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Cholesterol succinyl chitosan anchored liposomes: preparation, characterization, physical stability, and drug release behavior
BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 471 – 477
Original Article www.nanomedjournal.com
Cholesterol succinyl chitosan anchored liposomes: preparation, characterization, physical stability, and drug release behavior
Yinsong Wang, MDa,⁎, Shaoli Tu, MSb , Rongshan Li, MSa , XiaoYing Yang, PhDa , Lingrong Liu, MDb , Qiqing Zhang, PhDb,c a
b
College of Pharmacy, Tianjin Medical University, Tianjin, People's Republic of China Institute of Biomedical Engineering, Chinese Academy of Medical Science, Peking Union Medical College, Tianjin, People's Republic of China c Research Center of Biomedical Engineering Medical School Xiamen University, Xiamen, People's Republic of China Received 2 June 2009; accepted 16 September 2009
Abstract The purpose of this study was to prepare cholesterol succinyl chitosan anchored liposomes (CALs) and to investigate their characterization, physical stability, and drug release behavior in vitro. Three cholesterol succinyl chitosan (CHCS) conjugates with different substitution degrees (DS) of the cholesterol moiety were synthesized and used as the anchoring materials to coating on the liposome surface by the incubation method. CALs were almost spherical and had a classic shell-core structure. Compared with plain liposomes and chitosancoated liposomes (CCLs), CALs had larger sizes, higher zeta potentials, and better physical stability after storage at 4 ± 2°C and 25 ± 2°C. Epirubicin, as a model drug, was effectively loaded into CALs and exhibited the more sustained release in both phosphate buffer solution (pH 7.4) and 1% (vol/vol) aqueous fetal bovine serum compared to plain liposomes and CCLs. From the Clinical Editor: Cholesterol succinyl chitosan anchored liposomes (CAL) as delivery vehicles are characterized in this work, including their physical stability and drug release behavior in vitro. Epirubicin as a model drug, was effectively loaded into CALs, and exhibited sustained release behavior both in phosphate buffer solution (PBS, pH 7.4) and 1% (V/V) aqueous fetal bovine serum (FBS). © 2010 Elsevier Inc. All rights reserved. Key words: Cholesterol succinyl chitosan; Drug carrier; Epirubicin; Polysaccharide anchored liposome
Liposomes, the lipid bilayer vesicles, have gained attention as drug carriers because they can reduce the toxicity and increase the therapeutic efficacy of various drugs.1 However, the applications of liposomes in drug delivery systems are currently rather limited because of their relatively short blood circulation time,2 and therefore various liposome formulations with prolonged circulation time in blood have been studied.3-5 Recently, many investigations have shown that some hydrophobically modified polysaccharides such as palmitoylated pullulan6 and amylopectin derivatives7,8 can anchor their hydrophobic modified groups into the phospholipid bilayer of liposomes by hydrophobic interaction and form a hydrophilic shell on the liposome surface. This novel kind of liposome, termed polysaccharide anchored liposome, has attracted increasing interest for its following advantages in drug delivery systems9,10: The hydrophilic polysaccharide shell can not only This work was supported by the National Natural Science Foundation of China (grant no. 30900303). ⁎Corresponding author: College of Pharmacy, Tianjin Medical University, Tianjin 300070, People's Republic of China. E-mail address: [email protected] (Y.S. Wang).
increase the physical stability of liposomes but also provide steric protection for liposomes to escape the adsorption of opsonins and the phagocytosis of mononuclear macrophage, thereby prolonging their circulation time in blood. Moreover, there are many functional groups such as hydroxyl, amino, and carboxyl groups in polysaccharide molecules, so that some biologically active molecules (eg, ligand, monoclonal antibody and biosensor) can be introduced to the polysaccharide anchored liposomes by covalent bonds. Chitosan, a homopolymer of (1,4)-linked 2-amino-2-deoxyβ-glucan, is produced by the deacetylation of chitin, which is the second most abundant, renewable natural polysaccharide after cellulose. Chitosan and its derivatives have been used in many biomedical applications because of their excellent properties such as biocompatibility, biodegradability, nontoxicity, and bioadhesivity.11-13 In this study, chitosan was selected as the polysaccharide material and was hydrophobically modified by cholesterol to obtain the amphiphilic cholesterol succinyl chitosan (CHCS) conjugates, which were used as the anchoring materials to coat on the liposome surface by the incubation method. The characterization and the physical stability in vitro of chitosan anchored liposomes (CALs) were studied. Furthermore,
1549-9634/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2009.09.005 Please cite this article as: Y.S. Wang, S.L. Tu, R.S. Li, X.Y. Yang, L.R. Liu, Q.Q. Zhang, Cholesterol succinyl chitosan anchored liposomes: preparation, characterization, physical stability, and drug release behavior. Nanomedicine: NBM 2010;6:471-477, doi:10.1016/j.nano.2009.09.005
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epirubicin (EPB) was chosen as a model drug to assess the drug loading and release behavior of CALs.
Methods Materials Biomedical grade chitosan (deacetylation degree was 92%, viscosity average molecular weight was 8.0 × 104 Da) was supplied by Yuhuan Ocean Biochemical Co., Ltd. (Zhejiang, China). Three CHCS conjugates were synthesized by the method that we previously reported.14 The substitution degrees (DS) of the cholesterol moiety of CHCS, defined as the amount of cholesterol moieties per 100 glucosamine units of chitosan, was determined by the colloid titration method,15 and the DS values of three CHCS conjugates were 2.80% (CHCS-1), 5.58% (CHCS-2), and 8.00% (CHCS-3), respectively. EPB was supplied by Hisun Pharmaceutical Co. (Zhejiang, China). Phosphatidylcholine (Lipoid S100) was purchased from Lipoid (Ludwigshafen, Germany). Cholesterol was purchased from DingGuo Biotechnology Co., Ltd. (Beijing, China). Aqueous fetal bovine serum (FBS) was purchased from the Institute of Blood Disease of Peking Union Medicine College (Beijing, China). Sephadex G-50 was purchased from Amersham Biosciences (Uppsala, Sweden). Preparation of CALs The thin-film hydration method16 was developed to prepare the plain liposomes. Phosphatidylcholine (50 mg) and cholesterol (15 mg) were dissolved in chloroform. The mixture was dried under reduced pressure using a Eyela rotary evaporator (model N-1000; Eyela, Tokyo, Japan) at 40°C to form a thin lipid film, and then trace solvent was removed by holding the lipid film under high vacuum overnight. The lipid film was hydrated by the addition of 5.0 mL 0.3 M citric acid buffer solution (pH 4.0) and then vortexed and ultrasonicated for 9 minutes (with two intervals of 2 minutes after every 3 minutes) using an ultrasonic processor (model UH-500A; Automatic Science Instrument Co., Ltd, Tianjin, China) at 40 W to produce multilamellar vesicles (MLVs). Then, MLVs were respectively filtered through 0.45 μm and 0.22 μm porosity membrane filters to obtain small-size liposomes. CHCS-1, CHCS-2, and CHCS-3 anchored liposomes, respectively named as CAL-1, CAL-2, and CAL-3, were prepared by the incubation method. Briefly, different amounts of CHCS (12.5, 33, 75, and 200 mg) dissolved in 2% aqueous acetic acid solutions were respectively added to 5 mL of the above plain liposomal suspensions, and the polysaccharide/lipid weight ratios were 1/4, 2/3, 3/2, and 4/1. Then, the mixture solutions were incubated at 20°C with gentle shaking for 1 hour and followed by storage at 4°C for 24 hours until use. Furthermore, CCLs as the control were also prepared by the same method as above. Characterization of CALs The sizes and size distributions of CALs were measured by dynamic laser light scattering (DLLS) with a Brookhaven digital
autocorrelator (model BI-90 Plus; Brookhaven Instruments, Holtsville, New York) at a scattering angle of 90 degrees, a wavelength of 633 nm, and a temperature of 25 ± 0.1°C. The zeta potentials of CALs were determined using a Brookhaven electrophoretic light-scattering spectrometer (model BI-Zetaplus; Brookhaven Instruments, Holtsville, New York). Transmission electron microscopy (TEM) observations were performed after negative staining of CALs with phosphomolybdic acid. Briefly, the liposome samples were dropped onto carbon-coated grids (100 mesh) and drawn off with a piece of filter paper. Then, the grids were immersed for 1 minute in 2% phosphomolybdic acid aqueous solution and washed twice with 200 mL distilled water. Finally, the grids were dried and imaged using a Philips transmission electron microscope (model EM400ST; Philips, Eindhoven, The Netherlands). Study of physical stability of CALs CALs were stored at 4 ± 2°C, 25 ± 2°C, and 37 ± 2°C for a period of 5, 10, 15, 20, and 30 days. As above, the sizes and size distributions of CALs were evaluated by DLLS using a Brookhaven digital autocorrelator (model BI-90 Plus; Brookhaven Instruments). The morphologic changes of CALs were observed after 30 days of storage by a Philips transmission electron microscope (model EM400ST; Philips). Drug loading and release studies EPB was first loaded into the plain liposomes by the modified pH gradient method.17 Briefly, 5 mL of the plain liposome suspension prepared above was applied to a Sephadex G-50 column (2 × 60 cm) and eluted with phosphate buffer solution (PBS; pH 7.4) to obtain liposomes with a pH gradient between their interior and exterior. Then, different volumes (62.5, 125, 250, and 500 μL) of 10 mg/mL EPB in PBS (pH 7.4) were added to these pH gradient liposomes and incubated at 4°C for 12, 24, 36, and 48 hours, respectively. The resulting liposome dispersion passed through the Sephadex G-50 column with PBS (pH 7.4) as the eluent to separate free EPB from the plain EPB-loaded liposomes (PELs). The fluorescence intensity of free EPB was measured by a Shimadzu fluorescence spectrophotometer (model RF-4500; Shimadzu, Kyoto, Japan). The excitation wavelength (λex) and the emission wavelength (λem) were set at 470 and 585 nm, respectively. The entrapment efficiency of EPB in liposomes was calculated according to the following equation: EE =
ðW0 W1 Þ 100k W0
where EE is the entrapment efficiency, W0 is the total amount of EPB initially added, and W1 is the amount of free EPB. Then, PELs were incubated with CHCS conjugates to prepare CHCS anchored EPB-loaded liposomes (CAELs) with the polysaccharide/lipid weight ratio of 3/2 using the same method for preparing CALs. At the same time, chitosan-coated EPBloaded liposomes (CCELs) as the control were also prepared as the same method for preparing CCL. In vitro release behaviors of EPB from CAELs were studied by the dialysis method18 both in PBS (pH 7.4) and 1% (vol/vol) aqueous FBS. Briefly, 2 mL liposome suspension was placed
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into the dialysis tube (molecular weight cutoff 8 to 14 kDa; Millipore, Bedford, Massachusetts) and dialyzed against the above release media (10 mL) at 37 ± 0.5°C in an air-bath shaker at 100 rpm. At scheduled time intervals, the whole of the release media was collected and then the fresh release media were added. The release amount of EPB was determined by fluorescence spectrophotometry as described earlier. The accumulative release percentage of EPB (RE %) was calculated according to the following equation:
D0t RE ðkÞ = D0
100
where D0-tis the amount of drug released from liposome suspension from the beginning to the scheduled time, and D0 is the total amount of drug in liposome suspension. Results Preparation and characterization of CALs In our study, the different polysaccharide/lipid weight ratios were investigated to prepare CAL. Figure 1 shows the effects of polysaccharide/lipid weight ratio on the size (Figure 1, A) and the zeta potential (Figure 1, B) of CAL, and the same trend occurred among CALs with different DS of the cholesterol moiety. Compared with the plain liposomes (size 148.2 ± 3.0 nm and zeta potential −4.75 mV), CALs had significantly larger sizes and positive zeta potentials, which indicated that CHCS conjugates with different DS of the cholesterol moiety successfully coated on the surface of the plain liposomes to form the polysaccharide shells. The sizes and the zeta potentials of CALs evidently increased with the polysaccharide/lipid weight ratio increasing from 0 to 3/2 but slightly changed when the polysaccharide/lipid weight ratio continued increasing, which indicated that CHCS conjugates coating on the liposome surface reached a saturation state. Therefore, 3/2 was believed to be the optimal polysaccharide/lipid weight ratio to prepare CALs. Moreover, as shown in Figure 1, CALs had evidently larger sizes and significantly higher zeta potentials compared with those of CCL under the same polysaccharide/lipid weight ratio. For example, the zeta potentials of CAL-1, CAL-2, and CAL-3 were respectively +25.48 mV, +22.06 mV, and +21.09 mV at the polysaccharide/lipid weight ratio of 3/2, whereas the zeta potential of CCL was only +7.81 mV. Therefore, it could be deduced that CALs had better stability than CCL due to their higher zeta potentials. Figure 1, A also shows that the size of CAL increased when the DS of the cholesterol moiety of CHCS increased from 2.8% to 8.0%; for example, the size of CAL-3 was 262.5 ± 6.5 nm under the polysaccharide/lipid weight ratio of 2/3, evidently larger than the size of CAL-1 (205.8 ± 1.2 nm) and the size of CAL-2 (232.5 ± 9.1 nm), which suggested that CHCS with larger DS of the cholesterol moiety could be easier to coat on the surface of the plain liposomes. Figure 2 shows TEM images of the plain liposomes (Figure 2, A1 and A2), CAL-2 with the polysaccharide/lipid weight ratio of 3/2 (Figure 2, B1 and B2), and the physical mixture of plain liposomes and CHCS-2 with the same polysaccharide/lipid
Figure 1. Effects of polysaccharide/lipid weight ratio (wt/wt) on the (A) size and (B) zeta potential of liposomes. Each data point represents the mean of at least three independent experiments (-●-, CCL; -○-, CAL-1; -■-, CAL-2; -□-, CAL-3).
weight ratio of CAL-2 (Figure 2, C). Morphologically, plain liposomes were nearly spherical (Figure 2, A1) and had an obvious multilamellar (onion-like) structure characterized by multiple membrane bilayers, each separated from the next by an aqueous layer (Figure 2, A2); CAL-2 also had a spherical shape (Figure 2, B1) and a classic shell-core structure (Figure 2, B2) with a shell thickness of about 30 nm. However, the plain liposomes were wrapped with a thin polymer film formed by CHCS-2 under the TEM observation (Figure 2, C) when they were simply mixed with CHCS-2. Furthermore, the size of CAL2 determined by DLLS was about 245.4 ± 8.1 nm, obviously larger than the size determined by TEM images. We believed this was because the polysaccharide shell of CAL-2 was highly hydrophilic in nature and would be likely to swell in aqueous media, thus the size determined by DLLS was a hydrodynamic diameter and was larger than the size measured by TEM in the dried state. Other CALs had similar morphologic characteristics
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Figure 2. TEM images of (A1, A2) plain liposomes, (B1, B2) CAL-2, and (C) the physical mixture of liposomes with CHCS-2.
to CAL-2. All the above results suggested that CHCS conjugates could effectively be coated on the surface of liposomes by the incubation method in this study. The physical stability of CAL CALs were stored at 4 ± 2°C, 25 ± 2°C, and 37 ± 2°C for a period of 5, 10, 15, 20, and 30 days and then were determined by DLLS and TEM to evaluate their physical stability. The plain liposomes and CCL were also investigated as the controls. Figure 3 shows the effect of storage conditions on the size of liposomes. An insignificant difference (P N .05) was found in the sizes of all liposomal formulations stored at 4 ± 2°C (Figure 3, A) for 30 days. But a significant (P b .05) increase in the size of the plain liposomes was observed after 30-day storage both at 25 ± 2°C (Figure 3, B) and 37 ± 2°C (Figure 3, C), which was due to the fusion of the plain liposomes. No significant (P N .05) changes in the sizes of CALs and CCL were observed when they were stored at 25 ± 2°C up to 30 days (Figure 3, B). However, the sizes of CALs and CCL all significantly (P b .05) increased after 30-day storage at 37 ± 2°C. These results indicated that CALs and CCL exhibited good stability on storage for at least 30 days at 4 ± 2°C and 25 ± 2°C.
Figure 3. Particle sizes of plain liposomes, CCL, and CALs on time of storage at (A) 4 ± 2°C, (B) 25 ± 2°C, and (C) 37 ± 2°C. All values are expressed as mean ± SD (n = 3).
Figure 4 shows the morphologies of CAL-2 and CCL after 30-day storage at 25 ± 2°C. CAL-2 (Figure 4, A) retained its spherical shape, and no evident aggregation phenomenon was observed; the other two CALs had similar morphology to CAL2. CCLs (Figure 4, B) were irregularly spherical, and a significant aggregation phenomenon took place. Therefore, it could be concluded that CALs had better storage stability than CCL, which was perhaps due to their higher zeta potentials compared with that of CCL as discussed earlier. Drug loading and release behaviors of CALs EPB, as a weak base (pKa = 7.7), was first loaded into the plain liposomes by the modified pH gradient method.17 This method is often used to load weak bases such as doxorubicin and
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Figure 5. Effect of preparation conditions on the encapsulation efficiency of EPB. Each data point represents the mean of at least three independent experiments (-○-, incubation time 12 hours; -■-, incubation time 24 hours; -●-, incubation time 36 hours; -□-, incubation time 48 hours).
Figure 4. TEM images of (A) CAL-2 and (B) CCL after 30-day storage at 25 ± 2°C.
vincristine,19-21 which coexist in aqueous solutions in neutral and charged forms, into liposomes using pH gradient as a driving force. In this study, the preparation conditions such as the weight ratio of EPB to lipid and the incubation time were investigated to obtain an optimal drug encapsulation. As shown in Figure 5, EPB encapsulation efficiency increased with the weight ratio of EPB to lipid increasing from 0.01 to 0.025, and then decreased with the weight ratio further increasing, which suggested that the ability of liposomes to load EPB reached saturation. Figure 5 also shows there was no significant difference (P N .05) of EPB encapsulation efficiency among the incubation times (12 to 48
hours) when the weight ratio of EPB to lipid was less than 0.05, but an obvious increase of EPB encapsulation efficiency occurred with the incubation time increasing when the weight ratio of EPB to lipid was larger than 0.05. However, when the incubation time was longer than 36 hours, a significantly larger size of liposomes was observed, which was perhaps due to the fusion of liposomes. Therefore, considering the preparation conditions prescribed above, 0.025 (EPB to lipid weight ratio) and 24 hours (incubation time) were chosen to prepare PELs, and EPB encapsulation efficiency was high, to 96.8%. Then, PELs were incubated with CHCS conjugates to prepare CAELs at the polysaccharide/lipid weight ratio of 3/2. CAELs had similar morphology to CALs such as the spherical shape and the classic shell-core structure under TEM observation, but a slight decrease on EPB content was measured after PEL surface coating with CHCS conjugates. We believed this was because EPB passively leaked out of liposomes in the incubation period, and EPB leakage rates were 2.65%, 1.98%, and 3.04% for CAEL-1, CAEL-2, and CAEL-3, respectively. EPB release behavior from CAEL was studied in vitro by the dialysis method in PBS (pH 7.4) and 1% aqueous FBS, respectively. EPB release from PELs and CCELs was also investigated as the controls. The cumulative EPB release profiles are shown in Figure 6. In PBS (pH 7.4) (Figure 6, A), EPB release from PEL showed a rapid pattern, about 52% EPB released in 24 hours; but only about 37%, 33%, and 41% EPB released respectively from CAEL-1, CAEL-2, and CAEL-3, indicating that CAEL significantly sustained the release of EPB. In 1% FBS (Figure 6, B), EPB release rates from PELs and CAELs markedly accelerated; nearly 100% EPB released from all liposome formulations in 24 hours. We believe this was because the bioactive substances such as proteins and enzymes in FBS destroyed the liposome structure and polysaccharide coating shell. However, CAELs also showed a significant sustained drug release in the first 12 hours, about 73.6%, 67.7%, and 79.5% EPB released respectively from CAEL-1, CAEL-2, and CAEL-3
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Figure 6. Dynamic release profiles of EPB from PEL (-●-), CCEL (-○-), CAEL-1 (-■-), CAEL-2 (-□-), and CAEL-3 (-▲-) in (A) PBS (pH 7.4) and (B) 1% FBS.
compared with 100% EPB from PELs in 1% FBS (Figure 6, B). The above results suggested that CAELs had better stability than PELs in vitro due to CHCS shells on the surface of PELs. Moreover, compared with CCEL, EPB release from CAELs more or less slowed both in PBS (pH 7.4) and 1% FBS, which indicated that CHCS coating shells were more stable than chitosan coating shell on the liposome surface.
Discussion Recently, many investigations into CCL have been performed to enhance the stability of liposomes and increase their intestinal uptake.22-24 We previously hoped to use CCL as the carrier of anticancer drugs, but the chitosan layer formed by the electrostatic attractive interaction between the positively charged amino groups in chitosan molecules and the negatively charged surface of liposomes was not effective enough to significantly stabilize liposomes in vitro, so that the anchoring method using hydrophobically modified chitosan derivatives (eg, CHCS and
palmitoyl chitosan conjugates) as anchoring materials was then applied to increase the stability of liposomes. In our later research, cholesterol modified groups showed better hydrophobic anchoring properties for preparation of surface coating liposomes compared with alkylated modified groups, which was consistent with the previous report of Kang et al.10 Compared with CCL, CALs had larger sizes, higher zeta potentials, better physical stability, and more sustained drug release behavior under the same study conditions. We believe this was due to the difference of coating mechanisms between CHCS conjugates and chitosan on the liposome surface. It was clear that chitosan coating on the liposome surface mainly depended on the electrostatic attraction force. The amino groups of chitosan molecules carrying positive charges neutralized the negative surface charges of liposomes, so that the zeta potential of liposomes changed from negative to positive after chitosan coating, and its absolute value was lower than that of some chitosan particles (eg, +28.44 mV previously reported).25 However, in addition to the electrostatic attraction force, the process of CHCS coating also involved the cholesterol modified groups anchoring into the phospholipid bilayer of liposome, thus a thicker and more stable polysaccharide shell formed on the liposome surface. Comparison of drug release profiles of CAELs both in PBS (pH 7.4) and 1% FBS (Figure 6) showed that EPB release behavior from CAEL was significantly influenced by the DS of the cholesterol moiety of CHCS. EPB release rate from CAEL decreased with the DS of the cholesterol moiety increasing from 2.8% (CHCS-1) to 5.6% (CHCS-2). This was because CHCS with higher DS of the cholesterol moiety had more cholesterol modified groups to anchor into the liposome phospholipid bilayer, and thus a more stable polysaccharide coating shell was obtained. However, with DS of the cholesterol moiety increasing from 5.6% (CHCS-2) to 8.0% (CHCS-3), EPB release rate evidently increased, such as EPB release from CAEL-3 being even faster than that from CAEL-1 both in PBS (pH 7.4) and in 1% FBS. We believed this was perhaps because more cholesterol modified moieties anchoring into the liposome phospholipid bilayer resulted in a higher liposome membrane permeability. Therefore, CHCS-2 with DS of the cholesterol moiety of 5.6% was considered an optimal hydrophobically modified chitosan derivative to prepare polysaccharide anchored liposomes in this study. EPB was chosen as a model anticancer drug to assess the potential of CAL as a novel carrier of anticancer drugs. As an anthracycline anticancer agent, EPB has a wide range of antitumor activity and is used to treat various carcinomas. However, EPB therapy may cause some serious side effects such as allergic reactions, cardiotoxicity, and blood problems.26,27 Therefore, CAL was used as a carrier of EPB in an attempt to sustain its release, prolong its circulation time, enhance its therapeutic index, and decrease its toxic effects. Our current results clearly showed that CHCS anchoring on the surface of PELs could significantly improve their physical stability and sustain the release of EPB in vitro, but investigations in vivo are required to further confirm the advantages of CAL as the carrier of EPB over the plain liposomes and CCL, and related experiments are now in progress.
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Acknowledgment The authors thank Drs. Xindu Yang, Wenzhi Yang, and Hongli Chen for their helpful discussion and suggestions.
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Original Article www.nanomedjournal.com
Cholesterol succinyl chitosan anchored liposomes: preparation, characterization, physical stability, and drug release behavior
Yinsong Wang, MDa,⁎, Shaoli Tu, MSb , Rongshan Li, MSa , XiaoYing Yang, PhDa , Lingrong Liu, MDb , Qiqing Zhang, PhDb,c a
b
College of Pharmacy, Tianjin Medical University, Tianjin, People's Republic of China Institute of Biomedical Engineering, Chinese Academy of Medical Science, Peking Union Medical College, Tianjin, People's Republic of China c Research Center of Biomedical Engineering Medical School Xiamen University, Xiamen, People's Republic of China Received 2 June 2009; accepted 16 September 2009
Abstract The purpose of this study was to prepare cholesterol succinyl chitosan anchored liposomes (CALs) and to investigate their characterization, physical stability, and drug release behavior in vitro. Three cholesterol succinyl chitosan (CHCS) conjugates with different substitution degrees (DS) of the cholesterol moiety were synthesized and used as the anchoring materials to coating on the liposome surface by the incubation method. CALs were almost spherical and had a classic shell-core structure. Compared with plain liposomes and chitosancoated liposomes (CCLs), CALs had larger sizes, higher zeta potentials, and better physical stability after storage at 4 ± 2°C and 25 ± 2°C. Epirubicin, as a model drug, was effectively loaded into CALs and exhibited the more sustained release in both phosphate buffer solution (pH 7.4) and 1% (vol/vol) aqueous fetal bovine serum compared to plain liposomes and CCLs. From the Clinical Editor: Cholesterol succinyl chitosan anchored liposomes (CAL) as delivery vehicles are characterized in this work, including their physical stability and drug release behavior in vitro. Epirubicin as a model drug, was effectively loaded into CALs, and exhibited sustained release behavior both in phosphate buffer solution (PBS, pH 7.4) and 1% (V/V) aqueous fetal bovine serum (FBS). © 2010 Elsevier Inc. All rights reserved. Key words: Cholesterol succinyl chitosan; Drug carrier; Epirubicin; Polysaccharide anchored liposome
Liposomes, the lipid bilayer vesicles, have gained attention as drug carriers because they can reduce the toxicity and increase the therapeutic efficacy of various drugs.1 However, the applications of liposomes in drug delivery systems are currently rather limited because of their relatively short blood circulation time,2 and therefore various liposome formulations with prolonged circulation time in blood have been studied.3-5 Recently, many investigations have shown that some hydrophobically modified polysaccharides such as palmitoylated pullulan6 and amylopectin derivatives7,8 can anchor their hydrophobic modified groups into the phospholipid bilayer of liposomes by hydrophobic interaction and form a hydrophilic shell on the liposome surface. This novel kind of liposome, termed polysaccharide anchored liposome, has attracted increasing interest for its following advantages in drug delivery systems9,10: The hydrophilic polysaccharide shell can not only This work was supported by the National Natural Science Foundation of China (grant no. 30900303). ⁎Corresponding author: College of Pharmacy, Tianjin Medical University, Tianjin 300070, People's Republic of China. E-mail address: [email protected] (Y.S. Wang).
increase the physical stability of liposomes but also provide steric protection for liposomes to escape the adsorption of opsonins and the phagocytosis of mononuclear macrophage, thereby prolonging their circulation time in blood. Moreover, there are many functional groups such as hydroxyl, amino, and carboxyl groups in polysaccharide molecules, so that some biologically active molecules (eg, ligand, monoclonal antibody and biosensor) can be introduced to the polysaccharide anchored liposomes by covalent bonds. Chitosan, a homopolymer of (1,4)-linked 2-amino-2-deoxyβ-glucan, is produced by the deacetylation of chitin, which is the second most abundant, renewable natural polysaccharide after cellulose. Chitosan and its derivatives have been used in many biomedical applications because of their excellent properties such as biocompatibility, biodegradability, nontoxicity, and bioadhesivity.11-13 In this study, chitosan was selected as the polysaccharide material and was hydrophobically modified by cholesterol to obtain the amphiphilic cholesterol succinyl chitosan (CHCS) conjugates, which were used as the anchoring materials to coat on the liposome surface by the incubation method. The characterization and the physical stability in vitro of chitosan anchored liposomes (CALs) were studied. Furthermore,
1549-9634/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2009.09.005 Please cite this article as: Y.S. Wang, S.L. Tu, R.S. Li, X.Y. Yang, L.R. Liu, Q.Q. Zhang, Cholesterol succinyl chitosan anchored liposomes: preparation, characterization, physical stability, and drug release behavior. Nanomedicine: NBM 2010;6:471-477, doi:10.1016/j.nano.2009.09.005
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epirubicin (EPB) was chosen as a model drug to assess the drug loading and release behavior of CALs.
Methods Materials Biomedical grade chitosan (deacetylation degree was 92%, viscosity average molecular weight was 8.0 × 104 Da) was supplied by Yuhuan Ocean Biochemical Co., Ltd. (Zhejiang, China). Three CHCS conjugates were synthesized by the method that we previously reported.14 The substitution degrees (DS) of the cholesterol moiety of CHCS, defined as the amount of cholesterol moieties per 100 glucosamine units of chitosan, was determined by the colloid titration method,15 and the DS values of three CHCS conjugates were 2.80% (CHCS-1), 5.58% (CHCS-2), and 8.00% (CHCS-3), respectively. EPB was supplied by Hisun Pharmaceutical Co. (Zhejiang, China). Phosphatidylcholine (Lipoid S100) was purchased from Lipoid (Ludwigshafen, Germany). Cholesterol was purchased from DingGuo Biotechnology Co., Ltd. (Beijing, China). Aqueous fetal bovine serum (FBS) was purchased from the Institute of Blood Disease of Peking Union Medicine College (Beijing, China). Sephadex G-50 was purchased from Amersham Biosciences (Uppsala, Sweden). Preparation of CALs The thin-film hydration method16 was developed to prepare the plain liposomes. Phosphatidylcholine (50 mg) and cholesterol (15 mg) were dissolved in chloroform. The mixture was dried under reduced pressure using a Eyela rotary evaporator (model N-1000; Eyela, Tokyo, Japan) at 40°C to form a thin lipid film, and then trace solvent was removed by holding the lipid film under high vacuum overnight. The lipid film was hydrated by the addition of 5.0 mL 0.3 M citric acid buffer solution (pH 4.0) and then vortexed and ultrasonicated for 9 minutes (with two intervals of 2 minutes after every 3 minutes) using an ultrasonic processor (model UH-500A; Automatic Science Instrument Co., Ltd, Tianjin, China) at 40 W to produce multilamellar vesicles (MLVs). Then, MLVs were respectively filtered through 0.45 μm and 0.22 μm porosity membrane filters to obtain small-size liposomes. CHCS-1, CHCS-2, and CHCS-3 anchored liposomes, respectively named as CAL-1, CAL-2, and CAL-3, were prepared by the incubation method. Briefly, different amounts of CHCS (12.5, 33, 75, and 200 mg) dissolved in 2% aqueous acetic acid solutions were respectively added to 5 mL of the above plain liposomal suspensions, and the polysaccharide/lipid weight ratios were 1/4, 2/3, 3/2, and 4/1. Then, the mixture solutions were incubated at 20°C with gentle shaking for 1 hour and followed by storage at 4°C for 24 hours until use. Furthermore, CCLs as the control were also prepared by the same method as above. Characterization of CALs The sizes and size distributions of CALs were measured by dynamic laser light scattering (DLLS) with a Brookhaven digital
autocorrelator (model BI-90 Plus; Brookhaven Instruments, Holtsville, New York) at a scattering angle of 90 degrees, a wavelength of 633 nm, and a temperature of 25 ± 0.1°C. The zeta potentials of CALs were determined using a Brookhaven electrophoretic light-scattering spectrometer (model BI-Zetaplus; Brookhaven Instruments, Holtsville, New York). Transmission electron microscopy (TEM) observations were performed after negative staining of CALs with phosphomolybdic acid. Briefly, the liposome samples were dropped onto carbon-coated grids (100 mesh) and drawn off with a piece of filter paper. Then, the grids were immersed for 1 minute in 2% phosphomolybdic acid aqueous solution and washed twice with 200 mL distilled water. Finally, the grids were dried and imaged using a Philips transmission electron microscope (model EM400ST; Philips, Eindhoven, The Netherlands). Study of physical stability of CALs CALs were stored at 4 ± 2°C, 25 ± 2°C, and 37 ± 2°C for a period of 5, 10, 15, 20, and 30 days. As above, the sizes and size distributions of CALs were evaluated by DLLS using a Brookhaven digital autocorrelator (model BI-90 Plus; Brookhaven Instruments). The morphologic changes of CALs were observed after 30 days of storage by a Philips transmission electron microscope (model EM400ST; Philips). Drug loading and release studies EPB was first loaded into the plain liposomes by the modified pH gradient method.17 Briefly, 5 mL of the plain liposome suspension prepared above was applied to a Sephadex G-50 column (2 × 60 cm) and eluted with phosphate buffer solution (PBS; pH 7.4) to obtain liposomes with a pH gradient between their interior and exterior. Then, different volumes (62.5, 125, 250, and 500 μL) of 10 mg/mL EPB in PBS (pH 7.4) were added to these pH gradient liposomes and incubated at 4°C for 12, 24, 36, and 48 hours, respectively. The resulting liposome dispersion passed through the Sephadex G-50 column with PBS (pH 7.4) as the eluent to separate free EPB from the plain EPB-loaded liposomes (PELs). The fluorescence intensity of free EPB was measured by a Shimadzu fluorescence spectrophotometer (model RF-4500; Shimadzu, Kyoto, Japan). The excitation wavelength (λex) and the emission wavelength (λem) were set at 470 and 585 nm, respectively. The entrapment efficiency of EPB in liposomes was calculated according to the following equation: EE =
ðW0 W1 Þ 100k W0
where EE is the entrapment efficiency, W0 is the total amount of EPB initially added, and W1 is the amount of free EPB. Then, PELs were incubated with CHCS conjugates to prepare CHCS anchored EPB-loaded liposomes (CAELs) with the polysaccharide/lipid weight ratio of 3/2 using the same method for preparing CALs. At the same time, chitosan-coated EPBloaded liposomes (CCELs) as the control were also prepared as the same method for preparing CCL. In vitro release behaviors of EPB from CAELs were studied by the dialysis method18 both in PBS (pH 7.4) and 1% (vol/vol) aqueous FBS. Briefly, 2 mL liposome suspension was placed
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into the dialysis tube (molecular weight cutoff 8 to 14 kDa; Millipore, Bedford, Massachusetts) and dialyzed against the above release media (10 mL) at 37 ± 0.5°C in an air-bath shaker at 100 rpm. At scheduled time intervals, the whole of the release media was collected and then the fresh release media were added. The release amount of EPB was determined by fluorescence spectrophotometry as described earlier. The accumulative release percentage of EPB (RE %) was calculated according to the following equation:
D0t RE ðkÞ = D0
100
where D0-tis the amount of drug released from liposome suspension from the beginning to the scheduled time, and D0 is the total amount of drug in liposome suspension. Results Preparation and characterization of CALs In our study, the different polysaccharide/lipid weight ratios were investigated to prepare CAL. Figure 1 shows the effects of polysaccharide/lipid weight ratio on the size (Figure 1, A) and the zeta potential (Figure 1, B) of CAL, and the same trend occurred among CALs with different DS of the cholesterol moiety. Compared with the plain liposomes (size 148.2 ± 3.0 nm and zeta potential −4.75 mV), CALs had significantly larger sizes and positive zeta potentials, which indicated that CHCS conjugates with different DS of the cholesterol moiety successfully coated on the surface of the plain liposomes to form the polysaccharide shells. The sizes and the zeta potentials of CALs evidently increased with the polysaccharide/lipid weight ratio increasing from 0 to 3/2 but slightly changed when the polysaccharide/lipid weight ratio continued increasing, which indicated that CHCS conjugates coating on the liposome surface reached a saturation state. Therefore, 3/2 was believed to be the optimal polysaccharide/lipid weight ratio to prepare CALs. Moreover, as shown in Figure 1, CALs had evidently larger sizes and significantly higher zeta potentials compared with those of CCL under the same polysaccharide/lipid weight ratio. For example, the zeta potentials of CAL-1, CAL-2, and CAL-3 were respectively +25.48 mV, +22.06 mV, and +21.09 mV at the polysaccharide/lipid weight ratio of 3/2, whereas the zeta potential of CCL was only +7.81 mV. Therefore, it could be deduced that CALs had better stability than CCL due to their higher zeta potentials. Figure 1, A also shows that the size of CAL increased when the DS of the cholesterol moiety of CHCS increased from 2.8% to 8.0%; for example, the size of CAL-3 was 262.5 ± 6.5 nm under the polysaccharide/lipid weight ratio of 2/3, evidently larger than the size of CAL-1 (205.8 ± 1.2 nm) and the size of CAL-2 (232.5 ± 9.1 nm), which suggested that CHCS with larger DS of the cholesterol moiety could be easier to coat on the surface of the plain liposomes. Figure 2 shows TEM images of the plain liposomes (Figure 2, A1 and A2), CAL-2 with the polysaccharide/lipid weight ratio of 3/2 (Figure 2, B1 and B2), and the physical mixture of plain liposomes and CHCS-2 with the same polysaccharide/lipid
Figure 1. Effects of polysaccharide/lipid weight ratio (wt/wt) on the (A) size and (B) zeta potential of liposomes. Each data point represents the mean of at least three independent experiments (-●-, CCL; -○-, CAL-1; -■-, CAL-2; -□-, CAL-3).
weight ratio of CAL-2 (Figure 2, C). Morphologically, plain liposomes were nearly spherical (Figure 2, A1) and had an obvious multilamellar (onion-like) structure characterized by multiple membrane bilayers, each separated from the next by an aqueous layer (Figure 2, A2); CAL-2 also had a spherical shape (Figure 2, B1) and a classic shell-core structure (Figure 2, B2) with a shell thickness of about 30 nm. However, the plain liposomes were wrapped with a thin polymer film formed by CHCS-2 under the TEM observation (Figure 2, C) when they were simply mixed with CHCS-2. Furthermore, the size of CAL2 determined by DLLS was about 245.4 ± 8.1 nm, obviously larger than the size determined by TEM images. We believed this was because the polysaccharide shell of CAL-2 was highly hydrophilic in nature and would be likely to swell in aqueous media, thus the size determined by DLLS was a hydrodynamic diameter and was larger than the size measured by TEM in the dried state. Other CALs had similar morphologic characteristics
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Figure 2. TEM images of (A1, A2) plain liposomes, (B1, B2) CAL-2, and (C) the physical mixture of liposomes with CHCS-2.
to CAL-2. All the above results suggested that CHCS conjugates could effectively be coated on the surface of liposomes by the incubation method in this study. The physical stability of CAL CALs were stored at 4 ± 2°C, 25 ± 2°C, and 37 ± 2°C for a period of 5, 10, 15, 20, and 30 days and then were determined by DLLS and TEM to evaluate their physical stability. The plain liposomes and CCL were also investigated as the controls. Figure 3 shows the effect of storage conditions on the size of liposomes. An insignificant difference (P N .05) was found in the sizes of all liposomal formulations stored at 4 ± 2°C (Figure 3, A) for 30 days. But a significant (P b .05) increase in the size of the plain liposomes was observed after 30-day storage both at 25 ± 2°C (Figure 3, B) and 37 ± 2°C (Figure 3, C), which was due to the fusion of the plain liposomes. No significant (P N .05) changes in the sizes of CALs and CCL were observed when they were stored at 25 ± 2°C up to 30 days (Figure 3, B). However, the sizes of CALs and CCL all significantly (P b .05) increased after 30-day storage at 37 ± 2°C. These results indicated that CALs and CCL exhibited good stability on storage for at least 30 days at 4 ± 2°C and 25 ± 2°C.
Figure 3. Particle sizes of plain liposomes, CCL, and CALs on time of storage at (A) 4 ± 2°C, (B) 25 ± 2°C, and (C) 37 ± 2°C. All values are expressed as mean ± SD (n = 3).
Figure 4 shows the morphologies of CAL-2 and CCL after 30-day storage at 25 ± 2°C. CAL-2 (Figure 4, A) retained its spherical shape, and no evident aggregation phenomenon was observed; the other two CALs had similar morphology to CAL2. CCLs (Figure 4, B) were irregularly spherical, and a significant aggregation phenomenon took place. Therefore, it could be concluded that CALs had better storage stability than CCL, which was perhaps due to their higher zeta potentials compared with that of CCL as discussed earlier. Drug loading and release behaviors of CALs EPB, as a weak base (pKa = 7.7), was first loaded into the plain liposomes by the modified pH gradient method.17 This method is often used to load weak bases such as doxorubicin and
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Figure 5. Effect of preparation conditions on the encapsulation efficiency of EPB. Each data point represents the mean of at least three independent experiments (-○-, incubation time 12 hours; -■-, incubation time 24 hours; -●-, incubation time 36 hours; -□-, incubation time 48 hours).
Figure 4. TEM images of (A) CAL-2 and (B) CCL after 30-day storage at 25 ± 2°C.
vincristine,19-21 which coexist in aqueous solutions in neutral and charged forms, into liposomes using pH gradient as a driving force. In this study, the preparation conditions such as the weight ratio of EPB to lipid and the incubation time were investigated to obtain an optimal drug encapsulation. As shown in Figure 5, EPB encapsulation efficiency increased with the weight ratio of EPB to lipid increasing from 0.01 to 0.025, and then decreased with the weight ratio further increasing, which suggested that the ability of liposomes to load EPB reached saturation. Figure 5 also shows there was no significant difference (P N .05) of EPB encapsulation efficiency among the incubation times (12 to 48
hours) when the weight ratio of EPB to lipid was less than 0.05, but an obvious increase of EPB encapsulation efficiency occurred with the incubation time increasing when the weight ratio of EPB to lipid was larger than 0.05. However, when the incubation time was longer than 36 hours, a significantly larger size of liposomes was observed, which was perhaps due to the fusion of liposomes. Therefore, considering the preparation conditions prescribed above, 0.025 (EPB to lipid weight ratio) and 24 hours (incubation time) were chosen to prepare PELs, and EPB encapsulation efficiency was high, to 96.8%. Then, PELs were incubated with CHCS conjugates to prepare CAELs at the polysaccharide/lipid weight ratio of 3/2. CAELs had similar morphology to CALs such as the spherical shape and the classic shell-core structure under TEM observation, but a slight decrease on EPB content was measured after PEL surface coating with CHCS conjugates. We believed this was because EPB passively leaked out of liposomes in the incubation period, and EPB leakage rates were 2.65%, 1.98%, and 3.04% for CAEL-1, CAEL-2, and CAEL-3, respectively. EPB release behavior from CAEL was studied in vitro by the dialysis method in PBS (pH 7.4) and 1% aqueous FBS, respectively. EPB release from PELs and CCELs was also investigated as the controls. The cumulative EPB release profiles are shown in Figure 6. In PBS (pH 7.4) (Figure 6, A), EPB release from PEL showed a rapid pattern, about 52% EPB released in 24 hours; but only about 37%, 33%, and 41% EPB released respectively from CAEL-1, CAEL-2, and CAEL-3, indicating that CAEL significantly sustained the release of EPB. In 1% FBS (Figure 6, B), EPB release rates from PELs and CAELs markedly accelerated; nearly 100% EPB released from all liposome formulations in 24 hours. We believe this was because the bioactive substances such as proteins and enzymes in FBS destroyed the liposome structure and polysaccharide coating shell. However, CAELs also showed a significant sustained drug release in the first 12 hours, about 73.6%, 67.7%, and 79.5% EPB released respectively from CAEL-1, CAEL-2, and CAEL-3
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Figure 6. Dynamic release profiles of EPB from PEL (-●-), CCEL (-○-), CAEL-1 (-■-), CAEL-2 (-□-), and CAEL-3 (-▲-) in (A) PBS (pH 7.4) and (B) 1% FBS.
compared with 100% EPB from PELs in 1% FBS (Figure 6, B). The above results suggested that CAELs had better stability than PELs in vitro due to CHCS shells on the surface of PELs. Moreover, compared with CCEL, EPB release from CAELs more or less slowed both in PBS (pH 7.4) and 1% FBS, which indicated that CHCS coating shells were more stable than chitosan coating shell on the liposome surface.
Discussion Recently, many investigations into CCL have been performed to enhance the stability of liposomes and increase their intestinal uptake.22-24 We previously hoped to use CCL as the carrier of anticancer drugs, but the chitosan layer formed by the electrostatic attractive interaction between the positively charged amino groups in chitosan molecules and the negatively charged surface of liposomes was not effective enough to significantly stabilize liposomes in vitro, so that the anchoring method using hydrophobically modified chitosan derivatives (eg, CHCS and
palmitoyl chitosan conjugates) as anchoring materials was then applied to increase the stability of liposomes. In our later research, cholesterol modified groups showed better hydrophobic anchoring properties for preparation of surface coating liposomes compared with alkylated modified groups, which was consistent with the previous report of Kang et al.10 Compared with CCL, CALs had larger sizes, higher zeta potentials, better physical stability, and more sustained drug release behavior under the same study conditions. We believe this was due to the difference of coating mechanisms between CHCS conjugates and chitosan on the liposome surface. It was clear that chitosan coating on the liposome surface mainly depended on the electrostatic attraction force. The amino groups of chitosan molecules carrying positive charges neutralized the negative surface charges of liposomes, so that the zeta potential of liposomes changed from negative to positive after chitosan coating, and its absolute value was lower than that of some chitosan particles (eg, +28.44 mV previously reported).25 However, in addition to the electrostatic attraction force, the process of CHCS coating also involved the cholesterol modified groups anchoring into the phospholipid bilayer of liposome, thus a thicker and more stable polysaccharide shell formed on the liposome surface. Comparison of drug release profiles of CAELs both in PBS (pH 7.4) and 1% FBS (Figure 6) showed that EPB release behavior from CAEL was significantly influenced by the DS of the cholesterol moiety of CHCS. EPB release rate from CAEL decreased with the DS of the cholesterol moiety increasing from 2.8% (CHCS-1) to 5.6% (CHCS-2). This was because CHCS with higher DS of the cholesterol moiety had more cholesterol modified groups to anchor into the liposome phospholipid bilayer, and thus a more stable polysaccharide coating shell was obtained. However, with DS of the cholesterol moiety increasing from 5.6% (CHCS-2) to 8.0% (CHCS-3), EPB release rate evidently increased, such as EPB release from CAEL-3 being even faster than that from CAEL-1 both in PBS (pH 7.4) and in 1% FBS. We believed this was perhaps because more cholesterol modified moieties anchoring into the liposome phospholipid bilayer resulted in a higher liposome membrane permeability. Therefore, CHCS-2 with DS of the cholesterol moiety of 5.6% was considered an optimal hydrophobically modified chitosan derivative to prepare polysaccharide anchored liposomes in this study. EPB was chosen as a model anticancer drug to assess the potential of CAL as a novel carrier of anticancer drugs. As an anthracycline anticancer agent, EPB has a wide range of antitumor activity and is used to treat various carcinomas. However, EPB therapy may cause some serious side effects such as allergic reactions, cardiotoxicity, and blood problems.26,27 Therefore, CAL was used as a carrier of EPB in an attempt to sustain its release, prolong its circulation time, enhance its therapeutic index, and decrease its toxic effects. Our current results clearly showed that CHCS anchoring on the surface of PELs could significantly improve their physical stability and sustain the release of EPB in vitro, but investigations in vivo are required to further confirm the advantages of CAL as the carrier of EPB over the plain liposomes and CCL, and related experiments are now in progress.
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Acknowledgment The authors thank Drs. Xindu Yang, Wenzhi Yang, and Hongli Chen for their helpful discussion and suggestions.
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