Bioconjugate Chem. 2008, 19, 1491–1495
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Minimal Chemical Modification of Reductive End of Dextran to Produce an Amphiphilic Polysaccharide Able to Incorporate onto Lipid Nanocapsules Antoine Richard,† Alexandre Barras,† Amena Ben Younes,‡ Nicole Monfilliette-Dupont,*,† and Patricia Melnyk† Laboratoire de Chimie et MicroNanotechnologie a` Vise´e The´rapeutique, UMR 8161 CNRS-Universite´ de Lille 2-Universite´ de Lille 1-Institut Pasteur de Lille, and INSERM IFR 142 Institut Pasteur de Lille-Institut de Biologie de Lille, Institut de Biologie de Lille, 1 Rue du Pr. Calmette, 59021 Lille cedex, France. Received December 4, 2007; Revised Manuscript Received May 19, 2008
In order to graft an amphiphilic polysaccharide to lipid nanocapsules, we present here a new method of dextran lipidation. The lipidation strategy is based on the formation of an oxime linkage between the amphiphilic hydroxylamine C16E20ONH2 and the reductive end of a 40 kDa dextran. This chemoselective reaction allows us to control the lipidation site and the number of lipid introduced on the dextran molecule. This new amphiphilic dextran was used to coat the surface of lipid nanocapsules. The coating efficiency was followed by dynamic light scattering and the presence of the polysaccharide was confirmed by 1H NMR and observed by electronic microscopy.
INTRODUCITON Since the development of liposomes, many other drug carriers in the nanometer range have been in development: polymer particles, solid lipid nanoparticles, lipid nanocapsules, and dendrimers (1). In order to extend the release of a drug and/or to address nanoparticles to the target cells or organ to treat, much attention has been devoted to increasing the blood halftime of these nanocarriers. Indeed, due to their interaction with the reticulo-endothelial system, their blood half-life is very low (2, 3). Toward this aim, nanoparticles were usually covered by a hydrophilic polymer, often a poly(ethylene glycol) (PEG). The longer the PEG, the more the blood half-time increases. Usually, medium molecular weight PEGs (2000-5000 Da) were used (2, 3). Instead of PEG, biopolymers like polysaccharides can be used. Some authors reported the coating of liposome with dextran or chitosan (4–9). In order to anchor the polymer onto the liposome surface, a chemical modification of the polysaccharide with a hydrophobic function is necessary. These hydrophobic moieties, such as cholesteryl derivatives, were generally grafted in a statistically random manner on hydroxyl or amine functional groups present on the carbohydrate (4, 5, 10). By modulating the polysaccharide/lipophilic molecule ratio, it is possible to control the number of lipid moieties attached to the polysaccharide. Nevertheless, the reaction could occur anywhere on the reactive functions of the polymer. We propose here a lipidation method of a dextran to attach a single lipid molecule at the polysaccharide reductive end. This coupling was achieved by forming an oxime bond as described by Dumy et al. (11–13) The oxime formation was achieved by reaction of a hydroxylamine with native dextran in aqueous medium. This chemoselective method can be used while maintaining biological properties (aqueous solubility, molecular recognition, etc.) and chemical functions which can be present on the polysaccharide chain. Moreover, by anchoring the sugar by one of its ends on the particle, we take advantage of its full length, which is impossible with statistical “lipidation” on the polymer. * To whom correspondence should be addressed. Phone: +33 3 20 87 12 20. Fax +33 3 20 87 12 33. E-mail:
[email protected]. † UMR 8161 CNRS. ‡ INSERM IFR 142.
As a proof of concept, the amphiphilic derivative 3 of dextran T40 (40 kDa) was synthesized and was anchored onto lipid nanocapsules. These nanocapsules consist of an oily liquid triglyceride core surrounded by a tensioactive cohesive interface. This tensioactive cohesive interface was mainly constituted of solutol HS15 which exposes a medium PEG chain containing an average of 15 ethylene glycol units (14, 15). To minimize the nanocapsule disturbance by the carbohydrate moiety, a PEG chain of an average of 20 ethylene glycol units was introduced between the polysaccharide and a C16 alkyl chain.
EXPERIMENTAL SECTION Chemicals and Materials. Mass spectra were recorded on a Perspective Biosystems Voyager-DE STR, Biospectrometry Workstation MALDI-TOF spectrometer, and measurements were acquired after deposition on a dihydroxybenzoic acid (DHB) matrix. NMR spectra were recorded on a 300 MHz Bruker spectrometer. Chemicals reagents were purchased from Sigma-Aldrich. Lipid nanocapsules were made of labrafac lipophile WL 1349, soy lecithin Phospholipon 90G, and Solutol HS 15 were generously provided by Gattefosse´ SA, Phospholipid, and BASF, respectively. Anchor Synthesis. Compound 1: Under nitrogen atmosphere, 1 g (0.89 mmol, 1 equiv) of Brij-58P, 0.698 g (2.66 mmol, 3 equiv) of PPh3, and 0.349 g (2.14 mmol, 2.4 equiv) of N-hydroxyphthalimide were dissolved in 20 mL of THF freshly distilled over sodium in the presence of benzophenone. 560 µL (2.66 mmol, 3 equiv) of DIAD were then added dropwise. The reaction medium was stirred for 3 h under nitrogen atmosphere and was concentrated under vacuum. The residue was precipitated in 50 mL of dry diethyl ether at 4 °C and then centrifuged. The resulting solid was collected and triturated twice with diethyl ether. The white solid obtained was dried under vacuum in the presence of P2O5 overnight. 952 mg of the phthalimide derivative was collected. Yield: 84%. 1H NMR, CDCl3, ppm: 7.83 (m, 2H); 7.76 (m, 2H); 4.38 (t, 4.5 Hz, 2H); 3.88 (t, 5 Hz, 2H); 3.65 (s, 42H); 3.44 (t, 6.8 Hz, 2H); 1.57 (m, 2H); 1.25 (s, 28H); 0.88 (t, 6.7 Hz, 3H). Compound 2: 952 mg (0.75 mmol, 1 equiv) of compound 1 was dissolved in 20 mL of absolute ethanol and 1 mL (20 mmol, 27 equiv) of hydrazine hydrate was added. After 4 h, the reaction medium was filtered and the solvent was evaporated under reduced pressure. The crude product was dissolved in diethyl
10.1021/bc700444t CCC: $40.75 2008 American Chemical Society Published on Web 06/21/2008
1492 Bioconjugate Chem., Vol. 19, No. 7, 2008
ether, precipitated at 4 °C, and centrifuged. The solid was collected, dissolved in CH2Cl2, and precipitated twice with diethyl ether. The white solid obtained was then dried overnight under vacuum in the presence of P2O5. 789 mg of the hydroxylamine derivative 2 was collected. Yield: 92%. MALDITOF MS: Two Gaussian distributions of peaks separated by m/z ) 44 corresponding to ethylene glycol monomer. The first Gaussian corresponds to the [M + H+] centered on m/z ) 1138.9 and the second one corresponds to the [M + Na+] centered on m/z ) 1160.9. 1H NMR, CDCl3, ppm: 3.84 (t, 4.6 Hz, 2H); 3.65 (s, 88H); 3.57, (m, 2H); 3.44 (t, 6.8 Hz, 2H); 1.54 (m, 2H); 1.25 (s, 28H); 0.88 (t, 6.7 Hz, 3H). 13C NMR, CDCl3, ppm: 74.9; 71.7; 70.7; 70.2; 69.7; 32.1; 29.8; 29.6. 29.5; 26.2; 22.8; 14.3. Dextran Coupling. Compound 3: 1.6 g (40 µmol, 1 equiv) of dextran T40 and 174 mg (140 µmol, 3.5 equiv) of 2 were dissolved in 3 mL of a 100 mM acetate buffer pH 5.5. After 72 h, the reaction medium was poured onto 50 mL of methanol and centrifuged. The solid was dissolved twice in water, precipitated again in methanol, and centrifuged. Finally, the amphiphilic dextran was dissolved in water and lyophilized. 1.531 g of a white powder was collected. Yield: 93%. 1H NMR: 7.47 (d, 6.2 Hz, 0.8H); 6.83 (d, 6.6 Hz, 0.2H); 4.85 (d, 3.3 Hz, 247H); 3.8 (m, 443H); 3.6 (m, 564H); 3.4 (m, 400H); 1.45 (s, 2H); 1.18 (s, 30H); 0.79 (t, 6.1 Hz, 3H). Nanocapsules. The preparation of lipid nanocapsules was based on a phase inversion method that allows the preparation of very small nanocapsules by thermal manipulation of an oil/ water system as described by Heurtault et al. (14). Briefly, 420 mg of labrafac, 37.5 mg of phospholipon P90, 240 mg of solutol, 22 mg of NaCl, and 540 µL of distilled water were mixed at 80 °C. The mixture was subjected to 3 temperature cycles from 70 to 90 °C under magnetic stirring; then, it was cooled to 78 °C, 3.3 mL of 0 °C water were added, and the suspension was stirred at room temperature. Lipodextran Insertions into Preformed Nanocapsules. Lipodextran nanocapsules were obtained by two postinsertion methods. First method: plain nanocapsules (15 mg LN/mL) were incubated for 24 h with lipodextran (10 mg/mL of LN) under magnetic stirring at room temperature. Second method: plain nanocapsules (15 mg LN/mL) were incubated for 120 min with 100 µL/mL of LN of an aqueous micellar solution of lipodextran (100 mg/mL) under magnetic stirring at 60 °C. The suspension was then dialyzed (pore size: 300 000 Da) against distilled water overnight to remove any uninserted lipodextran from the external volume of nanoparticles. Lipid Nanoparticle Characterization. The average diameter and polydispersity of the particle were determined by photon
Richard et al. Scheme 1. Synthesis of the Amphiphilic Hydroxylamine C16E20ONH2
Scheme 2. Coupling of Dextran to Amphiphilic Hydroxylamine 2
correlation spectroscopy at 25 °C using a DynaProMS800. Nanoparticle suspensions were diluted at 1/2000 (v/v) in distilled water and filtered over 0.22 µm. Mean results were given for 100 acquisitions, and measurements were performed in triplicate. The zeta potential was determined with a Malvern Zetasizer Nano ZS (Malvern Instruments S.A., Worcestershire, UK). Electron Microscopy. Samples were studied using a slightly modified version of the negative staining method as previously described by Op De Beeck et al. (16); 20 µL droplets of free and covered nanocapsule preparations were adsorbed for 1 min onto Formvar carbon-coated nickel grids (400 mesh, Electron Microscopy Science, Washington, PA). The grids were dried by absorption with a filter paper, stained for 1 min on a drop of 2% uranyl acetate stain, air-dried for less than 2 min, and immediately examined under a transmission electron microscope (TEM, Hitachi 7500, Japan) at 80 kV and HR conditions. Because of their rapid degradation, every sample is processed for electron microscopy and immediately examined under TEM.
RESULTS AND DISCUSSION Synthesis. The amphiphilic hydroxylamine moiety 2 was synthesized in two steps. First, the hydroxylamine function was introduced by coupling the N-hydroxyphthalimide by means of a Mitsunobu reaction. Second, the phthalimide group was
Figure 1. Analysis of oxime 1H of amphiphilic dextran 3 by NMR spectroscopy.
Technical Notes
Bioconjugate Chem., Vol. 19, No. 7, 2008 1493
Figure 2. (a) Size distribution evolution of lipid nanocapsules in presence of amphiphilic dextran 3. -- t ) 0 h, s t ) 4 h, · · · · t ) 24 h, - · - · t ) 72 h. (b) Evolution of the mean diameter of lipid nanocapsules with time at room temperature. (c) Evolution of the mean diameter of lipid nanocapsules with time at 60 °C. Table 1. Fitting Results of the Evolution of Nanocapsule Mean Diameter with Eq 1 in the Presence of Amphiphilic Dextran 3 postinsertion
60 °C
room temperature
parameter
value
error
value
error
A B τ R2
58.03 nm 18.3 nm 5.4 h 0.9978
0.7 nm 0.9 nm 0.7 h -
58.53 nm 13.7 nm 0.2 h 0.9945
0.6 nm 0.7 nm 0.03 h -
removed by hydrazinolysis (Scheme 1). The starting product was the commercial nonionic detergent Brij 58P which consists of a C16 alkyl chain bearing a PEG chain of an average of 20 ethylene glycol units with a free hydroxyl group at the chain end. The coupling of hydroxylamine 2 with dextran T40 occurs in an aqueous solvent at high concentration (at less than 10 mM) with an excess of compound 2 (Scheme 2). The hydroxylamine reacts with the reductive end of dextran in 72 h at room temperature to form the corresponding oxime compound. Taking advantage of dextran insolubility in methanol, buffer salt and excess reagent can be removed by precipitation in methanol to give the amphiphilic dextran 3 in an overall yield of 72% for the three steps. 1 H NMR of compound 3 shows the presence of a doublet at 7.47 ppm and another one at 6.83 ppm, which confirms the presence of the oxime link between the dextran and the hydroxylamine 2 (Figure 1). Moreover, taking the acetal proton (4.85 ppm) of the glucose unit of dextran (247 H for Dextran T40) as integration reference, the doublet at 7.47 ppm integrates for roughly 0.8 H and the one at 6.83 ppm for roughly 0.2 H. It is thus concluded that the oxime link is in E configuration at 80% and in Z at 20%.
Figure 3. Evolution of nanocapsules mean diameter as a function of dextran concentration: •, amphiphilic dextran; 9, commercial dextran of 40 kDa.
Table 2. Lipid Nanoparticles Characterization (NP, lipid nanoparticles without amphiphilic dextran; and Dextran NP, lipid nanoparticles with amphiphilic dextran). zeta potential
NP Dextran NP (RT) Dextran NP (60 °C)
mean particle size (nm)
polydispersity (nm)
peak (mV)
width (mV)
59.2 ( 2.9 71.9 ( 1.5 73.8 ( 2.1
6.8 ( 1.6 6.2 ( 1.4 9.4 ( 1.4
-4.08 -2.86 -1.67
10.4 6.0 3.5
After three consecutive dialyses (1:1000 in water), NMR spectra do not change (data not shown), which allows us to conclude that the oxime link is stable. Dextran Incorporation onto Nanocapsules. The dextran T40 average diameter was measured by dynamic light scattering at 9 nm. So, if we anchor this polysaccharide uniformly on the nanocapsules surface, their mean diameter should grow by approximately 20 nm. The size of our nanocapsules, initially close to 58 nm, which were suspended in an amphiphilic dextran solution at 10 mg/mL in water, was in accordance with this hypothesis (Figure 2). The increase of particle mean diameter with time could be well fitted by a monoexponential curve as defined in eq 1 t (1) dj ) A + B · 1 - exp τ in which d is the mean diameter, A the starting diameter of nanoparticles (without dextran), B the mean diameter raise, and τ the characteristic time of dextran insertion. The fitting results of this equation are given in Table 1. We found an increase in diameter of 18.3 ( 0.9 nm. This is in accordance with a full coverage of nanocapsules by a 40 kDa dextran. The anchorage of amphiphilic dextran was complete in 24 h when postinsertion was realized at room temperature with a characteristic time of 5 h 20 min ( 42 min and was complete in 45 min when postinsertion was realized at 60 °C with characteristic time of 12 min ( 1.8 min. For a second time, taking advantage of the hydroxylamine reactivity in aqueous medium, we tried to form the oxime 3 directly on to the nanoparticles. This strategy was already employed to link peptides to lipid vesicles by an oxime bond (17). To this end, compound 2 was first incorporated during the formulation of nanocapsules. These nanocapsules were suspended in dextran solution, but the dextran concentration required (around 10 mM) for the reaction to occur with the hydroxylamine carried by particles was incompatible with nanocapsule stability. Furthermore, the coformulation of the amphiphilic dextran 3 with other components does not yield the expected particles. Thus, the postinsertion method seems to be the most efficient to coat these lipid nanocapsules with our dextran.
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Richard et al.
Figure 4. TEM visualization of lipid nanocapsules. Bar scale represents 100 nm. (a) Lipid nanocapsules without dextran. (b) Lipid nanocapsules in the presence of 5 mg/mL of amphiphilic dextran 3. (c) Enlargement of (b); white arrows show a deposit attributed to dextran.
The presence of the C16 alkyl chain is a necessary condition to achieve the nanoparticles coverage: only polysaccharide 3 brings about a significant increase of nanocapsules diameter. But, as can be seen in Figure 3, a sufficient polysaccharide concentration was needed to obtain a size increase of 18 nm, corresponding to an entire coverage of particles by dextran. In our case, the concentration should be greater than 5 mg/mL to complete the postinsertion. As shown in Table 2, the zeta potential of the nanoparticles measured was slightly influenced by the presence of dextran on the surface. Zeta potential of almost neutral value (-2.86 and -1.67 mV for postinsertion at room temperature and 60 °C, respectively) was measured for the nanoparticles prepared by postinsertion with amphiphilic dextran, which is a neutral polysaccharide. To confirm this result, the nanocapsules were observed by transmission electron microscopy using the negative staining method (Figure 4). A dark deposit dense to electrons was observed on the nanocapsules surface only if the nanocapsules were incubated with amphiphilic dextran. The same experiment was carried out with the nanocapsules in the presence of lipodextran without incubation or free dextran T40 and no dark deposit dense to electrons was observed. This observation suggests that the amphiphilic dextran coats the surface of lipid nanocapsules. In addition, the size of this deposit, as determined from the TEM micrograph, corresponds to the size of 40 kDa dextran. To corroborate our direct observation by electron microscopy, we wanted to have a spectrometric signature of the dextran presence on the nanocapsules surface. Thus after gel filtration through a sephacryl 500HR column, a 1H NMR spectrum of the nanocapsule fraction in H2O/D2O 95/5 solvent was acquired. As the NMR spectra show (Figure 5), specific signals of dextran were detected (in the region between 3.8 and 4.0 ppm) with amphiphilic dextran. In the control experiment with commercial dextran T40, no dextran peak was found on nanoparticles NMR spectrum. Thus, the lipid tail link to dextran allows the polysaccharide fixation onto the surface of nanocapsules. The quantification of lipodextran has been investigated by the 1H NMR method (see Supporting Information for quantitative analysis of lipodextran incorporation onto nanocapsules by 1H NMR). Integration of the signals indicates the quantity of lipodextran on the surface of nanocapsules (5.1% mass incorporation of nanocapsules at RT and 10.1% mass incorporation of nanocapsules at 60 °C). The results indicate that the postinsertion method at 60 °C allows us to introduce approximately twice as much lipodextran on the surface of nanocapsules as at RT.
Figure 5. NMR spectra in H2O/D2O 95/5 of (a) nanocapsules in the presence of amphiphilic dextran 3 after gel filtration, (b) commercial dextran T40, and (c) nanocapsules in the presence of commercial dextran T40 after gel filtration.
Full Conclusion. We have developed here an efficient and convenient method to anchor a polysaccharide onto the surface of lipid nanocapsules. The formation of amphiphilic dextran as well as the formulation of nanocapsules were performed under aqueous conditions without organic solvent. This is an important parameter when biological or pharmaceutical applications are engaged. We have shown that only one alkyl C16 chain is sufficient to anchor a 40 kDa dextran. Moreover, the postinsertion of this carbohydrate can be achieved under mild conditions: at room temperature in less than one day. This method could constitute an attractive alternative to the use of
Technical Notes
PEG and could be useful for decorating lipid particles with functionalized polysaccharide with the aim of active cell targeting.
ACKNOWLEDGMENT This work was financially supported by “La ligue contre le cancer” and “La Cance´ropoˆle Nord-Ouest”. We gratefully acknowledge CNRS, Universite´ de Lille 2, Universite´ de Lille 1, Institut Pasteur de Lille for logistical support, Herve´ Drobecq for MALDI-TOF acquisitions, Ge´rard Montagne for NMR acquisitions, Ludivine Houelrenault for TEM micrographs, and Clement Roux for proof reading. Supporting Information Available: Details of the quantification of lipodextran by 1H NMR method. This material is available free of charge via the Internet at http://pubs.acs.org.
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