Synthesis of Sulfated Cyclodextrin Amphiphiles with Liposomal Encapsulation Properties

A novel class of amphiphiles with sulfate groups at the C-6 position and palmitoyl groups at the C-2, 3 positions of α-, β-, and γ-cyclodextrin (CD) were efficiently synthesized. These compounds formed stable monolayers with high collapse pressures at the air-water interface. The mixed monolayer behaviors of the 6-O-sulfated CD amphiphiles (SO3-CDC16) in the presence of dipalmitoyl phosphatidylcholine (DPPC) and cholesterol were discussed using the surface pressure-molecular area (π-A) isotherms. The collapse pressures showed maxima at molar ratios of SO3-CDC16 lower than 10 mol%. A morphological analysis of the liposomes containing DPPC and 4 mol% SO3-CDC16 formed in PBS was carried out using transmission electron microscopy with negative staining, and vesicles with maximum diameters of 350 500 nm were observed. Moreover, the releasing ability of these liposomes was examined using a fluorescent compound, calcein. It was clearly shown that liposomes containing SO3-CDC16 could release encapsulated calcein more easily than liposomes consisting only of DPPC, and that the release rate depended on the phase transition temperature of the SO3-CDC16 included in the liposome membrane.


Introduction
α-, β-, and γ-cyclodextrins (CDs) are cyclic oligosaccharides consisting of six, seven, and eight D-glucopyranose residues, respectively, which are linked by α-1,4 glycosidic bonds to form macrocycles.CDs have a hydrophilic outer surface and a hydrophobic central cavity and have the ability to form inclusion complexes with specific guests.Therefore, CDs have the potential to alter the physical, chemical, and biological properties of various organic compounds [1]- [3].The most common pharmaceutical application of CDs is to improve the solubility, stability, and bioavailability of various hydrophobic drugs [4].However, natural CDs have relatively low solubility in both water and organic solvents, thus limiting their use in pharmaceutical formulations.Recently, various kinds of CD derivatives have been prepared to extend the physicochemical properties and inclusion capacity of natural CDs as novel drug carriers [5] [6].Many researchers have synthesized CD derivatives bearing saccharides, which are used to target sugar recognition molecules and improve drug delivery capability, and which are expected to have a glycoside cluster effect [7]- [14].However, the high external hydrophilicity of these CD derivatives results in a lack of affinity of the CD-based supramolecules with biological membranes.This problem is one of the reasons why researchers have been interested in CD derivatives with a relatively hydrophobic exterior.As a result, CD derivatives with increased affinity for biological membranes have been obtained by introducing fatty acids onto the hydroxyl groups of CDs.Thus, it is suggested that drug delivery to organs, tissues, and cells could become sufficiently possible.Such amphiphilic CDs have been prepared by grafting hydrocarbon chains on the hydroxyl groups of α-, β-, or γ-CD.For example, a Medusa-like CD was obtained by grafting hydrophobic groups onto all the primary hydroxyl groups of a β-CD [15]- [19].Skirt-shaped CDs were obtained corresponding to esterification or alkylation of all the secondary hydroxyl groups [20] [21].These various CD derivatives do not differ only in the position and length of the alkyl chains, but also in the nature of the chemical bond.
The monolayer behaviors of synthetic CD amphiphiles have been studied at the air-water interface [22]- [28].The surface pressure-molecular area (π-A) isotherms of the monolayers disclose the size of the molecules, as well as the stability and phase behaviors of the monolayer films.The miscibility of mixed monolayers composed of two lipids with various concentration ratios can be elucidated from the π-A isotherms.A correspondence between the ideal line and a plot of molecular area at a fixed surface pressure versus the molar fraction of two lipids shows complete miscibility, whereas deviation from the ideal line represents partial miscibility.Liposomes are artificial cell-like materials and have been considered as a strong candidate for a drug delivery carrier [29]- [33].Drug delivery carriers are required to keep drugs inside their vehicle while traveling through the blood stream and to release the drugs after binding to the target cell.
In a previous paper, we reported the synthesis of 6-O-sulfated CD amphiphiles (SO 3 -CDC 16 ) and preliminary data on the formation of liposomes containing these CD derivatives [34].Herein, the miscibility of monolayers composed of SO 3 -CDC 16 with dipalmitoyl phosphatidylcholine (DPPC) and cholesterol was investigated in detail.Moreover, the formation of these liposomes was confirmed by transition electron microscopy (TEM) using negative staining.To test the encapsulation ability of the liposomes, 3,3'-bis [N,N-bis(carboxymethyl) aminomethyl]fluoroscein (calcein), an anionic dye was captured in the liposomes and changes in fluorescence intensity were used to monitor the time dependence of dye leakage.

Materials and Methods
Unless otherwise stated, all commercially available solvents and reagents were used without further purification. 1H-and 13 C-NMR were recorded at 600 MHz or 150 MHz on a BRUKER 600 spectrometer.Assignment of the ring-protons was made by first-order analysis of the spectra, and was confirmed by H-H COSY and HMQC spectra.Elemental analyses were performed with a Yanako CHN recorder MT-6.Optical rotations were determined with a Perkin-Elmer 241 polarimeter for samples in a 10 cm cell at ambient temperature (22˚C ± 2˚C).Fluorescent measurements were carried out with a Perkin-Elmer Luminescence spectrometer LS-50B.Transmission Electron Microscope (TEM) images were obtained using JEOL JEM-1210 after negative straining with uranyl acetate.Differential Scanning Calorimetry (DSC) was recorded at a Perkin-Elmer DSC 6000.Each sample was heated from 10˚C to 80˚C with at a heating rate of 5˚C/min.Chemical reactions were monitored by thinlayer chromatography (TLC) on precoated plates of silica gel 60F254 (layer thickness, 0.25 mm; E. Merck, Darmstadt).Column chromatography was performed on silica gel (Silica gel 60; 0.015 -0.040 mm, E. Merck).

Surface Pressure-Molecular Area Isotherms for Monomolecular Layers
The π-A isotherms for monomolecular layers were obtained from experiments performed on a Langmuir-type film balance.The water subphase was obtained through reverse osmosis using a Milli-Ro Plus 3 water purification system (Millipore, USA).A mixture of DPPC and cholesterol containing 0 -100 mol% SO 3 -CDC 16 10-12 was spread at the air-water interface from mixed chloroform solutions (0.2 mg/mL) by using a microsyringe (Hamilton).After complete evaporation of the organic solvent, the measurement was performed using a Langmuir-type film balance (U.S.I., Japan) located at the air-water interface at 23˚C.The temperature was controlled by a thermostated bath.The monolayers were compressed at a rate of 60 mm 2 /min.The π-A isotherms were determined at least three times.

Preparation of Liposomes by Reverse-Phase Evaporation
SO 3 -CDC 16 10-12 in diethyl ether was added to a 25 mL round-bottom flask with a cap, and the solvent was removed under reduced pressure.The amphiphile was redissolved in diethyl ether, and pure water was added to the solution for the formation of reverse-phase vesicles (diethyl ether:water = 3:1, v/v).The resulting two-phase system was briefly sonicated in a bath-type sonicator (NEY 28H, USA: 120 W, 45 kHz) under a nitrogen atmosphere until the mixture forms a homogeneous opalescent dispersion.Liposomes consisting of only SO 3 -CDC 16 were purified by column chromatography on a Sephadex G-50 column (30 cm × 1.5 cm i.d.) and eluted with pure water.
DPPC and cholesterol were added to SO 3 -CDC 16 to increase the stability of the liposomes.The mole ratio of DPPC, cholesterol, and SO 3 -CDC 16 10-12 in the liposomes was 100:10:4.The amphiphilic mixtures dissolved in dichloromethane were put into a 25 mL round-bottom flask, and phosphate buffered saline (PBS, pH 7.4) solution was added in the absence or presence of calcein (0.1 mM) (dichloromethane:PBS = 3:1).The mixtures underwent ultrasonication for 5 min until they formed a homogeneous opalescent dispersion.In these suspensions, which were used to form reverse-phase vesicles, dichloromethane was gently removed with a rotary evaporator under reduced pressure.As the majority of the solvent was removed, the material first formed a viscous gel.After destruction of the gel using a vortex mixer for 1 min, dichloromethane was removed completely from the mixture to give a liposome solution.The desired liposomes were separated by column chromatography on a Sephadex G-50 column (30 cm × 1.5 cm i.d.), eluting with PBS at 4˚C.

Determination of Calcein Loading and Releasing Amounts
The amount of calcein trapped in the liposomes was measured according to the method of Oku et al. [35] or Sakai et al. [36] The membrane integrity of the liposome composed of DPPC containing 10 mol% cholesterol and 4 mol% SO 3 -CDC 16 10-12 after incubation in PBS at 37˚C was evaluated by calculating the percentage of retained calcein encapsulated in the liposome.Initially, 0.1 mM calcein in PBS was encapsulated in the.At different time intervals (up to 48 h), the retention of calcein was estimated by mixing 20 μL from each incubation tube with 2.0 mL of PBS (pH 7.4).A fluorescence spectrometer was used to measure the fluorescence intensity of calcein (λ ex = 490 nm, λ em = 520 nm) before (F total ) and after (F in ) the addition of 10 mM CoCl 2 solution (20 μL).Then, 20% Triton X-100 solution (20 μL) was added to release the entrapped calcein, and the fluorescence intensity was measured again to determine the background fluorescence intensity (F q ).The trapped volume (%) of calcein was calculated according to Equation (1): Trapped volume % of total 100 where r is the dilution factor due to the addition of the CoCl 2 and Triton X-100 solutions.Herein, r was 1.02.
Recently, it has been reported by Lesieur et al. that the esterification of CD derivatives using hexanoyl chloride in the presence of 4-dimethylaminopyridine (DMAP) results in over-acylation [28]  performed with sulfur trioxide-trimethylamine complex in a mixed solvent of toluene and DMF over 48 h under a nitrogen atmosphere, at 80˚C in the case of the β-and γ-CD amphiphiles and at 100˚C in the case of the α-CD amphiphile [38].Purification using a Sephadex LH-20 column gave the desired SO 3 -α-, β-, and γ-CDC 16 10-12 bearing palmitoyl groups.A shift of ~4 ppm in the low magnetic field for the C-6 position in the 13 C-NMR spectra showed that the primary hydroxyl groups were sulfated efficiently.The chemical structures of each CD derivative prepared here were elucidated using 1 H and 13 C NMR data and elemental analyses.

Monolayer Behaviors of SO3-CDC16 at the Air-Water Interface
The monolayer behaviors of SO 3 -CDC 16 10-12 at the air-water interface were evaluated.The π-A isotherms recorded for 10-12 on pure water are shown in Figure 2. The isotherms indicated that each SO 3 -CDC 16 forms a stable monolayer with a high collapse pressure (40 mN/m) at the air-water interface.The observed molecular areas at the collapse pressure were 2.45, 2.98, and 3.20 nm 2 for 10, 11, and 12, respectively.The corresponding mean areas per single lipid chain were 0.204, 0.213, and 0.200 nm 2 for 10, 11 and 12, respectively.These values are independent of the size of the cyclodextrin and close to the calculated hydrocarbon cross-sectional area of 0.200 nm 2 .This result indicates that the hydrophobic chains of SO 3 -CDC 16 are well-packed and form dense monolayers.Similarly, the π-A isotherms for the DPPC-cholesterol monolayer (mole ratio of 100:10) containing 10-12 (0 to 50 mol%) were investigated.Figure 3 shows the π-A isotherms at the air-water interface for DPPCcholesterol, 11, and their mixtures.The films containing between 4 and 8 mol% of 11 formed liquid condensed films with high collapse pressures (max 65 mN/m).This collapse pressure is close to the surface tension of water (72.5 mN/m, 25˚C).With more than 27 mol% of 11-12, the collapse pressures of the mixed films were close  to those of pure SO 3 -CDC 16 (~40 mN/m), and the shape of the isotherms was similar.The molecular areas at a pressure of 30 mN/m are plotted as a function of SO 3 -CDC 16 concentration in Figure 4.For most of the mixed ratios for each SO 3 -CDC 16 , the plots of the experimental molecule area at 30 mN/m were located on the ideal straight line.However, slight positive deviations from the ideal straight line were observed when the molecular fraction of SO 3 -CDC 16 was less than 8 mol%.The variation of the collapse pressures for 10-12 is shown in Figure 5.The collapse pressures showed maxima between 2 and 8 mol%.These results indicate that SO 3 -CDC 16 forms stable monolayers at low concentrations by interacting with DPPC-cholesterol.A possible interaction between SO 3 -CDC 16 and the matrix lipids is an electrostatic interaction between the positive charge of DPPC and the negative charge of the sulfonic moieties in SO 3 -CDC 16 .For example, ~20 DPPC molecules surround a single CD derivative when the concentration of 11 is 4 mol%.In this work, good miscibility of the monolayers with up to 8 mol% of 10 and 11 supports this suggestion.However, the decrease of collapse pressure at 8 mol% of 12 may be caused by an insufficient number of DPPC molecules to encircle 12.
These results show that the mixed monolayers containing no more than 8 mol% of 10 and 11 or less than 8 mol% of 12 have higher collapse pressures than those of only DPPC-cholesterol or only SO 3 -CDC 16 , and therefore form more stable monolayers than SO 3 -CDC 16 alone.The more stable mixed films are formed because there are a sufficient number of DPPC molecules to entirely surround SO 3 -CDC 16 .Thus, various activity measurements were performed with 4 mol% of SO 3 -CDC 16 , as this is sufficient for DPPC to encircle SO 3 -CDC 16 .

Morphological Analysis of Liposomes Composed of SO3-CDC16
A morphological analysis of liposomes composed of SO 3 -CDC 16 was performed by TEM after negative staining with a 2% uranyl acetate solution.First, the preparation of liposomes composed only of SO 3 -CDC 16 , in the  absence of other amphiphiles was carried out by the reverse-phase evaporation method in water [39]- [41].The TEM images of the liposomes are shown in Figure 6.The liposomes composed of 10 (α-liposome) were observed as spherical particles with a diameter of ~150 nm at the maximum and tended to be smaller than those of the other SO 3 -CDC 16 derivatives.The liposomes composed of 11 (β-liposome) had a diameter of ~350 nm at the maximum, whereas, the liposomes composed of 12 (γ-liposome) had a diameter of 50 -120 nm at the maximum.The γ-liposomes tended to aggregate with each other.However, these liposomes composed only of SO 3 -CDC 16 could not be prepared in buffer with high ion concentrations.In order to make liposomes that would be stable in a living body as drug delivery carriers, SO 3 -CDC 16 were introduced into liposomal membranes composed of phospholipids.Generally, liposomes composed of the CD amphiphiles were added to other amphiphiles, such as surfactants.Based on the results of the π-A isotherms, monolayers containing 4 mol% of SO 3 -CDC 16 were the most stable.Therefore, liposomes with the same components are expected to be stable in buffer or other media.Figure 7 shows spherical particles composed of DPPC and cholesterol (100:10, mole ratio) containing 4 mol% of SO 3 -CDC 16 .The liposomes containing 4 mol% of 10 (4 mol% α-liposome) had diameters of around 50 -250 nm with 400 nm at the maximum.In liposomes containing 4 mol% of 11 (4 mol% β-liposome), the diameters were about 50 -400 nm.Similarly, the size of the liposomes containing 4 mol% of 12 (4 mol% γ-liposome) had diameters of 80 -500 nm.

Release of Calcein Encapsulated in Liposomes
The fluorescence of calcein in buffered solution is reduced substantially and rapidly by the addition of Co 2+ ions.In a solution containing calcein encapsulated in liposomes, the observed decrease in fluorescence intensity due to quenching of free calcein by Co 2+ is completed within a few seconds of adding Co 2+ .The remaining fluorescence is not significantly affected by further additions of Co 2+ ions.As revealed by a second decrease in fluorescence intensity, the subsequent addition of detergent allows Co 2+ ions access to the calcein that was sequestered within the liposomes.The remaining fluorescence represents the sum of the fluorescence of the small amount of free calcein and the very low chelate.This can be reduced somewhat by using a very large excess of Co 2+ ions, but the simplest procedure is to take the fluorescence after the detergent addition as the baseline.The fraction of the total volume that is trapped within the liposomes is given as the ratio difference between the initial and final values of fluorescence.
The retention time of vesicles encapsulating calcein was monitored after incubation in PBS for 48 h at 37˚C. Figure 8 shows the retention of calcein in the liposome composed of DPPC and cholesterol (100:10, mole ratio) with and without 4 mol% SO 3 -CDC 16 .More than 20% of the total calcein was released from the liposomes within 2 h at 37˚C; however, after that, the release rate was slow.The time at which half the total amount of   calcein was released was 26, 32, and 42 h for liposomes containing 10, 11, and 12, respectively.Even after 48 h, liposomes that did not contain SO 3 -CDC 16 still retained 60% or more calcein.The liposomal membranes containing SO 3 -CDC 16 are more stable than that consisting only of DPPC; however, the retention time of calcein encapsulated in liposomes containing SO 3 -CDC 16 is shorter.The relation between liposomal membrane stability and retention capacity is not directly proportional.The release of molecules encapsulated in liposomes is related to the membrane mobility caused by the phase transition temperature of the compounds.The phase transition temperatures of DPPC, 10, 11, and 12 are 42˚C, 41.0˚C, 37.2˚C, and 36.6˚C,respectively.When the phase transition temperature is lower than the temperature of the buffered solution, the release of encapsulated calcein from the liposome is accelerated due to turbulence of the membrane.It has been suggested that the retention capability of the liposome could be controlled by changing the kind of CD.

Conclusion
SO 3 -CDC 16 bearing sulfate groups and long acyl chains were synthesized efficiently using α-, β-, and γ-CDs as a starting material.The obtained amphiphiles formed stable monolayers in the presence of DPPC and cholesterol at the air-water interface, and the collapse pressures were maximized at molar ratios of SO 3 -CDC 16 lower than 10 mol%.Moreover, liposomes with DPPC containing 4 mol% SO 3 -CDC 16 formed in PBS could be observed as vesicles with diameters of 350 -500 nm.The ability of these liposomes to release calcein was also investigated, and the liposomes containing SO 3 -CDC 16 were clearly shown to release encapsulated calcein more easily than the liposomes consisting only of DPPC.It was suggested that the release rate depended on the phase transition temperature of the SO 3 -CDC 16 derivative present in the liposome membrane.We are currently investigating the ability of the liposomes containing SO 3 -CDC 16 as drug delivery carriers, and the results will be reported as soon as possible.

Figure 1 .
Figure1shows the synthetic method for SO 3 -α-, β-, and γ-CDC16 .First, selective silylation at the primary hydroxyl groups of α-, β-, and γ-CD was carried out with tert-butyldimethylsilyl chloride (TBDMS-Cl) according to the method described by Ashton et al.[37] to give silylated α-, β-, and γ-CD derivatives 1-3, respectively.Recently, it has been reported byLesieur et al.  that the esterification of CD derivatives using hexanoyl chloride in the presence of 4-dimethylaminopyridine (DMAP) results in over-acylation[28] [29].In fact, the preparation of CD palmitate under this condition leads to asymmetric CD derivatives.Therefore, the use of palmitoyl anhydride could efficiently introduce a palmitoyl groups on the secondary face of CDs.The esterification of silylated CDs 1-3 was performed by adding two equivalents of palmitoyl anhydride and one equivalent of DMAP per hydroxyl group in dry pyridine.The reaction mixture was stirred for 48 h at 70˚C and gave the desired CD palmitates 4-6.Next, desilylation occurred in the presence of boron trifluoride etherate (BF 3 •Et 2 O) at room temperature for 6 h.Desilylated derivatives 8 and 9 were purified by gel filtration chromatography with a Sephadex LH-20 column.Because the desilylated α-CD derivative 7 was very moisture sensitive and more unstable than the other CD derivatives, it was evaporated under reduced pressure and used in the next step without further purification.Finally, sulfation of the free primary hydroxyl groups of the obtained CD amphiphiles 7-9 was

Figure 5 .
Figure 5. Relationships between SO 3 -CDC 16 molar fraction and collapse pressure for mixed monolayers.