- Email: [email protected]
Liposomal doxorubicin-loaded chitosan microspheres capable of controlling release of doxorubicin for anti-cancer chemoembolization: in vitro characteristics
J. DRUG DEL. SCI. TECH., 23 (3) 283-286 2013
Liposomal doxorubicin-loaded chitosan microspheres capable of controlling release of doxorubicin for anti-cancer chemoembolization: in vitro characteristics Hyeong-Min Kim1, Ga-Hyeon Lee1, Hyo-Jeong Kuh2, Byung Kook Kwak3, Jaehwi Lee1* 1 College of Pharmacy, Chung-Ang University, 84 Heuksuk-ro, Dongjak-gu, Seoul 156-756, South Korea Department of Biomedical Science, College of Medicine, The Catholic University of Korea, Seoul 137-701, South Korea 3 Department of Radiology, Chung-Ang University Hospital, Seoul 156-755, South Korea *Correspondence: [email protected]
2
The aim of this study was to develop liposomal doxorubicin-loaded chitosan microspheres (LDox-CSMS) for anti-cancer chemoembolization. LDox-CSMS and doxorubicin-loaded CSMS (Dox-CSMS) devoid of liposomal carrier were prepared with an emulsification and cross-linking method. Various stirring rates were required to obtain large fractions of CSMS in the size range of 300-500 µm. Drug loading amounts were considerably influenced by the amount of doxorubicin-loaded liposomes added. Drug release from LDox-CSMS was controlled by the drug loading amount, which was varied by the addition of liposomal doxorubicin. These results indicate that LDox-CSMS can be used as an advanced chemoembolic agent that can control drug release behaviors. Key words: Liposomal doxorubicin – Chemoembolization – Chitosan – Release.
Arterial embolization treats cancers such as hepatoma by occluding the blood vessels connected to tumor tissues, thereby blocking the supply of oxygen and nutrients necessary for the survival of tumor tissues [1]. For many years, polymeric microparticles with irregular shapes, such as polyvinyl alcohol (PVA) particles (Contour PVA Embolization Particles, Boston Scientific Co., Nanterre, France), have frequently been used in clinical practice [2]. Currently, microspheres with narrow size distributions have gradually replaced irregular microparticles due to improved embolization efficiency and reduced side effects, including decreased inflammation reactions between blood vessel walls and microparticles [3]. Moreover, multifunctional embolic materials such as drug-eluting embolic microspheres (DC Bead, Biocompatibles, Surrey, UK; Quadrasphere, BioSphere Medical Inc., MA, United States) have recently been commercialized for their combined anti-cancer effects of blood vessel occlusion and local delivery of chemotherapeutics [4, 5]. The main drawback associated with current drug-releasing embolic microspheres is that drug loading onto microbeads is typically accomplished with an ion-exchange reaction by immersing the microspheres in drug solution for more than 30 min [6]. Additionally, control of drug release may not be possible since drug release from the microspheres depends on the exchange of drugs with endogenous ions in the blood vessels [6]. Thus, drug-preloaded embolic microspheres would be an advanced system compared to those requiring time-consuming drug loading before intervention in the clinical setting. In the present study, deformable chitosan embolic microspheres loaded with liposomal doxorubicin were developed as a novel and multifunctional anti-cancer embolic material. The drug was encapsulated into liposomes and then loaded in microspheres in order to control the drug release profile, which might be needed in specific clinical situations [7]. Chitosan was used as the principal embolic material and the platform for drug loading. Although chitosan is an ideal biomaterial due to its non-toxicity, biocompatibility and biodegradability, it is not suitable for chemoembolization because of its poor physicochemical properties such as brittleness [8]. However, we modified the material’s physicochemical properties to give deformability; thus, chitosan was successfully used for chemoembolization [6].
Chitosan from crab shells (over 75 % deacetylated, product number 417963), glutaraldehyde (25 wt % aqueous biological grade), sorbitan sesquioleate, cholesterol, ammonium sulfate, phosphate buffered saline (PBS) and lysozyme from chicken egg whites (product number L6876) were purchased from Sigma-Aldrich Company (St. Louis, MO, United States). Phosphatidylcholine (PC) from soybeans was purchased from Lipoid GmbH (Ludwigshafen, Germany). Doxorubicin hydrochloride (Dox) was obtained from Dong-A Pharmaceutical Company (Seoul, Korea). Glacial acetic acid, acetone, liquid paraffin, petroleum ether, toluene, and chloroform were purchased from Daejung Chemicals (Siheung, Korea). Methanol was purchased from JT Baker Chemical Company (Phillipsburg, NJ, United States). Sucrose was purchased from Samchun Chemicals (Yeosu, Korea). Distilled and deionized water was used for the preparation of all solutions. A 1:1 mixture of methanol and chloroform dissolved a mixture of soybean PC and cholesterol (6:4, w/w). Thin lipid films were formed by a rotary vacuum evaporator (HS-2000NS, Hanshin Scientific Co., Jeonju-si, Korea) at 37 °C and hydrated with 250 mM ammonium sulfate. The liposomes were extruded 10 times using an extruder (Avanti Mini-Extruder, Avanti Polar Lipids Inc., Alabaster, AL, United States) and dialyzed 4 times with a dialysis bag (MWCO 12-14K) for 24 h to establish a transmembrane ammonium sulfate gradient in 20 % (w/v) sucrose solution [9]. Dox solution was added to the liposome suspensions to achieve a Dox concentration of 1 mg/mL. The mixture was then incubated at 37 ± 0.5 °C for 2 h. To calculate drug loading efficiency and amount of Dox, unencapsulated Dox in the liposomal suspension was separated by centrifugation at 2,000 g for 30 min after the liposomal suspension was put into a filter-equipped conical tube (Millipore Amicon Ultra Centrifugal Filters, MWCO = 100 K, Millipore Korea Co. Ltd., Seoul, Korea) [10]. The amount of Dox that passed through the filter membrane (i.e., unentrapped Dox) was monitored by measuring fluorescence levels of Dox at 480 nm excitation and 550 nm emission wavelength using a microplate reader (Synergy H1 Hybrid Reader, BioTek, Seoul, Korea) [11]. The drug loading efficiency and amount of Dox in the liposomal suspension were then determined using the equations reported elsewhere [12]. 283
J. DRUG DEL. SCI. TECH., 23 (3) 283-286 2013
Liposomal doxorubicin-loaded chitosan microspheres capable of controlling release of doxorubicin for anti-cancer chemoembolization: in vitro characteristics Hyeong-Min Kim, Ga-Hyeon Lee, Hyo-Jeong Kuh, Byung Kook Kwak, Jaehwi Lee
added to the chitosan solution. The reason for this increased loading efficiency can be explained by the increased viscosity caused by increased amounts of the liposomal Dox preventing the dispersion of the liposomal Dox to the outer phases [14]. The loading amounts of Dox in LDox-CSMS were measured to be 0.19 (LDox10), 1.09 (LDox30), and 1.88 (LDox50) %, respectively. The loading efficiency and loading amount of Dox for Dox-CSMS were 56.74 and 2.84 %, respectively (Table I). The prepared CSMS were classified according to size with a sieving technique. To obtain LDox-CSMS in the range of 300-500 μm in size, which is the size generally used for chemoembolization in hepatocellular carcinoma [15], controlling the stirring rate of the reaction solution was critical because the stirring rate is regarded as one of the key factors affecting the size distribution of microspheres produced by emulsion-based microencapsulation [16]. Figure 1A indicates the size distribution of various CSMS prepared at optimal stirring rates that produced the largest quantities of CSMS in the range of 300-500 μm. Larger proportions of various CSMS with sizes of 300-500 μm were obtained at stirring rates of 620, 750, 785, and 850 rpm for Dox-CSMS, LDox10-CSMS, LDox30-CSMS, and LDox50-CSMS, respectively. Indeed, when the stirring rate was set at 620 rpm, about 80 % of the Dox-CSMS ranged in size between 300 and 500 μm, while only 17-28 % of the three types of LDox-CSMS were within the same size range. The reason for this finding might be the increased viscosity of the media with increasing amounts of the liposomal Dox [17]. A faster stirring rate was also required to obtain larger fractions of LDox-CSMS in the size range of 300-500 μm. In practice, for LDox30-CSMS, an increased stirring rate caused more LDox-CSMS to be produced in the size range of 300 and 500 μm (Figure 1B).
Table I - Properties of liposomal doxorubicin (Dox), Dox-CSMS* and LDox-CSMS**. Group
Liposomes added to CSMS (mg)
Drug/ Chitosan (w/w)
Loading efficiency (%)
Loading amount (%)
Liposomal doxorubicin
-
-
98.48
4.92
Dox-CSMS
-
6/120
56.74
2.84
LDox10CSMS
10
0.5/120
60.73
0.19
LDox30CSMS
30
1.5/120
91.09
1.09
LDox50CSMS
50
2.5/120
93.91
1.88
*Dox-CSMS indicates Dox-added chitosan microspheres. **LDox-CSMS indicates liposomal Dox-loaded chitosan microspheres.
Dox-loaded chitosan microspheres (Dox-CSMS) and liposomal Dox-loaded chitosan microspheres (LDox-CSMS) were prepared using an emulsification and cross-linking method [6]. An aqueous chitosan solution (4 %, w/v) was prepared with acetic acid (5 %, v/v) and the liposomal suspension prepared above was added to the chitosan solution. The quantities of the liposomal Dox added to the chitosan solution are shown in Table I. The liposomal suspension-chitosan solution mixture was then added to a mixture of liquid paraffin and petroleum ether (7:5, 30 mL) containing sorbitan sesquioleate (3 mL) as an emulsifier. The emulsion formed was stirred for 20 min at stirring rates of 750, 785, and 850 rpm for LDox10-CSMS, LDox30-CSMS, and LDox50CSMS, respectively. Subsequently, glutaraldehyde-saturated toluene (4 mL) was added to each emulsion drop by drop for cross-linking the chitosan microdroplets for 60 min. The cross-linked chitosan microspheres were washed with petroleum ether three times, acetone once, and distilled water three times. The washed LDox-CSMS was dried at room temperature for 24 h in a desiccator. Separately, free Dox (1 mg/mL) was added to chitosan solution and the mixture was emulsified at 620 rpm. Then, the same procedures were applied to produce Dox-CSMS. The quantity of Dox unincorporated in LDox-CSMS and DoxCSMS was measured by fluorescence detection of the oil phase. Then, drug loading efficiency and amount for Dox-CSMS and LDox-CSMS were determined. An in vitro release study was carried out in PBS (pH=6.0) with lysozyme (50,000 units/vessel) for 2 days [6]. Membrane tubes containing 20 mg each of Dox-CSMS and LDox-CSMS were immersed in release medium (30 mL) placed in conical tubes. Then, the tubes were stored at 37 ± 0.5 °C and shaken at 80 rpm in a shaking incubator. The release medium (0.2 mL) was taken out at predetermined time intervals (0.5, 1, 2, 4, 6, 24, 48 hours) and was replaced with the same volume of fresh release medium. Released Dox was measured by fluorescence detection as described above. The mean particle size, polydispersity index, and zeta potential values of the liposomal Dox were measured to be 149.9 nm, 0.134, and -20.35 mV, respectively. The loading efficiency of Dox in the liposomal Dox was about 98 % greater than values reported elsewhere [13] due to the transmembrane ammonium sulfate gradient between the liposomal core and the external medium [9]. The loading amount of Dox in the liposomal Dox was measured to be 4.92 %. The liposomal Dox was then loaded into the CSMS. In the case of LDox-CSMS, the loading efficiencies of Dox were measured to be 60.73 (LDox10), 91.09 (LDox30), and 93.91 (LDox50) %, respectively. Loading efficiency increased as increased amounts of the liposomal Dox were
Figure 1 - Panel A illustrates the size distributions of Dox-CSMS and DoxL-CSMS prepared with stirring rates (rpm) of 620 (Dox-CSMS), 750 (LDox10-CSMS), 785 (LDox30-CSMS), and 850 (LDox50-CSMS). Panel B demonstrates the effect of stirring rate on the weight ratio of LDox30-CSMS (300-500 μm, %). Values indicate means ± SD (n = 3). 284
Liposomal doxorubicin-loaded chitosan microspheres capable of controlling release of doxorubicin for anti-cancer chemoembolization: in vitro characteristics Hyeong-Min Kim, Ga-Hyeon Lee, Hyo-Jeong Kuh, Byung Kook Kwak, Jaehwi Lee
The release of Dox from the CSMS was monitored in PBS buffer (pH 6.0) containing lysozyme (50,000 units/vessel) because the extracellular pH of most tumor tissues is generally more acidic than that of normal tissues [18], and lysozyme existing in plasma causes the degradation of chitosan [19]. The rank order of the amount of Dox released over 48 h was as follows: Dox-CSMS > LDox50-CSMS > LDox30-CSMS and > LDox10-CSMS (Figure 2). The reason for this rank might be the difference in loading amount [20]. The loading amount of Dox in LDox-CSMS varied according to the amount of liposomal Dox added, with more liposomal Dox producing greater loading amounts. This result indicates that drug loading into the Dox-CSMS could be modified, and that Dox release could also be controlled. The release and tissue distribution of Dox from drug-eluting beads have been evaluated with results revealing that drug levels in tissue had a considerable effect on coagulative necrosis of liver parenchyma after embolization [21]. However, it is our understanding that the optimum rate and mode of drug delivery from drug-loaded embolic microparticles have not yet been elucidated. Thus, to study these effects, the preparation of embolic microspheres with various drug release kinetics is considered crucial. However, as reported in our previous publication, adding much more drug to the chitosan solution alone did not significantly change the drug release kinetics that exhibited initial burst release [6]. Thus, we employed nanocarriers embedded in microspheres since drug release can be controlled by both nanocarriers and micromatrices. Indeed, the release profiles of Dox were varied depending on the amount of liposomal Dox added. We selected liposomes to control drug release profiles as Dox is hydrophilic and can be encapsulated inside the liposomes, and thereby the release of the drug was efficiently modified probably by lipid bilayer and amount of liposomes incorporated into the chitosan microspheres. There have been other nanoparticular carriers, such as micelles, solid lipid nanoparticles and nanostructured lipid carriers. However, the reason for the use of liposomes was because hydrophilic drug such as Dox cannot effectively be loaded into these nanoparticles and they were not compatible with hydrophilic polymers like chitosan.
Figure 2 - In vitro release of Dox from LDox-CSMS and Dox-CSMS. Data are expressed as amounts. Values indicate means ± SD (n = 3).
7.
8.
9.
10.
11.
* In conclusion, drug release could be modified by the incorporation of liposomal Dox in CSMS and thereby various release kinetics were achieved, which might be useful for specific clinical situations. It therefore seems likely that LDox-CSMS can be developed as an advanced chemoembolic agent providing controlled release of Dox.
12.
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J. DRUG DEL. SCI. TECH., 23 (3) 283-286 2013
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Liposomal doxorubicin-loaded chitosan microspheres capable of controlling release of doxorubicin for anti-cancer chemoembolization: in vitro characteristics Hyeong-Min Kim, Ga-Hyeon Lee, Hyo-Jeong Kuh, Byung Kook Kwak, Jaehwi Lee
ACKNOWLEDGEMENT
biodegradable microspheres loaded with famotidine. - Trop. J. Pharmaceut. Res., 10, 309-316, 2011. Namur J., Wassef M., Millot J.M., Lewis A.L., Manfait M., Laurent A. - Drug-eluting beads for liver embolization: concentration of doxorubicin in tissue and in beads in a pig model. - J. Vasc. Interv. Radiol., 21, 259-267, 2010.
This work was supported by the Mid-career Researcher Program through NRF grant funded by the MEST (No. 2010-0027798).
MANUSCRIPT Received 3 September 2012, accepted for publication 24 November 2012.
286
Liposomal doxorubicin-loaded chitosan microspheres capable of controlling release of doxorubicin for anti-cancer chemoembolization: in vitro characteristics Hyeong-Min Kim1, Ga-Hyeon Lee1, Hyo-Jeong Kuh2, Byung Kook Kwak3, Jaehwi Lee1* 1 College of Pharmacy, Chung-Ang University, 84 Heuksuk-ro, Dongjak-gu, Seoul 156-756, South Korea Department of Biomedical Science, College of Medicine, The Catholic University of Korea, Seoul 137-701, South Korea 3 Department of Radiology, Chung-Ang University Hospital, Seoul 156-755, South Korea *Correspondence: [email protected]
2
The aim of this study was to develop liposomal doxorubicin-loaded chitosan microspheres (LDox-CSMS) for anti-cancer chemoembolization. LDox-CSMS and doxorubicin-loaded CSMS (Dox-CSMS) devoid of liposomal carrier were prepared with an emulsification and cross-linking method. Various stirring rates were required to obtain large fractions of CSMS in the size range of 300-500 µm. Drug loading amounts were considerably influenced by the amount of doxorubicin-loaded liposomes added. Drug release from LDox-CSMS was controlled by the drug loading amount, which was varied by the addition of liposomal doxorubicin. These results indicate that LDox-CSMS can be used as an advanced chemoembolic agent that can control drug release behaviors. Key words: Liposomal doxorubicin – Chemoembolization – Chitosan – Release.
Arterial embolization treats cancers such as hepatoma by occluding the blood vessels connected to tumor tissues, thereby blocking the supply of oxygen and nutrients necessary for the survival of tumor tissues [1]. For many years, polymeric microparticles with irregular shapes, such as polyvinyl alcohol (PVA) particles (Contour PVA Embolization Particles, Boston Scientific Co., Nanterre, France), have frequently been used in clinical practice [2]. Currently, microspheres with narrow size distributions have gradually replaced irregular microparticles due to improved embolization efficiency and reduced side effects, including decreased inflammation reactions between blood vessel walls and microparticles [3]. Moreover, multifunctional embolic materials such as drug-eluting embolic microspheres (DC Bead, Biocompatibles, Surrey, UK; Quadrasphere, BioSphere Medical Inc., MA, United States) have recently been commercialized for their combined anti-cancer effects of blood vessel occlusion and local delivery of chemotherapeutics [4, 5]. The main drawback associated with current drug-releasing embolic microspheres is that drug loading onto microbeads is typically accomplished with an ion-exchange reaction by immersing the microspheres in drug solution for more than 30 min [6]. Additionally, control of drug release may not be possible since drug release from the microspheres depends on the exchange of drugs with endogenous ions in the blood vessels [6]. Thus, drug-preloaded embolic microspheres would be an advanced system compared to those requiring time-consuming drug loading before intervention in the clinical setting. In the present study, deformable chitosan embolic microspheres loaded with liposomal doxorubicin were developed as a novel and multifunctional anti-cancer embolic material. The drug was encapsulated into liposomes and then loaded in microspheres in order to control the drug release profile, which might be needed in specific clinical situations [7]. Chitosan was used as the principal embolic material and the platform for drug loading. Although chitosan is an ideal biomaterial due to its non-toxicity, biocompatibility and biodegradability, it is not suitable for chemoembolization because of its poor physicochemical properties such as brittleness [8]. However, we modified the material’s physicochemical properties to give deformability; thus, chitosan was successfully used for chemoembolization [6].
Chitosan from crab shells (over 75 % deacetylated, product number 417963), glutaraldehyde (25 wt % aqueous biological grade), sorbitan sesquioleate, cholesterol, ammonium sulfate, phosphate buffered saline (PBS) and lysozyme from chicken egg whites (product number L6876) were purchased from Sigma-Aldrich Company (St. Louis, MO, United States). Phosphatidylcholine (PC) from soybeans was purchased from Lipoid GmbH (Ludwigshafen, Germany). Doxorubicin hydrochloride (Dox) was obtained from Dong-A Pharmaceutical Company (Seoul, Korea). Glacial acetic acid, acetone, liquid paraffin, petroleum ether, toluene, and chloroform were purchased from Daejung Chemicals (Siheung, Korea). Methanol was purchased from JT Baker Chemical Company (Phillipsburg, NJ, United States). Sucrose was purchased from Samchun Chemicals (Yeosu, Korea). Distilled and deionized water was used for the preparation of all solutions. A 1:1 mixture of methanol and chloroform dissolved a mixture of soybean PC and cholesterol (6:4, w/w). Thin lipid films were formed by a rotary vacuum evaporator (HS-2000NS, Hanshin Scientific Co., Jeonju-si, Korea) at 37 °C and hydrated with 250 mM ammonium sulfate. The liposomes were extruded 10 times using an extruder (Avanti Mini-Extruder, Avanti Polar Lipids Inc., Alabaster, AL, United States) and dialyzed 4 times with a dialysis bag (MWCO 12-14K) for 24 h to establish a transmembrane ammonium sulfate gradient in 20 % (w/v) sucrose solution [9]. Dox solution was added to the liposome suspensions to achieve a Dox concentration of 1 mg/mL. The mixture was then incubated at 37 ± 0.5 °C for 2 h. To calculate drug loading efficiency and amount of Dox, unencapsulated Dox in the liposomal suspension was separated by centrifugation at 2,000 g for 30 min after the liposomal suspension was put into a filter-equipped conical tube (Millipore Amicon Ultra Centrifugal Filters, MWCO = 100 K, Millipore Korea Co. Ltd., Seoul, Korea) [10]. The amount of Dox that passed through the filter membrane (i.e., unentrapped Dox) was monitored by measuring fluorescence levels of Dox at 480 nm excitation and 550 nm emission wavelength using a microplate reader (Synergy H1 Hybrid Reader, BioTek, Seoul, Korea) [11]. The drug loading efficiency and amount of Dox in the liposomal suspension were then determined using the equations reported elsewhere [12]. 283
J. DRUG DEL. SCI. TECH., 23 (3) 283-286 2013
Liposomal doxorubicin-loaded chitosan microspheres capable of controlling release of doxorubicin for anti-cancer chemoembolization: in vitro characteristics Hyeong-Min Kim, Ga-Hyeon Lee, Hyo-Jeong Kuh, Byung Kook Kwak, Jaehwi Lee
added to the chitosan solution. The reason for this increased loading efficiency can be explained by the increased viscosity caused by increased amounts of the liposomal Dox preventing the dispersion of the liposomal Dox to the outer phases [14]. The loading amounts of Dox in LDox-CSMS were measured to be 0.19 (LDox10), 1.09 (LDox30), and 1.88 (LDox50) %, respectively. The loading efficiency and loading amount of Dox for Dox-CSMS were 56.74 and 2.84 %, respectively (Table I). The prepared CSMS were classified according to size with a sieving technique. To obtain LDox-CSMS in the range of 300-500 μm in size, which is the size generally used for chemoembolization in hepatocellular carcinoma [15], controlling the stirring rate of the reaction solution was critical because the stirring rate is regarded as one of the key factors affecting the size distribution of microspheres produced by emulsion-based microencapsulation [16]. Figure 1A indicates the size distribution of various CSMS prepared at optimal stirring rates that produced the largest quantities of CSMS in the range of 300-500 μm. Larger proportions of various CSMS with sizes of 300-500 μm were obtained at stirring rates of 620, 750, 785, and 850 rpm for Dox-CSMS, LDox10-CSMS, LDox30-CSMS, and LDox50-CSMS, respectively. Indeed, when the stirring rate was set at 620 rpm, about 80 % of the Dox-CSMS ranged in size between 300 and 500 μm, while only 17-28 % of the three types of LDox-CSMS were within the same size range. The reason for this finding might be the increased viscosity of the media with increasing amounts of the liposomal Dox [17]. A faster stirring rate was also required to obtain larger fractions of LDox-CSMS in the size range of 300-500 μm. In practice, for LDox30-CSMS, an increased stirring rate caused more LDox-CSMS to be produced in the size range of 300 and 500 μm (Figure 1B).
Table I - Properties of liposomal doxorubicin (Dox), Dox-CSMS* and LDox-CSMS**. Group
Liposomes added to CSMS (mg)
Drug/ Chitosan (w/w)
Loading efficiency (%)
Loading amount (%)
Liposomal doxorubicin
-
-
98.48
4.92
Dox-CSMS
-
6/120
56.74
2.84
LDox10CSMS
10
0.5/120
60.73
0.19
LDox30CSMS
30
1.5/120
91.09
1.09
LDox50CSMS
50
2.5/120
93.91
1.88
*Dox-CSMS indicates Dox-added chitosan microspheres. **LDox-CSMS indicates liposomal Dox-loaded chitosan microspheres.
Dox-loaded chitosan microspheres (Dox-CSMS) and liposomal Dox-loaded chitosan microspheres (LDox-CSMS) were prepared using an emulsification and cross-linking method [6]. An aqueous chitosan solution (4 %, w/v) was prepared with acetic acid (5 %, v/v) and the liposomal suspension prepared above was added to the chitosan solution. The quantities of the liposomal Dox added to the chitosan solution are shown in Table I. The liposomal suspension-chitosan solution mixture was then added to a mixture of liquid paraffin and petroleum ether (7:5, 30 mL) containing sorbitan sesquioleate (3 mL) as an emulsifier. The emulsion formed was stirred for 20 min at stirring rates of 750, 785, and 850 rpm for LDox10-CSMS, LDox30-CSMS, and LDox50CSMS, respectively. Subsequently, glutaraldehyde-saturated toluene (4 mL) was added to each emulsion drop by drop for cross-linking the chitosan microdroplets for 60 min. The cross-linked chitosan microspheres were washed with petroleum ether three times, acetone once, and distilled water three times. The washed LDox-CSMS was dried at room temperature for 24 h in a desiccator. Separately, free Dox (1 mg/mL) was added to chitosan solution and the mixture was emulsified at 620 rpm. Then, the same procedures were applied to produce Dox-CSMS. The quantity of Dox unincorporated in LDox-CSMS and DoxCSMS was measured by fluorescence detection of the oil phase. Then, drug loading efficiency and amount for Dox-CSMS and LDox-CSMS were determined. An in vitro release study was carried out in PBS (pH=6.0) with lysozyme (50,000 units/vessel) for 2 days [6]. Membrane tubes containing 20 mg each of Dox-CSMS and LDox-CSMS were immersed in release medium (30 mL) placed in conical tubes. Then, the tubes were stored at 37 ± 0.5 °C and shaken at 80 rpm in a shaking incubator. The release medium (0.2 mL) was taken out at predetermined time intervals (0.5, 1, 2, 4, 6, 24, 48 hours) and was replaced with the same volume of fresh release medium. Released Dox was measured by fluorescence detection as described above. The mean particle size, polydispersity index, and zeta potential values of the liposomal Dox were measured to be 149.9 nm, 0.134, and -20.35 mV, respectively. The loading efficiency of Dox in the liposomal Dox was about 98 % greater than values reported elsewhere [13] due to the transmembrane ammonium sulfate gradient between the liposomal core and the external medium [9]. The loading amount of Dox in the liposomal Dox was measured to be 4.92 %. The liposomal Dox was then loaded into the CSMS. In the case of LDox-CSMS, the loading efficiencies of Dox were measured to be 60.73 (LDox10), 91.09 (LDox30), and 93.91 (LDox50) %, respectively. Loading efficiency increased as increased amounts of the liposomal Dox were
Figure 1 - Panel A illustrates the size distributions of Dox-CSMS and DoxL-CSMS prepared with stirring rates (rpm) of 620 (Dox-CSMS), 750 (LDox10-CSMS), 785 (LDox30-CSMS), and 850 (LDox50-CSMS). Panel B demonstrates the effect of stirring rate on the weight ratio of LDox30-CSMS (300-500 μm, %). Values indicate means ± SD (n = 3). 284
Liposomal doxorubicin-loaded chitosan microspheres capable of controlling release of doxorubicin for anti-cancer chemoembolization: in vitro characteristics Hyeong-Min Kim, Ga-Hyeon Lee, Hyo-Jeong Kuh, Byung Kook Kwak, Jaehwi Lee
The release of Dox from the CSMS was monitored in PBS buffer (pH 6.0) containing lysozyme (50,000 units/vessel) because the extracellular pH of most tumor tissues is generally more acidic than that of normal tissues [18], and lysozyme existing in plasma causes the degradation of chitosan [19]. The rank order of the amount of Dox released over 48 h was as follows: Dox-CSMS > LDox50-CSMS > LDox30-CSMS and > LDox10-CSMS (Figure 2). The reason for this rank might be the difference in loading amount [20]. The loading amount of Dox in LDox-CSMS varied according to the amount of liposomal Dox added, with more liposomal Dox producing greater loading amounts. This result indicates that drug loading into the Dox-CSMS could be modified, and that Dox release could also be controlled. The release and tissue distribution of Dox from drug-eluting beads have been evaluated with results revealing that drug levels in tissue had a considerable effect on coagulative necrosis of liver parenchyma after embolization [21]. However, it is our understanding that the optimum rate and mode of drug delivery from drug-loaded embolic microparticles have not yet been elucidated. Thus, to study these effects, the preparation of embolic microspheres with various drug release kinetics is considered crucial. However, as reported in our previous publication, adding much more drug to the chitosan solution alone did not significantly change the drug release kinetics that exhibited initial burst release [6]. Thus, we employed nanocarriers embedded in microspheres since drug release can be controlled by both nanocarriers and micromatrices. Indeed, the release profiles of Dox were varied depending on the amount of liposomal Dox added. We selected liposomes to control drug release profiles as Dox is hydrophilic and can be encapsulated inside the liposomes, and thereby the release of the drug was efficiently modified probably by lipid bilayer and amount of liposomes incorporated into the chitosan microspheres. There have been other nanoparticular carriers, such as micelles, solid lipid nanoparticles and nanostructured lipid carriers. However, the reason for the use of liposomes was because hydrophilic drug such as Dox cannot effectively be loaded into these nanoparticles and they were not compatible with hydrophilic polymers like chitosan.
Figure 2 - In vitro release of Dox from LDox-CSMS and Dox-CSMS. Data are expressed as amounts. Values indicate means ± SD (n = 3).
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* In conclusion, drug release could be modified by the incorporation of liposomal Dox in CSMS and thereby various release kinetics were achieved, which might be useful for specific clinical situations. It therefore seems likely that LDox-CSMS can be developed as an advanced chemoembolic agent providing controlled release of Dox.
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Liposomal doxorubicin-loaded chitosan microspheres capable of controlling release of doxorubicin for anti-cancer chemoembolization: in vitro characteristics Hyeong-Min Kim, Ga-Hyeon Lee, Hyo-Jeong Kuh, Byung Kook Kwak, Jaehwi Lee
ACKNOWLEDGEMENT
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This work was supported by the Mid-career Researcher Program through NRF grant funded by the MEST (No. 2010-0027798).
MANUSCRIPT Received 3 September 2012, accepted for publication 24 November 2012.
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