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Gas-forming liposomes prepared using a liposomal magnetoporation method
Accepted Manuscript Title: Gas-forming liposomes prepared using a liposomal magnetoporation method Authors: Jae Min Lee, Dong Sup Kwag, Yu Seok Youn, Eun Seong Lee PII: DOI: Reference:
S0927-7765(17)30204-7 http://dx.doi.org/doi:10.1016/j.colsurfb.2017.04.017 COLSUB 8484
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
19-1-2017 6-4-2017 10-4-2017
Please cite this article as: Jae Min Lee, Dong Sup Kwag, Yu Seok Youn, Eun Seong Lee, Gas-forming liposomes prepared using a liposomal magnetoporation method, Colloids and Surfaces B: Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.04.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Gas-forming liposomes prepared using a liposomal magnetoporation method Jae Min Lee a, Dong Sup Kwag a, Yu Seok Youn b, Eun Seong Lee a, *
a
Department of Biotechnology, The Catholic University of Korea, 43-1 Yeokgok 2-dong, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea b
School of Pharmacy, Sungkyunkwan University, 300 Chonchon-dong, Jangan-ku, Suwon, Gyeonggi-do 440-746, Republic of Korea
* To whom correspondence should be addressed. Tel: +82-2-2164-4921 Fax: +82-2-2164-4865 E-mail: [email protected] (E.S. Lee)
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Graphical abstract
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Research Highlights ► We developed gas-forming liposomes using magnetoporated liposomes ► The liposomes induced hyperthermia-dependent drug release and enhanced tumor ablation. ► The liposomes hold great promise for use in the selective delivery of drugs to tumor sites under local hyperthermia.
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Abstract In this study, we report a gas-forming drug carrier engineered using the liposomal magnetoporation method. The liposomes that were magnetoporated under a magnetic shear stress possessed an opened lipid bilayer hole. A photosensitizing model drug (chlorin e6: Ce6) and 1H-1H-2H-perfluoro-1hexene (PFH, as a volatile gas-forming agent) were efficiently loaded into the opened holes of the magnetoporated liposomes. PFH in the liposomes is vaporized at 50 C and can initiate liposome destabilization. The experimental results demonstrated that the liposomes were destabilized at 50 C efficiently released Ce6 and enhanced Ce6-mediated phototoxicity against KB tumor cells. As a result, these liposomes induced a significantly increased in vitro and in vivo photodynamic tumor inhibition.
Keywords: Liposomal magnetoporation, gas-forming, perfluorohexene, chlorin e6, photodynamic therapy
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1. Introduction An important purpose of advanced drug delivery systems is to suitably deliver drugs to a particular position in the body, which demands the use of proper vehicles that efficiently encapsulate drugs and release drugs in response to biological signals [1-3]. Over the last few decades, liposomes consisting of lipid bilayer containers have been used as carriers for chemical or biological drugs and have received much attention as essential tools for successful drug delivery [1-6]. In particular, various investigations for flexible bilayer membranes and dynamic properties (such as fusion, fission, and shape deformation) of liposomes offer various possibilities for developing advanced drug carrying systems [7]. Indeed, such trials have extended liposome applications into diverse fields such as pharmaceutics and cosmetics [3,8,9]. Recently, our group developed partially uncapped liposomes by applying magnetic shear stress to liposomes containing Fe3O4 nanoparticles [7]. These liposomes displayed an opened lipid bilayer hole in a liposome membrane as a result of the leakage of Fe3O4 nanoparticles. This magnetoporation method is a valuable tool for preparing porous liposomes and can provide an opened entrance for chemicals or biological drugs into the liposome before the natural recovery (closing) of the lipid bilayer hole [7]. Perfluorohexene [1H-1H-2H-perfluoro-1-hexene (PFH)] is stable liquid oil at room temperature. However, PFH is easily vaporized near its boiling point at 50 C [2,5,10-12]. Importantly, PFH can be used to prepare nano/micro-sized bubbles and to provide high vapor pressure (at the hyperthermia condition, ~50 C) in nano-sized containers for drug release. However, it is difficult to load PFH into nano-sized containers because of its high lipophilicity and liquid oil state [2,5,11,12]. In this study, we utilized magnetoporated liposomes (prepared using the magnetoporation method) as an opened liposomal template for preparing gas (PFH)-forming liposomes and for establishing hyperthermia-sensitive drug-encapsulating liposome. Here, we encapsulated a photosensitizing antitumor drug (chlorin e6: Ce6) and PFH into the magnetoporated liposomes. It is 5
anticipated that the efficiently loaded PFH in the magnetoporated liposomes would be vaporized at 50 C and destabilize the liposomes, resulting in an increased drug release rate (Fig. 1). We preferentially investigated hyperthermia-induced liposomal destabilization, drug release behaviors, and Ce6-mediated phototoxicity of KB tumors.
2. Materials and methods 2.1. Materials Phosphatidylcholine (PC), dimethylsulfoxide (DMSO), chloroform, acetone, poloxamer 188, 1H-1H-2H-perfluoro-1-hexene (PFH), iron oxide (II, III) magnetic nanoparticles (average 5 nm in diameter), and 9,10-dimethylanthracene were obtained from Sigma-Aldrich (USA). Chlorin e6 (Ce6) was obtained from Frontier Scientific Inc. (USA). RPMI-1640, fetal bovine serum (FBS), penicillin, trypsin, ethylenediaminetetraacetic acid (EDTA), and streptomycin were purchased from Welgene Inc. (Korea). Wheat germ agglutinin Alexa Fluor® 488 conjugate (WGA-Alexa Fluor® 488) was purchased from Life Technologies (USA). A Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies Inc. (Japan) [7,13-16].
2.2. Liposome preparation using the magnetoporation method PC (20 mg) dissolved in chloroform (5 mL) was added to a round-bottomed flask. The solvent in the round-bottomed flask was removed using a rotary evaporator (EYELA, N-1000, Fisher Scientific Inc., USA) [7]. The obtained thin PC film was rehydrated with 150 mM PBS (pH 7.4, 10 mL) containing Fe3O4 nanoparticles (0.5 mg, dispersed in 0.1 mL of toluene) using a sonicator (60 Hz for 5 min) at 25 C. The toluene in the round-bottomed flask was removed using a rotary evaporator. The resulting liposomes were stirred using a custom-made magnetic impeller (radius of 6
30 mm and thickness of 2 mm) [7] at 1500 rpm for 1 min to produce the magnetoporated liposomes. Under the magnetic shear stress, the Fe3O4 nanoparticles in the liposomes perforated a liposome membrane, resulting in holes in the lipid bilayer. The Fe3O4 nanoparticles that leaked from the magnetoporated liposomes were removed using ring-shaped, neodymium rare-earth magnets (radius of 10 mm and thickness of 10 mm) attached to the bottom of a flask [7]. Next, during the recovery period of lipid bilayer hole, the magnetoporated liposomes were mixed with poloxamer (16 mg), Ce6 [8 mg or 0 mg (as a control)] or PFH [48 mg or 0 mg (as a control)] in 150 mM PBS (pH 7.4, 5 mL) for 2 h at 25 C and then the opened pores of the magnetoporated liposomes were recovered at 37 C for 1 h, finally yielding liposomes encapsulating Ce6 and PFH (hereafter denoted as mLipo-CP). In addition, we prepared liposomes that encapsulated only Ce6 (hereafter denoted as mLipo-C) and liposomes without Ce6 and PFH (hereafter denoted as mLipo) as control groups. The resulting solution was transferred to a pre-swollen dialysis membrane tube (Spectra/Por MWCO 1K) and dialyzed against deionized water for 24 h and then lyophilized [13,14,16-20]. The loading of Ce6 in the liposomes was measured using a fluorescence RF-5301PC spectrofluorometer (Shimadzu, Japan) at λex 450 nm and λem 670 using a 90/10 (vol %) solution of DMSO/PBS (150 mM, pH 7.4) [15,21,22]. Loading of the PFH in the liposomes was measured using a Lambda1050 UV/Vis spectrophotometer (Perkin Elmer, USA) at 210 nm after extracting PFH from the lipophilized liposomes using acetone. The Ce6 or PFH loading efficiency (%) was defined as the weight percentage of Ce6 or PFH entrapped in the liposomes relative to the initial amount of Ce6 or PFH. The Ce6 or PFH loading content (%) was calculated as the weight percent ratio of Ce6 or PFH in the liposomes [16,20,21,23].
2.3. Characterization of liposome The particle size distribution of liposomes (0.1 mg/mL, PBS) preheated at 37-50 C for 30 min was measured using a Zetasizer 3000 instrument (Malvern Instruments, USA) at each temperature, 7
which was equipped with a He-Ne laser beam at a wavelength of 633 nm and a fixed scattering angle of 90°. The morphologies of the liposomes (0.1 mg/mL) that were exposed at 37-50 C for 30 min were confirmed using a JEM 1010 transmission electron microscope (TEM, JEOL, Japan) [7,13,14,16,17,20,24].
2.4. In vitro Ce6 release The liposomes (equivalent Ce6 1 mg/mL) were suspended in 1 mL of PBS (150 mM, pH 7.4) and preheated at 37 or 50 C for 30 min [7,13,14,16,17,20,24]. The liposome solution was added to a pre-swollen dialysis membrane tube (Spectra/Por MWCO 1000K), and immersed in 15 mL of fresh PBS (150 mM, pH 7.4) [7,13,14,16,17,20,24]. The solution was placed in a shaking water bath at 70 rpm and each temperature of 37 °C or 50 °C. To preserve the sink condition of Ce6, the outer phase was replaced with fresh buffer solution at each time point. The amount of Ce6 released in each solution was monitored using a fluorescence spectrophotometer at 450 and 670 nm (fluorescence) wavelengths using a 90/10 (vol %) solution of DMSO/PBS (150 mM, pH 7.4) as a co-solvent [1316,21,24,25].
2.5. Singlet oxygen generation Generation of singlet oxygen from the liposomes (equivalent Ce6 10 g/mL) or free Ce6 (10 g/mL) at 37 C or 50 C was confirmed using 9,10-dimethylanthracene. Before the test, each sample was preheated at 37 C or 50 C for 30 min. 9,10-dimethylanthracene (20 mmol) was mixed with each type of fabricated liposomes (equivalent Ce6 10 g/mL) or free Ce6 (10 g/mL) in PBS (150 mM, pH 7.4). The solution was illuminated at a light intensity of 5.2 mW/cm2 using a 670 nm laser source for 10 min. When the 9,10-dimethylanthracene fluorescence intensity (measured using a fluorescence RF-5301PC spectrofluorometer at λex 360 nm and λem 380-550 nm) reached a plateau after 1 h, the change in 9,10-dimethylanthracene fluorescence intensity (Ff - Fs) was plotted after 8
subtracting the fluorescence intensity (Fs) of each sample from the full 9,10-dimethylanthracene fluorescence intensity (without Ce6, indicating no singlet oxygen, Ff) [15,22,26].
2.6. In vitro cellular uptake The liposomes were preheated at 50 °C for 30 min and then 37 °C for 30 min. The cells were incubated with the liposomes (equivalent Ce6 10 μg/mL) or free Ce6 (10 μg/mL) dispersed in RPMI1640 at 37 °C, for 4 h. The treated cells were stained using DAPI and WGA-Alexa Fluor® 488 to visualize the cell nuclei and cell membranes. The cells were washed three times with fresh PBS (pH 7.4) and then examined using a confocal laser-scanning microscope (Carl Zeiss LSM710, Germany). The cellular uptake of the liposomes (4 h incubation) was quantified by flow cytometry (BD FACS Canto II, BD Biosciences, USA) and the data were analyzed using FACS Diva software (BD Biosciences, USA) [14-16,18,20,21].
2.7. In vitro phototoxicity Human nasopharyngeal epidermal carcinoma KB tumor cells (from the Korean Cell Line Bank) were maintained in RPMI-1640 medium with 1 % penicillin-streptomycin, and 10 % FBS in a humidified standard incubator with a 5 % CO2 atmosphere at 37°C. Prior to testing, KB tumor cells (1105 cells/mL) suspended in RPMI-1640 medium were seeded onto well plates and cultured for 24 h. The phototoxicity of each liposome or free Ce6 with light illumination was tested in the KB tumor cells. Before dispersing in RPMI-1640, each liposome preparation was preheated at 50 °C for 30 min and then 37 °C for 30 min. The liposomes (equivalent Ce6 10 μg/mL) or free Ce6 (10 μg/mL) dispersed in RPMI-1640 medium was administered to cells cultured in 96-well plates. After incubation for 4 h, the cells were washed three times with PBS (pH 7.4). The cells were illuminated or not illuminated at a light intensity of 5.2 mW/cm2 using a 670 nm NIR laser for 10 min and then further incubated for 12 h. Cell viability was tested using a Cell Counting Kit-8 (CCK-8 assay) [139
16,20,21,27].
2.8. In vivo tumor inhibition In vivo studies were conducted in 6- to 7-week-old female nude mice (BALB/c nu/nu mice, Institute of Medical Science, Tokyo). The nude mice were maintained under the guidelines of an approved protocol from the Institutional Animal Care and Use Committee (IACUC) of the Catholic University of Korea (Republic of Korea) [16,20,25]. The liposomes (equivalent Ce6 10 mg/kg, preheated at 50 °C for 30 min and then 37 °C for 30 min) or free Ce6 (10 mg/kg) were intratumorally injected into the KB tumor-bearing nude mice (tumor volume ~ 150 mm3). At 4 h post-injection of each sample, the tumor site of the nude mice was illuminated at a light intensity of 5.2 mW/cm2 with a 670 nm NIR laser for 30 min. Tumor volume was calculated using the formula: tumor volume=length×(width)2/2 [13,16,20,24-26].
2.9. Statistical evaluation All of the results were analyzed via Student’s t-test or ANOVA at a significance level of p < 0.01 (**). The MINITAP® release 14 statistical software program was used for all statistical analysis [13,21,26].
3. Results and Discussion 3.1. Preparation of mLipo-CP In this study, we prepared gas-form liposomes using the magnetoporation method described in our previous report [7]. The magnetoporated liposomes obtained using superparamagnetic Fe3O4 nanoparticles and a magnetic impeller with a tailor-made magnet (radius of 30 mm and thickness of 2 mm) [7] were utilized to encapsulate Ce6 and PFH. The opened holes of the magnetoporated 10
liposomes enabled a facile encapsulation of Ce6 and PFH before the natural recovery (closing) of the liposomal membranes (Fig. 1). As a result, the loading efficiency and loading contents of Ce6 in mLipo-CP were 86.1 wt.% and 9.8 wt.%, respectively. The loading efficiency and loading content of the PFH in mLipo-CP were 24.3 wt.% and 15.9 wt.%, respectively. The loading efficiency and loading contents of Ce6 in mLipo-C were 83.4 wt.% and 9.5 wt.%, respectively. These results are comparable with that of the conventional liposome (prepared via film rehydration of the lipid film using PBS containing PFH at 60 Hz sonication) [7], which exhibited 5-7 % PFH loading efficiency (data not shown). In particular, the resulting liposomes are expected to respond to external stimuli (i.e., hyperthermia condition) and accelerate the drug release rate as a result of their destabilization due to the vaporization of encapsulated PFH [2,5,12] at 50 C (Fig. 1).
3.2. Characterization of mLipo-CP To verify our proposed mechanism (Fig. 1), we firstly investigated the changes in particle size and morphology of the liposomes according to the temperature of the solution. The average particle sizes of the liposomes at 37 C were approximately 72 nm (mLipo-CP), 50 nm (mLipo-C), and 47 nm (mLipo) in diameter (Fig. 2). It was observed that the particle size of mLipo and mLipo-C were not significantly different at 37-50 C (Fig. 2a). However, the particle size of mLipo-CP (preheated at 50 C for 30 min) rapidly increased to an average diameter of 500 nm upon elevating the temperature from 37 C to 50 C (Fig. 2b). In additon, the particle size of mLipo-CP preheated at 40 C for 30 min was not significantly different with that of mLipo-CP preheated at 37 C for 30 min (data not shown). It is thought that the vaporization of PFH at 50 C caused volumetric expansion of liposomes. Indeed, the TEM images demonstrated that mLipo-CP at 50 C was destabilized and volumetrically expanded, but the other liposomes (mLipo-C) showed negligible change in their morphologies at 37 and 50 C (Fig. 3). The results suggest that the vaporization of PFH at 50 C can influence the drug release rate from the mLipo-CP. 11
3.3. In vitro Ce6 release In vitro drug release profiles from mLipo-CP, mLipo-C as a function of the time were monitored in PBS incubated at 37 C. As shown in Fig. 4, at 4 h incubation, approximately 40 % and 30 % of Ce6 was passively released from mLipo-CP and mLipo-C, respectively (Fig. 4a). However, the hyperthermia (50 C for 30 min) generated a 70 % (from mLipo-CP) and 30 % (from mLip-C) Ce6 release at 4 h incubation (Fig. 4b). These data support that mLipo-CP responded to the hyperthermia and actively released Ce6. In addition, mLipo-CP and mLipo-C reached a drug release plateau in 8 h at both 37 C and 50 C. Fig. 5 shows the singlet oxygen generation from the liposomes during light illumination. To estimate their tendency for photo-induced singlet oxygen generation, the liposomes (mLipo-CP and mLipo-C) or free Ce6 (as a control group) dispersed in PBS were exposed to light illumination (5.2 mW/cm2 at 670 nm for 10 min) [13,15,18,20-23]. As expected, mLipo-C showed low singlet oxygen generation due to the auto-quenching effect [15,20,23] of the Ce6 molecules densely populated in the compacted liposomal core. However, mLipo-CP (preheated at 50 C for 30 min) resulted in tremendous singlet oxygen generation, probably due to the Ce6 release and the Ce6 distributed in enlarged liposomal core at 50 C. In addition, free Ce6 showed less singlet oxygen generation due to its self-aggregation (i.e., auto-quenched Ce6 molecules) in PBS [14,18,20,23-26].
3.4. In vitro phototoxicity of mLipo-CP Fig. 6 shows the extensive internalization of Ce6 molecules entrapped in mLipo-CP for KB tumor cells. The confocal images revealed that free Ce6 was passively diffused to the cells and resulted in relatively low cellular uptake. However, mLipo-CP (preheated at 50 C for 30 min) showed extensive internalization in KB tumor cells, indicating that the lipophilic interaction between the cells and the liposomes [1,4,8,9] (damaged after pretreatment at 50 C for 30 min) 12
facilitated the cellular uptake of Ce6 molecules. Importantly, Ce6 molecules entrapped in the compacted liposomal cores of mLipo-C were assumed to be at the auto-quenched condition [15,20,22,23], resulting in relatively low Ce6 fluorescence (Fig. 6a). The quantitative results of the cellular uptake of the liposomes (using a FACSCalibur™ flow cytometer) [13,16,20,26] demonstrated that the average fluorescence intensity of mLipo-CP (preheated at 50 C for 30 min) was approximately 5.6 times and 10.4 times higher than that observed for free Ce6 and mLipo-C (preheated at 50 C for 30 min), respectively (Fig. 6b). These results strongly support that mLipoCP is effective in improving Ce6 uptake in KB tumors cells. Fig. 7a shows the phototoxic activity of mLipo-CP for KB tumor cells. Under light illumination (5.2 mW/cm2 at 670 nm for 10 min) [14-16,21,22], free Ce6 and mLipo-C induced less phototoxicity in KB tumors cells as a result of the down-regulated singlet oxygen generation [15,17,20,25]. However, mLipo-CP was active in ablating KB tumor cells. mLipo-CP (preheated at 50 C for 30 min) showed 2.8 and 3.3 times higher phototoxicities than free Ce6 and mLipo-C (preheated at 50 C for 30 min), respectively. Prior to light illumination, mLipo-C and mLipo-CP did not show any significant cytotoxicity (up to 100 g/mL) against the KB tumor cells (Fig. 7b), indicating the non-toxicity of the liposomes [15,17,18,20,22,25,26].
3.5. In vivo phototoxicity of mLipo-CP Fig. 8 shows the in vivo results for the anti-tumor activity of Ce6 carried by the mLipo-CP. Here, the liposomes (preheated at 50 °C for 30 min and then 37 °C for 30 min) or free Ce6 were intratumorally injected into the KB tumor-bearing nude mice. In particular, the intratumoral injection route was utilized to evaluate the antitumor efficacy of the preheated mLipo-CP in local tumor environment that can be heated using external ultrasound systems [28]. At 4 h post-injection of each sample, solid tumors were locally illuminated at a light intensity of 5.2 mW/cm2 using a 670 nm laser source for 30 min, and then tumor volume regression was monitored daily for 6 days. 13
Here, any skin-burning morphology was not observed for the treated nude mice during light illumination. The data demonstrated that the administration of mLipo-CP (preheated at 50 C for 30 min) led to significant regression of the KB tumors. At 6 days post-injection, the tumor volumes of the mice treated with mLipo-CP (preheated at 50 C for 30 min) were approximately 1.7 times and 2.7 times smaller than those treated with free Ce6 and mLipo-C, respectively. These results are consistent with the in vitro cell viability tests (Fig. 6). Neither complete tumor growth regression nor toxicity-induced death was observed in any of the mice treated with the liposomes or free Ce6 (data not shown) [13-18,20].
Conclusion In this study, the magnetoporated liposomes prepared using superparamagnetic Fe3O4 nanoparticles and a magnetic impeller were optimized to efficiently encapsulate a photosensitizing drug (Ce6) and a volatile gas-forming oil (PFH) through the lipid bilayer holes in magnetoporated liposome membranes. The resulting liposomes induced hyperthermia-dependent drug release and improved in vivo photodynamic therapy at the tumor site. The collective results from a series of both in vitro and in vivo studies strongly suggest that these liposomes hold great promise for use in the selective delivery of drugs to tumor sites under local hyperthermia.
Acknowledgment This work was financially supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: no HI14C1835).
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Figure Captions
Fig. 1. Schematic concept of the gas-forming liposomes. Fig. 2. Changes in the particle size distributions of (a) mLipo-C and (b) mLipo-CP dispersed in PBS (150 mM, pH 7.4) according to the temperature (37-50 C). Fig. 3. TEM images of (a) mLipo-C and (b) mLipo-CP at 37 C and 50 C. Fig. 4. Cumulative Ce6 release profiles from mLipo-C or mLipo-CP (equivalent Ce6 1 mg/mL) preheated at (a) 37 C or (b) 50 C for 30 min (SD, n=3). Fig. 5. The 9,10-dimethylanthracene fluorescence change (at λex 360 nm and λem 380-550 nm) of the liposomes (equivalent Ce6 10 g/mL) and free Ce6 (10 g/mL) in PBS (150 mM, pH 7.4) preheated at (a) 37 C or (b) 50 C for 30 min. Singlet oxygen generation is indicated by the change in 9,10dimethylanthracene fluorescence intensity (Ff-Fs). Fig. 6. (a) Confocal images and (b) flow cytometry analysis of KB tumor cells treated with the liposomes (equivalent Ce6 10 μg/mL) or free Ce6 (10 μg/mL) for 4 h at 37 C. Before adding the liposomes to the cells, the liposomes were preheated at 50 C for 30 min and then at 37 C for 30 min. Fig. 7. (a) Phototoxicity and cell viability determined by a CCK-8 assay of KB cells treated with the liposomes (equivalent Ce6 1-10 μg/mL) or free Ce6 (1-10 μg/mL). Before adding the liposomes to the cells, the liposomes were preheated at 50 C for 30 min and then at 37 C for 30 min. All of the cells were illuminated for 10 min at a light intensity of 5.2 mW/cm2 using a 670 nm laser source (SD, n=7) (** p < 0.01 compared to free Ce6). (b) Cell viability of the KB cells treated with the liposomes (1-100 μg/mL) without light illumination for 24 h (SD, n=7). Fig. 8. Tumor volume changes in the KB-tumor-bearing nude mice locally irradiated (at 4 h postinjection) with each liposome (preheated at 50 C for 30 min and then at 37 C for 30 min, equivalent Ce6 10 mg/kg) or free Ce6 (10 mg/kg) (SD, n=5) (** p < 0.01 compared to free Ce6).
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S0927-7765(17)30204-7 http://dx.doi.org/doi:10.1016/j.colsurfb.2017.04.017 COLSUB 8484
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
19-1-2017 6-4-2017 10-4-2017
Please cite this article as: Jae Min Lee, Dong Sup Kwag, Yu Seok Youn, Eun Seong Lee, Gas-forming liposomes prepared using a liposomal magnetoporation method, Colloids and Surfaces B: Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.04.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Gas-forming liposomes prepared using a liposomal magnetoporation method Jae Min Lee a, Dong Sup Kwag a, Yu Seok Youn b, Eun Seong Lee a, *
a
Department of Biotechnology, The Catholic University of Korea, 43-1 Yeokgok 2-dong, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea b
School of Pharmacy, Sungkyunkwan University, 300 Chonchon-dong, Jangan-ku, Suwon, Gyeonggi-do 440-746, Republic of Korea
* To whom correspondence should be addressed. Tel: +82-2-2164-4921 Fax: +82-2-2164-4865 E-mail: [email protected] (E.S. Lee)
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Graphical abstract
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Research Highlights ► We developed gas-forming liposomes using magnetoporated liposomes ► The liposomes induced hyperthermia-dependent drug release and enhanced tumor ablation. ► The liposomes hold great promise for use in the selective delivery of drugs to tumor sites under local hyperthermia.
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Abstract In this study, we report a gas-forming drug carrier engineered using the liposomal magnetoporation method. The liposomes that were magnetoporated under a magnetic shear stress possessed an opened lipid bilayer hole. A photosensitizing model drug (chlorin e6: Ce6) and 1H-1H-2H-perfluoro-1hexene (PFH, as a volatile gas-forming agent) were efficiently loaded into the opened holes of the magnetoporated liposomes. PFH in the liposomes is vaporized at 50 C and can initiate liposome destabilization. The experimental results demonstrated that the liposomes were destabilized at 50 C efficiently released Ce6 and enhanced Ce6-mediated phototoxicity against KB tumor cells. As a result, these liposomes induced a significantly increased in vitro and in vivo photodynamic tumor inhibition.
Keywords: Liposomal magnetoporation, gas-forming, perfluorohexene, chlorin e6, photodynamic therapy
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1. Introduction An important purpose of advanced drug delivery systems is to suitably deliver drugs to a particular position in the body, which demands the use of proper vehicles that efficiently encapsulate drugs and release drugs in response to biological signals [1-3]. Over the last few decades, liposomes consisting of lipid bilayer containers have been used as carriers for chemical or biological drugs and have received much attention as essential tools for successful drug delivery [1-6]. In particular, various investigations for flexible bilayer membranes and dynamic properties (such as fusion, fission, and shape deformation) of liposomes offer various possibilities for developing advanced drug carrying systems [7]. Indeed, such trials have extended liposome applications into diverse fields such as pharmaceutics and cosmetics [3,8,9]. Recently, our group developed partially uncapped liposomes by applying magnetic shear stress to liposomes containing Fe3O4 nanoparticles [7]. These liposomes displayed an opened lipid bilayer hole in a liposome membrane as a result of the leakage of Fe3O4 nanoparticles. This magnetoporation method is a valuable tool for preparing porous liposomes and can provide an opened entrance for chemicals or biological drugs into the liposome before the natural recovery (closing) of the lipid bilayer hole [7]. Perfluorohexene [1H-1H-2H-perfluoro-1-hexene (PFH)] is stable liquid oil at room temperature. However, PFH is easily vaporized near its boiling point at 50 C [2,5,10-12]. Importantly, PFH can be used to prepare nano/micro-sized bubbles and to provide high vapor pressure (at the hyperthermia condition, ~50 C) in nano-sized containers for drug release. However, it is difficult to load PFH into nano-sized containers because of its high lipophilicity and liquid oil state [2,5,11,12]. In this study, we utilized magnetoporated liposomes (prepared using the magnetoporation method) as an opened liposomal template for preparing gas (PFH)-forming liposomes and for establishing hyperthermia-sensitive drug-encapsulating liposome. Here, we encapsulated a photosensitizing antitumor drug (chlorin e6: Ce6) and PFH into the magnetoporated liposomes. It is 5
anticipated that the efficiently loaded PFH in the magnetoporated liposomes would be vaporized at 50 C and destabilize the liposomes, resulting in an increased drug release rate (Fig. 1). We preferentially investigated hyperthermia-induced liposomal destabilization, drug release behaviors, and Ce6-mediated phototoxicity of KB tumors.
2. Materials and methods 2.1. Materials Phosphatidylcholine (PC), dimethylsulfoxide (DMSO), chloroform, acetone, poloxamer 188, 1H-1H-2H-perfluoro-1-hexene (PFH), iron oxide (II, III) magnetic nanoparticles (average 5 nm in diameter), and 9,10-dimethylanthracene were obtained from Sigma-Aldrich (USA). Chlorin e6 (Ce6) was obtained from Frontier Scientific Inc. (USA). RPMI-1640, fetal bovine serum (FBS), penicillin, trypsin, ethylenediaminetetraacetic acid (EDTA), and streptomycin were purchased from Welgene Inc. (Korea). Wheat germ agglutinin Alexa Fluor® 488 conjugate (WGA-Alexa Fluor® 488) was purchased from Life Technologies (USA). A Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies Inc. (Japan) [7,13-16].
2.2. Liposome preparation using the magnetoporation method PC (20 mg) dissolved in chloroform (5 mL) was added to a round-bottomed flask. The solvent in the round-bottomed flask was removed using a rotary evaporator (EYELA, N-1000, Fisher Scientific Inc., USA) [7]. The obtained thin PC film was rehydrated with 150 mM PBS (pH 7.4, 10 mL) containing Fe3O4 nanoparticles (0.5 mg, dispersed in 0.1 mL of toluene) using a sonicator (60 Hz for 5 min) at 25 C. The toluene in the round-bottomed flask was removed using a rotary evaporator. The resulting liposomes were stirred using a custom-made magnetic impeller (radius of 6
30 mm and thickness of 2 mm) [7] at 1500 rpm for 1 min to produce the magnetoporated liposomes. Under the magnetic shear stress, the Fe3O4 nanoparticles in the liposomes perforated a liposome membrane, resulting in holes in the lipid bilayer. The Fe3O4 nanoparticles that leaked from the magnetoporated liposomes were removed using ring-shaped, neodymium rare-earth magnets (radius of 10 mm and thickness of 10 mm) attached to the bottom of a flask [7]. Next, during the recovery period of lipid bilayer hole, the magnetoporated liposomes were mixed with poloxamer (16 mg), Ce6 [8 mg or 0 mg (as a control)] or PFH [48 mg or 0 mg (as a control)] in 150 mM PBS (pH 7.4, 5 mL) for 2 h at 25 C and then the opened pores of the magnetoporated liposomes were recovered at 37 C for 1 h, finally yielding liposomes encapsulating Ce6 and PFH (hereafter denoted as mLipo-CP). In addition, we prepared liposomes that encapsulated only Ce6 (hereafter denoted as mLipo-C) and liposomes without Ce6 and PFH (hereafter denoted as mLipo) as control groups. The resulting solution was transferred to a pre-swollen dialysis membrane tube (Spectra/Por MWCO 1K) and dialyzed against deionized water for 24 h and then lyophilized [13,14,16-20]. The loading of Ce6 in the liposomes was measured using a fluorescence RF-5301PC spectrofluorometer (Shimadzu, Japan) at λex 450 nm and λem 670 using a 90/10 (vol %) solution of DMSO/PBS (150 mM, pH 7.4) [15,21,22]. Loading of the PFH in the liposomes was measured using a Lambda1050 UV/Vis spectrophotometer (Perkin Elmer, USA) at 210 nm after extracting PFH from the lipophilized liposomes using acetone. The Ce6 or PFH loading efficiency (%) was defined as the weight percentage of Ce6 or PFH entrapped in the liposomes relative to the initial amount of Ce6 or PFH. The Ce6 or PFH loading content (%) was calculated as the weight percent ratio of Ce6 or PFH in the liposomes [16,20,21,23].
2.3. Characterization of liposome The particle size distribution of liposomes (0.1 mg/mL, PBS) preheated at 37-50 C for 30 min was measured using a Zetasizer 3000 instrument (Malvern Instruments, USA) at each temperature, 7
which was equipped with a He-Ne laser beam at a wavelength of 633 nm and a fixed scattering angle of 90°. The morphologies of the liposomes (0.1 mg/mL) that were exposed at 37-50 C for 30 min were confirmed using a JEM 1010 transmission electron microscope (TEM, JEOL, Japan) [7,13,14,16,17,20,24].
2.4. In vitro Ce6 release The liposomes (equivalent Ce6 1 mg/mL) were suspended in 1 mL of PBS (150 mM, pH 7.4) and preheated at 37 or 50 C for 30 min [7,13,14,16,17,20,24]. The liposome solution was added to a pre-swollen dialysis membrane tube (Spectra/Por MWCO 1000K), and immersed in 15 mL of fresh PBS (150 mM, pH 7.4) [7,13,14,16,17,20,24]. The solution was placed in a shaking water bath at 70 rpm and each temperature of 37 °C or 50 °C. To preserve the sink condition of Ce6, the outer phase was replaced with fresh buffer solution at each time point. The amount of Ce6 released in each solution was monitored using a fluorescence spectrophotometer at 450 and 670 nm (fluorescence) wavelengths using a 90/10 (vol %) solution of DMSO/PBS (150 mM, pH 7.4) as a co-solvent [1316,21,24,25].
2.5. Singlet oxygen generation Generation of singlet oxygen from the liposomes (equivalent Ce6 10 g/mL) or free Ce6 (10 g/mL) at 37 C or 50 C was confirmed using 9,10-dimethylanthracene. Before the test, each sample was preheated at 37 C or 50 C for 30 min. 9,10-dimethylanthracene (20 mmol) was mixed with each type of fabricated liposomes (equivalent Ce6 10 g/mL) or free Ce6 (10 g/mL) in PBS (150 mM, pH 7.4). The solution was illuminated at a light intensity of 5.2 mW/cm2 using a 670 nm laser source for 10 min. When the 9,10-dimethylanthracene fluorescence intensity (measured using a fluorescence RF-5301PC spectrofluorometer at λex 360 nm and λem 380-550 nm) reached a plateau after 1 h, the change in 9,10-dimethylanthracene fluorescence intensity (Ff - Fs) was plotted after 8
subtracting the fluorescence intensity (Fs) of each sample from the full 9,10-dimethylanthracene fluorescence intensity (without Ce6, indicating no singlet oxygen, Ff) [15,22,26].
2.6. In vitro cellular uptake The liposomes were preheated at 50 °C for 30 min and then 37 °C for 30 min. The cells were incubated with the liposomes (equivalent Ce6 10 μg/mL) or free Ce6 (10 μg/mL) dispersed in RPMI1640 at 37 °C, for 4 h. The treated cells were stained using DAPI and WGA-Alexa Fluor® 488 to visualize the cell nuclei and cell membranes. The cells were washed three times with fresh PBS (pH 7.4) and then examined using a confocal laser-scanning microscope (Carl Zeiss LSM710, Germany). The cellular uptake of the liposomes (4 h incubation) was quantified by flow cytometry (BD FACS Canto II, BD Biosciences, USA) and the data were analyzed using FACS Diva software (BD Biosciences, USA) [14-16,18,20,21].
2.7. In vitro phototoxicity Human nasopharyngeal epidermal carcinoma KB tumor cells (from the Korean Cell Line Bank) were maintained in RPMI-1640 medium with 1 % penicillin-streptomycin, and 10 % FBS in a humidified standard incubator with a 5 % CO2 atmosphere at 37°C. Prior to testing, KB tumor cells (1105 cells/mL) suspended in RPMI-1640 medium were seeded onto well plates and cultured for 24 h. The phototoxicity of each liposome or free Ce6 with light illumination was tested in the KB tumor cells. Before dispersing in RPMI-1640, each liposome preparation was preheated at 50 °C for 30 min and then 37 °C for 30 min. The liposomes (equivalent Ce6 10 μg/mL) or free Ce6 (10 μg/mL) dispersed in RPMI-1640 medium was administered to cells cultured in 96-well plates. After incubation for 4 h, the cells were washed three times with PBS (pH 7.4). The cells were illuminated or not illuminated at a light intensity of 5.2 mW/cm2 using a 670 nm NIR laser for 10 min and then further incubated for 12 h. Cell viability was tested using a Cell Counting Kit-8 (CCK-8 assay) [139
16,20,21,27].
2.8. In vivo tumor inhibition In vivo studies were conducted in 6- to 7-week-old female nude mice (BALB/c nu/nu mice, Institute of Medical Science, Tokyo). The nude mice were maintained under the guidelines of an approved protocol from the Institutional Animal Care and Use Committee (IACUC) of the Catholic University of Korea (Republic of Korea) [16,20,25]. The liposomes (equivalent Ce6 10 mg/kg, preheated at 50 °C for 30 min and then 37 °C for 30 min) or free Ce6 (10 mg/kg) were intratumorally injected into the KB tumor-bearing nude mice (tumor volume ~ 150 mm3). At 4 h post-injection of each sample, the tumor site of the nude mice was illuminated at a light intensity of 5.2 mW/cm2 with a 670 nm NIR laser for 30 min. Tumor volume was calculated using the formula: tumor volume=length×(width)2/2 [13,16,20,24-26].
2.9. Statistical evaluation All of the results were analyzed via Student’s t-test or ANOVA at a significance level of p < 0.01 (**). The MINITAP® release 14 statistical software program was used for all statistical analysis [13,21,26].
3. Results and Discussion 3.1. Preparation of mLipo-CP In this study, we prepared gas-form liposomes using the magnetoporation method described in our previous report [7]. The magnetoporated liposomes obtained using superparamagnetic Fe3O4 nanoparticles and a magnetic impeller with a tailor-made magnet (radius of 30 mm and thickness of 2 mm) [7] were utilized to encapsulate Ce6 and PFH. The opened holes of the magnetoporated 10
liposomes enabled a facile encapsulation of Ce6 and PFH before the natural recovery (closing) of the liposomal membranes (Fig. 1). As a result, the loading efficiency and loading contents of Ce6 in mLipo-CP were 86.1 wt.% and 9.8 wt.%, respectively. The loading efficiency and loading content of the PFH in mLipo-CP were 24.3 wt.% and 15.9 wt.%, respectively. The loading efficiency and loading contents of Ce6 in mLipo-C were 83.4 wt.% and 9.5 wt.%, respectively. These results are comparable with that of the conventional liposome (prepared via film rehydration of the lipid film using PBS containing PFH at 60 Hz sonication) [7], which exhibited 5-7 % PFH loading efficiency (data not shown). In particular, the resulting liposomes are expected to respond to external stimuli (i.e., hyperthermia condition) and accelerate the drug release rate as a result of their destabilization due to the vaporization of encapsulated PFH [2,5,12] at 50 C (Fig. 1).
3.2. Characterization of mLipo-CP To verify our proposed mechanism (Fig. 1), we firstly investigated the changes in particle size and morphology of the liposomes according to the temperature of the solution. The average particle sizes of the liposomes at 37 C were approximately 72 nm (mLipo-CP), 50 nm (mLipo-C), and 47 nm (mLipo) in diameter (Fig. 2). It was observed that the particle size of mLipo and mLipo-C were not significantly different at 37-50 C (Fig. 2a). However, the particle size of mLipo-CP (preheated at 50 C for 30 min) rapidly increased to an average diameter of 500 nm upon elevating the temperature from 37 C to 50 C (Fig. 2b). In additon, the particle size of mLipo-CP preheated at 40 C for 30 min was not significantly different with that of mLipo-CP preheated at 37 C for 30 min (data not shown). It is thought that the vaporization of PFH at 50 C caused volumetric expansion of liposomes. Indeed, the TEM images demonstrated that mLipo-CP at 50 C was destabilized and volumetrically expanded, but the other liposomes (mLipo-C) showed negligible change in their morphologies at 37 and 50 C (Fig. 3). The results suggest that the vaporization of PFH at 50 C can influence the drug release rate from the mLipo-CP. 11
3.3. In vitro Ce6 release In vitro drug release profiles from mLipo-CP, mLipo-C as a function of the time were monitored in PBS incubated at 37 C. As shown in Fig. 4, at 4 h incubation, approximately 40 % and 30 % of Ce6 was passively released from mLipo-CP and mLipo-C, respectively (Fig. 4a). However, the hyperthermia (50 C for 30 min) generated a 70 % (from mLipo-CP) and 30 % (from mLip-C) Ce6 release at 4 h incubation (Fig. 4b). These data support that mLipo-CP responded to the hyperthermia and actively released Ce6. In addition, mLipo-CP and mLipo-C reached a drug release plateau in 8 h at both 37 C and 50 C. Fig. 5 shows the singlet oxygen generation from the liposomes during light illumination. To estimate their tendency for photo-induced singlet oxygen generation, the liposomes (mLipo-CP and mLipo-C) or free Ce6 (as a control group) dispersed in PBS were exposed to light illumination (5.2 mW/cm2 at 670 nm for 10 min) [13,15,18,20-23]. As expected, mLipo-C showed low singlet oxygen generation due to the auto-quenching effect [15,20,23] of the Ce6 molecules densely populated in the compacted liposomal core. However, mLipo-CP (preheated at 50 C for 30 min) resulted in tremendous singlet oxygen generation, probably due to the Ce6 release and the Ce6 distributed in enlarged liposomal core at 50 C. In addition, free Ce6 showed less singlet oxygen generation due to its self-aggregation (i.e., auto-quenched Ce6 molecules) in PBS [14,18,20,23-26].
3.4. In vitro phototoxicity of mLipo-CP Fig. 6 shows the extensive internalization of Ce6 molecules entrapped in mLipo-CP for KB tumor cells. The confocal images revealed that free Ce6 was passively diffused to the cells and resulted in relatively low cellular uptake. However, mLipo-CP (preheated at 50 C for 30 min) showed extensive internalization in KB tumor cells, indicating that the lipophilic interaction between the cells and the liposomes [1,4,8,9] (damaged after pretreatment at 50 C for 30 min) 12
facilitated the cellular uptake of Ce6 molecules. Importantly, Ce6 molecules entrapped in the compacted liposomal cores of mLipo-C were assumed to be at the auto-quenched condition [15,20,22,23], resulting in relatively low Ce6 fluorescence (Fig. 6a). The quantitative results of the cellular uptake of the liposomes (using a FACSCalibur™ flow cytometer) [13,16,20,26] demonstrated that the average fluorescence intensity of mLipo-CP (preheated at 50 C for 30 min) was approximately 5.6 times and 10.4 times higher than that observed for free Ce6 and mLipo-C (preheated at 50 C for 30 min), respectively (Fig. 6b). These results strongly support that mLipoCP is effective in improving Ce6 uptake in KB tumors cells. Fig. 7a shows the phototoxic activity of mLipo-CP for KB tumor cells. Under light illumination (5.2 mW/cm2 at 670 nm for 10 min) [14-16,21,22], free Ce6 and mLipo-C induced less phototoxicity in KB tumors cells as a result of the down-regulated singlet oxygen generation [15,17,20,25]. However, mLipo-CP was active in ablating KB tumor cells. mLipo-CP (preheated at 50 C for 30 min) showed 2.8 and 3.3 times higher phototoxicities than free Ce6 and mLipo-C (preheated at 50 C for 30 min), respectively. Prior to light illumination, mLipo-C and mLipo-CP did not show any significant cytotoxicity (up to 100 g/mL) against the KB tumor cells (Fig. 7b), indicating the non-toxicity of the liposomes [15,17,18,20,22,25,26].
3.5. In vivo phototoxicity of mLipo-CP Fig. 8 shows the in vivo results for the anti-tumor activity of Ce6 carried by the mLipo-CP. Here, the liposomes (preheated at 50 °C for 30 min and then 37 °C for 30 min) or free Ce6 were intratumorally injected into the KB tumor-bearing nude mice. In particular, the intratumoral injection route was utilized to evaluate the antitumor efficacy of the preheated mLipo-CP in local tumor environment that can be heated using external ultrasound systems [28]. At 4 h post-injection of each sample, solid tumors were locally illuminated at a light intensity of 5.2 mW/cm2 using a 670 nm laser source for 30 min, and then tumor volume regression was monitored daily for 6 days. 13
Here, any skin-burning morphology was not observed for the treated nude mice during light illumination. The data demonstrated that the administration of mLipo-CP (preheated at 50 C for 30 min) led to significant regression of the KB tumors. At 6 days post-injection, the tumor volumes of the mice treated with mLipo-CP (preheated at 50 C for 30 min) were approximately 1.7 times and 2.7 times smaller than those treated with free Ce6 and mLipo-C, respectively. These results are consistent with the in vitro cell viability tests (Fig. 6). Neither complete tumor growth regression nor toxicity-induced death was observed in any of the mice treated with the liposomes or free Ce6 (data not shown) [13-18,20].
Conclusion In this study, the magnetoporated liposomes prepared using superparamagnetic Fe3O4 nanoparticles and a magnetic impeller were optimized to efficiently encapsulate a photosensitizing drug (Ce6) and a volatile gas-forming oil (PFH) through the lipid bilayer holes in magnetoporated liposome membranes. The resulting liposomes induced hyperthermia-dependent drug release and improved in vivo photodynamic therapy at the tumor site. The collective results from a series of both in vitro and in vivo studies strongly suggest that these liposomes hold great promise for use in the selective delivery of drugs to tumor sites under local hyperthermia.
Acknowledgment This work was financially supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: no HI14C1835).
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Figure Captions
Fig. 1. Schematic concept of the gas-forming liposomes. Fig. 2. Changes in the particle size distributions of (a) mLipo-C and (b) mLipo-CP dispersed in PBS (150 mM, pH 7.4) according to the temperature (37-50 C). Fig. 3. TEM images of (a) mLipo-C and (b) mLipo-CP at 37 C and 50 C. Fig. 4. Cumulative Ce6 release profiles from mLipo-C or mLipo-CP (equivalent Ce6 1 mg/mL) preheated at (a) 37 C or (b) 50 C for 30 min (SD, n=3). Fig. 5. The 9,10-dimethylanthracene fluorescence change (at λex 360 nm and λem 380-550 nm) of the liposomes (equivalent Ce6 10 g/mL) and free Ce6 (10 g/mL) in PBS (150 mM, pH 7.4) preheated at (a) 37 C or (b) 50 C for 30 min. Singlet oxygen generation is indicated by the change in 9,10dimethylanthracene fluorescence intensity (Ff-Fs). Fig. 6. (a) Confocal images and (b) flow cytometry analysis of KB tumor cells treated with the liposomes (equivalent Ce6 10 μg/mL) or free Ce6 (10 μg/mL) for 4 h at 37 C. Before adding the liposomes to the cells, the liposomes were preheated at 50 C for 30 min and then at 37 C for 30 min. Fig. 7. (a) Phototoxicity and cell viability determined by a CCK-8 assay of KB cells treated with the liposomes (equivalent Ce6 1-10 μg/mL) or free Ce6 (1-10 μg/mL). Before adding the liposomes to the cells, the liposomes were preheated at 50 C for 30 min and then at 37 C for 30 min. All of the cells were illuminated for 10 min at a light intensity of 5.2 mW/cm2 using a 670 nm laser source (SD, n=7) (** p < 0.01 compared to free Ce6). (b) Cell viability of the KB cells treated with the liposomes (1-100 μg/mL) without light illumination for 24 h (SD, n=7). Fig. 8. Tumor volume changes in the KB-tumor-bearing nude mice locally irradiated (at 4 h postinjection) with each liposome (preheated at 50 C for 30 min and then at 37 C for 30 min, equivalent Ce6 10 mg/kg) or free Ce6 (10 mg/kg) (SD, n=5) (** p < 0.01 compared to free Ce6).
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