determined. Specific fluorescence intensity (Fsp) for the bound ANS was calculated as Fmax/C1ANS. In the second titration, increasing concentrations of ANS (C2ANS) were added to a constant concentration of polymer (CdHPG) and the fluorescence intensity (F) measured. The concentration of ANS bound (CboundANS) was calculated as F/Fsp and that of free ANS (Cfree ANS) as (C2ANS - CboundANS). Plots of CdHPG/ CboundANS against 1/Cfree ANS and linear regression gave the values for Kb and n according to the following equation:
CdHPG bound C ANS
)
1 1 + free n Kb × n × C ANS
Paclitaxel Binding and Release Studies. Preparation of Paclitaxel Encapsulated dHPG-C. Paclitaxel and dHPG-C (1:1 mol/ mol) were dissolved in a small amount of acetonitrile in 20 mL vials and dried in an oven at 60 °C for 1 h, then flashed with a nitrogen stream to eliminate traces of the organic solvent. The resulting HPG/ paclitaxel matrix was hydrated with 2 mL of 10 mM phosphate buffered saline (pH 7.4), vortexed for 2 min, and incubated in the oven (60 °C) for 1 h. The resulting dHPG/paclitaxel solution was generally a clear solution. In those cases where a white precipitation was observed, the solution was centrifuged (18000g for 10 min) and the supernatant was transferred into new vials and kept in a cool place until use. HPG/ paclitaxel solutions were stable at room temperature for two weeks. Quantification of Paclitaxel. The amount of paclitaxel incorporated in dHPG-C was determined by reversed-phase HPLC as established previously.27 Drug content analysis was performed using a symmetry C18 column (Waters Nova-Pak C18 column) with a mobile phase containing a mixture of acetonitrile, water, and methanol (58:37:5, v/v/ v) at a flow rate of 1 mL/min. Sample injection volumes were 20 µL, and paclitaxel detection was performed using a UV detector at a wavelength of 232 nm. Total run time was set to 5 min, and paclitaxel retention time was 2.5 min. Peak area was recorded and processed. Drug Release Study. Paclitaxel released from dHPG-C was determined by the dialysis method. Briefly, 2 mL of HPG/paclitaxel solution was transferred into dialysis membrane tubing (MWCO 6000–8000) and then transferred into bottles containing 500 mL of 10 mM phosphate buffered saline (pH ) 7.4). The total volume of the release medium was chosen such that when paclitaxel was completely released, its concentration was below its solubility in water (0.3 µg/mL). The bottles were incubated at 37 °C with slight agitation (100 rpm). At different time points, the dialysis tubes were removed and transferred into fresh phosphate buffered saline bottles. The amount of paclitaxel released was extracted by dichloromethane (20 mL) and assayed by HPLC method as described above. At the end of the release study (after 15 days), the residual paclitaxel was also determined. The extraction efficiency for paclitaxel is 95%.
Results and Discussion Human serum albumin is the most abundant plasma protein in the body and has two principle physiological functions.28 It provides ∼80% of the colloid osmotic pressure that balances the hydrostatic pressure in the vascular tree. Second, it acts as a carrier of fatty acids, bilirubin, hormones, drugs, and metal ions by reversibly binding these agents. Its replacement under conditions of blood loss (surgery, acute injury) is critical clinically, which is the reason for the large quantity of HSA used annually.29 The polysaccharide and gelatin-based materials, which are currently used as plasma expanders, do not perform any function of albumin other than the maintenance of osmotic
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Kainthan et al.
Figure 1. Structure of derivatized hyperbranched polyglycerols (dHPG). Table 1. Physical Characteristics of the dHPG Polymers polymer
C18 mol %
MPEG mol %
glycidol mol %
no. of C18a
Mn × 10-4
Mw/Mn
[η] (mL/g)
Rh nm (QELS)
dHPG-A dHPG-B dHPG-C
2.0 1.9 3.2
16.8 26.7 27.7
81.2 71.4 69.1
6.8 9.5 15
4.4 8.5 7.8
2.2 1.5 3.2
7.2 7.9 8.2
6.5 7.7 8.4
a Number of C18 chains per molecule; Mn number average molecular weight; Mw/Mn polydispersity; [η] intrinsic viscosity; QELS quasi-elastic light scattering; Rh average hydrodynamic radius.
pressure. One of the major functions of HSA is the binding and transport of fatty acids. Typically, one to two long chain fatty acids are bound per albumin molecule, although up to six binding sites, some with weaker binding constants, are described. Recently, we reported the development of a second-generation synthetic substitute for albumin based on derivatized hyperbranched polyglycerol (HPG-C18-PEG) that not only has superior volume replacement properties due to a low intrinsic viscosity and high biocompatibility but closely mimics the binding and transport properties of the natural material.23 Unlike the current plasma expanders, these derivatized HPGs (dHPGs) bind hydrophobic molecules such as fatty acids and other hydrophobic molecules. They belong to the class of “unimolecular micelles” and therefore can be used as general drug carriers. Several HPGs derivatized with hydrophobic groups and methoxy polyethylene glycol (MPEG) chains were synthesized in a simple single pot synthetic procedure based on ring-opening polymerization of epoxides. Details of the synthesis and polymer characteristics of have been described earlier.23 The structure of the copolymer is shown in Figure 1. Briefly, a HPG of molecular weight 7000 g/mol was prepared by anionic ringopening multibranching polymerization of glycidol from partially deprotonated trimethylolpropane using potassium methylate.17 HPG has numerous hydroxyl end groups, the number per molecule being roughly equal to the degree of polymeri-
zation. Some of these were modified with C18 alkyl chains (2-5%) and MPEG-350 chains (mol wt 350) (20-40%) by sequential addition of 1,2-epoxyoctadecane and MPEG-epoxide. The unreacted alkyl epoxide was removed from the methanolic polymer solution by extraction with hexane. The final polymer product was purified by dialysis against water to remove any unreacted PEG chains and then freeze-dried. The HPG-C18-PEG polymers bind hydrophobic molecules in water due to the presence of a hydrophobic binding pocket formed by the alkyl chains inside the polymer molecule. The hydrophobically modified HPGs are not fully soluble in water so the PEG chains are added to enhance the solubility as well as to protect these alkyl chains from interacting with biological structures such as proteins or cells. The molecular characteristics for the three selected polymers designated as dHPG-A, dHPGB, and dHPG-C are summarized in Table 1. A detailed study of the binding and release properties of these was conducted. Polymers with unimolecular micellar properties have been reported to have different conformations depending on the polarity of the solvent. To check the interaction between the hydrophobic core and solvent, NMR spectra were recorded in CDCl3, DMSO-d6, and D2O. The spectra are shown in Figure 2 and Figure 3. The protons from glycerol and PEG segments were observed without any changes in both the solvents. The proton peaks from the alkyl chains were broad in an aqueous environment whereas they were sharp in CDCl3 and DMSO-
Unimolecular Micelles from Hyperbranched Polyglycerols
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Figure 2. 1H NMR spectrum of dHPG-C in D2O.
Figure 3. 1H NMR spectrum of dHPG-C in DMSO-d6.
d6. This suggests that in water the alkyl chains have reduced mobility, likely due to their hydrophobic association.30 However, the peak areas were similar in both the solvents, suggesting that they have a more open and flexible structure than other core–shell structured dendritic polymers reported earlier31,32 and implies that the interior of the polymer is easily accessible for solvent and other ligand molecules. Part of the reason for the flexible nature might be due to the imperfection in branching and the presence of flexible ether bonds in the polymer.
Hydrodynamic Size of the Polymer Molecules. The average hydrodynamic sizes of the dHPG polymers in 0.1 N aqueous NaNO3 solutions were measured while flowing (0.8 mL/min) through a GPC system outfitted with a Viscotek triple detector and quasi-elastic light scattering (QELS) detector. The viscosity average sizes and the hydrodynamic sizes were determined using the viscosity and the QELS detector, respectively. The viscosity average radii and hydrodynamic radii obtained for all the three polymers were below 10 nm, ruling out the formation of
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Figure 4. Disaggregation of the polymer molecules (dHPG-A) through pyrene encapsulation.
aggregates (Table 1).23 However, the GPC results correspond to only very dilute polymer solutions. One of the intended uses of this class of polymers is as plasma expanders, which are typically infused at higher concentrations, and the peak polymer concentration could be as high as 10 mg/mL plasma. Therefore, lack of aggregation at these concentrations is a prerequisite for such applications. Quiescent QELS batch measurements were then made using polymer solutions of higher concentrations. The average Rh values of dHPG-A, dHPG-B, and dHPG-C (10 mg/mL solutions) were 10.1, 10.0, and 14.5 nm in DMF and were 6.7, 8 and 10.5 nm in MeOH, respectively. Although the polymer sizes were smaller in MeOH, the generally low values are indicative of the absence of large aggregates in these solutions. Similarly, HPG-PEG copolymer without alkyl chains showed Rh of
