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Controls on Polymer Molecular Weight May Be Used To Control the Size of Palmitoyl Glycol Chitosan Polymeric Vesicles Wei Wang,† Anne Marie McConaghy,† Laurence Tetley,‡ and Ijeoma F. Uchegbu*,† Department of Pharmaceutical Sciences, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, Glasgow G4 0NR, U.K., and Electron Microscopy Unit, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, U.K. Received July 28, 2000. In Final Form: October 16, 2000 For the first time, to our knowledge, it has been demonstrated that polymeric vesicle size may be controlled by controls on polymer molecular weight. A direct relationship exists between the square root of the palmitoyl glycol chitosan molecular weight and sonicated polymeric vesicle z-average mean diameter (r ) 0.95). Glycol chitosan samples of varying molecular weight were prepared by hydrolysis with 4 M hydrochloric acid and palmitoyl glycol chitosan samples of varying molecular weight synthesized by reacting glycol chitosan with palmitic acid N-hydroxysuccinimide ester. Polymer characterization was carried out by gel permeation chromatography/laser light scattering and 1H NMR. Vesicles produced from the various palmitoyl glycol chitosan samples by probe sonication in the presence of cholesterol were sized and imaged by transmission electron microscopy. Palmitoyl glycol chitosan samples of MW 276 000, 134 000, 89 000, 28 000, and 31 000 produced unilamellar polymeric vesicles with a z-average mean diameter of 481, 429, 384, 221, and 206 nm, respectively. In addition palmitoyl glycol chitosan vesicles could be prepared from the low molecular weight polymer (MW 28 000) alone in the absence of cholesterol.
Introduction Small molecular weight amphiphiles such as phospholipids1,2 and synthetic surfactants3,4 have been known to assemble into bilayer vesicles and have been used as drug delivery systems.5,6 To improve the stability of these supramolecular structures, a number of groups have prepared polymeric vesicles from either polymerizable monomers which were subsequently polymerized7-11 or polymers in which the polymer backbone is separated from the hydrophobic side chains by oligoethylene spacer groups.12-14 Previously the latter were thought to be the * To whom correspondence should be addressed. E-mail:
[email protected]. Phone: +44 141 548 3895. Fax: +44 141 552 6443. † University of Strathclyde. ‡ University of Glasgow. (1) Gregoriadis, G., Ed. Liposome Technology; CRC Press: Boca Raton, FL, 1993. (2) Bangham, A. D.; Standish, M. M.; Watkins, J. C. J. Mol. Biol. 1965, 13, 238-252. (3) Handjani-Vila, R.-M.; RIbier, A.; Rondot, B.; Vanlerberghe, G. Int. J. Cosmetic Sci. 1979, 1, 303-314. (4) Uchegbu, I. F., Ed. Synthetic surfactant vesiclessan introduction to niosomes and other nonphospholipid systems, 1st ed.; Harwood Academic Publishers: Amsterdam, 1999. (5) Harrison, M.; Tomlinson, D.; Stewart, S. J. Clin. Oncol. 1995, 13, 914-920. (6) Uchegbu, I. F.; Double, J. A.; Turton, J. A.; Florence, A. T. Pharm. Res. 1995, 12, 1019-24. (7) Bader, H.; Ringsdorf, H.; Skura, J. Angew. Chem., Int. Ed. Engl. 1981, 20, 91-92. (8) Fendler, J.; Tundo, P. Acc. Chem. Res. 1984, 17, 3-8. (9) Samuel, N.; Singh, M.; Yamaguchi, K.; Regen, S. J. Am. Chem. Soc. 1985, 107, 42-47. (10) Cho, I.; Chung, K. C. Macromolecules 1988, 21, 565-571. (11) Cho, I.; Kim, Y.-D. Macromol. Rapid Commun. 1998, 19, 2730. (12) Kunitake, T.; Nakashima, N.; Takarabe, K.; Nagai, M.; Tsuge, A.; Yanagi, H. J. Am. Chem. Soc. 1981, 103, 5945-5947. (13) Elbert, R.; Laschewsky, A.; Ringsdorf, H. J. Am. Chem. Soc. 1985, 107, 4134-4141. (14) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988, 27, 113-158.
only types of preformed polymers that could produce vesicle bilayers due to the perceived necessity to decouple the polymer backbone from the bilayer-forming amphiphile.12-14 However, fairly recently our work has established that polymeric unilamellar vesicles may be prepared from the amphiphilic polymer palmitoyl glycol chitosan (PGC) (Scheme 1) in which the hydrophobic pendant group is directly attached to the hydrophilic polymer backbone.15 In addition recently block copolymers such as polystyreneb-poly(ethylene oxide) diblock copolymers16 and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymers17 have also been found to form bilayer vesicles. It is apparent that the architectural spectrum of vesicle-forming polymers is indeed wide. Polymeric vesicles show increased retention of dissolved solutes18 and an increased resistance to solubilization by alcohol19 or bile salts.15 Additionally polymeric vesicles prepared from palmitoyl glycol chitosan have recently been encapsulated within phosphatidylcholine liposomes and show a reduced degree of lipid mixing with these liposomes when compared to nonionic surfactant vesicles (niosomes) prepared from small molecular weight amphiphiles.20 Vesicles obtained from palmitoyl glycol chitosan with a degree of polymerization of 800 were 300-600 nm in size15 with a z-average mean diameter of 420 nm. Limitations were observed on the lower size limit that could be achieved with these polymeric vesicles, and even the extrusion of bleomycin palmitoyl glycol chitosan (15) Uchegbu, I. F.; Scha¨tzlein, A. G.; Tetley, L.; Gray, A. I.; Sludden, J.; Siddique, S.; Mosha, E. J. Pharm. Pharmacol. 1998, 50, 453-8. (16) Yu, K.; Eisenberg, A. Macromolecules 1998, 31, 3509-3518. (17) Schille´n, K.; Bryskhe, K.; Mel’nikova, Y. S. Macromolecules 1999, 32, 6885-6888. (18) Ringsdorf, H.; Schlarb, B.; Tyminski, P. N.; O’Brien, D. F. Macromolecules 1988, 21, 671-677. (19) Roks, M. F. M.; Visser, H. G. J.; Zwikker, J. W.; Verkley, A. J.; Nolte, R. J. M. J. Am. Chem. Soc. 1983, 105, 4507-4510. (20) McPhail, D.; Tetley, L.; Dufes, C.; Uchegbu, I. F. Int. J. Pharm. 2000, 200, 73-86.
10.1021/la001078w CCC: $20.00 © 2001 American Chemical Society Published on Web 01/05/2001
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Scheme 1. Synthesis of Palmitoyl Glycol Chitosan
(degree of polymerization 800) vesicles through a 200 nm filter reduced these vesicles from a mean diameter of 669 nm to a minimum mean diameter of 290 nm.21 It is proposed that restrictions on curvature posed by the length of the polymer backbone prevent the realization of small vesicle sizes and hypothesized that reductions in the length of the polymer backbone would result in an increase in vesicle curvature. The current work demonstrates that, by reducing the molecular weight of the vesicle-forming polymer palmitoyl glycol chitosan 10-fold, it is possible to reduce the vesicle size by half and hence produce polymeric vesicles in the 200 nm size range by probe sonication alone. It is also possible to produce vesicles from palmitoyl glycol chitosan in the absence of cholesterol. Vesicle size is a crucial determinant of drug biodistribution. Liposomes of more than 300 nm diameter accumulate in the spleen and liver while liposomes of 150-200 nm circulate for longer periods and are not rapidly cleared by the liver and spleen.22 Long blood circulation times ultimately enable drug targeting to remote sites such as solid tumors.23 Experimental Section Materials. Glycol chitosan and palmitic acid N-hydroxysuccinimide were obtained from Sigma Chemical Co., U.K. Hydrochloric acid was obtained from Merck, U.K. Acid Degradation of Glycol Chitosan. Glycol chitosan (2 g) was dissolved in hydrochloric acid (4 M, 150 mL) and the solution filtered to remove insoluble impurities. The filtered solution was placed in a preheated water bath at 50 °C. At the time points 4, 8, 24, and 48 h the reaction was stopped and the products were isolated and purified as described below. The reaction solution was exhaustively dialyzed (Visking seamless cellulose tubing, molecular weight cutoff 12 400 for cytochrome c) against distilled water (5 L with six changes over 24 h). The dialysate at the end of the dialysis procedure had a neutral pH. The dialysate was subsequently freeze-dried, and the product was recovered as a cream-colored cotton-wool-like material. Synthesis of Palmitoyl Glycol Chitosan. Synthesis was carried out as previously described by reacting glycol chitosan with palmitic acid N-hydroxysuccinimide.15 (21) Sludden, J. A.; Uchegbu, I. F.; Schatzlein, A. G. J. Pharm. Pharmacol. 2000, 52, 377-382. (22) Litzinger, D.; Buiting, A. M. J.; Rooijen, N.v.; Huang, L. Biochim. Biophys. Acta 1994, 1190, 99-107. (23) Gabizon, Price, D. C.; Huberty, J.; Bresalier, R. S.; Papahajopoulos, D. Cancer Res. 1990, 50, 6371-6378.
1H NMR. 1H NMR analysis (with integration) and 1H correlation spectroscopy experiments were also performed as previously described.15 Laser Light Scattering (LLS) and Gel Permeation Chromatography (GPC) Measurements. GPC/LLS experiments (MiniDAWN detector equipped with a 20 mV semiconductor diode laser, vertically polarized, λ ) 690 nm) with refractive index (RI) detection (Waters 2410 refractive index detector, λ ) 850 nm) were carried out on glycol chitosan and palmitoyl glycol chitosan using as both solvent and mobile phase 0.3 M CH3COONa/0.2 M CH3COOH (pH 4.4) for glycol chitosan and the mixed solvent 0.3 M CH3COONa/0.2 M CH3COOH (pH 4.4)methanol (1:1) for palmitoyl glycol chitosan. GPC was achieved using a PSS Hema-Bio 300 column (300 × 8 mm, particle size 10 µm, exclusion limit for dextran 500 000) and a PSS Hema-Bio 40 column (300 × 8 mm, particle size 10 µm, exclusion limit for dextran 3 000 000). All the sample solutions were filtered (0.2 µm), and 200 µL of each sample solution was injected onto the columns at a loading concentration of 1-3 mg mL-1 using a Waters 717 plus autosampler. All measurements were performed at room temperature and data processed using Astra for Windows 4.70 software. Refractive index increments (dn/dc) of glycol chitosan and palmitoyl glycol chitosan solutions in their respective solvents were determined on a Waters 2410 refractive index detector (λ ) 850 nm). The data were processed using DNDC for Windows 5.10 software. Preparation of Palmitoyl Glycol Chitosan Vesicles. Palmitoyl glycol chitosan vesicles were prepared by probe sonication of the polymer (8 mg) and cholesterol (4 mg, Sigma, U.K.) in water (4 mL) or by probe sonication of the polymer alone (8 mg) in water (4 mL) until a homogeneous dispersion was obtained (∼10 min, Soniprep 150 probe sonicator) with the instrument set at 15% of its maximum output. Sonication was continued until a limiting size distribution was obtained (determined as described below) and did not normally exceed 10 min. Vesicle Sizing. Vesicle size and size distribution were measured by photon correlation spectroscopy (Malvern Zetasizer 1000) at 25 °C at a wavelength of 633 nm and the data analyzed using the Contin method of data analysis. Measurements were performed in triplicate. Electron Microscopy. Transmission electron microscopy with negative staining was performed as follows. Carbon-coated 200 mesh copper grids were glow discharged and vesicle suspensions applied, followed by the application of methylamine tungstate negative stain. The grids were dried and imaged using a LEO 902 electron microscope at 80 kV.
Size of PGC Polymeric Vesicles
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Figure 1. Influence of degradation time on the acid degradation of glycol chitosan. Table 1. Degradation of Glycol Chitosan glycol chitosan sample
degradation time (h)
MW
Mw/Mn
GC1 GC2 GC3 GC4 GC5
0 4 8 24 48
242 000 105 000 62 000 26 000 20 000
1.07 1.04 1.12 1.25 1.30
Freeze-fracture electron microscopy was carried out as previously described.15
Results and Discussion Preparations of Different Molecular Weight Chitosan and Palmitoyl Glycol Chitosan Samples. Acid hydrolysis of glycol chitosan resulted in materials of different molecular weights (Table 1, Figure 1). The dn/dc value of glycol chitosan was determined as 0.08 mL g-1, and the GPC/LLS method used to determine the molecular weight of the degraded samples discriminated effectively between the various samples. The dn/dc values were measured at a higher wavelength than the light scattering determination. dn/dc decreases slightly with increasing wavelength,24 and hence a slight error is introduced into the determination of the absolute molecular weight of all the samples. However, the comparisons made in the present communication are still valid as all samples were measured using the same technique. The slight magnitude of the error in the determination of the absolute molecular weight can be appreciated if results for a polystyrene standard are considered. The dn/dc of a polystyrene standard (Fluka, U.K.) with a manufacturer’s stated MW of 32 000 was measured and found to be 0.148 mL g-1 at 850 nm (0.161 mL g-1 at 436 nm).24 The molecular weight of this material was determined as 32 400 in our laboratories on the same instrumentation used for the present study. Increasing the acid degradation time decreased the molecular weight of the resulting polymer, and the commercially supplied glycol chitosan sample GC1 with a molecular weight of 242 000 could be degraded to onetenth of its molecular weight after 24 h, yielding a sample with a molecular weight of 26 000. This degradation while initially proceeding at a fast rate became limiting as time progressed and the number of cleavable sites diminished (Figure 1). The molecular weight distribution (Mw/Mn) shows an increase with an increase in degradation time as the degradation of the samples occurs at random sites. Degradation of chitosan has been attempted with hydrochloric acid,25 phosphoric acid,26 and nitrous acid.27 (24) Michielsen. S. Specific Refractive Index Increments of Polymers in Dilute Solution. In Polymer Handbook; Brandrup, J., Immergut, E. H., Grulke, E. A., Eds.; John Wiley & Sons: New York. 1999; pp 547627. (25) Richardson, S. C. W.; Kolbe, H. J. V.; Duncan, R. Int. J. Pharm. 1999, 178, 231-243. (26) Hasegawa, M.; Isogai, A.; Onabe, F. Carbohydr. Polym. 1993, 20, 279-283. (27) Varum, K. M.; Anthonsen, M. W.; Grasdalen, H.; Smidsrod, O. Carbohydr. Res. 1991, 211, 17-23.
Figure 2. GPC/LLS-RI chromatogram of PGC5. See the Experimental Section for chromatography conditions.
However, reports detailing the degradation of glycol chitosan could not be found in the literature. The method used is an adaptation of the method reported by Richardson and others to degrade chitosan25 except that in our system a lower temperature was used (50 °C instead of 100 °C), there was no purging of the reaction mixture with nitrogen, and the finished product was isolated by dialysis. The molecular weight cutoff of the dialysis membrane used to isolate the product was 12 400; thus, small molecular weight fractions (
