Novel analytical method to evaluate the surface condition of polyethylene glycol-modified liposomes

Colloids and Surfaces A: Physicochem. Eng. Aspects 397 (2012) 73–79

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa

Novel analytical method to evaluate the surface condition of polyethylene glycol-modified liposomes Keisuke Yoshino a,∗ , Kyouko Taguchi a , Mayu Mochizuki a , Shigenori Nozawa a , Hiroaki Kasukawa a , Kenji Kono b a b

Terumo Corporation R&D center, 1500 inokuchi, Nakai-machi, Ashigarakami-gun, Kanagawa 259-0151, Japan Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University,1-1, Gakuen-cho, Sakai, Osaka 599-8531, Japan

a r t i c l e

i n f o

Article history: Received 22 October 2011 Received in revised form 7 January 2012 Accepted 27 January 2012 Available online 6 February 2012 Keywords: Liposomes Polyethylene glycol (PEG) Long circulation Deviation of PEG-lipid Liposome preparation method

a b s t r a c t In liposomal drug delivery, polyethylene glycol (PEG) modification is known to prolong the circulation time of liposomes in the blood stream, and hence, this method has been employed for liposomes in clinical use. To achieve effective prolongation of liposomal circulation time using PEG-modification, it is of importance to control not only the average amount of PEG chains incorporated in the liposomes but also the deviation in PEG amount among PEG-modified liposomes (PEGylated liposomes). Therefore, evaluation of PEG-modification is essential for the quality control of PEGylated liposomes. In this study, we developed a novel method to estimate PEG-modification for PEGylated liposomes using chromatography with an anion-exchange column. We applied this method to liposomes with varying PEG-lipid contents and found that the chromatography could successfully separate PEGylated liposomes on the basis of the PEG-lipid content of their liposome membranes. Results demonstrate that the established method can be potentially used to control the quality of PEGylated liposomes not only for research and but also for manufacturing processes. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Currently, studies on drug delivery systems have great attention to achieve the selective delivery and distribution of drugs to the target disease sites for safety and effectiveness. Therefore, feasibility of various types of particulate systems such as liposomes, emulsions, lipid microspheres, and polymeric nanoparticles has been evaluated as effective drug delivery systems [1–3]. The clearance of liposomes has been considered to occur through their capture by the reticuloendothelial system (RES), which takes up liposomes circulating in the blood stream and removes them from the blood. To deliver drug molecules encapsulated in liposomes selectively to target site, the avoidance of recognition of liposomes by RES and the control of their pharmacokinetics are essential. Especially, it is important to avoid capture by the phagocytic cells in the liver and spleen for prolongation of liposome circulation time in the blood. Although various factors of liposomes, such as chain length, unsaturation of lipids, lipid composition, size, and zeta potential, affect the circulation time of liposomes [6–8], surface modification of liposome membranes with monosialoganglioside GM1 or polyethylene glycol (PEG)-

∗ Corresponding author. Tel.: +81 465 81 4187; fax: +81 465 81 4114. E-mail address: Keisuke [email protected] (K. Yoshino). 0927-7757/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2012.01.035

conjugated lipid (PEG-lipid) has been shown to greatly improve their circulation time [4,5]. Based on these findings, the surface coating approach has been actively investigated to develop effective liposome-based drug delivery systems. PEG is known as a highly hydrophilic polymer with very low toxicity, and hence, PEG and its derivatives have been widely used to improve the stability and pharmacokinetics of drug carriers and parent drug [9]. In liposomal drug delivery, PEG-lipid has been widely used for surface coating of liposomes to achieve prolongation of their circulation time in the blood. This technique has been already employed for the preparation of liposomal drug delivery systems, which are known as PEGylated liposomes [10–23]. Especially, doxorubicin-loaded PEGylated liposomes, which are named Doxil® , show high efficacy and low toxicity. Therefore, these have been widely used in clinical applications and approved in more than 80 countries for the treatment of cancer [24,25]. These facts clearly demonstrate the importance of PEG modification techniques for the production of efficient liposomal drug delivery systems. Physiological and physicochemical stabilities and pharmacokinetics of PEGylated liposomes are affected by both the average amount of PEG attached to the liposome surface and deviation of PEG amounts among PEGylated liposomes. For this reason, analytical methods have become very important to estimate whether PEG-lipids are appropriately and uniformly incorporated in liposomes. Analytical methods such as high

74

K. Yoshino et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 397 (2012) 73–79

performance liquid chromatography (HPLC) generally provide information on PEG-modification from the average amount of PEG-lipids contained in liposomes, because the analyses are usually carried out using PEGylated liposomes dissolved in organic solvent. Therefore, these analytical methods cannot be used for the estimation of the deviation in PEG-lipid amounts among PEGylated liposomes; however, information on the deviation in PEG-modification is of great importance for quality control of PEGylated liposomes. In this study, we attempt to evaluate not only the average amount of PEG-lipid incorporated in liposomes but also deviations in PEG-lipid amounts among PEGylated liposomes using a novel analytical method that is based on chromatography with an anionexchange column. We demonstrate that the combination of the electrostatic interaction of anion exchange gels with PEGylated liposomes and linear ion gradient achieved efficient separation of PEGylated liposomes with varying PEG-lipid contents. 2. Material and methods 2.1. Materials Hydrogenated soybean lecithin (HSPC) was purchased from Lipoid GmbH (Ludwigshafen, Germany). Cholesterol (Chol) was purchased from Solvay pharmaceuticals (Nieuweweg, Netherlands). Dehydrated ethanol and ammonium sulfate were purchased from Wako Pure Chemical Co. Ltd. (Osaka, Japan). Polyethylene glycol5000 -distearoyl phosphoethanolamine (PEGlipid) was purchased from NOF Co. Ltd. (Tokyo, Japan). Doxorubicin hydrochloride (DXR) was purchased from Boryung Pharmaceuticals Co. Ltd. (Seoul, Korea). All other chemicals were purchased from Kanto Chemicals Co., Inc. (Tokyo, Japan). 2.2. Preparation of liposomes PEGylated liposomes were prepared by using 2 different techniques, namely the pre- and post-modification methods, as described below (Fig. 1). 2.2.1. Preparation of PEGylated liposomes by the pre-modification method PEGylated liposomes containing HSPC and Chol (molar ratio 54:46) and a given mol% of PEG-lipid (0.5, 1.0, 1.5, and 2.0 mol%) were prepared as follows. These lipids (0.7 g HSPC, 0.3 g Chol, 0.05–0.69 g PEG-lipid) were dissolved in ethanol (1 ml) at 68 ◦ C and hydrated in 250 mM aqueous solution of ammonium sulfate (9 ml) for 15 min at 68 ◦ C to afford crude liposomes. The obtained crude liposomes were extruded through 2 stacked polycarbonate membranes with pore sizes of 200 nm and 100 nm using Extruder T10. 2.2.2. Preparation of PEGylated liposomes by the post-modification method Liposomes containing HSPC and Chol (molar ratio, 54:46) were prepared as follows. HSPC (7.0 g) and Chol (2.9 g) were dissolved in ethanol (10 ml) at 68 ◦ C and hydrated in 250 mM aqueous solution of ammonium sulfate (90 ml) for 15 min at 68 ◦ C to afford crude liposomes. The obtained crude liposomes were extruded through 2 stacked polycarbonate membranes with pore sizes of 200 nm and 100 nm using Extruder T100. Then, a given volume of PEG-lipid aqueous solution (36.74 mg/ml) was added to the liposome suspension and heated at 65 ◦ C for 30 min to afford PEGylated liposomes with a desired PEG-lipid mol% (0.25, 0.5, 0.75, 1.0, and 2.0 mol%). It has been confirmed that PEG-lipid molecules were completely incorporated to liposomal membrane under the experimental condition.

2.2.3. DXR loading into liposomes DXR loading into liposomes was performed according to a previous report [26]. Briefly, PEGylated liposomes obtained by the above method were applied to a Sepharose 4 Fast Flow column (GE Healthcare, UK) pre-conditioned by 10% sucrose and 10 mM Tris solution (pH 9.0), affording PEGylated liposomes with pH gradient. An aqueous DXR solution (10 mg/ml) was added to PEGylated liposomes at DXR/total lipid molar ratio of 0.16 and incubated at 65 ◦ C for 30 min. Unloaded DXR was removed using the Sepharose 4 Fast Flow column by using 10% sucrose and 10 mM histidine solution (pH 6.5) as an eluent. Finally, sterile filtration using a 0.2 ␮m membrane filter (Minisart Plus, Sartorius, Goettingen, Germany)) was carried out for PEGylated liposomes loaded with DXR. 2.3. Characterization of DXR-loaded liposomes 2.3.1. Determination of lipid component PEGylated liposomes (2 ml) were dissolved in a mixture (9 ml) of water, chloroform, and 2-propanol. A small aliquot (30 ␮l) of the liposome solution was applied to an HPLC system equipped with an Inertsil Ph column (GL Science, Tokyo, Japan) and a differential refractometer. The mobile phase was a mixed solution of acetate buffer, methanol, and ethanol. Flow rate was 1 ml/min. 2.3.2. Determination of DXR concentration DXR concentration was determined by measuring the absorbance. Liposomes (40 ␮l) were dissolved in 2 ml methanol, and absorbance of the solution at 480 nm was measured using UV-2400PC (Shimadzu, Kyoto, Japan). 2.3.3. PEG surface evaluation using an anion-exchange column and linear ion gradient The HPLC system (Shimadzu, Kyoto, Japan) was equipped with TSK gel DEAE-5PW (75 mm × 7.5 mm) (Tosoh Biosciences, Tokyo, Japan), UV detector (SPD-10Avp), pump (LC-10ADvp), system controller (SCL-10Avp), and auto sampler (SIL-10ADvp). The flow rate was 1.0 ml/min. The analysis was carried out by following the absorbance at 280 nm. The mobile phase gradient was programmed as follows: total flow rate: 1 ml/min and linear ion gradient program: (1) 100% flow of pH 9.0, 20 mM Tris buffer till 0.5 min, (2) a linear gradient of pH 9.0, 20 mM Tris buffer containing 0.5 mM NaCl from 0.5 min to 35 min, and (3) 100% flow of pH 9.0, 20 mM Tris buffer containing 0.5 mM NaCl from 35 min to 40 min. This analytical condition was optimized through preliminary experiments. Other mobile phase programs are shown in Fig. 2. 2.3.4. Other methods HSPC concentration was determined using the Phospholipid C-test Wako® (Wako Pure Chemicals Ltd., Tokyo, Japan). Particle size and zeta potential were determined using Zetasizer 3000HS (Malvern Instruments, UK). 3. Results and discussion 3.1. Chromatographic analysis of liposomes PEGylated liposomes with varying PEG-lipid contents were prepared according to the pre-and post-modification methods (Fig. 1). Composition, PEG-lipid content, size, and zeta-potential of these PEGylated liposomes are listed in Table 1. These liposomes had similar diameters of around 110–130 nm. Bare liposomes exhibited a highly negative zeta potential because HSPC was partly hydrolyzed into lysophosphatidylcholine and fatty acids during the DXR loading process at high temperature and low pH [27]. In contrast, PEGylated liposomes showed low negative zeta potential values,

K. Yoshino et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 397 (2012) 73–79

75

Fig. 1. Preparation procedures of polyethylene glycol-modified (PEGylated) liposomes by the pre- and post-modification methods.

probably because hydrated PEG chains covered the lipid membrane and shielded the negatively charged surface [28,29]. First, we examined separation of the liposomes using an anionexchange column because their surface was negatively charged

and hence had affinity to positively charged column gels. In addition, we used an eluent with an ion gradient for column-mediated liposome separation because we expected the ion gradient to magnify subtle difference in the electrostatic interactions between

Fig. 2. Chromatograms for bare, Po-0.25, Po-0.5, Po-0.75, Po-1.0, and Po-2.0 liposomes under various programs of linear ion gradients. Each program of ion gradient is shown in the figure.

76

K. Yoshino et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 397 (2012) 73–79

Table 1 Characterization of liposomes. Lot No.

PEG modification method

HSPC:Chol:PEG (molar ratio)

PEG-lipid content (mol%)

DXR conc. (mg/ml)

Diameter (nm)

Zeta potential (mV)

Bare Po-0.25 Po-0.5 Po-0.75 Po-1.0 Po-2.0 Pre-0.5 Pre-1.0 Pre-1.5 Pre-2.0

– Post Post Post Post Post Pre Pre Pre Pre

54:46:0 54:46:0.25 54:46:0.5 54:46:0.75 54:46:1.0 54:46:2.0 54:46:0.5 54:46:1.0 54:46:1.5 54:46:2.0

0 0.27 0.54 0.78 1.08 2.14 0.48 1.00 1.47 2.02

1.53 1.61 1.6 1.55 1.53 1.77 1.53 1.57 1.54 1.42

122.5 120.3 122.4 124.1 126.2 130.5 113.4 112.1 112.7 105.9

−12.9 −7.3 −4.8 −4.3 −5.3 −3.6 −7.6 −5.4 −4.4 −4.3

these liposomes and column gels. Fig. 2 depicts chromatograms for PEGylated liposomes with varying PEG-lipid contents under different ion gradient conditions. As Fig. 2A shows, these liposomes were eluted quite quickly and did not show significant difference in the elution volume under the steep ion gradient condition. However, when ion concentration was increased slowly, then the chromatograms exhibited significant difference in the elution volume among the liposomes (Fig. 2B), and a highly different elution volume of the PEGylated liposomes was achieved using chromatography under very slow increase of ion concentration increase (Fig. 2C). As seen in Table 1, liposomes with high content of PEG-lipid tend to have low negative charge density and decreased electrostatic interaction with a positively charged column gel. Highly hydrated PEG chains attached to the liposome surface also might reduce the adsorption of liposomes to the column gels. These situations should induce a PEG-lipid contentdependent interaction between PEGylated liposomes and column gels. In addition, very slow increase in the ion concentration of the eluent could magnify the PEG-lipid content-dependent interaction. As a result, chromatography with an anion-exchange column under an ion gradient condition achieved significantly different retention times for PEGylated liposomes depending on PEGylation extent. Because excellent retention time differences among these PEGylated liposomes were achieved under the condition shown in Fig. 2C, chromatography experiments were carried out under that condition thereafter. To obtain information on the separation capability of a chromatography system with an anion-exchange column for PEGylated liposomes, the relationship between retention time and PEG-lipid content was investigated. As is apparent in Fig. 3A, the retention time of the PEGylated liposomes decreased with increasing PEGlipid content from 0 mol% to 0.75 mol%. However, further increase of PEG-lipid content of the liposomes resulted in no change in the retention time of the liposomes.

It is known that more than 0.5 mol% of PEG-lipid content is required for the PEGylated liposomes to exhibit excellent longcirculating ability in the blood (see Fig. S1). On the contrary, liposomes with PEG-lipid content of less than 0.5 mol% cannot exhibit long circulation performance. Based on Fig. 3A and Fig. S1, our analytic method can distinguish the presence of PEGylated liposomes having insufficient circulating capability (PEG-lipid content is less than 0.5 mol%). To obtain information on the electrostatic interaction during elution, relationship between the retention time and zeta potential of the liposomes was also investigated. As Fig. 3B shows, the retention time of PEGylated liposomes linearly increased with increasing negative value of zeta potential. As the PEG-lipid content of the liposomes increases, an increase of PEG chain amount and an increase of negative charges are induced concomitantly, and both of them should affect the affinity of the liposomes to the column gels. However, the correlation between the zeta potential of liposomes and their retention time strongly suggests that electrostatic interaction plays an important role in the PEG-lipid content-dependent elution of PEGylated liposomes. 3.2. Separation of PEGylated liposomes by chromatography Capability of our chromatography to separate PEGylated liposomes based on the PEG-lipid content was examined. Equivalent amounts of each type of PEGylated liposomes, namely, bare, Po0.25, Po-0.5, Po-0.75, Po-1.0, and Po-2.0 (Table 1) were mixed so that their average PEG-lipid content was 0.75 mol%. The mixed liposome suspension was analyzed using our chromatography (Fig. 4). The obtained chromatogram displayed 3 peaks around 8, 11, and 16 min in addition to a sharp peak around 7 min. Taking into account the chromatograms for these liposomes shown in Fig. 2C, the peaks at 8, 11, and 16 min might correspond to the Po-0.5, Po0.25 and bare liposomes, respectively, and the large peak at 7 min

Fig. 3. Chromatography for PEGylated liposomes. Correlations of PEG-lipid content (A) and zeta potential (B) with retention time.

K. Yoshino et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 397 (2012) 73–79

77

mAU

500

250

0 0

2

4

6

8

10

12

14

16

18

20

Retention time (min) Fig. 4. Chromatogram of a mixed suspension of bare, Po-0.25, Po-0.5, Po-0.75, Po1.0, and Po-2.0 liposomes.

is derived from Po-0.75, Po-1.0, and Po-2.0 liposomes. The result indicates that our chromatography was actually able to separate PEGylated liposomes with PEG-lipid contents below 0.75 mol%. As comparison, we measured the mean particle size or zeta potential of the mixed liposome suspension. As a result, the mean diameter and zeta potential were 123.9 nm and −5.9 mV respectively. The high heterogeneity of the mixed liposome could be identified with our analytical method (Fig. 4), however the heterogeneity which the mixed liposome has could not be judged only by seeing from these values. Indeed, the mixed liposome suspension as a whole had an average PEG-lipid content of 0.75 mol%, which provides long-circulating property to liposomes. Hence, such a liposome suspension may be judged to have sufficient blood circulation capability on the basis of the conventional analysis of liposomes, in which the evaluation of PEG-lipid content is carried out using liposomes dissolved in organic solvents. In contrast, if the present method is used for the analysis of the same liposome suspension, the liposomes with PEGlipid content below 0.5 mol% can be distinguished from those with PEG-lipid content above 0.75 mol%, and it can be observed that a half of the liposomes contained in the mixed suspension were not able to show required circulation capability. Therefore, the present method can provide valuable information on not only the mean value of the PEG-lipid content of liposomes but also the deviation in PEG-lipid content among PEGylated liposomes. 3.3. Comparison between liposomes prepared by the pre- and post-modification methods So far, we examined our chromatography using PEGylated liposomes prepared by the post-modification method, which gave principally liposomes having PEG chains only on the outer leaflet of the liposome membrane (Fig. 1). However, another type of PEGylated liposomes, which have PEG chains on both the sides of the membrane, can be obtained by the pre-modification method. Therefore, we investigated how such difference of PEG-modification may affect the separation performance of our chromatography. We carried out our analysis for PEGylated liposomes, namely, Pre-0.5, Pre-1.0, Pre-1.5, and Pre-2.0 (Table 1) and their chromatograms were compared to those of liposomes prepared by the post-modification methods. We already showed that electrostatic interaction between the liposome surface and column gels plays an important role in the separation of liposomes in our chromatography (Fig. 3). Therefore, we measured their zeta potentials and plotted the values

Fig. 5. Chromatography for PEGylated liposomes prepared by the post-modification (circles) and pre-modification (squares) methods. Correlation of the zeta potentials of liposomes with the PEG-lipid contents of liposomes.

against their PEG-lipid contents (Fig. 5). As is seen in Fig. 5, the PEGylated liposomes prepared by the pre-modification method increased their zeta potential with increasing PEG-lipid content and reached a constant value of about −4 mV when the PEG-lipid content was 1.5 mol%. In contrast, the PEGylated liposomes prepared by the post-modification method increased their zeta potential more significantly with PEG-lipid content and reached the same constant value at PEG-lipid content of 0.75 mol%. The result indicates that PEG chains were specifically attached to the outer surface of the liposomes for those prepared by the post-modification method. Based on the result of Fig. 5, surface properties of PEGylated liposomes Pre-0.5, Pre-1.0, and Pre-1.5 might be similar to those of those of PEGylated liposomes Po-0.25, Po-0.5, and Po-0.75, respectively. Therefore, these liposomes were applied to our chromatography, and their chromatograms were compared. As Fig. 6 shows, Pre-0.5 and Po-0.25 (Fig. 6A), Pre-1.0 and Po-0.5 (Fig. 6B), and Pre-1.5 and Po-0.75 (Fig. 6C) showed peaks at roughly the same retention time of 10–12 min, 8 min, and 7 min, respectively. The result indicates that Pre-0.5 and Po-0.25 liposomes, Pre-1.0 and Po-0.5 liposomes, and Pre-1.5 and Po-0.75 liposomes have similar surface properties on an average, irrespective of their preparation methods. However, when widths of these peaks were compared, the liposomes prepared by the pre-modification method exhibited relatively broad peaks compared to those prepared by the post-modification method. This fact suggests that the former liposomes have more heterogeneous surface properties than the latter liposomes. The post-modification method could produce PEGylated liposomes with smaller deviation in PEG chain amounts on the surface compared to those prepared by the pre-modification method. 3.4. Analysis of the PEGylation process in the post-modification method Finally, we attempted to follow the process of PEGylation of liposomes in the post-modification method using our chromatography (Fig. 7). PEG-lipid suspension only was not detected in this analytical method. As shown in Fig. 2, the bare liposomes were eluted at about 16 min in our chromatography. However, when the PEGlipid suspension was added to the bare liposome suspension and immediately the mixed suspension was applied to the chromatography, a sharp peak with broad shoulder was observed between 6.5 and 13 min of elution time in the chromatogram (Fig. 7A). Based on the result of Fig. 3, the sharp peaks at 6.5–7 min and a broad peak between 7 and 13 min corresponded to PEGylated liposomes with PEG-lipid content of more than 0.75 mol% and those with

78

K. Yoshino et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 397 (2012) 73–79

(B)

(C)

500

500

500

250

250

mAU

(A)

250

Pre-0.5

Po-0.25

0

4

8

12

16

Retention time (min)

Pre-1.0

Po-0.5

0 20

Pre-1.5

0 0

4

Po-0.75

8

12

16

20

Retention time (min)

0 0

4

8

12

16

20

Retention time (min)

Fig. 6. Chromatograms for Po-0.25 and Pre-0.5 liposomes (A), Po-0.5 and Pre-1.0 liposomes (B), and Po-0.75 and Pro-1.5 liposomes (C).

Fig. 7. Chromatography for a mixed suspension of bare liposome and PEG-lipid. Chromatograms were taken just after mixing bare liposome and PEG-lipid (A), or at 5 min (B), 15 min (C), and 30 min after their mixing and incubation at 65 ◦ C.

PEG-lipid content below 0.5 mol%, respectively. The broad shoulder became smaller and shifted to 7–11 min of elution time in the chromatogram of the liposome suspension after a 5 min incubation (Fig. 7B), and finally, the shoulder peak was integrated with the sharp peak after a 15 min incubation (Fig. 7C and D). This result indicates that insertion of PEG-lipid into the liposome membrane took place immediately after mixing, and the modification of liposomes with PEG-lipid was completed in 15 min. To confirm actual modification efficiency of PEGylated liposomes during PEG modification process, further examination was taken separately. The result is shown in Fig. S2. As shown in Fig. S2, PEG-lipid was modified within 7.5 min with more than 90% of modification efficiency (calculated concentration/actual measured concentration) at 65 ◦ C. From this result, it was confirmed that PEGylated liposomes (eluted at 6.5–7 min) was surely modified by 0.75 mol% of PEG-lipid with high modification efficiency through the PEG modification process. To our knowledge, it might be the first time to show the process of

PEG-modification after mixing of liposomes and PEG-lipid. Indeed, this information might be of great importance for the production of quality-controlled PEGylated liposomes. 4. Conclusions We established a novel chromatography for the evaluation of PEGylated liposomes using anion-exchange column and linear ion gradient. Our chromatography successfully separated PEGylated liposomes on the basis of their PEG-lipid contents. In addition, the chromatography enabled the evaluation of the deviation in PEG-lipid contents among PEGylated liposomes. So far, PEGylated liposomes have been characterized from the viewpoints of lipid composition, particle size, zeta potential, and PEG-lipid content because these characters affect the pharmacokinetics of PEGylated liposomes in the body [30,11]. Regarding the PEG-lipid content of liposomes, both the average amount of PEG-lipid and deviation in

K. Yoshino et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 397 (2012) 73–79

PEG-lipid amounts among PEGylated liposomes are important to control the quality of PEGylated liposomes, although conventional methods cannot evaluate the deviation in PEGylation. Our chromatography was able to separate PEGylated liposomes by their PEG-lipid content and hence provide information on the deviation in the PEG-lipid amounts among PEGylated liposomes. In addition, it revealed the process of PEGylation during the incubation of liposomes with PEG-lipid in an aqueous solution. We believe that our chromatography is a novel method that provides important information for the production of PEGylated liposomes of high quality, and hence can be used as a valuable tool not only for research and but also for manufacturing processes. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.colsurfa.2012.01.035. References [1] Y. Nishioka, H. Yoshino, Lymphatic targeting with nanoparticulate system, Adv. Drug Deliv. Rev. 1 (2001) 55–64. [2] R.H. Müller, K. Mäder, S. Gohla, Solid lipid nanoparticles (SLN) for controlled drug delivery – a review of the state of the art, Eur. J. Pharm. Biopharm. 50 (2000) 161–177. [3] D.B. Fenske, P.R. Cullis, Liposomal nanomedicines, Expert Opin. Drug Deliv. 5 (2008) 25–44. [4] T.M. Allen, C. Hansen, J. Rutledge, Liposomes with prolonged circulation times: factors affecting uptake by reticuloendothelial and other tissues, Biochim. Biophys. Acta 981 (1989) 27–35. [5] D. Liu, A. Mori, L. Huang, Role of liposomes size and RES blockade in controlling biodistribution and tumor uptake of GM1-containing liposomes, Biochim. Biophys. Acta 1104 (1992) 95–101. [6] D.V. Devine, K. Wong, K. Serrano, A. Chonn, P.R. Cullis, Liposome-complement interactions in rat serum: implications for liposome survival studies, Biochim. Biophys. Acta 1191 (1994) 43–51. [7] M.I. Papisov, Theoretical consideration of RES-avoiding liposomes: molecular mechanics and chemistry of liposome interactions, Adv. Drug Deliv. Rev. 32 (1998). [8] L.D. Mayer, L.C. Tai, D.S. Ko, D. Masin, R.S. Ginsberg, P.R. Cullis, M.B. Bally, Influence of vesicle size, lipid composition, and drug-to-lipid ratio on the biological activity of liposomal doxorubicin in mice, Cancer Res. 49 (1989) 5922–5930. [9] J.N. Harris, N.E. Martin, M. Mode, Pegylation, Clin. Pharmacokinet. 40 (2001) 539–551. [10] G. Blume, G. Cevc, Liposome for the sustained drug release in vivo, Biochim. Biophys. Acta 1029 (1990) 91–97. [11] M.C. Woodle, D.D. Lasic, Sterically stabilized liposomes, Biochim. Biophys. Acta 1113 (1992) 171–199. [12] D.D. Lasic, D. Needlham, The stealth liposome: a prototypical biomaterial, Chem. Rev. 95 (1995) 2601–2628. [13] F.K. Bedu-Addo, L. Huang, Interaction of PEG-phospholipid conjugates with phospholipids: implications in liposomal drug delivery, Adv. Drug Deliv. Rev. 16 (1995) 235–247.

79

[14] F.K. Bedu-Addo, P. Tang, Y. Xu, L. Huang, Effects of polyethylene-glycol chain length and phospholipids acyl chain composition on the interaction of polyethyleneglycol–phospholipid conjugates with phospholipids: implications in liposomal drug delivery, Pharm. Res. 13 (1996) 710–717. [15] H. Du, P. Chandaroy, S.W. Hui, Grafted poly-(ethylene glycol) on lipid surfaces inhibits protein adsorption and cell adhesion, Biochim. Biophys. Acta 1326 (1997) 236–248. [16] M.E. Price, R.M. Cornelius, J.L. Brash, Protein adsorption to polyethylene glycol modified liposomes from fibrinogen solution and from plasma, Biochim. Biophys. Acta 1512 (2001) 191–205. [17] D.A. Auguste, R.K. Prud’homme, P.L. Ahl, P. Meers, J. Kohn, Association of hydrophobically-modified poly(ethylene glycol) with fusogenic liposomes, Biochim. Biophys. Acta 1616 (2003) 184–195. [18] T.M. Allen, C. Hamsem, F. Martin, C. Redemann, A. Yau-Young, Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo, Biochim. Biophys. Acta 1066 (1991) 29–36. [19] M.C. Woodle, K.K. Matthay, M.S. Newman, J.E. Hidayat, L.R. Collins, C. Redemann, F.J. Martin, D. Papahadjopoulos, Versatility in lipid compositions showing prolonged circulation with sterically stabilized liposomes, Biochim. Biophys. Acta 1105 (1992) 193–200. [20] D.C. Litzinger, L. Huang, Amphipathic poly(ethylene glycol) 5000-stabilized dioleoylphosphatidylethanolamine liposomes accumulate in spleen, Biochim. Biophys. Acta 1127 (1992) 249–254. [21] K. Maruyama, T. Yuda, A. Okamoto, S. Kojima, A. Suginaka, M. Iwatsuru, Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoyl phosphatidylcholine and cholesterol containing amphipathic poly(ethylene glycol), Biochim. Biophys. Acta 1128 (1992) 44–49. [22] D.C. Litzinger, A.M. Buiting, N.V. Rooijen, L. Huang, Effect of liposome size on the circulation time and intraorgan distribution of amphipathic poly(ethylene glycol)-containing liposomes, Biochim. Biophys. Acta 1190 (1994) 99–107. [23] N.D. Santos, C. Allen, A.M. Doppen, M. Anantha, K.A. Cox, R.C. Gallagher, G. Karlsson, K. Edwards, G. Kenner, L. Samuels, M.S. Webb, M.B. Bally, Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: relating plasma circulation lifetime to protein binding, Biochim. Biophys. Acta 1768 (2007) 1367–1377. [24] D.C. Drummond, O. Meyer, K. Hong, D.B. Kirpotin, D. Papahadjopoulos, Optimizing liposomes for delivery of chemotherapeutic agent to solid tumors, Pharmacol. Rev. 51 (1999) 691–743. [25] A. Gabizon, H. Shmeeda, Y. Barenholz, Pharmacokinetics of pegylated liposomal doxorubicin: review of animal and human studies, Clin. Pharmacokinet. 42 (2003) 419–436. [26] G. Haran, R. Cohen, L.K. Bar, Y. Barenholz, Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases, Biochim. Biophys. Acta 1151 (1993) 201–215. [27] M. Grit, W.J.M. Underberg, D.J.A. Crommelin, Hydrolysis of saturated soybean phosphatidylcholine in aqueous liposome dispersions, J. Pharm. Sci. 82 (4) (April 1993). [28] Y. Sadzuka, A. Nakade, R. Hirama, A. Miyagishima, Y. Nozawa, S. Hirota, T. Sonobe, Effects of mixed polyethyleneglycol modification on fixed aqueous layer thickness and antitumor activity of doxorubicin containing liposome, Int. J. Pharm. 238 (2002) 171–180. [29] D.Z. Liu, Y.L. Hsieh, S.Y. Chang, W.Y. Chen, Microcalorimetric studies on the physical stability of poly-ethylene glycol-grafted liposome, Colloids Surf. A 212 (2003) 227–234. [30] W.C. Zamboni, K. Yoshino, Formulation and physiological factors affecting the pharmacokinetics and pharmacodynamics of liposomal anticancer agents, Drug Deliv. Syst. 25 (2010) 58–70.