Efficient Loading into and Controlled Release of Lipophilic Compound from Liposomes by Using Cyclodextrin as Novel Trapping Agent

Abstract

Lipid bilayer vesicles, liposomes are representative drug delivery carriers. High encapsulation efficiency and release control of drugs are essential for clinical application of liposomes. For efficient drug loading into liposomes, remote loading method using driving force like transmembrane gradients of pH and ions are utilized. Ions are called as “trapping agents,” which are also critical for the controlled release of drugs loaded into liposomes inside. It is difficult to apply ions as trapping agents to various drugs because of limited physicochemical compatibility between drugs and ions. Cyclodextrins (CDs) with hydrophobic cavity can make inclusion complexes with various hydrophobic compounds. Therefore, we aimed to evaluate the potential of CDs as a novel trapping agent using sulfobutylether-β-cyclodextrin (SBE-β-CD) and ibuprofen (IB), a weak acid hydrophobic drug. Encapsulation efficiency of IB in liposomes with pH gradient was approximately 27%, and it was enhanced by intraliposomal SBE-β-CD inclusion in addition to pH gradient, which was SBE-β-CD concentration-dependent. In liposomes with pH gradient, a large fraction of IB was released in a short time. This early-stage rapid IB release was significantly suppressed by the inclusion of SBE-β-CD inside liposomes. Thus, novel remote loading technology by intraliposomal SBE-β-CD enabled the efficient encapsulation of the hydrophobic drug into the aqueous phase of liposomes as well as their controlled release. This technology should be applied to various drugs that can be included into CDs in order to enhance their therapeutic benefits.

INTRODUCTION

Liposomes are lipid bilayer vesicles surrounding an aqueous core.¹⁾ Liposomes have been widely applied as drug delivery carriers since these structures enable incorporation of both hydrophilic and hydrophobic drugs. High encapsulation efficiency and release control of drugs are essential for clinical application of liposomes. Active remote loading of drugs by transmembrane gradients is utilized to achieve efficient drug entrapment.¹⁾ Major driving forces for remote loading are transmembrane gradients of pH and ions. In order to stably retain drugs inside liposomes after loading, appropriate pH and ions need to be selected depending on the physicochemical properties of drugs so that the precipitation of drug-ion salt can be formed.^2,3) Ions used for remote loading are called as “trapping agents” since ions help drugs retain inside liposomes and control the release of drugs from liposomes. If liposomes contain no trapping agents inside, intraliposomal drugs should be released rapidly from liposomes. Burst release of drugs in early stage is not preferable since drugs loaded into liposomes should be retained within liposomes until reaching target organs. Therefore, appropriate selection of a trapping reagent is critical for successful preparation of liposomes by remote loading method.

Doxil^® and Onivyde^® approved by U.S. Food and Drug Administration (FDA) are liposomal formulations developed by remote loading method.⁴⁾ These formulations could not be realized without optimal trapping agents. However, each ion used for these formulations as a trapping agent is not always suitable for other drugs since each drug requires an individual trapping agent to form stable drug precipitation. Hence, it is difficult to apply ions as trapping agents to other various drugs.^5,6)

Cyclodextrins (CDs) are cyclic oligosaccharides that have hydrophobic cavity and hydrophilic surface.⁷⁾ CD cavities are able to include various hydrophobic compounds by host (CDs)-guest (included compounds) interactions.^7,8) If CDs are used as trapping agents, hydrophobic compounds loaded into liposomes would make inclusion complexes with CDs inside liposomes, which may increase the loading of hydrophobic compounds into liposomes and control their release from liposomes. Therefore, CDs could make it possible to apply remote loading methods to numerous hydrophobic compounds. However, CDs have yet to be tried as trapping agents for the improvement of drug loading efficiency into and drug release profile from liposomes. In this study, in order to apply remote loading techniques to various hydrophobic drugs, we aimed to develop a novel drug loading technology using CDs as intraliposomal trapping agents.

MATERIALS AND METHODS

Materials

1,2-Distearoyl-sn-glycero-3-phosphatidylcholine (DSPC) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Ibuprofen (IB) and indomethacin were purchased from Nacalai Tesque Inc. (Kyoto, Japan). Cholesterol was purchased from Sigma (St. Louis, MO, U.S.A.). 1,2-Distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG-2000-DSPE) was purchased from NOF Corporation (Tokyo, Japan). Sulfobutylether-β-cyclodextrin (SBE-β-CD) was purchased from Angene International Limited (London, U.K.). All other reagents were commercial products of the highest grade.

IB-Loaded Liposome Preparation

Thin-film consisting of DSPC : cholesterol : PEG-2000-DSPE (6 : 4: 0.03, molar ratio) same as Onivyde^®9) was hydrated in 100 mM calcium acetate (pH 7.4) ± 50–200 mM SBE-β-CD under mechanical agitation. The obtained suspension was sonicated for 20 min at 60W. Unentrapped SBE-β-CD was removed by a PD-10 column eluted with 17 mM PIPES-145 mM NaCl (pH 6.0). For the remote loading of IB into liposomes, the mixture of IB solution and liposome suspension (SBE-β-CD : IB = 25 : 1, 50 : 1, 100 : 1, molar ratio) was heated for 30 min at 60 °C. Non-encapsulated IB was removed using a Sephadex G25 column. Particle size, polydispersity index and zeta potential were determined by a dynamic light scattering spectrophotometer (Zetasizer Advance, Malvern instruments Ltd., Worcester-shire, U.K.).

In the case of IB partition into lipid membrane by conventional hydration method, thin-film consisting of DSPC : cholesterol:PEG-2000-DSPE : IB (6 : 4 : 0.03 : 1, molar ratio) was hydrated in 149 mM phosphate-buffered saline (PBS) (pH 7.4) and sonicated followed by gel filtration under the same conditions above.

Encapsulation Efficiency

IB-loaded liposomes were mixed with acetonitrile followed by rotation for 20 min and centrifugation at 5500 × g for 15 min at room temperature. The supernatant was measured at excitation 230 nm/emission 285 nm using a spectrofluorophotometer (RF-5300PC, Shimadzu Corporation, Kyoto, Japan). The IB encapsulation efficiency was calculated by the following formula:

  

Release Study

IB release from liposomes was determined by dialysis method according to our previous study.¹⁰⁾ Equal volume mixture of IB-loaded liposomes (50 µg as IB) and fetal bovine serum in a dialysis tube (Spectra/Por4 Membrane, MWCO: 12000–14000, Spectrum Laboratories) was incubated in PBS (pH 7.4) containing 3% bovine serum albumin (BSA) at 37 °C at 100 rpm. Collected samples from outer phase were mixed with acetonitrile containing indomethacin (internal standard) followed by rotation and centrifugation described above. The supernatant was analyzed by HPLC (Shimadzu LC-20A system, Shimadzu Corporation). HPLC conditions were as follows: column, COSMOSIL 5C18-MS-II (Nacalai Tesque, Kyoto, Japan); temperature, 30 °C; flow rate, 1.0 mL/min; detection, UV 220 nm; mobile phase, 0.1% acetic acid/acetonitrile (40/60, v/v).

Statistical Analysis

Multiple comparisons were performed by non-repeated measures ANOVA followed by SNK test. p-Values <0.05 were considered statistically significant.

RESULTS AND DISCUSSION

In this study, IB as a lipophilic model drug and SBE-β-CD as a CD were used. In our preliminary experiments, stability constants between IB and various CDs were determined by solubility method. The stability constant in β-CD was much larger than α-CD and γ-CD (α-CD, 20; β-CD, 1700; γ-CD, 70). In addition, the stability constant in methyl-β-CD was 3900, the largest among various β-CD derivatives. However, methyl-β-CD-containing liposomes could not be formed because methyl-β-CD would destabilize liposomes. CDs are known to make inclusion complexes with cholesterol, which is a major component of liposomes.^11,12) The complexation efficiency of methyl-β-CD with cholesterol was much larger than other β-CD derivatives since methyl-β-CD has more hydrophobic cavity compared with other β-CDs.¹²⁾ Moreover, methyl-β-CD was demonstrated to be the only CD to impair liposomal membrane integrity, resulting in the destabilization of liposomes.^11,12) Our preliminary data also showed that the stability constant with IB was the second largest in SBE-β-CD next to methyl-β-CD. Therefore, we selected SBE-β-CD as a CD.

Encapsulation Efficiency of IB

In this study, pH values outside and inside of liposomes were 6 and 7.4, respectively. Molecular form of IB (weak acid lipophilic drug, pK_a value 4.5) was efficiently loaded into liposomes by this pH gradient since larger amount of molecular form of IB was existing outside (pH 6) than inside (pH 7.4) of liposomes.¹³⁾ Regarding the pH of liposomes outside, pH value higher than 4.5 is preferable in order to solubilize IB in outer phase of liposomes. However, too high pH value leads to the decrease in the ratio of molecular form of IB outside liposomes, which is able to pass through lipid bilayer. Thus, we decided outer pH value to pH 6.0 in this study. In remote loading method, intraliposomal pH is generally set in order to contain more ionic form inside than outside of liposomes since ionic form, which is unable to pass through lipid bilayer, is easily retained inside liposomes.^9,13) Thus, inner pH value was set at 7.4.

All particles were monodispersed (polydispersity index < 0.25) and particle size was larger in SBE-β-CD containing-liposomes than liposomes without SBE-β-CD (Table 1). When IB was partitioned into lipid bilayer by conventional hydration method, only a small amount of IB was incorporated into liposomes (7%) (Table 1). However, when IB was loaded into aqueous phase by remote loading method, encapsulation efficiency of IB in liposomes with pH gradient (inner pH 7.4/outer pH 6.0) was approximately 27%, which was significantly increased to approximately 80% by intraliposomal SBE-β-CD inclusion in addition to pH gradient (Table 1). This increase was concentration-dependent (50 < 100 < 200 mM SBE-β-CD). After IB was entrapped inside liposomes, IB formed the inclusion complexes with SBE-β-CD keeping the concentration gradient of molecular form of IB across liposomal membrane, resulting in continuous incorporation of IB from liposomes outside.

Table 1. Physicochemical Properties and Encapsulation Efficiency of Prepared Liposomes

Loading method	Remote loading (loading into aqueous phase)				Passive loading by hydration (partition into lipid membrane)
pH (inner/outer)	7.4/6.0				7.4/7.4
CD in aqueous phase	—	50 mM SBE-β-CD	100 mM SBE-β-CD	200 mM SBE-β-CD	—
Particle size (nm)	82.3 ± 0.96	117.9 ± 4.4**	145.6 ± 9.4**, ††	133.7 ± 3.0**, ††	79.4 ± 1.7
Polydispersity index	0.22 ± 0.03	0.21 ± 0.03	0.24 ± 0.04	0.12 ± 0.03	0.23 ± 0.03
Zeta potential (mV)	−4.5 ± 0.11	−9.4 ± 4.7	−5.8 ± 2.0	−10.2 ± 2.9	−4.2 ± 0.94
Encapsulation efficiency (%)	26.7 ± 3.6	47.5 ± 5.6**	53.0 ± 2.8**	79.6 ± 5.1**, ‡‡	7.3 ± 0.8**

Each value represents the mean ± S.D. of at least three experiments. ** p < 0.01 (vs. pH 7.4/6.0, SNK test), ^†† p < 0.01 (vs. pH 7.4/6.0–50 mM SBE-β-CD, SNK test), ^‡‡ p < 0.01 (vs. pH 7.4/6.0–100 mM SBE-β-CD, SNK test).

Considering that molecular form may make more stable inclusion complex than ionic form, we also examined inner pH of 4.0 using 200 mM SBE-β-CD-containing liposomes, at which more molecular form of IB is present than its ionic form. As a result, IB was hardly entrapped into SBE-β-CD containing-liposomes at pH 4.0 (inner)/6.0 (outer) (IB encapsulation efficiency, 6%), suggesting that intraliposomal molecular form of IB may be quickly released to liposomes outside through lipid bilayer membrane rather than be included in SBE-β-CD. Thus, regarding intraliposomal pH, pH value higher than 6.0 (outer pH) would be better. In addition, in the case of SBE-β-CD-containing liposomes without pH gradient, IB encapsulation efficiency was extremely low and was found to be 8% at both pH 7.4 (inner)/7.4 (outer) and pH 6.0 (inner)/6.0 (outer). These results indicated that pH gradient is indispensable for IB to efficiently enter into aqueous phase. Therefore, both pH gradient and CD inclusion are necessary for efficient drug loading.

Release Profile of IB

Using only pH gradient, a large fraction of IB was released in a short time (Fig. 1; 4 h, 62%; 24 h, 92%). This early-stage rapid IB release in serum was significantly suppressed by intraliposomal SBE-β-CD inclusion, which was equivalent to IB release from lipid bilayer of liposomes prepared by conventional hydration method. Thus, the inclusion of CDs is also important for controlling the release of intraliposomal drugs. Inclusion complexes of IB and SBE-β-CD are difficult to be released from liposomes through lipid bilayer due to the large molecular size of complexes. In addition, the outside of CDs is hydrophilic. Thus, IB needs to dissociate from CD complexes before its release from liposomes. Dissociation of drugs from CD complexes is considered to be much slower than the drug release through lipid bilayer.¹⁴⁾ Therefore, the inclusion of CDs inside liposomes would have improved the retention of IB within liposomes, leading to its more favorable release profiles.

Fig. 1. IB Release Profile from Liposomes

IB release from liposomes was determined by the dialysis method. The dialysis tube containing IB-loaded liposomes and serum was incubated in PBS containing BSA at 37 °C at 100 rpm. ○, ●, □, ■: IB was loaded into liposomes inside by remote loading method. pH 7.4/pH 6.0 shows inner/outer pH of liposomes. SBE-β-CD was included in aqueous phase of liposomes. ▲: IB was partitioned into lipid membrane by hydration method. Insert shows the extended figure in a short-term (approx. 2 h). ** p < 0.01 (vs. pH 7.4/pH 6.0, SNK test). Each point represents the mean ± standard deviation (S.D.) of at least three experiments.

High-concentration SBE-β-CD is considered to possibly influence the membrane integrity of liposomes and hence IB release from liposomes by extracting cholesterol to some extent. In this study, however, IB release from 200 mM SBE-β-CD containing liposomes was not significantly different from that from 50 or 100 mM SBE-β-CD containing liposomes (Fig. 1), suggesting that SBE-β-CD had little effects on membrane integrity of liposomes. This result is in accordance with the previous reports demonstrating that SBE-β-CD hardly permeabilizes liposomal membrane.^12,15) These reports have also shown that hydroxypropyl-β-CD, as well as SBE-β-CD, did not significantly increase the content leakage from liposomes. Therefore, we consider that hydroxypropyl-β-CD is also available as an intraliposomal trapping agent.

In conclusion, we succeeded in developing novel remote loading technology using SBE-β-CD as an intraliposomal trapping agent. Intraliposomal SBE-β-CD was able to enhance the encapsulation efficiency of the hydrophobic drug into the aqueous phase of liposomes as well as control the drug release. Since a lot of drugs, such as sorafenib,¹⁶⁾ curcumin¹⁷⁾ and celecoxib,¹⁸⁾ have been reported to form the stable inclusion complexes with SBE-β-CD, we consider that the present remote loading method can be applied to other various drugs.

Conflict of Interest

The authors declare no conflict of interest.

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