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Enhanced circulation time and antitumor activity of doxorubicin by comblike polymer-incorporated liposomes
Journal of Controlled Release 120 (2007) 161 – 168 www.elsevier.com/locate/jconrel
Enhanced circulation time and antitumor activity of doxorubicin by comblike polymer-incorporated liposomes Hee Dong Han a,b , Aeri Lee a , Taewon Hwang a , Chung Kil Song a , Hasoo Seong a , Jinho Hyun c , Byung Cheol Shin a,⁎ a
b
Bioactive Molecules Delivery and Control Research Team, Korea Research Institute of Chemical Technology, P.O. Box 107, Yuseong, Daejeon 305-600, South Korea Laboratory of Infection and Immunology, Graduate School of Medicine, Korea University, Ansan-si, Gyeonggi-do 425-707, South Korea c School of Biological Resources and Materials Engineering, Seoul National University, Seoul 151-742, South Korea Received 2 February 2006; accepted 29 March 2007 Available online 4 April 2007
Abstract Polymer incorporation on liposomal membranes has been extensively studied as a method of enhancing the circulation time of liposomes in the bloodstream. In this study, we investigated the in vitro and in vivo characteristics of liposomes whose surface was modified using a comblike polymer comprised of a poly(methyl methacrylate) (PMMA) backbone and short poly(ethylene oxide) (PEO) side chains. Doxorubicin (DOX)loaded liposomes incorporating with the comblike polymer were prepared and their circulation time, biodistribution and antitumor activity were evaluated in B16F10 melanoma tumor-bearing mice. The circulation half-life time in the bloodstream of the comblike polymer-incorporated liposomes (CPILs) was approximately 14- or 2-fold higher than those of the conventional or polyethyleneglycol-fixed liposomes (PEGliposomes), respectively. Additionally, in the biodistribution assay, the accumulation of the CPILs in the tumor was higher than those of the other liposomes. Based on this result, the antitumor activities of the CPILs were higher than those of conventional liposome formulation of DOX or free DOX due to the higher passive targeting efficiency of the long-circulating CPILs to tumor. This study suggests that the incorporation of the comblike polymer on the liposomal membrane is a promising tool to further improve circulation time of liposomes in tumor-bearing mice. © 2007 Published by Elsevier B.V. Keywords: Comblike polymer; Liposomes; Circulation time; Biodistribution
1. Introduction Liposomes have been extensively studied in an attempt to enhance the therapeutic efficacy of anticancer drug in the field of cancer chemotherapy [1]. However, the conventional liposomes have been found to be plagued by rapid opsonization and taken up by the reticuloendothelial system (RES), resulting in their circulation time being shortened [2]. This is an obstacle to the effective delivery of drugs using liposomes. In previous studies, these problems associated with the delivery and circulation of liposomes after their intravenous injection were resolved by incorporating lipid-grafted polyethyleneglycol (PEG) into the
⁎ Corresponding author. Tel.: +82 42 860 7223; fax: +82 42 861 4151. E-mail address: [email protected] (B.C. Shin). 0168-3659/$ - see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.jconrel.2007.03.020
liposomal membrane, thereby reducing their opsonization and increasing their circulation time [3–5]. In spite of these advances, however, the current clinically approved liposomal formulations have still some problems associated with their circulation time, resulting in side effects such as cardiotoxicity, myelosuppression, alopecia and nausea often occurring in the field of clinical cancer chemotherapy. Recently, many researchers have studied the side effect of liposomal DOX, DOXIL® (Alza Co., USA), which use PEG-coated liposomes as a DOX formulation. Though DOXIL® has a high therapeutic efficacy against Kaposi's sarcoma and other solid tumors, some side effects such as stomatitis, morbilliform, hand-foot syndrome, mild myelosuppression and vomiting are often reported [6–9]. To overcome these side effects, many researchers still attempt to further enhance the circulation time of carrier which is considered as main reasons of side effects. In this view point, our study attempted to prepare a new PEG-coated
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liposomes using comblike polymer incorporation on liposomal surface and investigate antitumor effect of the prepared liposomes. For the purpose of increasing the circulation time of liposomes, a number of liposomes with various functionalities such as temperature sensitive polymer conjugation or polymer–lipid conjugation have been developed [10–14]. One of the potential approaches to prolong the circulation time of the liposomes in clinical trials is polymer conjugation on the surface of liposomes [15,16]. In this study, the comblike polymer as a surface modifier was incorporated into the liposomal bilayer by hydrophobic interaction. The comblike polymer was composed of hydrophobic methyl methacrylate (MMA) and the hydrophilic PEG derivatives, poly(ethylene glycol) methacrylate (hydroxyl-poly(oxyethylene) methacrylate, HPOEM) and poly(ethylene glycol) methyl ether methacrylate (poly(oxyethylene) methacrylate, POEM). Comblike polymers are one of the most potent materials that can be used to prolong the stability and circulation time of liposomes and their application in the field of biomaterials has been extensively studied. Moreover, comblike polymers are used as a resistant material in biosensors, biochips or patterning for the purpose of inhibiting the adsorption of conflicting protein signals from plasma [17,18]. Notably, the incorporation of a comb polymer on liposomal membranes prevents the adhesion of cell or plasma proteins, however, this subject has not been sufficiently studied. Therefore, we newly designed sterically-stabilized liposomes in order to prolong their circulation time in the bloodstream by incorporating a comblike polymer onto the liposomal membrane. Moreover, it is postulated that the prolongation of the circulation time of the liposomes induces a steric barrier against protein binding in the serum, due to the PEG derivatives in the comblike polymer. In our comblike polymer-incorporated liposomes (CPILs), the MMA hydrophobic chains in the comblike polymer can be anchored within the hydrophobic lipid bilayer of the liposomes and the hydrophilic chains composed of PEG derivatives can protrude into the aqueous medium. The objective of this study was to evaluate whether the incorporation of a comblike polymer as a polymeric modifier on the liposomal surface could enhance the circulation time of liposomal DOX. Furthermore, the in vivo pharmacokinetics, biodistribution and antitumor activity were investigated in accordance with the circulation time of the CPILs using murine B16F10 tumor-bearing mice. 2. Materials and methods 2.1. Materials L-α-phosphatidylcholine(soy-hydrogenated) (HSPC), cholesterol (CHOL) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2000) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Doxorubicin (DOX) was purchased from Boryung Pharm. Co. (South Korea). Methyl methacrylate (MMA), poly (ethylene glycol) methacrylate (hydroxyl-poly(oxyethylene) methacrylate, HPOEM), poly(ethylene glycol) methyl ether methacrylate (poly(oxyethylene) methacrylate, POEM), tetrahydrofuran (THF), azo(bis)-isobutyronitrile (AIBN) and 1-methox-
yphenol were purchased from Aldrich Co. (Alabaster, AL, USA). Dulbecco's modified Eagle medium (DMEM) and aqueous fetal bovine serum (FBS) were purchased from Bio-Tech. Inc. (Parker Ford, USA). All other materials were of analytical grade and used without further purification. 2.2. Synthesis of comblike polymer The comblike polymer, a random copolymer of MMA, HPOEM and POEM, was synthesized according to the method of Irvine et al. [17,19]; briefly, 21 ml of MMA (0.197 mol), 6.55 g of HPOEM (0.018 mol), 6.55 g of POEM (0.014 mol) and 0.239 g of AIBN (1.46 mmol) were dissolved in 500 ml of THF. The solution was degassed by bubbling nitrogen for 20 min, followed by refluxing at 70 °C for 18 h. The reaction was terminated by the addition of 20 mg of 1-methoxyphenol. The copolymer was purified by two precipitations in 8:1 (v/v) petroleum ether/methanol and then dried in a vacuum at 25 °C for 24 h. The molecular weight of the copolymer was determined by gel permeation chromatography (GPC, KF 804 column, intelligent refractive index detector (RI930), intelligent HPLC pump (PU980), Jasco). 2.3. Preparation of liposomes The DOX-loaded liposomes were prepared according to the remote loading method using an ammonium sulfate gradient [20]. The prepared liposomes and their lipid compositions were as follows; (1) conventional liposomes; HSPC:CHOL = 9.57:3.19 mg/ ml; (2) PEG-liposomes; HSPC:CHOL:DSPE-mPEG-2000 = 9.57:3.19:3.19 mg/ml as a DOXIL® formulation; (3) CPILs; HSPC:CHOL:Comblike polymer = 9.57:3.19:3.19 mg/ml. Briefly, the lipids with the above compositions were dissolved in chloroform, dried into a thin film on a rotary evaporator (Buchi Rotavapor R-200, Switzerland) and then suspended in 250 mM ammonium sulfate solution. The liposomal solution was extruded through a polycarbonate filter (pore size; 100 nm, Whatman, USA) using an extruder (Northern Lipids Inc. USA). The free ammonium sulfate was removed by dialysis for 48 h at 4 °C using cellulose dialysis tubing (MWCO 12,000, Viskase Co. Illinois, USA). The liposomal solution and 2 mg/ml DOX solution were mixed and then incubated for 2 h at 60 °C. The mixture was dialyzed to remove the free DOX. The DOX-loaded liposomes were stored at 4 °C until use. The size and zeta potential of the liposomes were measured by light scattering with a particle size analyzer (ELS-8000, Particle Analyzer, Otuska, Japan). The lipid concentration of liposomes was measured by phosphate assay method using PhosFree™ phosphate assay kit (#BK050, Cytoskeleton Inc., USA) [21,22]. The amount of DOX within the liposomes was measured by fluorescence spectrophotometry (Barnstead, Apogent Tech, USA) after destruction of liposomal vesicles with 10% Triton X-100 aqueous solution. The excitation and emission wavelengths were 490 nm and 590 nm, respectively. The loading efficiency of DOX within the liposomes was calculated by Eq. (1). Loading efficiency ðkÞ ¼ Fi =Ft 100
ð1Þ
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where Fi is the concentration of DOX loaded in the liposomes after their dissolution in 10% Triton X-100 and Ft is the initially added concentration of DOX. 2.4. Polymer amount on the surface of liposomes To confirm the incorporation of the comblike polymer on the surface of the liposomes, the amount of PEG in the PEGliposomes and CPILs was measured by the picrate assay method described by Sonobe et al. [23,24]. Briefly, 5 ml of the liposomal suspension was mixed with 10 ml of sodium nitrate– picrate solution and to the mixture was added 5 ml of 1, 2 dichloroethane. After vigorous shaking, the solution was centrifuged at 1500 ×g for 10 min. The organic layer was collected and the absorbance of the solution was measured at 380 nm using UV-spectrophotometry (UV-mini, Shimadzu Scientific Ins., Japan). 2.5. Cell line and mice B16F10, a murine melanoma cell line, was cultured in DMEM supplemented with 10% v/v heat-inactivated FBS and 10 μl/ml penicillin streptomycin. The cultures were sustained at 37 °C in a humidified incubator containing 5% CO2. The cells were maintained within their exponential growth phase. Female C57BL/6 mice (5 to 6 weeks old, 18–22 g) were purchased from Harlan Inc. (Indiana, USA). All of the procedures involved in the animal experiments were performed according to approved protocols and in accordance with the recommendations of the NIH guideline for the proper use and care of laboratory animals. 2.6. Pharmacokinetic studies In order to monitor the plasma levels of DOX in the mice, free or liposomal DOX was injected intravenously via the tail vein at a dose of 6 mg DOX/kg body weight (three mice per group). Drug-to-lipid ratios of conventional liposomes, PEGliposomes and CPILs were 0.15, 0.12 and 0.12 (wt/wt), respectively. At a predetermined time after the intravenous injection, the mice were sacrificed and blood was collected immediately by cardiac puncture of the mice. Two hundred microliters of blood were collected and mixed with an equal volume of normal saline. After centrifugation of the blood, the serum was divided into two equal aliquots. To the first aliquot 250 μl of 66 mM EDTA solution and isotonic 50 mM PBS buffer (pH 7.4) were added to make a final volume of 1 ml. The concentration of DOX was measured by fluorescence spectrophotometry at 590 nm (emission wavelength). To the second aliquot 250 μl of EDTA solution, 10 μl of Triton X-100 solution and PBS were added to make a final volume of 1 ml. The amount of DOX entrapped in the liposomes was calculated from the difference in the fluorescence intensity of the two aliquots. The difference of fluorescence intensity between the first aliquot and the second aliquot means that the drug release from liposomes indicates the stability of liposomal DOX in circulation. It was regarded, thus, that those data could show the
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DOX release from liposomes in vivo. The pharmacokinetic parameters were calculated from the average DOX concentrations in the bloodstream determined by a pharmacokinetic software BA calculator (Seoul National University, South Korea). 2.7. Biodistribution studies In the DOX biodistribution study, 5 × 105 cells of murine B16F10 melanoma were carefully inoculated into the right limb armpit of the mice subcutaneously. The administration of free DOX or liposomal DOX was started when the mean tumor volume reached approximately 10 mm3. The mice (three per group) were monitored for 64 h after the intravenous injection of the free DOX or liposomal DOX via the tail vein at a dose of 6 mg DOX/kg body weight. At a predetermined time after the intravenous injection, the mice were sacrificed and their liver, spleen, heart, lung, kidney and tumor were collected immediately. The organs and tumor were carefully washed with distilled water, weighed and homogenized with PBS solution. The DOX concentration in the homogenized tissue was measured by fluorescence spectrophotometry at 590 nm, as described in Section 2.6. 2.8. Antitumor activity 5 × 105 cells of murine B16F10 melanoma were carefully inoculated into the right limb armpits of the mice subcutaneously. Six days after the tumor inoculation, free DOX or liposomal DOX was injected intravenously via the tail vein at a dose of 6 mg DOX/kg body weight. The tumor volume was monitored for 16 days after a single intravenous injection of the different types of liposomal DOX. In order to determine the tumor volume, the size of each individual tumor was measured with a caliper and the tumor volume was calculated using Eq. (2). Tumor volume mm3 ¼ width length2 =2 ð2Þ 2.9. Statistical analysis All data expressed as means ± standard deviation (S.D.) are representative of at least three different experiments. When comparing more than two mean values of interest obtained from tumor treatment experiments of various liposomes or control groups, a one-way analysis of variance (ANOVA) was performed using SAS program. To find out whether the two values of interest were significantly different, Tukey's test was performed. All p values b0.05 were considered significant. 3. Results 3.1. Characteristics of comblike polymer The comblike polymer was synthesized by radical polymerization. The structure of the comblike polymer having an MMA backbone and short POEM side chains was confirmed by proton
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Fig. 1. Chemical structure (A) and 1H-NMR spectra of comblike polymer (B). The comblike polymer was synthesized by radical polymerization, and was composed of hydrophobic MMA and the hydrophilic PEG derivatives, HPOEM and POEM. The average molecular weight of the comblike polymer was ca 28,000 Da.
NMR analysis. Fig. 1 shows the chemical structure and 1HNMR spectra of the synthesized comblike polymer. As shown in Fig. 1B, the peaks for the ether protons (1), methacrylate protons (2) and methyl protons (3) were observed at 3.7 ppm, 0.5–2.0 ppm and 4.1 ppm, respectively. The average molecular weight of the polymer was found to be ca 28,000 Da by GPC analysis. 3.2. Characteristics of liposomes The physical properties of the various types of DOX-loaded liposomes are shown in Table 1. The mean particle size of the conventional liposomes, PEG-liposomes and CPILs was approximately 95–107 nm and the loading efficiency of DOX was 91–93%. Additionally, the zeta potential of the various liposomal formulations showed negative values ranging from − 9.02 ± 2.1 mV to − 3.88 ± 1.4 mV. It is generally known that surface charge of liposomes composed of phosphatidyl choline (PC) and CHOL is slightly negative and the zeta potential value of the liposomes decreases by addition of PEG–phosphatidyl
Fig. 2. Pharmacokinetic profiles of various liposomal DOX formulations after intravenous injection in tumor-bearing mice at a single dose of 6 mg DOX/kg body weight (⁎p b 0.01 vs. PEG-liposomes). The data represent the mean ± S.D. (n = 3).
ethanolamine (PEG–PE) due to the electronegativity of PEG– PE itself [25–27]. In our study, as the surface of CPIL was modified with comblike polymer having short PEG derivatives as side chain of the polymer, zeta potential value of CPILs decreased slightly from approximately − 3.88 mV to −9.02 mV. However, zeta potential value of CPILs was almost the same as that of PEG-liposomes indicating that they had similar characteristics with respect to physical and electrostatic properties. The amount of lipid that was incorporated in conventional liposomes, PEG-liposomes and CPILs is 9.41, 11.02 and 8.99 mg/ml liposome solution, respectively. The recovery rate of lipid in each liposomal formulation is 98.2, 86.3 and 93.8% based on the initial amount of lipid, respectively. The amounts of PEG and comblike polymer incorporated on the surface of the PEG-liposomes and CPILs were measured to be 1.2 ± 0.2 mg/ml and 1.5 ± 0.3 mg/ml, respectively, thus confirming that the PEG and comblike polymer were incorporated on the surface of respective liposomes. 3.3. Circulation time induced by polymer incorporation on the surface of liposomes in vivo The profiles of the DOX concentration in the bloodstream after the single intravenous injection of the conventional
Table 1 Physical properties of the various liposomal formulations Composition
Size [nm]
Conventional 107.8 ± 1.7 liposomes (HSPC:CHOL) PEG-liposomes 107.9 ± 0.3 (HSPC:CHOL: DSPE-mPEG) CPILs (HSPC: 95.8 ± 0.7 CHOL: Comblike polymer)
Zeta potential [mV]
Loading Polymer efficiency amount [%] [mg/ml]
Lipid amount [mg/ml]
− 3.88 ± 1.4
91
–
− 8.63 ± 4.3
93
1.2 ± 0.2
11.02
− 9.02 ± 2.1
93
1.5 ± 0.3
8.99
Table 2 Pharmacokinetic parameters of DOX after intravenous injection of various liposomal formulations in tumor-bearing mice at a dose of 6 mg DOX/kg body weight (n = 3)
9.41
AUC (μg h/ml) t1/2 (h) CL (ml/h) MRT (h)
Free DOX⁎
Conventional liposomes⁎⁎
PEGCPILs liposomes⁎⁎⁎
12.00 1.68 17.40 1.95
25.59 2.37 4.62 4.82
201.24 12.80 0.60 17.78
420.08 27.09 0.29 40.00
AUC: area under the curve, t1/2: half-life time, CL: clearance, MRT: mean residence time. ⁎pb 0.001 vs. CPILs, ⁎⁎pb 0.002 vs. CPILs, ⁎⁎⁎pb 0.001 vs. CPILs.
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free DOX (t1/2; 1.68 h, MRT; 1.95 h, ⁎p b 0.001 vs. CPILs), conventional liposomes (t1/2; 2.37 h, MRT; 4.82 h, ⁎⁎p b 0.002 vs. CPILs) or PEG-liposomes (t1/2; 12.80 h, MRT; 17.78 h, ⁎⁎⁎p b 0.001 vs. CPILs). The area under the curve (AUC) of the CPILs was higher (420.08 μg h/ml) than those of the free DOX (12.00 μg h/ml) or conventional liposomes (25.59 μg h/ml). In contrast, the clearance (CL) of the CPILs was lower (0.29 ml/h) than those of the free DOX (17.40 ml/h), conventional liposomes (4.62 ml/h) or PEG-liposomes (0.60 ml/h). These results indicate that the CPILs can circulate for a longer time in the blood than the free DOX, conventional liposomes or PEG-liposomes. 3.4. Tissue distribution of liposomes after intravenous injection in tumor-bearing mice
Fig. 3. Biodistribution of DOX after intravenous injection of DOX-loaded liposomal formulations in tumor-bearing mice at a single dose of 6 mg/kg body weight (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001). The data represent the mean ± S.D. (n = 3).
liposomes, PEG-liposomes and CPILs at a dose of 6 mg/kg are shown in Fig. 2. The free DOX rapidly disappeared from the circulation within 2 h due to its short half-life (t1/2: 1.68 h). In contrast, all of the DOX-loaded liposomes showed a longer circulation time than the free DOX. The DOX concentration of the CPILs in the bloodstream was the highest as compared with those of the other liposomes and the half-life of DOX was increased to 27.09 h. These results indicated that the CPILs could prolong the circulation time of DOX. These profiles confirmed that the incorporation of the comblike polymer was an effective method of improving the circulation time of liposomes in the bloodstream. Notably, the comblike polymer which was incorporated on the liposomal membrane prolonged the half-life of DOX as compared with that of the conventional liposomes (t1/2: 2.37 h) or PEG-liposomes (t1/2: 12.80 h). The pharmacokinetic parameters of the various types of liposomal DOX after their intravenous injection in the mice are summarized in Table 2. The CPILs significantly increased the half-life (t1/2; 27.09 h) and mean residence time (MRT; 40.00 h) of DOX during its circulation in the blood, as compared with the
To clarify the reduction in the RES uptake of liposomal DOX afforded by the incorporation of the comblike polymer, the DOX concentrations in the tissues were measured at predetermined time intervals after the intravenous injection of free or liposomal DOX. The DOX concentrations in the tumor, heart, liver, spleen, kidney and lung are described in Fig. 3. The concentrations of liposomal DOX in the tissues were significantly higher than those of the free DOX with the exception of the heart (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001). Notably, the DOX concentration of the CPILs in the tumor at 16 h was significantly higher than those of the other liposomal groups (Fig. 3A). The DOX concentration of the CPILs in the heart at 2 h was much lower than those of the free DOX or conventional liposomes (Fig. 3B). The DOX concentration in the heart is closely related to the inherent cardiac toxicity of DOX. Therefore, the liposomal DOX would tend to reduce the cardiac toxicity of DOX. The DOX concentrations of the CPILs in the liver and spleen were significantly lower than those of the conventional or PEG-liposomes (Fig. 3C, D). These results
Fig. 4. Tumor growth inhibition after intravenous injection of the free DOX and various liposomal DOX in tumor-bearing mice at a dose of 6 mg DOX/kg body weight. 5 × 105 murine B16F10 cells were inoculated into the armpits of the mice and the animals were treated with an intravenous injection of the free DOX, conventional liposomes, PEG-liposomes, CPILs or normal PBS solution as a control. The arrow indicates the injection day of the various liposomal DOX after tumor implantation. The data represent the mean ± S.D. (n = 6).
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clearly indicated that the RES uptake of the liposomes could be reduced by the incorporation of the comblike polymer into the liposomal membrane. The DOX concentrations of the CPILs in the kidney and lung were significantly lower than those of the conventional or PEG-liposomes (Fig. 3E, F). 3.5. Antitumor activity of liposomes induced by long-circulation property The antitumor activities of the DOX-loaded liposomes were evaluated in B16F10 tumor-bearing mice (six per group) after a single intravenous injection at a dose of 6 mg DOX/kg body weight. As shown in Fig. 4, the DOX-loaded liposomes suppressed the growth of the tumor as compared with the PBS control group. The tumor growth inhibition effects of free DOX and all of liposomal DOX were significant to the control PBS group (ANOVA test; p b 0.001). The CPILs showed higher tumor growth inhibition than the conventional liposomes or free DOX (Tukey's test; p b 0.001). Notably, the CPILs showed tumor growth inhibition effect comparable to PEG-liposomes that comprised a commercialized liposomal DOX, DOXIL®. The high antitumor activity of the CPILs suggested that the CPILs accumulated in the tumor tissue by passive targeting, because of their prolonged circulation time (Fig. 3A). Although some side effects might be expected to result from the use of polymeric modifiers, no severe sign of toxicity, such as fever, loss of weight, cachexia and myalgia, were observed in the mice treated with the liposomal DOX. 4. Discussion Polymer-incorporated liposomal drug delivery systems for cancer therapy have been developed and evaluated in many preclinical models [3,4,28]. Most of these previous studies have been largely focused on prolonging the circulation time of the liposomal DOX in the bloodstream [16,29]. To this end, lipidgrafted PEG-fixed liposomes (PEG-liposomes) have been developed. However, some problems were encountered when the current PEG-liposomes were used to deliver anticancer drugs in clinical trials [6–9]. These difficulties were associated with circulation of the liposomes in the bloodstream and the resulting side effects of DOX. Thus, we designed the new comblike polymer-incorporated CPILs, which prolonged the circulation time of the liposomes in the bloodstream as compared to that of the PEG-liposomes. The comblike polymer used in this study was synthesized by radical polymerization and applied to the liposomes as a surface modifier. To determine the amount of comblike polymer incorporated on the CPILs, the picrate assay was performed. The amount of comblike polymer incorporated in the CPILs was 1.5 ± 0.3 mg/ml (Table 1). This result indicated that the comblike polymer was incorporated on the liposomal bilayer via its hydrophobic interaction with lipid bilayers [30]. For the purpose of preparation of a more sterically-stabilized liposomes using comblike polymer as compared to PEG-liposomes, it appears that comblike polymer incorporated into the liposomal surface forms a hydrophilic steric barrier.
The circulation time of the liposomes in the bloodstream after the intravenous injection in the mice was evaluated by measuring the DOX concentration (Fig. 2). The CPILs showed a higher DOX concentration than the other liposomes. These profiles indicated that the incorporation of the comblike polymer improved the circulation of the liposomes in the bloodstream and, consequently, their half-life was increased. In contrast, the free DOX rapidly disappeared within 2 h. It was inferred from this that the effect of the comblike polymer incorporated into the liposomal membrane was to prolong the half-life of the liposomes by escaping their RES uptake. In addition, the comblike polymer incorporated on the liposomal surface might form a more stable steric barrier on the surface of CPIL than DSPE-PEG on the PEG-liposomes, resulting in the prolongation of circulation time of CPIL as compared to that of PEG-liposomes. Moreover, many researchers have reported various types of liposomes containing comblike polymer. They used the hydrophobically-modified polymers having molecular weight (Mw) of 25,000 or higher [30]. Takeuchi et al. also reported that circulation profiles of liposome coated with hydrophilic polymers having different Mw's in rats. They reported that PVA having Mw of 20,000 was more effective of prolongation of circulation time of liposomes than PVA having Mw of 6000 or 9000, even though amount of polymer incorporated on the liposomal surface was lower than other liposomes [4]. The studies of biodistribution suggested that, in the tumor (Fig. 3A), the CPILs showed a higher DOX concentration than the other liposomes over a long period of time. This result might reflect the increase in passive targeting via the enhanced permeation and retention (EPR) effect caused by the longcirculation ability of the CPILs in the blood. In the heart (Fig. 3B), the free DOX showed the highest drug level at 2 h. The CPILs and PEG-liposomes, however, reduced the drug level in comparison with the free DOX at 2 h, thus indicating that the lower concentration of DOX in the heart may lead to a lower cardiotoxicity of DOX. On the other hand, the lower drug level of the free DOX after 8 h than three of the liposomal DOX might be attributable to the rapid clearance of the free DOX as described in Fig. 2. In the liver (Fig. 3C), the CPILs showed a lower DOX concentration than the other liposomes. This result supported that the incorporation on the comblike polymer prevented the liposomes from trapping caused by the RES uptake. The antitumor activities of the CPILs were higher than that of conventional liposomes or free DOX (Fig. 4). The high antitumor activity of the CPILs is consistent with the higher accumulation of the CPILs in the tumor (Fig. 3A). This result showed that the therapeutic efficacy of DOX could be enhanced by EPR-mediated tumor targeting of the CPILs. Notably, the incorporation of the comblike polymer can prolong the circulation time in the blood (Fig. 2), which can be a contributory factor leading to an increase in passive targeting. The antitumor effects of CPILs were nearly the same against PEG-liposomes. These results demonstrated that the CPILs might augment the antitumor activity induced by enhanced circulation time through incorporation of comblike polymer
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onto the surface of liposomes. During this experimental procedure, all groups of tumor-bearing mice survived over the experiment period even though tumor mass of mice increased. Based on our study, a plausible drug release system derived from the CPILs can be described as follows; the CPILs, whose circulation time is prolonged by the incorporation of the comblike polymer, circulate continuously through the bloodstream. At the same time, the CPILs in the bloodstream can permeate through the angiogenic blood vessels of the tumor and, therefore, the CPILs are accumulated in the tumor tissue. Consequently, the DOX from the CPILs accumulated in the tumor tissue would be released. Mangesh et al. and Takeuchi et al. reported the polymer modified liposomes. In their studies, they used poly(methacrylate)-co-PEG having Mw 25,000 or modified PVA. However, they did not find any cytotoxicity of those polymers [31,32]. Thus, it is considered that comblike polymer may satisfy the toxicological issue in use. In addition, many researchers have studied polymer modified liposomes to enhance the liposome's circulation time or antitumor activity. Many scientific approaches related to the toxicology of polymers have also been challenged in the field of polymer modification on the drugloaded carrier. PMMA used in drug or protein-loaded carrier system has been found to be slowly degradable in the form of nanoparticles and to be safe as an efficient vaccine adjuvant without any observable in vivo side effect or toxic reaction [33,34]. On the other hand, PEG or PEG derivates having relatively high Mw of 6000–170,000 did not distribute significantly to heart, lung, liver, spleen, kidney, and thyroid gland when the polymers were administered intravenously, subcutaneously or intramuscularly: Rather they distributed to the gastrointestinal tract via the bile, and what had not been excreted in urine was excreted in feces [35]. In addition, PEG undergoes cytochrome P-450 oxidation, resulting in the formation of ketone, ester and aldehyde groups [35]. Thus, our comblike polymer composed of PMMA and PEG derivatives may be regarded to be non-toxic and to be degradable or excreted through urine or feces. In conclusion, the comblike copolymer that was composed of hydrophobic MMA chain and hydrophilic short PEG chain was incorporated in DOX-loaded liposomes for surface modification of the liposomes. In the in vivo circulation time measurement, the incorporation of the comblike polymer on the liposomal surface increased the half-life of the liposomes in the bloodstream after their intravenous injection. In addition, it can be inferred that the therapeutic efficacy of the CPILs toward the tumor was enhanced by EPR-mediated tumor targeting, because of the longest circulation time of the CPILs. Thus, our study suggests the potential of comblike polymer-incorporated liposomes for use as drug delivery systems to enhance the therapeutic efficacy of anticancer drugs administered by intravenous injection. Acknowledgement This work was supported by the Ministry of Commerce, Industry and Energy of Korea (10016573).
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Enhanced circulation time and antitumor activity of doxorubicin by comblike polymer-incorporated liposomes Hee Dong Han a,b , Aeri Lee a , Taewon Hwang a , Chung Kil Song a , Hasoo Seong a , Jinho Hyun c , Byung Cheol Shin a,⁎ a
b
Bioactive Molecules Delivery and Control Research Team, Korea Research Institute of Chemical Technology, P.O. Box 107, Yuseong, Daejeon 305-600, South Korea Laboratory of Infection and Immunology, Graduate School of Medicine, Korea University, Ansan-si, Gyeonggi-do 425-707, South Korea c School of Biological Resources and Materials Engineering, Seoul National University, Seoul 151-742, South Korea Received 2 February 2006; accepted 29 March 2007 Available online 4 April 2007
Abstract Polymer incorporation on liposomal membranes has been extensively studied as a method of enhancing the circulation time of liposomes in the bloodstream. In this study, we investigated the in vitro and in vivo characteristics of liposomes whose surface was modified using a comblike polymer comprised of a poly(methyl methacrylate) (PMMA) backbone and short poly(ethylene oxide) (PEO) side chains. Doxorubicin (DOX)loaded liposomes incorporating with the comblike polymer were prepared and their circulation time, biodistribution and antitumor activity were evaluated in B16F10 melanoma tumor-bearing mice. The circulation half-life time in the bloodstream of the comblike polymer-incorporated liposomes (CPILs) was approximately 14- or 2-fold higher than those of the conventional or polyethyleneglycol-fixed liposomes (PEGliposomes), respectively. Additionally, in the biodistribution assay, the accumulation of the CPILs in the tumor was higher than those of the other liposomes. Based on this result, the antitumor activities of the CPILs were higher than those of conventional liposome formulation of DOX or free DOX due to the higher passive targeting efficiency of the long-circulating CPILs to tumor. This study suggests that the incorporation of the comblike polymer on the liposomal membrane is a promising tool to further improve circulation time of liposomes in tumor-bearing mice. © 2007 Published by Elsevier B.V. Keywords: Comblike polymer; Liposomes; Circulation time; Biodistribution
1. Introduction Liposomes have been extensively studied in an attempt to enhance the therapeutic efficacy of anticancer drug in the field of cancer chemotherapy [1]. However, the conventional liposomes have been found to be plagued by rapid opsonization and taken up by the reticuloendothelial system (RES), resulting in their circulation time being shortened [2]. This is an obstacle to the effective delivery of drugs using liposomes. In previous studies, these problems associated with the delivery and circulation of liposomes after their intravenous injection were resolved by incorporating lipid-grafted polyethyleneglycol (PEG) into the
⁎ Corresponding author. Tel.: +82 42 860 7223; fax: +82 42 861 4151. E-mail address: [email protected] (B.C. Shin). 0168-3659/$ - see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.jconrel.2007.03.020
liposomal membrane, thereby reducing their opsonization and increasing their circulation time [3–5]. In spite of these advances, however, the current clinically approved liposomal formulations have still some problems associated with their circulation time, resulting in side effects such as cardiotoxicity, myelosuppression, alopecia and nausea often occurring in the field of clinical cancer chemotherapy. Recently, many researchers have studied the side effect of liposomal DOX, DOXIL® (Alza Co., USA), which use PEG-coated liposomes as a DOX formulation. Though DOXIL® has a high therapeutic efficacy against Kaposi's sarcoma and other solid tumors, some side effects such as stomatitis, morbilliform, hand-foot syndrome, mild myelosuppression and vomiting are often reported [6–9]. To overcome these side effects, many researchers still attempt to further enhance the circulation time of carrier which is considered as main reasons of side effects. In this view point, our study attempted to prepare a new PEG-coated
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liposomes using comblike polymer incorporation on liposomal surface and investigate antitumor effect of the prepared liposomes. For the purpose of increasing the circulation time of liposomes, a number of liposomes with various functionalities such as temperature sensitive polymer conjugation or polymer–lipid conjugation have been developed [10–14]. One of the potential approaches to prolong the circulation time of the liposomes in clinical trials is polymer conjugation on the surface of liposomes [15,16]. In this study, the comblike polymer as a surface modifier was incorporated into the liposomal bilayer by hydrophobic interaction. The comblike polymer was composed of hydrophobic methyl methacrylate (MMA) and the hydrophilic PEG derivatives, poly(ethylene glycol) methacrylate (hydroxyl-poly(oxyethylene) methacrylate, HPOEM) and poly(ethylene glycol) methyl ether methacrylate (poly(oxyethylene) methacrylate, POEM). Comblike polymers are one of the most potent materials that can be used to prolong the stability and circulation time of liposomes and their application in the field of biomaterials has been extensively studied. Moreover, comblike polymers are used as a resistant material in biosensors, biochips or patterning for the purpose of inhibiting the adsorption of conflicting protein signals from plasma [17,18]. Notably, the incorporation of a comb polymer on liposomal membranes prevents the adhesion of cell or plasma proteins, however, this subject has not been sufficiently studied. Therefore, we newly designed sterically-stabilized liposomes in order to prolong their circulation time in the bloodstream by incorporating a comblike polymer onto the liposomal membrane. Moreover, it is postulated that the prolongation of the circulation time of the liposomes induces a steric barrier against protein binding in the serum, due to the PEG derivatives in the comblike polymer. In our comblike polymer-incorporated liposomes (CPILs), the MMA hydrophobic chains in the comblike polymer can be anchored within the hydrophobic lipid bilayer of the liposomes and the hydrophilic chains composed of PEG derivatives can protrude into the aqueous medium. The objective of this study was to evaluate whether the incorporation of a comblike polymer as a polymeric modifier on the liposomal surface could enhance the circulation time of liposomal DOX. Furthermore, the in vivo pharmacokinetics, biodistribution and antitumor activity were investigated in accordance with the circulation time of the CPILs using murine B16F10 tumor-bearing mice. 2. Materials and methods 2.1. Materials L-α-phosphatidylcholine(soy-hydrogenated) (HSPC), cholesterol (CHOL) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2000) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Doxorubicin (DOX) was purchased from Boryung Pharm. Co. (South Korea). Methyl methacrylate (MMA), poly (ethylene glycol) methacrylate (hydroxyl-poly(oxyethylene) methacrylate, HPOEM), poly(ethylene glycol) methyl ether methacrylate (poly(oxyethylene) methacrylate, POEM), tetrahydrofuran (THF), azo(bis)-isobutyronitrile (AIBN) and 1-methox-
yphenol were purchased from Aldrich Co. (Alabaster, AL, USA). Dulbecco's modified Eagle medium (DMEM) and aqueous fetal bovine serum (FBS) were purchased from Bio-Tech. Inc. (Parker Ford, USA). All other materials were of analytical grade and used without further purification. 2.2. Synthesis of comblike polymer The comblike polymer, a random copolymer of MMA, HPOEM and POEM, was synthesized according to the method of Irvine et al. [17,19]; briefly, 21 ml of MMA (0.197 mol), 6.55 g of HPOEM (0.018 mol), 6.55 g of POEM (0.014 mol) and 0.239 g of AIBN (1.46 mmol) were dissolved in 500 ml of THF. The solution was degassed by bubbling nitrogen for 20 min, followed by refluxing at 70 °C for 18 h. The reaction was terminated by the addition of 20 mg of 1-methoxyphenol. The copolymer was purified by two precipitations in 8:1 (v/v) petroleum ether/methanol and then dried in a vacuum at 25 °C for 24 h. The molecular weight of the copolymer was determined by gel permeation chromatography (GPC, KF 804 column, intelligent refractive index detector (RI930), intelligent HPLC pump (PU980), Jasco). 2.3. Preparation of liposomes The DOX-loaded liposomes were prepared according to the remote loading method using an ammonium sulfate gradient [20]. The prepared liposomes and their lipid compositions were as follows; (1) conventional liposomes; HSPC:CHOL = 9.57:3.19 mg/ ml; (2) PEG-liposomes; HSPC:CHOL:DSPE-mPEG-2000 = 9.57:3.19:3.19 mg/ml as a DOXIL® formulation; (3) CPILs; HSPC:CHOL:Comblike polymer = 9.57:3.19:3.19 mg/ml. Briefly, the lipids with the above compositions were dissolved in chloroform, dried into a thin film on a rotary evaporator (Buchi Rotavapor R-200, Switzerland) and then suspended in 250 mM ammonium sulfate solution. The liposomal solution was extruded through a polycarbonate filter (pore size; 100 nm, Whatman, USA) using an extruder (Northern Lipids Inc. USA). The free ammonium sulfate was removed by dialysis for 48 h at 4 °C using cellulose dialysis tubing (MWCO 12,000, Viskase Co. Illinois, USA). The liposomal solution and 2 mg/ml DOX solution were mixed and then incubated for 2 h at 60 °C. The mixture was dialyzed to remove the free DOX. The DOX-loaded liposomes were stored at 4 °C until use. The size and zeta potential of the liposomes were measured by light scattering with a particle size analyzer (ELS-8000, Particle Analyzer, Otuska, Japan). The lipid concentration of liposomes was measured by phosphate assay method using PhosFree™ phosphate assay kit (#BK050, Cytoskeleton Inc., USA) [21,22]. The amount of DOX within the liposomes was measured by fluorescence spectrophotometry (Barnstead, Apogent Tech, USA) after destruction of liposomal vesicles with 10% Triton X-100 aqueous solution. The excitation and emission wavelengths were 490 nm and 590 nm, respectively. The loading efficiency of DOX within the liposomes was calculated by Eq. (1). Loading efficiency ðkÞ ¼ Fi =Ft 100
ð1Þ
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where Fi is the concentration of DOX loaded in the liposomes after their dissolution in 10% Triton X-100 and Ft is the initially added concentration of DOX. 2.4. Polymer amount on the surface of liposomes To confirm the incorporation of the comblike polymer on the surface of the liposomes, the amount of PEG in the PEGliposomes and CPILs was measured by the picrate assay method described by Sonobe et al. [23,24]. Briefly, 5 ml of the liposomal suspension was mixed with 10 ml of sodium nitrate– picrate solution and to the mixture was added 5 ml of 1, 2 dichloroethane. After vigorous shaking, the solution was centrifuged at 1500 ×g for 10 min. The organic layer was collected and the absorbance of the solution was measured at 380 nm using UV-spectrophotometry (UV-mini, Shimadzu Scientific Ins., Japan). 2.5. Cell line and mice B16F10, a murine melanoma cell line, was cultured in DMEM supplemented with 10% v/v heat-inactivated FBS and 10 μl/ml penicillin streptomycin. The cultures were sustained at 37 °C in a humidified incubator containing 5% CO2. The cells were maintained within their exponential growth phase. Female C57BL/6 mice (5 to 6 weeks old, 18–22 g) were purchased from Harlan Inc. (Indiana, USA). All of the procedures involved in the animal experiments were performed according to approved protocols and in accordance with the recommendations of the NIH guideline for the proper use and care of laboratory animals. 2.6. Pharmacokinetic studies In order to monitor the plasma levels of DOX in the mice, free or liposomal DOX was injected intravenously via the tail vein at a dose of 6 mg DOX/kg body weight (three mice per group). Drug-to-lipid ratios of conventional liposomes, PEGliposomes and CPILs were 0.15, 0.12 and 0.12 (wt/wt), respectively. At a predetermined time after the intravenous injection, the mice were sacrificed and blood was collected immediately by cardiac puncture of the mice. Two hundred microliters of blood were collected and mixed with an equal volume of normal saline. After centrifugation of the blood, the serum was divided into two equal aliquots. To the first aliquot 250 μl of 66 mM EDTA solution and isotonic 50 mM PBS buffer (pH 7.4) were added to make a final volume of 1 ml. The concentration of DOX was measured by fluorescence spectrophotometry at 590 nm (emission wavelength). To the second aliquot 250 μl of EDTA solution, 10 μl of Triton X-100 solution and PBS were added to make a final volume of 1 ml. The amount of DOX entrapped in the liposomes was calculated from the difference in the fluorescence intensity of the two aliquots. The difference of fluorescence intensity between the first aliquot and the second aliquot means that the drug release from liposomes indicates the stability of liposomal DOX in circulation. It was regarded, thus, that those data could show the
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DOX release from liposomes in vivo. The pharmacokinetic parameters were calculated from the average DOX concentrations in the bloodstream determined by a pharmacokinetic software BA calculator (Seoul National University, South Korea). 2.7. Biodistribution studies In the DOX biodistribution study, 5 × 105 cells of murine B16F10 melanoma were carefully inoculated into the right limb armpit of the mice subcutaneously. The administration of free DOX or liposomal DOX was started when the mean tumor volume reached approximately 10 mm3. The mice (three per group) were monitored for 64 h after the intravenous injection of the free DOX or liposomal DOX via the tail vein at a dose of 6 mg DOX/kg body weight. At a predetermined time after the intravenous injection, the mice were sacrificed and their liver, spleen, heart, lung, kidney and tumor were collected immediately. The organs and tumor were carefully washed with distilled water, weighed and homogenized with PBS solution. The DOX concentration in the homogenized tissue was measured by fluorescence spectrophotometry at 590 nm, as described in Section 2.6. 2.8. Antitumor activity 5 × 105 cells of murine B16F10 melanoma were carefully inoculated into the right limb armpits of the mice subcutaneously. Six days after the tumor inoculation, free DOX or liposomal DOX was injected intravenously via the tail vein at a dose of 6 mg DOX/kg body weight. The tumor volume was monitored for 16 days after a single intravenous injection of the different types of liposomal DOX. In order to determine the tumor volume, the size of each individual tumor was measured with a caliper and the tumor volume was calculated using Eq. (2). Tumor volume mm3 ¼ width length2 =2 ð2Þ 2.9. Statistical analysis All data expressed as means ± standard deviation (S.D.) are representative of at least three different experiments. When comparing more than two mean values of interest obtained from tumor treatment experiments of various liposomes or control groups, a one-way analysis of variance (ANOVA) was performed using SAS program. To find out whether the two values of interest were significantly different, Tukey's test was performed. All p values b0.05 were considered significant. 3. Results 3.1. Characteristics of comblike polymer The comblike polymer was synthesized by radical polymerization. The structure of the comblike polymer having an MMA backbone and short POEM side chains was confirmed by proton
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Fig. 1. Chemical structure (A) and 1H-NMR spectra of comblike polymer (B). The comblike polymer was synthesized by radical polymerization, and was composed of hydrophobic MMA and the hydrophilic PEG derivatives, HPOEM and POEM. The average molecular weight of the comblike polymer was ca 28,000 Da.
NMR analysis. Fig. 1 shows the chemical structure and 1HNMR spectra of the synthesized comblike polymer. As shown in Fig. 1B, the peaks for the ether protons (1), methacrylate protons (2) and methyl protons (3) were observed at 3.7 ppm, 0.5–2.0 ppm and 4.1 ppm, respectively. The average molecular weight of the polymer was found to be ca 28,000 Da by GPC analysis. 3.2. Characteristics of liposomes The physical properties of the various types of DOX-loaded liposomes are shown in Table 1. The mean particle size of the conventional liposomes, PEG-liposomes and CPILs was approximately 95–107 nm and the loading efficiency of DOX was 91–93%. Additionally, the zeta potential of the various liposomal formulations showed negative values ranging from − 9.02 ± 2.1 mV to − 3.88 ± 1.4 mV. It is generally known that surface charge of liposomes composed of phosphatidyl choline (PC) and CHOL is slightly negative and the zeta potential value of the liposomes decreases by addition of PEG–phosphatidyl
Fig. 2. Pharmacokinetic profiles of various liposomal DOX formulations after intravenous injection in tumor-bearing mice at a single dose of 6 mg DOX/kg body weight (⁎p b 0.01 vs. PEG-liposomes). The data represent the mean ± S.D. (n = 3).
ethanolamine (PEG–PE) due to the electronegativity of PEG– PE itself [25–27]. In our study, as the surface of CPIL was modified with comblike polymer having short PEG derivatives as side chain of the polymer, zeta potential value of CPILs decreased slightly from approximately − 3.88 mV to −9.02 mV. However, zeta potential value of CPILs was almost the same as that of PEG-liposomes indicating that they had similar characteristics with respect to physical and electrostatic properties. The amount of lipid that was incorporated in conventional liposomes, PEG-liposomes and CPILs is 9.41, 11.02 and 8.99 mg/ml liposome solution, respectively. The recovery rate of lipid in each liposomal formulation is 98.2, 86.3 and 93.8% based on the initial amount of lipid, respectively. The amounts of PEG and comblike polymer incorporated on the surface of the PEG-liposomes and CPILs were measured to be 1.2 ± 0.2 mg/ml and 1.5 ± 0.3 mg/ml, respectively, thus confirming that the PEG and comblike polymer were incorporated on the surface of respective liposomes. 3.3. Circulation time induced by polymer incorporation on the surface of liposomes in vivo The profiles of the DOX concentration in the bloodstream after the single intravenous injection of the conventional
Table 1 Physical properties of the various liposomal formulations Composition
Size [nm]
Conventional 107.8 ± 1.7 liposomes (HSPC:CHOL) PEG-liposomes 107.9 ± 0.3 (HSPC:CHOL: DSPE-mPEG) CPILs (HSPC: 95.8 ± 0.7 CHOL: Comblike polymer)
Zeta potential [mV]
Loading Polymer efficiency amount [%] [mg/ml]
Lipid amount [mg/ml]
− 3.88 ± 1.4
91
–
− 8.63 ± 4.3
93
1.2 ± 0.2
11.02
− 9.02 ± 2.1
93
1.5 ± 0.3
8.99
Table 2 Pharmacokinetic parameters of DOX after intravenous injection of various liposomal formulations in tumor-bearing mice at a dose of 6 mg DOX/kg body weight (n = 3)
9.41
AUC (μg h/ml) t1/2 (h) CL (ml/h) MRT (h)
Free DOX⁎
Conventional liposomes⁎⁎
PEGCPILs liposomes⁎⁎⁎
12.00 1.68 17.40 1.95
25.59 2.37 4.62 4.82
201.24 12.80 0.60 17.78
420.08 27.09 0.29 40.00
AUC: area under the curve, t1/2: half-life time, CL: clearance, MRT: mean residence time. ⁎pb 0.001 vs. CPILs, ⁎⁎pb 0.002 vs. CPILs, ⁎⁎⁎pb 0.001 vs. CPILs.
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free DOX (t1/2; 1.68 h, MRT; 1.95 h, ⁎p b 0.001 vs. CPILs), conventional liposomes (t1/2; 2.37 h, MRT; 4.82 h, ⁎⁎p b 0.002 vs. CPILs) or PEG-liposomes (t1/2; 12.80 h, MRT; 17.78 h, ⁎⁎⁎p b 0.001 vs. CPILs). The area under the curve (AUC) of the CPILs was higher (420.08 μg h/ml) than those of the free DOX (12.00 μg h/ml) or conventional liposomes (25.59 μg h/ml). In contrast, the clearance (CL) of the CPILs was lower (0.29 ml/h) than those of the free DOX (17.40 ml/h), conventional liposomes (4.62 ml/h) or PEG-liposomes (0.60 ml/h). These results indicate that the CPILs can circulate for a longer time in the blood than the free DOX, conventional liposomes or PEG-liposomes. 3.4. Tissue distribution of liposomes after intravenous injection in tumor-bearing mice
Fig. 3. Biodistribution of DOX after intravenous injection of DOX-loaded liposomal formulations in tumor-bearing mice at a single dose of 6 mg/kg body weight (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001). The data represent the mean ± S.D. (n = 3).
liposomes, PEG-liposomes and CPILs at a dose of 6 mg/kg are shown in Fig. 2. The free DOX rapidly disappeared from the circulation within 2 h due to its short half-life (t1/2: 1.68 h). In contrast, all of the DOX-loaded liposomes showed a longer circulation time than the free DOX. The DOX concentration of the CPILs in the bloodstream was the highest as compared with those of the other liposomes and the half-life of DOX was increased to 27.09 h. These results indicated that the CPILs could prolong the circulation time of DOX. These profiles confirmed that the incorporation of the comblike polymer was an effective method of improving the circulation time of liposomes in the bloodstream. Notably, the comblike polymer which was incorporated on the liposomal membrane prolonged the half-life of DOX as compared with that of the conventional liposomes (t1/2: 2.37 h) or PEG-liposomes (t1/2: 12.80 h). The pharmacokinetic parameters of the various types of liposomal DOX after their intravenous injection in the mice are summarized in Table 2. The CPILs significantly increased the half-life (t1/2; 27.09 h) and mean residence time (MRT; 40.00 h) of DOX during its circulation in the blood, as compared with the
To clarify the reduction in the RES uptake of liposomal DOX afforded by the incorporation of the comblike polymer, the DOX concentrations in the tissues were measured at predetermined time intervals after the intravenous injection of free or liposomal DOX. The DOX concentrations in the tumor, heart, liver, spleen, kidney and lung are described in Fig. 3. The concentrations of liposomal DOX in the tissues were significantly higher than those of the free DOX with the exception of the heart (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001). Notably, the DOX concentration of the CPILs in the tumor at 16 h was significantly higher than those of the other liposomal groups (Fig. 3A). The DOX concentration of the CPILs in the heart at 2 h was much lower than those of the free DOX or conventional liposomes (Fig. 3B). The DOX concentration in the heart is closely related to the inherent cardiac toxicity of DOX. Therefore, the liposomal DOX would tend to reduce the cardiac toxicity of DOX. The DOX concentrations of the CPILs in the liver and spleen were significantly lower than those of the conventional or PEG-liposomes (Fig. 3C, D). These results
Fig. 4. Tumor growth inhibition after intravenous injection of the free DOX and various liposomal DOX in tumor-bearing mice at a dose of 6 mg DOX/kg body weight. 5 × 105 murine B16F10 cells were inoculated into the armpits of the mice and the animals were treated with an intravenous injection of the free DOX, conventional liposomes, PEG-liposomes, CPILs or normal PBS solution as a control. The arrow indicates the injection day of the various liposomal DOX after tumor implantation. The data represent the mean ± S.D. (n = 6).
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clearly indicated that the RES uptake of the liposomes could be reduced by the incorporation of the comblike polymer into the liposomal membrane. The DOX concentrations of the CPILs in the kidney and lung were significantly lower than those of the conventional or PEG-liposomes (Fig. 3E, F). 3.5. Antitumor activity of liposomes induced by long-circulation property The antitumor activities of the DOX-loaded liposomes were evaluated in B16F10 tumor-bearing mice (six per group) after a single intravenous injection at a dose of 6 mg DOX/kg body weight. As shown in Fig. 4, the DOX-loaded liposomes suppressed the growth of the tumor as compared with the PBS control group. The tumor growth inhibition effects of free DOX and all of liposomal DOX were significant to the control PBS group (ANOVA test; p b 0.001). The CPILs showed higher tumor growth inhibition than the conventional liposomes or free DOX (Tukey's test; p b 0.001). Notably, the CPILs showed tumor growth inhibition effect comparable to PEG-liposomes that comprised a commercialized liposomal DOX, DOXIL®. The high antitumor activity of the CPILs suggested that the CPILs accumulated in the tumor tissue by passive targeting, because of their prolonged circulation time (Fig. 3A). Although some side effects might be expected to result from the use of polymeric modifiers, no severe sign of toxicity, such as fever, loss of weight, cachexia and myalgia, were observed in the mice treated with the liposomal DOX. 4. Discussion Polymer-incorporated liposomal drug delivery systems for cancer therapy have been developed and evaluated in many preclinical models [3,4,28]. Most of these previous studies have been largely focused on prolonging the circulation time of the liposomal DOX in the bloodstream [16,29]. To this end, lipidgrafted PEG-fixed liposomes (PEG-liposomes) have been developed. However, some problems were encountered when the current PEG-liposomes were used to deliver anticancer drugs in clinical trials [6–9]. These difficulties were associated with circulation of the liposomes in the bloodstream and the resulting side effects of DOX. Thus, we designed the new comblike polymer-incorporated CPILs, which prolonged the circulation time of the liposomes in the bloodstream as compared to that of the PEG-liposomes. The comblike polymer used in this study was synthesized by radical polymerization and applied to the liposomes as a surface modifier. To determine the amount of comblike polymer incorporated on the CPILs, the picrate assay was performed. The amount of comblike polymer incorporated in the CPILs was 1.5 ± 0.3 mg/ml (Table 1). This result indicated that the comblike polymer was incorporated on the liposomal bilayer via its hydrophobic interaction with lipid bilayers [30]. For the purpose of preparation of a more sterically-stabilized liposomes using comblike polymer as compared to PEG-liposomes, it appears that comblike polymer incorporated into the liposomal surface forms a hydrophilic steric barrier.
The circulation time of the liposomes in the bloodstream after the intravenous injection in the mice was evaluated by measuring the DOX concentration (Fig. 2). The CPILs showed a higher DOX concentration than the other liposomes. These profiles indicated that the incorporation of the comblike polymer improved the circulation of the liposomes in the bloodstream and, consequently, their half-life was increased. In contrast, the free DOX rapidly disappeared within 2 h. It was inferred from this that the effect of the comblike polymer incorporated into the liposomal membrane was to prolong the half-life of the liposomes by escaping their RES uptake. In addition, the comblike polymer incorporated on the liposomal surface might form a more stable steric barrier on the surface of CPIL than DSPE-PEG on the PEG-liposomes, resulting in the prolongation of circulation time of CPIL as compared to that of PEG-liposomes. Moreover, many researchers have reported various types of liposomes containing comblike polymer. They used the hydrophobically-modified polymers having molecular weight (Mw) of 25,000 or higher [30]. Takeuchi et al. also reported that circulation profiles of liposome coated with hydrophilic polymers having different Mw's in rats. They reported that PVA having Mw of 20,000 was more effective of prolongation of circulation time of liposomes than PVA having Mw of 6000 or 9000, even though amount of polymer incorporated on the liposomal surface was lower than other liposomes [4]. The studies of biodistribution suggested that, in the tumor (Fig. 3A), the CPILs showed a higher DOX concentration than the other liposomes over a long period of time. This result might reflect the increase in passive targeting via the enhanced permeation and retention (EPR) effect caused by the longcirculation ability of the CPILs in the blood. In the heart (Fig. 3B), the free DOX showed the highest drug level at 2 h. The CPILs and PEG-liposomes, however, reduced the drug level in comparison with the free DOX at 2 h, thus indicating that the lower concentration of DOX in the heart may lead to a lower cardiotoxicity of DOX. On the other hand, the lower drug level of the free DOX after 8 h than three of the liposomal DOX might be attributable to the rapid clearance of the free DOX as described in Fig. 2. In the liver (Fig. 3C), the CPILs showed a lower DOX concentration than the other liposomes. This result supported that the incorporation on the comblike polymer prevented the liposomes from trapping caused by the RES uptake. The antitumor activities of the CPILs were higher than that of conventional liposomes or free DOX (Fig. 4). The high antitumor activity of the CPILs is consistent with the higher accumulation of the CPILs in the tumor (Fig. 3A). This result showed that the therapeutic efficacy of DOX could be enhanced by EPR-mediated tumor targeting of the CPILs. Notably, the incorporation of the comblike polymer can prolong the circulation time in the blood (Fig. 2), which can be a contributory factor leading to an increase in passive targeting. The antitumor effects of CPILs were nearly the same against PEG-liposomes. These results demonstrated that the CPILs might augment the antitumor activity induced by enhanced circulation time through incorporation of comblike polymer
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onto the surface of liposomes. During this experimental procedure, all groups of tumor-bearing mice survived over the experiment period even though tumor mass of mice increased. Based on our study, a plausible drug release system derived from the CPILs can be described as follows; the CPILs, whose circulation time is prolonged by the incorporation of the comblike polymer, circulate continuously through the bloodstream. At the same time, the CPILs in the bloodstream can permeate through the angiogenic blood vessels of the tumor and, therefore, the CPILs are accumulated in the tumor tissue. Consequently, the DOX from the CPILs accumulated in the tumor tissue would be released. Mangesh et al. and Takeuchi et al. reported the polymer modified liposomes. In their studies, they used poly(methacrylate)-co-PEG having Mw 25,000 or modified PVA. However, they did not find any cytotoxicity of those polymers [31,32]. Thus, it is considered that comblike polymer may satisfy the toxicological issue in use. In addition, many researchers have studied polymer modified liposomes to enhance the liposome's circulation time or antitumor activity. Many scientific approaches related to the toxicology of polymers have also been challenged in the field of polymer modification on the drugloaded carrier. PMMA used in drug or protein-loaded carrier system has been found to be slowly degradable in the form of nanoparticles and to be safe as an efficient vaccine adjuvant without any observable in vivo side effect or toxic reaction [33,34]. On the other hand, PEG or PEG derivates having relatively high Mw of 6000–170,000 did not distribute significantly to heart, lung, liver, spleen, kidney, and thyroid gland when the polymers were administered intravenously, subcutaneously or intramuscularly: Rather they distributed to the gastrointestinal tract via the bile, and what had not been excreted in urine was excreted in feces [35]. In addition, PEG undergoes cytochrome P-450 oxidation, resulting in the formation of ketone, ester and aldehyde groups [35]. Thus, our comblike polymer composed of PMMA and PEG derivatives may be regarded to be non-toxic and to be degradable or excreted through urine or feces. In conclusion, the comblike copolymer that was composed of hydrophobic MMA chain and hydrophilic short PEG chain was incorporated in DOX-loaded liposomes for surface modification of the liposomes. In the in vivo circulation time measurement, the incorporation of the comblike polymer on the liposomal surface increased the half-life of the liposomes in the bloodstream after their intravenous injection. In addition, it can be inferred that the therapeutic efficacy of the CPILs toward the tumor was enhanced by EPR-mediated tumor targeting, because of the longest circulation time of the CPILs. Thus, our study suggests the potential of comblike polymer-incorporated liposomes for use as drug delivery systems to enhance the therapeutic efficacy of anticancer drugs administered by intravenous injection. Acknowledgement This work was supported by the Ministry of Commerce, Industry and Energy of Korea (10016573).
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