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In situ polycondensation of amino acid modified liposomes and their properties
REACTIVE
ELSEVIER
F”&ONAL POLYMERS
Reactive & Functional Polymers 30 (1996) 299-308
In situ polycondensation of amino acid modified liposomes and their properties Zi-Chen Li, Wei He, Fu-Mian Li * Department of Chemistry, Peking Universiq, Received 27 September
1995; accepted
Beijing 100871, China 14 November
1995
Abstract Three amphiphilic amino acids based on glutamic acid, i.e. S-[ 1-carboxy-2-( [N-bistetradecyl-L-glutamate]carbonyl)ethyl]cysteine (l), S-[l-carboxy-2-([N-bishexadecyl-L-glutamate]carbonyl)ethyl]cysteine (Z), S-[1-carboxy-2-([N-bisoctadecyl-L-glutamate]carbonyl)ethyl]cysteine (3), were synthesized. The aggregation behavior of them in water or buffer solution was studied. It was found that upon hydration and sonication in water, they could form stable liposomes. This kind of amino acid modified liposome was then polycondensed locally on the liposome surface to form a polypeptide-surfaced liposome and the peptide formation was detected by IT-IR, GPC, etc. The effect of polycondensation of amino acid on the properties of liposomes were studied by detecting the phase transition temperatures with DSC or measuring the leakage of the encapsulated fluorescent probe from the liposomes. It was observed that the phase transition temperatures of the peptide liposomes moved down and the polycondensation of amino acid moieties obviously increased the leakage of the encapsulated molecules. Keywords:
Liposome; Amino acid; Condensation; Peptide; Permeability
1. Introduction Polymerized liposomes have been studied extensively in recent years as stable biomembrane model and microstructures for applications concerning encapsulation, controlled release, biosensors, enzyme immobilization etc. Among these liposomes, the amphiphiles having unsaturated carbon-carbon bonds, such as diyne or diene, can be polymerized by UV irradiation and result in the stabilization of the self-assembled structures while preserving their morphological features [ 1,2]. However, the resulting polyolefins are difficult to degrade in vivo, and sometimes even cause toxicity, which greatly limit their uses as drug carriers. Modification of liposomes *Corresponding
author. Fax: +86 (IO) 275-1708.
1381-5148/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved. SSDI 1381-5148(95)00117-4
with amphiphilic amino acids followed by in situ polycondensation may be a potential choice to prepare stable liposomes, which are biodegradable and can be further modified [3-61. In this article, three amphiphilic amino acids based on glutamic acid, i.e. S-[1-carboxy-2-([N-bistetradecyl-L-glutamate]carbonyl)ethyl]cysteine (l), S-[1-car-boxy-2-([N-bishexadecyl-~-glutamate] carbonyl)ethyl] cysteine (2), and S-[ l-carboxy2-([N-bisoctadecyl-L-glutamate]carbonyl)ethyl] cysteine (3), were synthesized. The aggregation behavior of these three amphiphilic amino acids in water or buffer solutions were studied. They could all form stable liposomes. The in situ polycondensation of amino acid moieties in the liposomes and the effects of polycondensation on the properties of these liposomes will be reported here.
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2.2. Measurements
CH,U-Q,_, 0 L H, CHJCHJ,,
0F d
Infrared spectra were recorded on a Nicolet 750 FT-IR Spectrometer. ‘H-NMR spectra were recorded on a Bruke ARX-400 instrument. Chemical shifts are expressed in ppm downfield from TMS. FAB-MS spectra were measured with a ZAB-HS instrument.
CH,CHCOOH k CH, HCOOH F NH*
ic H*
n = 14, (1)
2.3. Synthesis of monomers
y1= 16, (2)
The three-step synthesis of 1, 2 and 3 is given in Scheme 1.
n = 18, (3) 2. Experimental 2. I. Materials
2.3.1. General procedure for the preparation of I, ZZand III
All reagents and chemicals were obtained from commercial sources and used without further purification. Tetradecanol, hexadecanol and octadecanol were recrystallized from methanol. Benzene was refluxed over Na and redistilled. Chloroform was dried with anhydrous sodium carbonate and redistilled. N-Cyclohexyl-N’-[B(N-methyl-morpholine)ethyl]carbodiimide-p-toluenesulfate (WSD) was purchased from Tokyo Kasei Co. 5(6)-Carboxyfluoresein (5(6)-CF) was purchased from Sigma Chemical co.
Amounts of 10 mmol of I_.-glutamic acid and 12 mmol of p-toluenesulfonic acid monohydrate were dissolved in 50 ml benzene and stirred at room temperature for 30 min. Then 22 mm01 of alkyl alcohol was added and the mixture was refluxed for 10 h, during which time the reaction water was collected in a trap. After benzene was removed under vacuum, the residue was recrystallized from methanol. Bistetradecyl-L-glutamate p-toluenesulfate (I): white powder with mp 51-52°C; 73% yield; ‘H NMR (400 MHz, CDCls): 6 0.88 (t, 6H, -CHs), 1.22-1.32 (broad, 44H,
HO& 7 HNH 2
TSOH,
7 HNH 2_TsOH
=H3w.&l-1o~
w
H,
CH,GH,&,__,OH +
benzene
reflux
HOfi E H2 0
0
I- III
R Maleic anhydride
CWCH~)~~oO~~~H,,JII+CHCOOH F * CH,
hHi
WO,
CH,GH,),, 0 $ 6 IV-VI
Scheme 1. Synthesis of 1,2 and 3.
Cysteine --
(I),
WY
(3)
-CH?-), 1.53 (t, 4H. -CH?-C-OOC-), 2.1-2.2 (m, 2H, -C-CHz-C(NH?)-COO-), 2.3 (s, 3H, -CHs, aromatic), 2.4-2.5 3.9-4.1 (m, 5H, (m, 2H, -CHz--COO-), -CH*-OOC-, -C-CH(NH?)-COO-), 7.17.8 (m, 4H, aromatic). p-toluenesulfate Bishexadecyl-L-glutamate (II): white powder with mp 58-59°C; 84% yield; ‘H NMR (400 MHZ, CDCls): 6 0.88 (t, 6H. -CH?), 1.22-1.32 (broad, 52H, -CH?-): 1.53 (t, 4H, -CH2-C-OOC-), 2.1-2.2 (m, 2H. -C-CH:-C(NHz)-COO-), 2.3 (s, 3H. -CH3, aromatic), 2.4-2.5 (m. 2H. -CH?-COO-), 3.9-4.1 (m, 5H, -CH?-OOC-, -C-CH(NH2)-COO-), 7.17.8 (m, 4H, aromatic). p-toluenesulfate Bisoctadecyl-L-glutamate (III): white powder with mp 67-69°C; 86% ‘H NMR (400 MHz, CDCls): 6 yield; 1.22-l .32 (broad, 60H, 0.88 (t, 6H. -CHj), -CH?-), 1.53 (t, 4H, -CH2-C-OOC-), 2.1-2.2 (m, 2H, -C-CH?-C(NHz)-COO-), 2.3 (s, 3H, -CH3, aromatic), 2.4-2.5 (m, 2H. -CH?-COO-), 3.9-4.1 (m, 5H, -CH?-OOC-, -C-CH(NH:!)-COO-), 7.17.8 (m, 4H, aromatic). 2.3.2. Generulprocedure,for the preparation of ZV, V and VI To a 6-mmol solution of maleic anhydride and 6 mmol of I (or II, III) in chloroform was added 12.5 mmol of anhydrous triethylamine at room temperature. The reaction mixture was stirred at 40°C for 5 h. After cooling, the solution was washed three times with 0.1 N HCl, dried over anhydrous MgS04 and then the solvent was removed by evaporation. The crude product was recrystallized from petroleum ether. Maleic acid (N-bistetradecyl-L-glutamate)monoamide (IV): white powder with mp 5152°C; 85% yield; ‘H NMR (400 MHz, CDC13): 6 0.88 (t, 6H, -CHj), 1.22-1.32 (broad, 44H, -CH?-), 1.65 (t, 4H. -CH?-C-OOC-), 2.1-2.3 (m, 2H, -C-CH?--C(NH2)-COO-), 2.4 (m, 2H, -CH?-COO-), 4.0-4.2 (m, 4H. -CH?-OOC-), 4.6 (m, 2H, -C-CH(NHz)-COO-), 6.3-6.4 (m, 2H, -CH=CH-). 7.9 (broad. lH, -NH-CO-).
Maleic acid (N-bishexadecyl L-glutamate)monoamide (V): white powder with mp 5859°C; 89% yield; ‘H NMR (400 MHz, CDCls): S 0.88 (t, 6H, -CH3), 1.22-1.32 (broad, 52H, -CH?-), 1.65 (t, 4H, -CH2-C-OOC-), 2.1-2.3 (m, 2H. -C-CH?--C(NHz)-COO-). 2.4 (m, 2H, -CH2-COO-), 4.0-4.2 -CH2-OOC-), 4.6 (m, 2H, (m, 4H, -C-CH(NH2)-COO-), 6.3-6.4 (m, 2H, -CH==CH-), 7.9 (broad, lH, -NH-CO-). Maleic acid (N-bisoctadecyl-L-glutamate)monoamide (VI): white powder with mp 6869°C; 92% yield; ‘H NMR (400 MHz, CDC13): 6 0.88 (t, 6H, -CHs), 1.22-1.32 (broad, 60H, -CH?-). 1.65 (t, 4H, -CH?-C-OOC-). 2.1-2.3 (m, 2H, -C-CHz--C(NHz)-COO-). 2.4 (m, 2H, -CH2-COO-), 4.0-4.2 -CH?-OOC-), 4.6 (m, 2H, (m, 4H, -C-CH(NH?)-COO-), 6.3-6.4 (m, 2H. -CH=CH-), 7.9 (broad, 1H. -NH-CO-). 2.3.3. Synthesis of the amphiphilic amino acids 1, 2 and 3 General procedure: Cysteine hydrochloride (5 mmol) was dissolved in 10 ml of water, and sodium hydrogen carbonate was added to raise the pH to 7-8, then 10 ml phosphate buffer solution (pH = 7.4) was added. This mixture was added to a hot propanol solution of the above maleic monoamide (4 mmol). The mixture was stirred under nitrogen atmosphere at 50°C for 8 h. After 80 ml 2 N HCl was added, the mixture was poured into 900 ml of ice water. The white precipitate was collected by suction and washed extensively with water. After drying, the crude product was recrystallized from chloroform/methanol. S - [ 1-carboxy-2-( [N-bistetradecyl-L-glutamate]carbonyl)ethyl]cysteine (1): white powder with mp 113-139°C liquid crystal; 87% yield; FAB-MS: (M + H)+ = 759; ‘H NMR (400 MHz, CDCl?): 6 0.88 (t, 6H, -CHs), 1.22-1.32 (broad, 44H, -CH:!-), 1.62 (t, 4H, -CH7-C-OOC-), 2.0-2.2 (m, 2H, -C-CH?-C(NH2)-COO-), 2.3 (m, 2H, -CHz-COO-), 2.4-4.0 (m, 6H, --N-CO-CHZ-CH-COOH, -SCH$ZH (NH:!)COOH), 4.0-4.2 (m, 4H, -CHz--OOC-), 4.5 (m, 1H. -C-CH(NHz)-COO-), 7.9
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(broad, lH, -NH-CO-). Analysis: Calculated for CaH74N209S: C, 63.35; H, 9.76; N, 3.69; S, 4.22. Found: C, 63.01; H, 9.86; N, 3.44; S, 4.18. S- [ 1-carboxy-2-( [N-bishexadecyl-L-glutamate]carbonyl)ethyl]cysteine (2): white powder with mp 119-150°C liquid crystal; 89% yield; FAB-MS: (M+H)+ = 815; ‘H NMR (400 MHz, CDC13): S 0.88 (t, 6H, -CHs), 1.22-l .32 (broad, 52H, -CH;!-), 1.62 (t, 4H, -CH2-C-OOC-), 2.0-2.2 (m, 2H, -C-CH2-C(NH*)-COO-), 2.4-4.0 (m, 2.3 (m, 2H, -CH2-COO-), 6H, -N-CO-CH2-CH-COOH, -SCH2CH (NHz)COOH), 4.0-4.2(m, 4H, -CH,-OOC-), 7.9 4.5 (m, lH, -C-CH(NH2)-COO-), Analysis: Calculated (broad, 1H, -NH-CO-). for CuHs2N209S: C, 64.89; H, 10.07; N, 3.44; S, 3.93. Found: C, 64.32; H, 10.22; N, 3.83; S, 3.87. S - [ 1-carboxy-2-( [ N-bisoctadecyl-L-glutamate]carbonyl)ethyl]cysteine (3): white powder with mp 117-145°C liquid crystal; 86% yield; FAB-MS: (M+H)+ = 871; ‘H NMR (400 MHz, CDCls): S 0.88 (t, 6H, -CH3), 1.22-1.32 (broad, 6OH, -CH2-), 1.62(t, 4H, -CH2-C-OOC-), 2.0-2.2 (m, 2H, -C-CH2-C(NH2)-COO-), 2.4-4.0 (m, 2.3 (m, 2H, -CH2-COO-), 6H, -N-CO-CH2-CH-COOH, -SCH&H (NHz)COOH), 4.0-4.2 (m, 4H, -CH2-OOC-), 7.9 4.5 (m, lH, -C-CH(NH2)-COO-), (broad, lH, -NH-CO-). Analysis: Calculated for C4sH9aN209S: C, 66.23; H, 10.34; N, 3.22; S, 3.68. Found: C, 65.55; H, 10.51; N, 3.78; S, 3.61. 2.4. Liposome preparation Typically, 10 mg of 1(2 or 3) was dissolved in 1 ml of chloroform. The solvent was thoroughly removed under vacuum for 24 h, 5 ml of buffer solution of different pH values (6.5,7.4, 8.5) was added, after vortexing for 10 min, the aqueous suspension was sonicated at 75°C for 20 min (60 W, bath type sonicator). For the preparation of uniform liposome, either of the following two methods may be used. The liposome solution was filtered through a 1 x 18 cm Sephadex G50 column or separated by ultracentrifugation (16,000 rpm, 10 min).
2.5. TEA4 observation
The liposome solution prepared by the above method was dropped onto a carbon-coated copper grid, excess liquid was blotted off, and air dried. Aqueous uranyl acetate was then dropped onto the dried specimen. After it was dried in vacua, the grid was observed on a JEOL-IOOCXII transmission electron microscope. 2.6. In situ polycondensation of amino acid modijied liposomes 1 (2 or 3) was sonicated with WSD in a buffer solution (70°C 60 W, 50 min), the molar ratio of WSD to lipid was varied from 0 to 3. The freshly prepared and cooled liposomal solution was ultracentrifuged and the upper layer of the resulting solution was used to evaluate the stability of liposomes by following the turbidity change at 400 nm (Schimadzu UV-2000 Spectrometer). The liposomal solution was allowed to stand at room temperature for 24 h and then observed on TEM to confirm the bilayer Iiposome structure.
2.7. Purification and characterization of the condensation product
The liposome prepared in a water-soluble carbodiimide (WSD) containing buffer solution was allowed to stand at room temperature for 24 h. Then equal volume of 1 N HCI was added to destroy the liposome structure. The precipitated residue was thoroughly washed with water and vacuum dried. The ET-IR spectrum was recorded on a Nicolet-750 Spectrometer. To measure the molecular weight of the product, part of the free acid groups must be previously converted to methyl esters to increase the solubility of product in THE So the product was dissolved in dry chloroform and methanol, dry HCI gas was introduced until the solution reached saturation. The reaction was kept at room temperature for 2 days. After removal of the solvent, it was dried. The molecular weight was measured on Waters 208 gel permeation chromatography (THF as eluent, polystyrene as standard).
Z.-C. Li et al. /Reactive & Functional Polymers 30 (1996) 299-308
2.8. Differential scanning calorimetry (DSC) 2-3 mg of lipid (or the condensation product) and 20-30 ~1 of water were placed into small pans and allowed to hydrate overnight at 70°C. The DSC analysis was performed on a Du Pont 1090 DSC from 20°C to 80°C with a scan rate of S”C/min. 2.9. Leakage of 5(6)-CF The lipids with or without WSD (molar ratio: WSD/lipid = 1.5) was sonicated in 100 mM 5(6)-CF, 50 mM Tris-HCl buffer (pH 8.5) for 20 min at 70°C. After cooling or standing at room temperature for 24 h, the liposome suspension was separated form the free dye on a Sephadex G-50 column (1 x 18 cm). The fractions containing the liposome was collected and diluted with the same buffer solution containing 100 mM NaCl to suitable concentrations. The leakage of CF was measured by following the increase of emission fluorescence intensity at 523 nm (excited at 493 nm) on a Hitachi 850 Fluorescence Spectrometer. 3. Results and discussion
3.1. Synthesis of 1,2 and3 In the first step of the synthesis, the amino group of glutamic acid was converted to ptoluenesulfate to reduce its reactivity with carboxylic acid. p-Toluenesulfonic acid also acted as a catalyst for the esterification of carboxylic acid with alkyl alcohol. The second step proceeded with ease. The addition of pyridine or triethylamine could turn the salts to free amino groups, and the subsequent reaction of amino groups with maleic anhydride at 50°C was completed in about 5 hours as detected by TLC, For the nucleophilic attack of the sulfur-containing amino acids on the double bonds of maleic amide, both -SH and -NH2 are possible, and the selectivity depends on the pH of the reaction media. Usually, in a low-pH medium, -SH is more active than -NH2. In this case, buffer solution of pH 7.4 was used to maintain the pH of the
303
reaction media [4,7]. 1, 2 and 3 displayed liquid crystalline properties during melting. 3.2. Liposome formation from the amphiphilic amino acids
1,2 and 3 belong to amphiphilic amino acids, just like the natural lipids, upon hydration in water or buffer solution, they can aggregate to form organized structures. For example, when they were hydrated in buffer solutions followed by sonication at 70°C for 20 min, clear liposomal suspensions with variable size particles (1000-3000 A) were formed. Prolonged sonication (usually 0.5 to 1 h) produced a suspension with an average particle size of 1000 A. They were identified as liposomes by their ability to entrap water-soluble markers and by TEM. Three typical electron microphotographs of 1, 2 and 3 are shown in Fig. 1. Since the ionization state of the head group-amino acid moiety is affected by the pH value of the buffer, which may affect their hydration ability and aggregation behavior of 1,2 and 3, three buffer solutions of different pH (6.5, 7.4, 8.5) were used to prepare liposomes. For these three amphiphilic amino acids, they could form stable liposomes in three buffer solutions of different pH as shown in Fig. 1. These amino acid modified liposomes served as the starting materials for the in situ polycondensation reaction of amino acid moieties and as the control for the evaluation of the effects of the polycondensation on the properties of liposomes. 3.3. Polycondensation of amino acid moieties on the surface of liposomes and characterization of the amphiphilic peptides Water-soluble carbodiimides have been extensively used in peptide synthesis [4,8]. The use of water-soluble carbodiimides for condensation of amino acids at the liposome/water interface was first reported by Neumann et al. [S] They also studied the optimum pH for the condensation reaction and found that at lower pH medium (for example 6.5) the condensation reaction proceeded effectively. When 1, 2 and 3 were sonicated in WSD-containing buffer solutions, the
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Z.-C. Li et al. /Reactive & Functional Polymers 30 (1996) 299-308 0.8
0
10
26 30 40 Tie (hr.)
50
60
Fig. 2. Time-dependent turbidity changes of liposomes from 3 (0.2 wt%) at 400 nm in 0.067 phosphate buffer solution (pH 6.5). Molar ratio of carbodiimide to 3: (0) 0, (W) l/l, (A) 1.50, (0) 2/l, (0) 3/l.
Fig. 1.Electron microphotographs of amino acid liposomes from 1, 2 and 3 in different buffer solutions. (a) 1 in 0.05 M borate buffer solution (pH 7.4), magnification: 33000x; (b) 2 in 0.05 M Tris-HCl buffer solution (pH 8.5). magnification: 24000x; (c) 3 in 0.067 M phosphate buffer solution (pH 6.5), magnification: 24000x.
amino acid condensed to form peptide. After 20 min of sonication, the starting material disappeared as detected by TLC. In order to completely convert the monomers to peptides, the condensation reaction required several additional hours incubation at room temperature. To clarify the effects of the WSD concentration and incubation time on the polycondensation reaction, the ratio of WSD to lipid was varied form 0 to 3. No precipitation was observed during the sonication. During the period when the suspension was incubated at 37°C for 48 h, the turbidity change was followed at 400 nm. The results are shown in Fig. 2. It can be seen that there was almost no change of turbidity during this period, which reflected high stability of liposomes. In fact, the liposome structures could still be seen by TEM after 24 h of incubation as shown in Fig. 3. This showed that the in situ polycondensation of 1, 2 and 3 occurred at the interface of liposome/ water. The amino acid liposomes and the peptide ones are stable against aggregation, which is due to the electrostatic repulsive force between the liposomes. After condensation, the liposomes were destroyed by adding diluted HCI. The residue was collected and washed with water, freeze-dried and its IR spectra were recorded. The results from the liposomes of 3 are shown in Fig. 4. Since there is a amide group (1650 cm-‘) in the amphiphilic amino acid, the formation of new amide bond due to the polycondensation was not
305
Z.-C. Li et al. /Reactive & Functional Polymers 30 (1996) 299-308 Table 1 Phase transition temperatures of phospholipid liposomes l-3 liposomes or of peptide liposomes from l-3
and of
Compound
Monomer
Peptide form
Phospholipid
1 2 3
40.8”C 54.9”C 67.3”C
33.2”C 47.O”C 59.2”C
23.9”C (DMPC) a 41 XC (DPPC) 54.9”C (DSPC)
a DMPC = dimyristoylphosphatidylcholine; DPPC = dipalmitoylphosphatidylcholine; DSPC = distearoylphosphatidylcholine.
clearly indicated that the polycondensation of amino acids was taken place. The process may be schemed as shown on Scheme 2. GPC measurement is a method frequently used to evaluate the molecular weight of a polymer. Because of the poor solubility of the condensation product in tetrahydrofuran, the carboxylic acid groups in the condensation product were in part converted to their methyl esters by the following procedure. The condensation product was dissolved in dry chloroform and methanol, and the solution was saturated by introducing dry HCl gas. The reaction mixture was kept at room temperature for 2 days. After the solvent was removed, the residue was dried and dissolved in THF for GPC measurement. From the GPC chart, peptide of more than 15 amphiphilic amino acids was observed, but most of the product were oligomers, the average polymerization degree was determined to be about 3-5. The reason for the low degree of polymerization has been discussed in the literature [5]. 3.4. Phase transition temperature of the amino acid liposomes
Fig. 3. Electron microphotographs of the peptide liposomes from 1, 2 and 3 in 0.05 M borate buffer solution (pH 7.4). Molar ratio of carbodiimide to lipid: 1.5, time: 24 h. (a) 1 (magnification: 24000x): (b) 2 (magnification: 49000x); (c) 3 (magnification: 24000x).
clear, but the peak of ucZo at 1650 cm-’ became wide, and it’s peak area became large. So the increase of the peak intensity of ~c,~/uc-n ratio from 0.108 of 3 to 0.432 of the peptide,
Phase transition temperature (7’J reflects the packing of amphiphiles in bilayer membrane and it is affected by the head groups and hydrophobic chains. For the three amphiphilic amino acids, the phase transition temperatures in swollen lamellar phases were determined by DSC measurements. The results are given in Table 1. For comparison, the phase transition temperatures of phospholipid with the same hydrophobic chain length as 1, 2 and 3 are also given. It can be seen clearly that the T, of the amino acid liposomes were higher than those of the phosphatidylcholines with the
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Z.-C. Li et al. /Reactive & Functional Polymers 30 (1996) 299-308
-nH20 WSD
@=hydrophil.ic unit (e.g. -COOH)
no precipitate due to additional hydrophibic unit
Scheme 2. Schematic representation of condensation polymerization of 1,2 and 3 liposomes.
Wavenumber (cm-l ) Fig. 4. FT-IR spectra of (A) 3 and (B) peptide from the polycondensation of 3 on the surface of liposomes (pH 6.5, time 24 h; [carbodiimde]/I3] = 1.50).
samehydrophobic chain lengths. It is assumed that the formation of hydrogen bonds between the amino acid head groups allows a better chain packing. When amino acid was condensed, Tc of the peptide liposomes moved to lower tem-
peratures as given in Table 1. The formation of peptides caused the shrinkage of the liposome surface and decrease of the arrangement order of hydrocarbon chains.
Z.-C. Li et al. /Reactive
0
& Functional Polymers 30 (1996)299-308
~-L-L 0
loo0
2ooo
3ooo
Time (min.) Fig. 5. Percent release of 5(6)-CF vs. time at 37°C from liposomes of 2 (0) and after 1 h (0) or 24 h (0) polycondensation. (pH 7.4, [carbodiimde]/[2] = 1.5/l).
307
of the surface caused the disorder of the bilayer membrane in the hydrophobic region. However, if the reaction is allowed to proceed for 24 h, the release of CF became slower, though it was still faster than in the case of the monomeric liposome. The longer reaction time leads the formation of larger peptides, which may account for this effect. For the other two amphiphilic amino acids, 1 and 3, similar results were obtained. This means that by controlling the condensation condition, the permeability of the amino acid liposomes can be adjusted, which make this kind of liposome good as controlled drug release system. 4. Conclusion
3.5. Leakage of encapsulated
molecules
An important property of liposome is that it can encapsulate water-soluble compounds in the inner aqueous phase. This is the basis for the use of liposome as drug carriers and releasing system. The leakage of encapsulated molecules from liposomes is frequently used as one of the conventional index for stability and permeability. 5(6)-CF is a commonly used probe. [9] The permeability of liposomes from 1, 2,3 and peptidemodified liposomes were examined by detecting the leakage of the encapsulated 5(6)-CF in a regular interval time (pH 7.4,37”C). Fig. 5 shows the increase of the fluorescence vs. time at 37°C for the liposomes from 2. It can be seen that there is very little increase of fluorescence with time (less than 10%) from the liposomes of 2 even after incubation at 37°C for 48 h. This result is similar to those obtained for the other kind of amino acid liposomes and is significantly less than that normally observed with liposomes from lipid. Since liposome from monomeric amphiphilic amino acids tend to condense and form peptide after sonication with WSD, the liposome prepared in this way in CF buffer solution was used to study the effects of condensation on the permeability of amino acid liposomes. After 1 h of the reaction, the leakage of CF became faster (Fig. 5). This may be caused by the decrease of the surface charge of the bilayer membrane after the condensation reaction. The other possible reason is that the shrinkage
Three novel amphiphilic amino acids based on glutamic acid were synthesized. They could form stable bilayer liposome in water and buffer solutions upon hydration and sonication. The amino acid groups, which located at the surface of liposomes, could be condensed to form oligopeptides. The phase transition temperatures of the amphiphilic amino acids were higher than those of the phospholipids with same hydrophobic chain length and moved down after polycondensation. The low permeability of these liposomes and the fact that the permeability can be adjusted by controlling the extent of condensation reaction make these compounds interesting alternatives to phospholipids for the long term encapsulation of various substances.
Acknowledgements This work was supported in part by the Doctoral Foundation of State Education Commission of China.
References [ 11 H. Bader, K. Dom, B. Hupfer and H. Ringsdorf, Adv. Polym. Sci., 65 (1985) 1. 121 A. Singh and J.M. Schnur, Polym. Adv. Tech., 5 (1994) 358. [3] A. Shibata, S. Yamashita, Y. Ito and T. Yamashita, Biochim. Biophys. Acta, 834 (1986) 147. 141 R. Neumann and H. Ringsdorf, J. Am. Chem. Sot., 108 (1986) 487. [S] R. Neumann, H. Ringsdorf, E.V. Patton and D.F. O’Brien, Biochim. Biophys. Acta, 898 (1987) 338.
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[6] N. Nishikama, M. Arai, M. Ono and I. Itoh, Chem. Lett.
(1993) 2017. [7] J.-H. Fuhrhop, H.-H. David, J. Mathieu, U. Liman, H.-J. Winter and E. Boekema, J. Am. Chem. Sot., 108 (1986) 1785.
[8] J.C. Sheehan and J.J. Hlavka, J. Org. Chem., 21 (1956) 439. [9] J.H. Weinstein, S. Yoshikami, P. Henkart, R. Blumenthal and W.A. Hargins, Science, 195 (1977) 489.
ELSEVIER
F”&ONAL POLYMERS
Reactive & Functional Polymers 30 (1996) 299-308
In situ polycondensation of amino acid modified liposomes and their properties Zi-Chen Li, Wei He, Fu-Mian Li * Department of Chemistry, Peking Universiq, Received 27 September
1995; accepted
Beijing 100871, China 14 November
1995
Abstract Three amphiphilic amino acids based on glutamic acid, i.e. S-[ 1-carboxy-2-( [N-bistetradecyl-L-glutamate]carbonyl)ethyl]cysteine (l), S-[l-carboxy-2-([N-bishexadecyl-L-glutamate]carbonyl)ethyl]cysteine (Z), S-[1-carboxy-2-([N-bisoctadecyl-L-glutamate]carbonyl)ethyl]cysteine (3), were synthesized. The aggregation behavior of them in water or buffer solution was studied. It was found that upon hydration and sonication in water, they could form stable liposomes. This kind of amino acid modified liposome was then polycondensed locally on the liposome surface to form a polypeptide-surfaced liposome and the peptide formation was detected by IT-IR, GPC, etc. The effect of polycondensation of amino acid on the properties of liposomes were studied by detecting the phase transition temperatures with DSC or measuring the leakage of the encapsulated fluorescent probe from the liposomes. It was observed that the phase transition temperatures of the peptide liposomes moved down and the polycondensation of amino acid moieties obviously increased the leakage of the encapsulated molecules. Keywords:
Liposome; Amino acid; Condensation; Peptide; Permeability
1. Introduction Polymerized liposomes have been studied extensively in recent years as stable biomembrane model and microstructures for applications concerning encapsulation, controlled release, biosensors, enzyme immobilization etc. Among these liposomes, the amphiphiles having unsaturated carbon-carbon bonds, such as diyne or diene, can be polymerized by UV irradiation and result in the stabilization of the self-assembled structures while preserving their morphological features [ 1,2]. However, the resulting polyolefins are difficult to degrade in vivo, and sometimes even cause toxicity, which greatly limit their uses as drug carriers. Modification of liposomes *Corresponding
author. Fax: +86 (IO) 275-1708.
1381-5148/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved. SSDI 1381-5148(95)00117-4
with amphiphilic amino acids followed by in situ polycondensation may be a potential choice to prepare stable liposomes, which are biodegradable and can be further modified [3-61. In this article, three amphiphilic amino acids based on glutamic acid, i.e. S-[1-carboxy-2-([N-bistetradecyl-L-glutamate]carbonyl)ethyl]cysteine (l), S-[1-car-boxy-2-([N-bishexadecyl-~-glutamate] carbonyl)ethyl] cysteine (2), and S-[ l-carboxy2-([N-bisoctadecyl-L-glutamate]carbonyl)ethyl] cysteine (3), were synthesized. The aggregation behavior of these three amphiphilic amino acids in water or buffer solutions were studied. They could all form stable liposomes. The in situ polycondensation of amino acid moieties in the liposomes and the effects of polycondensation on the properties of these liposomes will be reported here.
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2.2. Measurements
CH,U-Q,_, 0 L H, CHJCHJ,,
0F d
Infrared spectra were recorded on a Nicolet 750 FT-IR Spectrometer. ‘H-NMR spectra were recorded on a Bruke ARX-400 instrument. Chemical shifts are expressed in ppm downfield from TMS. FAB-MS spectra were measured with a ZAB-HS instrument.
CH,CHCOOH k CH, HCOOH F NH*
ic H*
n = 14, (1)
2.3. Synthesis of monomers
y1= 16, (2)
The three-step synthesis of 1, 2 and 3 is given in Scheme 1.
n = 18, (3) 2. Experimental 2. I. Materials
2.3.1. General procedure for the preparation of I, ZZand III
All reagents and chemicals were obtained from commercial sources and used without further purification. Tetradecanol, hexadecanol and octadecanol were recrystallized from methanol. Benzene was refluxed over Na and redistilled. Chloroform was dried with anhydrous sodium carbonate and redistilled. N-Cyclohexyl-N’-[B(N-methyl-morpholine)ethyl]carbodiimide-p-toluenesulfate (WSD) was purchased from Tokyo Kasei Co. 5(6)-Carboxyfluoresein (5(6)-CF) was purchased from Sigma Chemical co.
Amounts of 10 mmol of I_.-glutamic acid and 12 mmol of p-toluenesulfonic acid monohydrate were dissolved in 50 ml benzene and stirred at room temperature for 30 min. Then 22 mm01 of alkyl alcohol was added and the mixture was refluxed for 10 h, during which time the reaction water was collected in a trap. After benzene was removed under vacuum, the residue was recrystallized from methanol. Bistetradecyl-L-glutamate p-toluenesulfate (I): white powder with mp 51-52°C; 73% yield; ‘H NMR (400 MHz, CDCls): 6 0.88 (t, 6H, -CHs), 1.22-1.32 (broad, 44H,
HO& 7 HNH 2
TSOH,
7 HNH 2_TsOH
=H3w.&l-1o~
w
H,
CH,GH,&,__,OH +
benzene
reflux
HOfi E H2 0
0
I- III
R Maleic anhydride
CWCH~)~~oO~~~H,,JII+CHCOOH F * CH,
hHi
WO,
CH,GH,),, 0 $ 6 IV-VI
Scheme 1. Synthesis of 1,2 and 3.
Cysteine --
(I),
WY
(3)
-CH?-), 1.53 (t, 4H. -CH?-C-OOC-), 2.1-2.2 (m, 2H, -C-CHz-C(NH?)-COO-), 2.3 (s, 3H, -CHs, aromatic), 2.4-2.5 3.9-4.1 (m, 5H, (m, 2H, -CHz--COO-), -CH*-OOC-, -C-CH(NH?)-COO-), 7.17.8 (m, 4H, aromatic). p-toluenesulfate Bishexadecyl-L-glutamate (II): white powder with mp 58-59°C; 84% yield; ‘H NMR (400 MHZ, CDCls): 6 0.88 (t, 6H. -CH?), 1.22-1.32 (broad, 52H, -CH?-): 1.53 (t, 4H, -CH2-C-OOC-), 2.1-2.2 (m, 2H. -C-CH:-C(NHz)-COO-), 2.3 (s, 3H. -CH3, aromatic), 2.4-2.5 (m. 2H. -CH?-COO-), 3.9-4.1 (m, 5H, -CH?-OOC-, -C-CH(NH2)-COO-), 7.17.8 (m, 4H, aromatic). p-toluenesulfate Bisoctadecyl-L-glutamate (III): white powder with mp 67-69°C; 86% ‘H NMR (400 MHz, CDCls): 6 yield; 1.22-l .32 (broad, 60H, 0.88 (t, 6H. -CHj), -CH?-), 1.53 (t, 4H, -CH2-C-OOC-), 2.1-2.2 (m, 2H, -C-CH?-C(NHz)-COO-), 2.3 (s, 3H, -CH3, aromatic), 2.4-2.5 (m, 2H. -CH?-COO-), 3.9-4.1 (m, 5H, -CH?-OOC-, -C-CH(NH:!)-COO-), 7.17.8 (m, 4H, aromatic). 2.3.2. Generulprocedure,for the preparation of ZV, V and VI To a 6-mmol solution of maleic anhydride and 6 mmol of I (or II, III) in chloroform was added 12.5 mmol of anhydrous triethylamine at room temperature. The reaction mixture was stirred at 40°C for 5 h. After cooling, the solution was washed three times with 0.1 N HCl, dried over anhydrous MgS04 and then the solvent was removed by evaporation. The crude product was recrystallized from petroleum ether. Maleic acid (N-bistetradecyl-L-glutamate)monoamide (IV): white powder with mp 5152°C; 85% yield; ‘H NMR (400 MHz, CDC13): 6 0.88 (t, 6H, -CHj), 1.22-1.32 (broad, 44H, -CH?-), 1.65 (t, 4H. -CH?-C-OOC-), 2.1-2.3 (m, 2H, -C-CH?--C(NH2)-COO-), 2.4 (m, 2H, -CH?-COO-), 4.0-4.2 (m, 4H. -CH?-OOC-), 4.6 (m, 2H, -C-CH(NHz)-COO-), 6.3-6.4 (m, 2H, -CH=CH-). 7.9 (broad. lH, -NH-CO-).
Maleic acid (N-bishexadecyl L-glutamate)monoamide (V): white powder with mp 5859°C; 89% yield; ‘H NMR (400 MHz, CDCls): S 0.88 (t, 6H, -CH3), 1.22-1.32 (broad, 52H, -CH?-), 1.65 (t, 4H, -CH2-C-OOC-), 2.1-2.3 (m, 2H. -C-CH?--C(NHz)-COO-). 2.4 (m, 2H, -CH2-COO-), 4.0-4.2 -CH2-OOC-), 4.6 (m, 2H, (m, 4H, -C-CH(NH2)-COO-), 6.3-6.4 (m, 2H, -CH==CH-), 7.9 (broad, lH, -NH-CO-). Maleic acid (N-bisoctadecyl-L-glutamate)monoamide (VI): white powder with mp 6869°C; 92% yield; ‘H NMR (400 MHz, CDC13): 6 0.88 (t, 6H, -CHs), 1.22-1.32 (broad, 60H, -CH?-). 1.65 (t, 4H, -CH?-C-OOC-). 2.1-2.3 (m, 2H, -C-CHz--C(NHz)-COO-). 2.4 (m, 2H, -CH2-COO-), 4.0-4.2 -CH?-OOC-), 4.6 (m, 2H, (m, 4H, -C-CH(NH?)-COO-), 6.3-6.4 (m, 2H. -CH=CH-), 7.9 (broad, 1H. -NH-CO-). 2.3.3. Synthesis of the amphiphilic amino acids 1, 2 and 3 General procedure: Cysteine hydrochloride (5 mmol) was dissolved in 10 ml of water, and sodium hydrogen carbonate was added to raise the pH to 7-8, then 10 ml phosphate buffer solution (pH = 7.4) was added. This mixture was added to a hot propanol solution of the above maleic monoamide (4 mmol). The mixture was stirred under nitrogen atmosphere at 50°C for 8 h. After 80 ml 2 N HCl was added, the mixture was poured into 900 ml of ice water. The white precipitate was collected by suction and washed extensively with water. After drying, the crude product was recrystallized from chloroform/methanol. S - [ 1-carboxy-2-( [N-bistetradecyl-L-glutamate]carbonyl)ethyl]cysteine (1): white powder with mp 113-139°C liquid crystal; 87% yield; FAB-MS: (M + H)+ = 759; ‘H NMR (400 MHz, CDCl?): 6 0.88 (t, 6H, -CHs), 1.22-1.32 (broad, 44H, -CH:!-), 1.62 (t, 4H, -CH7-C-OOC-), 2.0-2.2 (m, 2H, -C-CH?-C(NH2)-COO-), 2.3 (m, 2H, -CHz-COO-), 2.4-4.0 (m, 6H, --N-CO-CHZ-CH-COOH, -SCH$ZH (NH:!)COOH), 4.0-4.2 (m, 4H, -CHz--OOC-), 4.5 (m, 1H. -C-CH(NHz)-COO-), 7.9
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(broad, lH, -NH-CO-). Analysis: Calculated for CaH74N209S: C, 63.35; H, 9.76; N, 3.69; S, 4.22. Found: C, 63.01; H, 9.86; N, 3.44; S, 4.18. S- [ 1-carboxy-2-( [N-bishexadecyl-L-glutamate]carbonyl)ethyl]cysteine (2): white powder with mp 119-150°C liquid crystal; 89% yield; FAB-MS: (M+H)+ = 815; ‘H NMR (400 MHz, CDC13): S 0.88 (t, 6H, -CHs), 1.22-l .32 (broad, 52H, -CH;!-), 1.62 (t, 4H, -CH2-C-OOC-), 2.0-2.2 (m, 2H, -C-CH2-C(NH*)-COO-), 2.4-4.0 (m, 2.3 (m, 2H, -CH2-COO-), 6H, -N-CO-CH2-CH-COOH, -SCH2CH (NHz)COOH), 4.0-4.2(m, 4H, -CH,-OOC-), 7.9 4.5 (m, lH, -C-CH(NH2)-COO-), Analysis: Calculated (broad, 1H, -NH-CO-). for CuHs2N209S: C, 64.89; H, 10.07; N, 3.44; S, 3.93. Found: C, 64.32; H, 10.22; N, 3.83; S, 3.87. S - [ 1-carboxy-2-( [ N-bisoctadecyl-L-glutamate]carbonyl)ethyl]cysteine (3): white powder with mp 117-145°C liquid crystal; 86% yield; FAB-MS: (M+H)+ = 871; ‘H NMR (400 MHz, CDCls): S 0.88 (t, 6H, -CH3), 1.22-1.32 (broad, 6OH, -CH2-), 1.62(t, 4H, -CH2-C-OOC-), 2.0-2.2 (m, 2H, -C-CH2-C(NH2)-COO-), 2.4-4.0 (m, 2.3 (m, 2H, -CH2-COO-), 6H, -N-CO-CH2-CH-COOH, -SCH&H (NHz)COOH), 4.0-4.2 (m, 4H, -CH2-OOC-), 7.9 4.5 (m, lH, -C-CH(NH2)-COO-), (broad, lH, -NH-CO-). Analysis: Calculated for C4sH9aN209S: C, 66.23; H, 10.34; N, 3.22; S, 3.68. Found: C, 65.55; H, 10.51; N, 3.78; S, 3.61. 2.4. Liposome preparation Typically, 10 mg of 1(2 or 3) was dissolved in 1 ml of chloroform. The solvent was thoroughly removed under vacuum for 24 h, 5 ml of buffer solution of different pH values (6.5,7.4, 8.5) was added, after vortexing for 10 min, the aqueous suspension was sonicated at 75°C for 20 min (60 W, bath type sonicator). For the preparation of uniform liposome, either of the following two methods may be used. The liposome solution was filtered through a 1 x 18 cm Sephadex G50 column or separated by ultracentrifugation (16,000 rpm, 10 min).
2.5. TEA4 observation
The liposome solution prepared by the above method was dropped onto a carbon-coated copper grid, excess liquid was blotted off, and air dried. Aqueous uranyl acetate was then dropped onto the dried specimen. After it was dried in vacua, the grid was observed on a JEOL-IOOCXII transmission electron microscope. 2.6. In situ polycondensation of amino acid modijied liposomes 1 (2 or 3) was sonicated with WSD in a buffer solution (70°C 60 W, 50 min), the molar ratio of WSD to lipid was varied from 0 to 3. The freshly prepared and cooled liposomal solution was ultracentrifuged and the upper layer of the resulting solution was used to evaluate the stability of liposomes by following the turbidity change at 400 nm (Schimadzu UV-2000 Spectrometer). The liposomal solution was allowed to stand at room temperature for 24 h and then observed on TEM to confirm the bilayer Iiposome structure.
2.7. Purification and characterization of the condensation product
The liposome prepared in a water-soluble carbodiimide (WSD) containing buffer solution was allowed to stand at room temperature for 24 h. Then equal volume of 1 N HCI was added to destroy the liposome structure. The precipitated residue was thoroughly washed with water and vacuum dried. The ET-IR spectrum was recorded on a Nicolet-750 Spectrometer. To measure the molecular weight of the product, part of the free acid groups must be previously converted to methyl esters to increase the solubility of product in THE So the product was dissolved in dry chloroform and methanol, dry HCI gas was introduced until the solution reached saturation. The reaction was kept at room temperature for 2 days. After removal of the solvent, it was dried. The molecular weight was measured on Waters 208 gel permeation chromatography (THF as eluent, polystyrene as standard).
Z.-C. Li et al. /Reactive & Functional Polymers 30 (1996) 299-308
2.8. Differential scanning calorimetry (DSC) 2-3 mg of lipid (or the condensation product) and 20-30 ~1 of water were placed into small pans and allowed to hydrate overnight at 70°C. The DSC analysis was performed on a Du Pont 1090 DSC from 20°C to 80°C with a scan rate of S”C/min. 2.9. Leakage of 5(6)-CF The lipids with or without WSD (molar ratio: WSD/lipid = 1.5) was sonicated in 100 mM 5(6)-CF, 50 mM Tris-HCl buffer (pH 8.5) for 20 min at 70°C. After cooling or standing at room temperature for 24 h, the liposome suspension was separated form the free dye on a Sephadex G-50 column (1 x 18 cm). The fractions containing the liposome was collected and diluted with the same buffer solution containing 100 mM NaCl to suitable concentrations. The leakage of CF was measured by following the increase of emission fluorescence intensity at 523 nm (excited at 493 nm) on a Hitachi 850 Fluorescence Spectrometer. 3. Results and discussion
3.1. Synthesis of 1,2 and3 In the first step of the synthesis, the amino group of glutamic acid was converted to ptoluenesulfate to reduce its reactivity with carboxylic acid. p-Toluenesulfonic acid also acted as a catalyst for the esterification of carboxylic acid with alkyl alcohol. The second step proceeded with ease. The addition of pyridine or triethylamine could turn the salts to free amino groups, and the subsequent reaction of amino groups with maleic anhydride at 50°C was completed in about 5 hours as detected by TLC, For the nucleophilic attack of the sulfur-containing amino acids on the double bonds of maleic amide, both -SH and -NH2 are possible, and the selectivity depends on the pH of the reaction media. Usually, in a low-pH medium, -SH is more active than -NH2. In this case, buffer solution of pH 7.4 was used to maintain the pH of the
303
reaction media [4,7]. 1, 2 and 3 displayed liquid crystalline properties during melting. 3.2. Liposome formation from the amphiphilic amino acids
1,2 and 3 belong to amphiphilic amino acids, just like the natural lipids, upon hydration in water or buffer solution, they can aggregate to form organized structures. For example, when they were hydrated in buffer solutions followed by sonication at 70°C for 20 min, clear liposomal suspensions with variable size particles (1000-3000 A) were formed. Prolonged sonication (usually 0.5 to 1 h) produced a suspension with an average particle size of 1000 A. They were identified as liposomes by their ability to entrap water-soluble markers and by TEM. Three typical electron microphotographs of 1, 2 and 3 are shown in Fig. 1. Since the ionization state of the head group-amino acid moiety is affected by the pH value of the buffer, which may affect their hydration ability and aggregation behavior of 1,2 and 3, three buffer solutions of different pH (6.5, 7.4, 8.5) were used to prepare liposomes. For these three amphiphilic amino acids, they could form stable liposomes in three buffer solutions of different pH as shown in Fig. 1. These amino acid modified liposomes served as the starting materials for the in situ polycondensation reaction of amino acid moieties and as the control for the evaluation of the effects of the polycondensation on the properties of liposomes. 3.3. Polycondensation of amino acid moieties on the surface of liposomes and characterization of the amphiphilic peptides Water-soluble carbodiimides have been extensively used in peptide synthesis [4,8]. The use of water-soluble carbodiimides for condensation of amino acids at the liposome/water interface was first reported by Neumann et al. [S] They also studied the optimum pH for the condensation reaction and found that at lower pH medium (for example 6.5) the condensation reaction proceeded effectively. When 1, 2 and 3 were sonicated in WSD-containing buffer solutions, the
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Z.-C. Li et al. /Reactive & Functional Polymers 30 (1996) 299-308 0.8
0
10
26 30 40 Tie (hr.)
50
60
Fig. 2. Time-dependent turbidity changes of liposomes from 3 (0.2 wt%) at 400 nm in 0.067 phosphate buffer solution (pH 6.5). Molar ratio of carbodiimide to 3: (0) 0, (W) l/l, (A) 1.50, (0) 2/l, (0) 3/l.
Fig. 1.Electron microphotographs of amino acid liposomes from 1, 2 and 3 in different buffer solutions. (a) 1 in 0.05 M borate buffer solution (pH 7.4), magnification: 33000x; (b) 2 in 0.05 M Tris-HCl buffer solution (pH 8.5). magnification: 24000x; (c) 3 in 0.067 M phosphate buffer solution (pH 6.5), magnification: 24000x.
amino acid condensed to form peptide. After 20 min of sonication, the starting material disappeared as detected by TLC. In order to completely convert the monomers to peptides, the condensation reaction required several additional hours incubation at room temperature. To clarify the effects of the WSD concentration and incubation time on the polycondensation reaction, the ratio of WSD to lipid was varied form 0 to 3. No precipitation was observed during the sonication. During the period when the suspension was incubated at 37°C for 48 h, the turbidity change was followed at 400 nm. The results are shown in Fig. 2. It can be seen that there was almost no change of turbidity during this period, which reflected high stability of liposomes. In fact, the liposome structures could still be seen by TEM after 24 h of incubation as shown in Fig. 3. This showed that the in situ polycondensation of 1, 2 and 3 occurred at the interface of liposome/ water. The amino acid liposomes and the peptide ones are stable against aggregation, which is due to the electrostatic repulsive force between the liposomes. After condensation, the liposomes were destroyed by adding diluted HCI. The residue was collected and washed with water, freeze-dried and its IR spectra were recorded. The results from the liposomes of 3 are shown in Fig. 4. Since there is a amide group (1650 cm-‘) in the amphiphilic amino acid, the formation of new amide bond due to the polycondensation was not
305
Z.-C. Li et al. /Reactive & Functional Polymers 30 (1996) 299-308 Table 1 Phase transition temperatures of phospholipid liposomes l-3 liposomes or of peptide liposomes from l-3
and of
Compound
Monomer
Peptide form
Phospholipid
1 2 3
40.8”C 54.9”C 67.3”C
33.2”C 47.O”C 59.2”C
23.9”C (DMPC) a 41 XC (DPPC) 54.9”C (DSPC)
a DMPC = dimyristoylphosphatidylcholine; DPPC = dipalmitoylphosphatidylcholine; DSPC = distearoylphosphatidylcholine.
clearly indicated that the polycondensation of amino acids was taken place. The process may be schemed as shown on Scheme 2. GPC measurement is a method frequently used to evaluate the molecular weight of a polymer. Because of the poor solubility of the condensation product in tetrahydrofuran, the carboxylic acid groups in the condensation product were in part converted to their methyl esters by the following procedure. The condensation product was dissolved in dry chloroform and methanol, and the solution was saturated by introducing dry HCl gas. The reaction mixture was kept at room temperature for 2 days. After the solvent was removed, the residue was dried and dissolved in THF for GPC measurement. From the GPC chart, peptide of more than 15 amphiphilic amino acids was observed, but most of the product were oligomers, the average polymerization degree was determined to be about 3-5. The reason for the low degree of polymerization has been discussed in the literature [5]. 3.4. Phase transition temperature of the amino acid liposomes
Fig. 3. Electron microphotographs of the peptide liposomes from 1, 2 and 3 in 0.05 M borate buffer solution (pH 7.4). Molar ratio of carbodiimide to lipid: 1.5, time: 24 h. (a) 1 (magnification: 24000x): (b) 2 (magnification: 49000x); (c) 3 (magnification: 24000x).
clear, but the peak of ucZo at 1650 cm-’ became wide, and it’s peak area became large. So the increase of the peak intensity of ~c,~/uc-n ratio from 0.108 of 3 to 0.432 of the peptide,
Phase transition temperature (7’J reflects the packing of amphiphiles in bilayer membrane and it is affected by the head groups and hydrophobic chains. For the three amphiphilic amino acids, the phase transition temperatures in swollen lamellar phases were determined by DSC measurements. The results are given in Table 1. For comparison, the phase transition temperatures of phospholipid with the same hydrophobic chain length as 1, 2 and 3 are also given. It can be seen clearly that the T, of the amino acid liposomes were higher than those of the phosphatidylcholines with the
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Z.-C. Li et al. /Reactive & Functional Polymers 30 (1996) 299-308
-nH20 WSD
@=hydrophil.ic unit (e.g. -COOH)
no precipitate due to additional hydrophibic unit
Scheme 2. Schematic representation of condensation polymerization of 1,2 and 3 liposomes.
Wavenumber (cm-l ) Fig. 4. FT-IR spectra of (A) 3 and (B) peptide from the polycondensation of 3 on the surface of liposomes (pH 6.5, time 24 h; [carbodiimde]/I3] = 1.50).
samehydrophobic chain lengths. It is assumed that the formation of hydrogen bonds between the amino acid head groups allows a better chain packing. When amino acid was condensed, Tc of the peptide liposomes moved to lower tem-
peratures as given in Table 1. The formation of peptides caused the shrinkage of the liposome surface and decrease of the arrangement order of hydrocarbon chains.
Z.-C. Li et al. /Reactive
0
& Functional Polymers 30 (1996)299-308
~-L-L 0
loo0
2ooo
3ooo
Time (min.) Fig. 5. Percent release of 5(6)-CF vs. time at 37°C from liposomes of 2 (0) and after 1 h (0) or 24 h (0) polycondensation. (pH 7.4, [carbodiimde]/[2] = 1.5/l).
307
of the surface caused the disorder of the bilayer membrane in the hydrophobic region. However, if the reaction is allowed to proceed for 24 h, the release of CF became slower, though it was still faster than in the case of the monomeric liposome. The longer reaction time leads the formation of larger peptides, which may account for this effect. For the other two amphiphilic amino acids, 1 and 3, similar results were obtained. This means that by controlling the condensation condition, the permeability of the amino acid liposomes can be adjusted, which make this kind of liposome good as controlled drug release system. 4. Conclusion
3.5. Leakage of encapsulated
molecules
An important property of liposome is that it can encapsulate water-soluble compounds in the inner aqueous phase. This is the basis for the use of liposome as drug carriers and releasing system. The leakage of encapsulated molecules from liposomes is frequently used as one of the conventional index for stability and permeability. 5(6)-CF is a commonly used probe. [9] The permeability of liposomes from 1, 2,3 and peptidemodified liposomes were examined by detecting the leakage of the encapsulated 5(6)-CF in a regular interval time (pH 7.4,37”C). Fig. 5 shows the increase of the fluorescence vs. time at 37°C for the liposomes from 2. It can be seen that there is very little increase of fluorescence with time (less than 10%) from the liposomes of 2 even after incubation at 37°C for 48 h. This result is similar to those obtained for the other kind of amino acid liposomes and is significantly less than that normally observed with liposomes from lipid. Since liposome from monomeric amphiphilic amino acids tend to condense and form peptide after sonication with WSD, the liposome prepared in this way in CF buffer solution was used to study the effects of condensation on the permeability of amino acid liposomes. After 1 h of the reaction, the leakage of CF became faster (Fig. 5). This may be caused by the decrease of the surface charge of the bilayer membrane after the condensation reaction. The other possible reason is that the shrinkage
Three novel amphiphilic amino acids based on glutamic acid were synthesized. They could form stable bilayer liposome in water and buffer solutions upon hydration and sonication. The amino acid groups, which located at the surface of liposomes, could be condensed to form oligopeptides. The phase transition temperatures of the amphiphilic amino acids were higher than those of the phospholipids with same hydrophobic chain length and moved down after polycondensation. The low permeability of these liposomes and the fact that the permeability can be adjusted by controlling the extent of condensation reaction make these compounds interesting alternatives to phospholipids for the long term encapsulation of various substances.
Acknowledgements This work was supported in part by the Doctoral Foundation of State Education Commission of China.
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[6] N. Nishikama, M. Arai, M. Ono and I. Itoh, Chem. Lett.
(1993) 2017. [7] J.-H. Fuhrhop, H.-H. David, J. Mathieu, U. Liman, H.-J. Winter and E. Boekema, J. Am. Chem. Sot., 108 (1986) 1785.
[8] J.C. Sheehan and J.J. Hlavka, J. Org. Chem., 21 (1956) 439. [9] J.H. Weinstein, S. Yoshikami, P. Henkart, R. Blumenthal and W.A. Hargins, Science, 195 (1977) 489.