Bioconjugate Chem. 2006, 17, 860−864
860
1H
NMR Spectroscopy as a Tool for Determining the Composition of Poly(hydroxyethyl-L-asparagine)-Coated Liposomes
Birgit Romberg,† J. Jantina Kettenes-van den Bosch,§ Tom de Vringer,‡ Gert Storm,† and Wim E. Hennink*,† Departments of Pharmaceutics and Biomedical Analysis, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands, and Astellas Pharma Europe B.V., Leiderdorp, The Netherlands. Received February 22, 2006; Revised Manuscript Received April 11, 2006
Liposomes coated with the poly(amino acid) poly(hydroxyethyl-L-asparagine) (PHEA) show long-circulation properties comparable to the frequently used PEG-liposomes. The pharmacokinetic characteristics of long-circulating liposomes are dependent on the density of the shielding polymer on the liposome surface. Therefore, it is necessary to know the exact composition of the liposomes including the amount of coating polymer present on the liposome surface. In this study, a 1H NMR method to establish the composition of liposomes coated with PHEA was developed and validated.
INTRODUCTION The therapeutic use of nanoparticulate carriers for the targeted delivery of drugs, for example, liposomes and polymeric nanoparticles, can be significantly improved by coating them with sterically stabilizing polymers such as poly(ethylene glycol) (PEG) (1, 2). Due to the hydrophilic PEG corona, these modified carriers display reduced recognition and uptake by cells of the mononuclear phagocyte system (MPS) and consequently show prolonged blood circulation times (3). Recently, we showed that liposome coatings based on poly(hydroxyethyl-L-asparagine) (PHEA) and poly(hydroxyethyl-L-glutamine) (PHEG) prolong liposome circulation to a similar extent as PEG (4). Such poly(amino acid) coatings are supposed to be biodegradable, as recently confirmed by the determination of the enzymatic degradation of PHEG (5). In general, the pharmacokinetic characteristics of longcirculating colloidal drug carriers depend on the molecular weight of the stabilizing polymer and its density on the carrier surface (6). To address the latter aspect, it is necessary to quantify the amount of polymer that is grafted onto the carrier. For example, the composition of liposomes that are prepared by the film-hydration method is not exactly known since it is not ensured that all components are quantitatively incorporated into the liposome bilayer. Reliable methods to study the composition of liposomes are scarce. Proton nuclear magnetic resonance (1H NMR) spectroscopy has been reported as an effective method for the quantitation and characterization of PEG-lipid conjugates in lipid mixtures (7), as well as on nanoparticle surfaces (8, 9). The PEG-lipid is relatively easy to quantify because of its simple 1H NMR spectrum. For the poly(amino acid) conjugates, however, quantitation is much more difficult because of strong peak overlap and partly broad signals of the polymer. In this study, a 1H NMR method was developed and validated to accurately determine the composition of coated liposomes formed by the film-hydration method with emphasis on the new coating conjugate PHEA-DODASuc (Figure 1). * To whom correspondence should be addressed. Tel: +31-(0)302536964. Fax: +31-(0)-2517839. E-mail: W.
[email protected]. † Department of Pharmaceutics, Utrecht University. § Department of Biomedical Analysis, Utrecht University. ‡ Astellas Pharma Europe B.V.
Figure 1. Molecular structure of DPPC, cholesterol, and PHEADODASuc (n ) 17).
EXPERIMENTAL PROCEDURES Materials. Poly(hydroxyethyl-L-asparagine)-N-succinyldioctadecylamine (PHEA-DODASuc) was synthesized as described previously (4). Dipalmitoylphosphatidylcholine (DPPC, Figure 1) was purchased from Lipoid GmbH, Ludwigshafen, Germany. R-Cyano-4-hydroxycinnaminic acid, cholesterol (Figure 1), deuteriochloroform (chloroform-d, 99.8 atom % D), and deuterated dimethyl sulfoxide (d6-DMSO) were obtained from Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands. All other reagents were of analytical grade. MALDI-ToF Mass Spectrometry of PHEA-DODASuc. PHEA-DODASuc was dissolved in demineralized water to a
10.1021/bc060045a CCC: $33.50 © 2006 American Chemical Society Published on Web 04/28/2006
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Technical Notes
Figure 2. Mass spectrum of PHEA-DODASuc. The three major peak populations are indicated by approximated curves: population 1, Na+ adduct of the conjugate; population 2, K+ adduct of the conjugate; population 3, Na+ adduct without the DODASuc anchor.
concentration of 1 mg/mL. The matrix compound R-cyano-4hydroxycinnaminic acid was dissolved to a concentration of 10 mg/mL in acetonitrile/water (1:1) containing 0.1% trifluoroacetic acid and mixed in a ratio of 3:1 (v/v) with the conjugate solution. Three microliters of this mixture was put onto a sample deposition plate and left at room temperature to dry. The spectrum was acquired in the positive ion mode on a VoyagerDE STR MALDI-ToF mass spectrometer (Applied Biosystems, Foster City, CA). Ionization was initiated with a nitrogen laser operating at 337 nm with a repetition rate of 7.6 Hz. Delayed extraction linear mode with an accelerating voltage of 20 kV was used, and the acquisition mass range was 700-7000 Da. The spectrum was analyzed with the Applied Biosystems Data Explorer Software. Sample Preparation for NMR Analysis. A mixture of CDCl3 and d6-DMSO in a ratio of 2:1 (v/v) was used for dissolving the samples containing DPPC, cholesterol, and PHEA-DODASuc. For signal assignment, DPPC and cholesterol were dissolved in CDCl3, and PHEA-DODASuc was dissolved in d6-DMSO (approximately 20 mg/mL). The lipid mixtures were prepared by mixing the required amounts of lipid stock solutions and adding CDCl3 and d6-DMSO to obtain the proper solvent ratio. For instance, for a lipid mixture of DPPC, cholesterol, and PHEA-DODASuc (molar ratio of 62:33:5), a stock solution of 81 µmol of DPPC and 43 µmol of cholesterol per milliliter was prepared in CDCl3, and PHEA-DODASuc was dissolved to a concentration of 13 µmol/mL in d6-DMSO. Two hundred microliters of the DPPC/cholesterol stock solution and 100 µL of the PHEA-DODASuc stock were mixed and further diluted with 200 µL of d6-DMSO and 400 µL of CDCl3. The total lipid concentration in the samples was approximately 20 mg/mL. Liposome Preparation. Lipids (DPPC, cholesterol, PHEADODASuc, total lipid ≈ 75 mg) at different ratios were dissolved in about 2 mL of a mixture of chloroform and methanol (1:1 v/v). A film was obtained by evaporation of the solvents under reduced pressure at 50 °C in a 50 mL roundbottom flask. After flushing with nitrogen, the lipid film was hydrated in 5 mM NaCl solution (total lipid concentration 30 µmol/mL). Liposomes were sequentially extruded through two stacked polycarbonate filters (Poretics, 400, 200, 100, and 50 nm) with a high-pressure extrusion device to a final mean size of approximately 100 nm (confirmed by dynamic light scattering measurements). One milliliter of the liposome dispersion was freeze-dried overnight and dried further in a vacuum oven at room temperature for several hours. The dry cake was dissolved
Table 1. Peak Assignment in the Mass Spectrum of PHEA-DODASuc population no.a 1 2 3 a
m/z
assignment of highest peak
3188.3 [DODASuc-(PHEA)16 + Na]+ 3204.1 [DODASuc-(PHEA)16 + K]+ 3060.0 [(PHEA)19 + Na]+
repeating unit (r.u.)
range (r.u.)
158 158 158
9-27 10-25 7-27
The peak population as depicted in Figure 2.
in a mixture of CDCl3/d6-DMSO (2:1) and subsequently analyzed by NMR. To separate liposomes from possible nonincorporated components, 1 mL of the liposome dispersion obtained after extrusion was subjected to ultracentrifugation at 200 000 g (30 min, 4 °C) using a Beckman LE-80K Ultracentrifuge. The supernatant was collected, freeze-dried, dissolved in d6-DMSO, and analyzed by NMR. The pellet was dispersed in 1 mL of 5 mM NaCl solution and subsequently freeze-dried and vacuumdried, dissolved in CDCl3/d6-DMSO (2:1), and analyzed by NMR. NMR Analysis. Homonuclear correlation (COSY, TOCSY) and unidimensional analysis of DPPC, cholesterol, and PHEADODASuc for signal assignment were carried out with a Varian INOVA 500 spectrometer (Varian, Palo Alto, CA) at 500 MHz. Signals were assigned based on chemical shifts, peak intensities, and spin-spin coupling. Spectra of lipid and liposomal mixtures were recorded with a Varian G-300 spectrometer (Varian, Palo Alto, CA) at 300 MHz. The number of accumulated transients was set to 16-32, the pulse angle was 62.5°, and the relaxation delay D1 was set to 10 s.
RESULTS AND DISCUSSION MALDI-ToF and NMR Analysis of PHEA-DODASuc. Figure 2 shows the mass spectrum of PHEA-DODASuc, and Table 1 gives the corresponding peak assignments. Three major peak populations are present in the spectrum. The most abundant population can be assigned to the Na+ adduct of the conjugate (Figure 2, population 1). The two less abundant populations belong to the K+ adduct of the conjugate (Figure 2, population 2) and the Na+ adduct of PHEA without the DODASuc anchor (Figure 2, population 3). The presence of the latter compound is likely due to incomplete coupling during synthesis of the conjugate. Due to differences in ionization efficiency, no quantitative data can be obtained from this spectrum. The number-average molecular weight of PHEA-DODASuc based on the peak population of the Na+ adduct of the conjugate was
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Figure 3. Typical 1H NMR spectrum of a mixture of DPPC/cholesterol/PHEA-DODASuc (molar ratio 62:33:5). Arrows indicate the peaks chosen for evaluation: (a) cholesterol; (b) DPPC; (c) PHEA-DODASuc. Table 2. Assignment of the Signals in the 1H NMR Spectrum of a Mixture of DPPC/Cholesterol/PHEA-DODASuc chemical shift (ppm)
assignment
0.6 0.8-2.3
18 cholesterol ω1, methylene bulk acyl chain, C2+C3 DPPC 1-2, 4, 7-17, 19-27 cholesterol ω1, methylene bulk acyl chain, C2 PHEA-DODASuc A3, E1, L2+L3 PHEA-DODASuc H3 DPPC, 3 cholesterol, A5, C1 PHEA-DODASuc A6 PHEA-DODASuc H2 DPPC G3 DPPC G1 DPPC H1 DPPC A2 PHEA-DODASuc G2 DPPC C6 cholesterol N PHEA-DODASuc (peptide, side chain and end group)
2.4-2.8 3.2 3.5 3.6 3.8 4.1 + 4.3 4.2 4.6 5.1 5.2 7.8-8.2
3300 Da and corresponds to an average degree of polymerization of 17. This is in good agreement with the molecular weight (Mn) of 4000, which was calculated after NMR analysis. NMR Signal Assignment. A typical spectrum of a mixture of DPPC, cholesterol, and PHEA-DODASuc is shown in Figure 3. Table 2 gives the peak assignments based on chemical shifts and intensities and comparison with literature (10-12). The cholesterol signal chosen for calculation of the ratio of the three compounds in the lipid mixture was at δ 5.2 (proton at C6 at the double bond); for DPPC the signal at δ 5.1 (proton at G2, the middle glycerol carbon atom) and for PHEA-DODASuc the signal at δ 3.5 (protons at C8, side chain-CH2 next to OH, two protons, for n ) 17, 2 × 17 ) 34 protons) were used. The chosen peaks are relatively narrow and sufficiently resolved to make correction for overlap unnecessary. NMR Analysis of Lipid Mixtures: Accuracy, Precision, and Linearity. Accuracy and precision of the NMR analysis was assessed by analyzing a single lipid mixture with a DPPC/ cholesterol/PHEA-DODASuc molar ratio of 62:34:4 on five consecutive days. The method allows for a relative quantitation of the lipid mixtures or, more precisely, assessing their molar composition. The integral values of the proton signals of DPPC (δ 5.1) and PHEA-DODASuc (δ 3.5, corrected for the number
Figure 4. Correlation between the percentage of PHEA-DODASuc (mol % of total lipid) in the feed and the calculated percentage of PHEA-DODASuc after NMR analysis.
of protons by division by 34) were compared with the value of the cholesterol integral (δ 5.2), which was set to one. Results were expressed as percentage of the feed fraction. The DPPC amount was determined to be 98% ( 5% of the feed fraction. In the case of PHEA-DODASuc 104% ( 5% of the feed fraction was found. Thus, these findings are in excellent agreement with the actual composition of the lipid mixture. Additionally, the accuracy and precision of the sample preparation were investigated. Five lipid mixtures with the same composition (DPPC/cholesterol/PHEA-DODASuc, 62:34:4) were prepared and analyzed on five subsequent days. For DPPC 100% ( 6% of the feed fraction was found. The spectrum of PHEADODASuc revealed 101% ( 4% of the feed fraction. Again, these values are in excellent agreement with the actual composition of the lipid mixture. Hence, the sample preparation does not influence the accuracy of the method. To study the linearity of the method, lipid mixtures with different amounts of PHEA-DODASuc were analyzed by NMR. Lipid ratios were chosen in a range relevant for liposome preparation (PHEA-DODASuc 3.0, 4.5, 6.1, and 9.2 mol % of total lipid). In Figure 4, the percentage of PHEA-DODASuc found after NMR analysis is plotted against the percentage of PHEA-DODASuc in the feed. This figure shows that the fraction of the coating conjugate in the mixture determined by NMR corresponds linearly with the feed fraction. The regression coefficient was >0.99. The NMR method is therefore suitable to accurately determine the composition of mixtures of DPPC, cholesterol, and PHEA-DODASuc.
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Technical Notes
Figure 5. 1H NMR spectrum of (a) native PHEA-DODASuc and (b) the freeze-dried supernatant of ultracentrifugation-treated liposomes, both in d6-DMSO. The arrows indicate peaks of the DODASuc anchor. Table 3. Amount of DPPC and PHEA-DODASuc Relative to Cholesterol in Two Liposomal Mixturesa lipid composition DPPC/chol/ PHEA-DODASuc 65:33:2 62:33:5
DPPC % of feed before after separationb separationb 96 ( 5 100 ( 5
102 ( 5 100 ( 5
PHEA-DODASuc % of feed before after separationb separationb 98 ( 5 107 ( 5
85 ( 5 88 ( 5
a The results are expressed as the calculated value ( the error. separation: removal of nongrafted conjugate from liposomes by ultracentrifugation (see Materials and Methods)
b
NMR Analysis of Liposomal Mixtures. Table 3 shows the results of the analysis of two freeze-dried liposomal dispersions. The compositions were determined before and after separation of nongrafted components from the liposomes by ultracentrifugation. The calculated fractions of DPPC, both before and after centrifugation, are in good agreement with the fraction in the feed. The fraction of PHEA-DODASuc in liposomes before ultracentrifugation is also in agreement with the feed fraction. This implies that the liposome preparation process does not alter the molar composition of the initial lipid mixture and the lipid film was fully converted into liposomes. After separation of nongrafted components by ultracentrifugation, however, the fractions of PHEA-DODASuc were 85% (17 nmol of PHEA/ µmol of total lipid) and 88% (44 nmol of PHEA/µmol of total lipid) of the feed fractions, respectively. This indicates that not all of the PHEA molecules that are used for liposome preparation were attached to the liposome surface. It is likely that the nonincorporated fraction is the fraction of the added PHEA molecules that lack the lipid anchor (DODASuc) and therefore is not grafted onto the liposome bilayer. To evaluate this, an NMR spectrum of the supernatant of liposomes obtained after ultracentrifugation and freeze-drying was recorded. Figure 5b
shows that the peaks that belong to the anchor part of the molecule at a chemical shift of δ 0.8-1.6 are absent in this spectrum (compare the spectrum of the native PHEA-DODASuc, Figure 5a). Whereas conjugate molecules with the anchor are likely quantitatively grafted onto the bilayers, the PHEA molecules without the anchor stay in solution. This means that approximately 85% of the PHEA molecules of the batch used in these experiments are attached to the DODASuc anchor and can be grafted onto the liposome bilayer. This is an important issue when studying the shielding properties of PHEA-DODASuc in relation to the grafting density. In summary, this paper presents a fast, reproducible, and relatively sensitive NMR-based analysis method for the quantitation of PHEA-DODASuc in liposomes.
LITERATURE CITED (1) Woodle, M. C., and Lasic, D. D. (1992) Sterically stabilized liposomes. Biochim. Biophys. Acta 1113, 171-199. (2) Torchilin, V. P., and Trubetskoy, V. S. (1995) Which polymers can make nanoparticulate drug carriers long-circulating? AdV. Drug DeliVery ReV. 16, 141-155. (3) Storm, G., Belliot, S. O., Daemen, T., and Lasic, D. D. (1995) Surface modification of nanoparticles to oppose uptake by the mononuclear phagocyte system. AdV. Drug DeliVery ReV. 17, 3148. (4) Metselaar, J. M., Bruin, P., de Boer, L. W., de Vringer, T., Snel, C., Oussoren, C., Wauben, M. H., Crommelin, D. J., Storm, G., and Hennink, W. E. (2003) A novel family of L-amino acid-based biodegradable polymer-lipid conjugates for the development of long-circulating liposomes with effective drug-targeting capacity. Bioconjugate Chem. 14, 1156-1164.
864 Bioconjugate Chem., Vol. 17, No. 3, 2006 (5) Romberg, B., Metselaar, J. M., de Vringer, T., Motonaga, K., Kettenes-van den Bosch, J. J., Oussoren, C., Storm, G., and Hennink, W. E. (2005) Enzymatic Degradation of Liposome-Grafted Poly(hydroxyethyl L-glutamine). Bioconjugate Chem. 16, 767774. (6) Torchilin, V. P., Omelyanenko, V. G., Papisov, M. I., Bogdanov, A. A., Jr., Trubetskoy, V. S., Herron, J. N., and Gentry, C. A. (1994) Poly(ethylene glycol) on the liposome surface: on the mechanism of polymer-coated liposome longevity. Biochim. Biophys. Acta 1195, 11-20. (7) Vernooij, E. A., Gentry, C. A., Herron, J. N., Crommelin, D. J., and Kettenes-van den Bosch, J. J. (1999) 1H NMR quantification of poly(ethylene glycol)-phosphatidylethanolamine in phospholipid mixtures. Pharm. Res. 16, 1658-1661. (8) Boada, J., Gallardo, M., and Estelrich, J. (1997) Determination of poly(ethylene glycol) activated with cyanuric chloride in liposomes. Anal. Biochem. 253, 33-36.
Romberg et al. (9) Garcia-Fuentes, M., Torres, D., Martin-Pastor, M., and Alonso, M. J. (2004) Application of NMR Spectroscopy to the Characterization of PEG-Stabilized Lipid Nanoparticles. Langmuir 20, 88398845. (10) Han, X., Chen, X., and Gross, R. W. (1991) Chemical and magnetic inequivalence of glycerol protons in individual subclasses of choline glycerophospholipids: implications for subclass-specific changes in membrane conformational states. J. Am. Chem. Soc. 113, 7104-7109. (11) Sparling, M. L., Zidovetzki, R., Muller, L., and Chan, S. I. (1989) Analysis of membrane lipids by 500 MHz 1H NMR. Anal. Biochem. 178, 67-76. (12) Spectral Database for Organic Compounds SDBS, National Institute of Advances in Industrial Science and Technology, 887HSP40-636 and 16108HSP-45-792. BC060045A
