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Folate receptor targeted biodegradable polymeric doxorubicin micelles
Journal of Controlled Release 96 (2004) 273 – 283 www.elsevier.com/locate/jconrel
Folate receptor targeted biodegradable polymeric doxorubicin micelles Hyuk Sang Yoo, Tae Gwan Park * Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea Received 12 November 2003; accepted 3 February 2004
Abstract Biodegradable polymeric micelles, self-assembled from a di-block copolymer of poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG), were prepared to achieve folate receptor targeted delivery of doxorubicin (DOX). In the diblock copolymer structure of PLGA – b-PEG, DOX was chemically conjugated to a terminal end of PLGA to produce DOX – PLGA – mPEG, and folate was separately conjugated to a terminal end of PEG to produce PLGA – PEG – FOL. The two diblock copolymers with different functional moieties at their chains ends were physically mixed with free base DOX in an aqueous solution to form mixed micelles. It was expected that folate moieties were exposed on the micellar surface, while DOX was physically and chemically entrapped in the core of micelles. Flow cytometry and confocal image analysis revealed that folate conjugated mixed micelles exhibited far greater extent of cellular uptake than folate unconjugated micelles against KB cells over-expressing folate receptors on the surface. They also showed higher cytotoxicity than DOX, suggesting that folate receptor medicated endocytosis of the micelles played an important role in transporting an increased amount of DOX within cells. In vivo animal experiments, using a nude mice xenograft model, demonstrated that when systemically administered, tumor volume was significantly regressed. Biodistribution studies also indicated that an increased amount of DOX was accumulated in the tumor tissue. D 2004 Elsevier B.V. All rights reserved. Keywords: Folate; Micelle; Doxorubicin; Targeting; PLGA
1. Introduction Folate receptor has been known to be vastly overexpressed in several human tumors [1 –3]. Folate has been popularly employed as a targeting moiety of various anti-cancer agents to avoid their non-specific attacks on normal tissues as well as to increase their cellular uptake within target cells, as studied in several * Corresponding author. Tel.: +82-42-869-2621; fax: +82-42869-2610. E-mail address: [email protected] (T.G. Park). 0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2004.02.003
previous studies [4– 7]. Folate was often covalently attached to a wide array of drug delivery carriers such as liposomes, polymer conjugates, and nano-particulates [2,6,8 –10]. Among them, anti-cancer liposomes with having surface exposed folate moieties have been extensively studied for passively targeting cancer cells. A folate group covalently attached to phospholipid or cholesterol was used to form doxorubicin (DOX) encapsulated liposomes [7,9,10]. This liposomal DOX formulation exhibited prolonged systemic circulation time and much lower cardiotoxicity than free DOX [10]. The folate-targeted liposomal DOX
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showed superior cytotoxicity to liposomal DOX formulation in cultured folate receptor (+) tumor cells in vitro. Furthermore, in a folate receptor (+) carcinoma murine xenograft model, the folate-targeted liposomal DOX demonstrated greater tumor inhibition effect with increasing the life-span of mice compared to the liposomal DOX [11,12]. Over the past decade, polymeric micelles have received much attention as an alternative delivery carrier for various drugs including anti-cancer agents [13 – 15]. Amphiphatic di-block copolymers self-assembled into polymeric micelles in an aqueous solution. The size of polymeric micelles is approximately less than 100 nm, which not only makes them ideal drug delivery carriers for escaping from renal exclusion and the reticulo-endothelial system, but also gives them an enhanced vascular permeability. Polymeric micelles are composed of a hydrophilic outer shell exposed to the aqueous phase and a hydrophobic inner core encapsulating drug molecules. For anticancer drugs such as DOX, biodegradable polymeric micelles were extensively utilized for passive targeting to solid tumors [16 – 18]. Very hydrophobic drugs such as paclitaxel were also successfully solubilized within the core of micelles to enhance their water solubility for the intravenous injection [19]. In this study, biodegradable DOX polymeric micelles having a targeting ability of folate receptor were prepared. DOX was chemically conjugated to the PLGA terminal end of a di-block copolymer composed of poly(D,L-lactic-co-glycolic acid) (PLGA)
and methoxy-poly(ethylene glycol) (mPEG). Folate was separately conjugated at the PEG terminal end of PEG –PLGA di-block copolymer. DOX conjugated PLGA – mPEG copolymer, folate conjugated PLGA – PEG copolymer, and unprotonated DOX were blended to form self-assembled micelles in aqueous solution. It was postulated that PLGA segments with terminally conjugated DOX were buried, along with physically entrapped free DOX, in the core, and PEG segments with terminally conjugated folate were oriented outside towards aqueous medium. This is schematically shown in Fig. 1. The formation of polymeric micelles was characterized and their cytotoxic effects against KB cells (folate receptor positive) were investigated in vitro. A flow cytometry study was also carried out to elucidate selective targeting capability of folate DOX micelles. The tumor suppression efficacy and biodistribution of the micelles were also evaluated on human tumor xenografted nude mice in vivo.
2. Materials and method 2.1. Materials PLGA (RG502H) (weight average MW: 8000) was purchased from Boehringer Ingelheim (Germany). Methoxy-poly(ethylene glycol)-amine (mPEG amine, CH 3 O-PEG-NH 2 ) and poly(ethylene glycol)-bisamine with MW 3400, N-hydroxysuccinimide (NHS)
Fig. 1. Schematic representation of DOX – PLGA – mPEG/PLGA – PEG – FOL mixed micelles that physically entrap free base DOX in the core while exposing folate on the surface.
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and dicyclohexylcarbodiimde (DCC), folate, DOX, and 3-(4,5-dimethylthiaol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO). Human squamous cell carcinoma cell line of the oral cavity (KB cell) was obtained from the Korean Cell Line Bank (KCLB). All other chemicals were of analytical grade.
gation percentage was calculated by determining the amount of DOX conjugated in DOX – PLGA –mPEG by a spectroscopic method. The conjugation process was monitored by a gel permeation chromatography and no detectable unreacted mPEG peak and PLGA peaks were found in the chromatogram. A synthetic scheme is shown in Fig. 2A.
2.2. Preparation of doxorubicin conjugated di-block copolymers
2.3. Preparation of folate-conjugated di-block copolymer
DOX was conjugated to a terminal hydroxyl group of PLGA in PLGA –mPEG di-block copolymer as described in the previous study with a minor modification [17]. Briefly, 5 g of PLGA dissolved in methylene chloride was activated by 206 mg of DCC and 115 mg of NHS at room temperature under nitrogen atmosphere for 24 h (PLGA/NHS/DCC stoichiometric molar ratio: 1/2/2). The resultant solution was filtered and precipitated by dropping into ice-cold diethyl ether and the activated PLGA was completely dried under vacuum. The activated PLGA (1 g) and mPEG amine (0.75 g) dissolved in 10 ml of methylene chloride were reacted at room temperature under nitrogen atmosphere for 3 h. The resultant solution was precipitated by dropping into ice-cold diethyl ether. The precipitated product, PLGA – mPEG was filtered and dried. One gram of PLGA – mPEG dissolved in 10 ml of methylene chloride was activated by adding 43 mg of p-nitrophenyl chloroformate and 29 mg of pyridine (PLGA – mPEG/p-nitrophenyl chloroformate/pyridine stoichiometric molar ratio: 1/ 2.8/4.8) at 0 jC. The reaction was carried out for 3 h at room temperature under nitrogen atmosphere. The activated PLGA –mPEG (0.1 g) dissolved in 3 ml of dimethylformamide (DMF) was reacted with 5.3 mg of DOX in the presence of 3.1 mg of triethylamine (TEA) for 24 h at room temperature under nitrogen atmosphere (stoichiometric molar ratio of activated PLGA – mPEG/DOX/TEA: 1/1.2/4). Unreacted DOX and other chemicals were removed by transfer of the organic phase into deionized water, which was followed by three subsequent dialyzing procedures against deionized water for 4 h (Spectra/Por 7, MWCO 10,000). The purified DOX – PLGA – mPEG was freeze-dried and stored at 20 jC for further use. The conjugation percentage of DOX to PLLA – PEG was 67.1% on a molar ratio basis. This conju-
Five gram of PLGA dissolved in methylene chloride was activated by 206 mg of DCC and 115 mg of NHS at room temperature under nitrogen atmosphere for 24 h (PLGA/NHS/DCC stoichiometric molar ratio: 1/2/2). The resultant solution was filtered and precipitated by dropping into ice-cold diethyl ether and the activated PLGA was completely dried under vacuum. The activated PLGA (1 g) dissolved in 8 ml of methylene chloride was added slowly to 2.1 g of PEG – bis-amine dissolved in 2 ml of methylene chloride in a drop-wise manner with gentle stirring. The reaction was carried out for 6 h under nitrogen atmosphere (PLGA/PEG – bis-amine stoichiometric molar ratio: 1/5) and the resultant solution was precipitated by addition of ice-cold diethyl ether. The precipitated product, amine-terminated di-block copolymer, PLGA – PEG –NH2 was filtered and dried. Folate-conjugated di-block copolymer was synthesized by coupling the PLGA – PEG – NH2 di-block copolymer to an activated folic acid as described in the previous studies with a minor modification [9,10]. Briefly, 500 mg of the di-block copolymer dissolved in 5 ml of DMSO was mixed with 13 mg of folic acid and 13 mg of DCC. The reaction was performed at room temperature for 7 h and then mixed with 50 ml of distilled water and centrifuged at 3000 rpm. After discarding the pellet, the supernatant was dialyzed and dried. A synthetic scheme is shown in Fig. 2B. The formation of folate-conjugated di-block copolymer, PLGA – PEG – FOL, was monitored and confirmed by a HPLC. The conjugation percentage of folate to PLGA –PEG was 44.8% on a molar ratio basis. The conjugation percentage was calculated by determining the amount of folic acid conjugated in PLGA – PEG – FOL. A known amount of dried PLGA – PEG –FOL was dissolved in dimethysulfoxide (DMSO) and an UV absorbance value at 365 nm was measured to
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determine the concentration of conjugated folic acid. Serially diluted concentrations of folic acid in DMSO were used to construct a calibration curve. 2.4. Preparation and size distribution of doxorubicin micelles Folate-targeted DOX micelles (DOX/FOL micelles) and DOX micelles (DOX micelles) were prepared by combining DOX – PLGA – mPEG, PLGA – PEG – FOL, and free DOX. For DOX/FOL micelles, 70 mg of DOX –PLGA –mPEG, 10 mg of PLGA – PEG –FOL and 20 mg of free DOX were dissolved in acetone in the presence of 6.8 mg of TEA. The organic phase was directly dispersed into aqueous phase with gentle stirring for 24 h to produce mixed micelles. Untrapped DOX and TEA were removed by extensive dialysis
against distilled water (SpectraPor 6, MWCO 10,000). The loading amount of DOX within the micelles was calculated by combining the amount of conjugated DOX plus the amount of physically entrapped DOX in the core. Size distribution of the DOX micelles was measured using a laser light scattering technique (ZetaPlus, Brookhaven Instrument, USA). 2.5. Flow cytometry, confocal image analysis, and cytotoxicity assay The extent of cellular uptake was compared for different micellar formulations using a flow cytometry (FACSCalibur, NJ). The cellular uptake was visualized by a confocal microscopy (Carl Zeiss LSM5100, Germany). MTT-based assay was performed to compare the cytotoxic effects of DOX/FOL and DOX micelles
Fig. 2. Synthetic schemes of (A) DOX – PLGA – mPEG and (B) PLGA – PEG – FOL.
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Fig. 2 (continued).
against KB cells in vitro according to the previously established method [16,17]. KB cells were maintained in RPMI1640 medium without folic acid, and with 10% fetal bovine serum (FBS) at 37 jC in 5% CO2 atmosphere. Cells harvested in a logarithmic growth phase were seeded on 96 wells at a cell density of 5 104 cells per ml. After incubating the cells in a logarithmic phase with various concentrations of DOX/FOL micelles, DOX micelles, or free DOX for 48 h, the MTT assay was performed and the percentage of cell viability was then determined.
micelles, or free DOX. DOX/FOL micelles, DOX micelles, or free DOX (an equivalent dose of DOX = 5 mg/kg) suspended in PBS were administered through the tail vein of animals at day 0 and 7. At predetermined time points, a major axis and a minor axis of tumors were measured using a caliper. Tumor volume was then calculated using the formula: (3/ 4)pa2b, where a and b are the length of the minor and major axis of tumor, respectively. All animals were accommodated in a pathogen-free laboratory environment throughout the experiments.
2.6. Animal experiments
2.7. Biodistribution of doxorubicin and doxorubicin micelles
Female athymic nude mice (nu/nu, body weight = 20 – 25 g) were subcutaneously implanted with a human epidermal carcinoma xenograft cell line, KB cells (1 106 cells per animal). After the implantation, tumors were allowed to grow for 21 days, followed by dosing with DOX/FOL micelles, DOX
Animals received a single intravenous (i.v.) injection of DOX/FOL micelles, DOX micelles, or free DOX at an equivalent DOX dose of 5 mg/kg. At the desired time, the animals were sacrificed and then liver, heart, spleen, lung, kidney, and tumor were removed
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without perfusion. The organs were freeze-dried and weighed. DOX distributed in different organs was extracted using chloroform and isopropanol as described in literature with a minor modification [19,20]. Briefly, lyophilized tissue was homogenized in 200 mM sodium phosphate buffer at pH 7.4, which was followed by an acidic hydrolysis in 2.0 M HCl solution at 80 jC for 10 min. Then, 0.9 ml of chloroform:isopropanol (3:1, v/v) was added to 0.7 ml of homogenized tissue in PBS (20 mg/ml). After vigorous vortex-mixing, an organic layer was collected by centrifugation at 16,000 g for 15 min at room temperature. The solution was completely dried under vacuum and re-dissolved in DDW. The recovery efficiency was >80% and could be corrected by using an internal standard. The recovered solution was analyzed by a HPLC system equipped with a fluorescence detector and a reversed phase C18 column. The mobile phase was composed of 0.01% trifluoroacetic acid aqueous solution and acetonitrile; increasing the percentage of acetonitrile from 5% to 45% in 40 min. A single DOX peak was detected at an excitation wavelength of 480 nm and an emission wavelength of 580 nm.
environment for physical entrapment of free base DOX within the core of micelles. It is also reported that DOX molecules are easily stacked together due to p – p interaction between aromatic multi-ring structures [21]. The size of mixed micelles containing DOX – PLGA – mPEG/PLGA – PEG – FOL was 104.9 F 11.5 nm and the loading amount of DOX was 19.6% (w/w). To comparatively estimate the targeting capacity of mixed micelles to folate receptor over-expressed cells, folate-deficient mixed micelles (DOX – PLGA – mPEG) were also prepared without mixing PLGA – PEG – FOL in the formulation. The
3. Results and discussion As shown in Fig. 2, DOX – PLGA – mPEG and PLGA – PEG – FOL were separately synthesized. The two di-block copolymers, at a weight ratio of DOX – PLGA – mPEG/PLGA – PEG –FOL = 7/1, were co-dissolved in acetone along with DOXHCl and TEA. The solution was dialyzed in aqueous solution to form mixed PLGA – PEG micelles entrapping free base DOX. As shown in Fig. 1, a folate moiety at the PEG end of PLGA – PEG – FOL is expected to be oriented outside, while a DOX moiety at the PLGA end of DOX – PLGA – mPEG is buried in the core. To increase the loading amount of DOX in the internal core of micelles, the mixed micelles were prepared with DOX and an excess amount of TEA in the acetone solution. In the presence of TEA, DOX was deprotonated and became hydrophobic. Free base DOX was likely to be partitioned to a greater extent into the hydrophobic core of micelles than its hydrophilic salt form of DOXHCl. Terminally conjugated DOX at the PLGA end, enriched in the core of micelles, might provide a more favorable partitioning
Fig. 3. Flow cytometry results of (A) DOX micelles and (B) DOX/ FOL micelles in the presence and absence of folate in the medium.
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size of DOX – PLGA – mPEG micelles was 111.7 F 17.2 nm and the loading amount of DOX was 19.2% (w/w). When DOX – PLGA –mPEG without incorporating the unprotonated DOX was used for the formation of micelles, the loading amount (chemically conjugated DOX) was 2.18% (w/w). When DOX hydrochloride was encapsulated within PLGA – mPEG micelles without using TEA, the loading amount (physically entrapped DOX) was merely about 0.5% (w/w) [17]. Thus it is obvious that DOX molecules loaded within the core of micelles were mostly in a physically entrapped state and they were aggregated in a nano-scale size. DOX – PLGA – mPEG micelles containing DOX nano-aggregates exhibited far improved physical stability compared to PLGA – mPEG micelles, suggesting that terminally conjugated
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DOX in the di-block copolymer was likely to stabilize the DOX aggregates. The p –p interaction occurring at the interface is expected to play a significant role in stabilizing hydrophobically aggregated DOX molecules in the core. Flow cytometry analysis was performed to compare endocytosis of folate-targeted DOX micelles (DOX – PLGA – mPEG/PLGA – PEG – FOL:DOX/ FOL micelles) and DOX micelles (DOX – PLGA – mPEG:DOX micelles) using a folate receptor positive cancer cell line, KB cell, in the presence and absence of folate in the incubation medium. This is shown in Fig. 3. When KB cells were incubated with DOX micelles, there was little difference in cellular uptake of DOX regardless of the addition of folate in the medium (A). However, when KB cells were incubated
Fig. 4. Confocal microscopic images of KB cells incubated with (A, B) DOX micelles and (C, D) DOX/FOL micelles in the presence and absence of folate in the medium.
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with DOX/FOL micelles, much greater cellular uptake of DOX was observed in the folate-free medium than in the folate medium. This suggests that free folate in the medium prevented DOX/FOL micelles from transporting into KB cells by competitive binding to folate receptors on the cell surface. The flow cytometry results directly indicate that the DOX/FOL micelles were transported within cells by a folate receptormediated endocytosis process. These results are consistent with those reported previously on the folate receptor-dependent cellular uptake of folate-coated liposomes for anti-cancer therapy [12,13]. Cellular uptake extents of DOX/FOL micelles were also evaluated by using confocal microscopy. For DOX micelles, there was no detectable change in fluorescent intensity of KB cells cultured in the presence and absence of folate in the medium. In contrast, for DOX/FOL micelles, more fluorescently labeled cells can be clearly visualized in the absence of folate in the medium, suggesting DOX/FOL micelles were endocytosed in a receptor-mediated manner. In accordance with the result from Fig. 3, Fig. 4 results confirmed that DOX/FOL micelles could be targeted to cancer cells over-expressing folate receptors on their surface. Employing folate as a targeting moiety was widely studied in various delivery vehicles, especially liposomes for anti-cancer therapy. In the present study, polymeric micelles with a targeting moiety were utilized as an alternative DOX delivery vehicle circumventing the instability concern occurring in the most cases of liposomes. The polymeric micelles not only provided a relatively large drug reservoir for the hydrophobic active ingredient, DOX, but also provided a specificity against certain types of cancer cells. Fig. 5 compared cytotoxic effects of free DOX, DOX micelles, and DOX/FOL micelles against KB cells. DOX micelles show a comparable cytotoxicity to free DOX. However, DOX/FOL micelles exhibit higher cytotoxicity than DOX micelles or free DOX. The IC 50 values of DOX/FOL micelles, DOX micelles, and free DOX were about 50, 70, and 75 AM, respectively. These IC50 values for KB cells are comparatively higher than the reported values of 1 – 10 AM for other cell lines, indicating that KB cells over-expressing folate receptors are more resistant to the treatment of DOX [22,23]. The results in Fig. 5 are consistent with those of Figs. 3 and 4. As a greater
Fig. 5. Cell viability results for free DOX, DOX micelles, and DOX/ FOL micelles.
amount of DOX could be intra-cellularly delivered into cells in the form of nano-sized micelles by endocytosis, the cells were more vulnerable to the cytotoxic effect of DOX. For free DOX, a multi-drug resistant effect, out-fluxing DOX through the p-glycoprotein pump, might play an additional role in decreasing the intracellular concentration of DOX. Thus, the higher cytotoxic result for DOX/FOL micelles can be attributed to the increased cellular uptake of DOX. In vivo animal studies were carried out to examine targeting and anti-tumor effects of DOX/FOL micelles using a KB cell xenografted nude mouse model. Fig. 6 shows the progress of tumor volume growth observed for 24 days with different formulations, free DOX, DOX micelles, and DOX/FOL micelles. It can be seen that DOX/FOL micelles suppressed the tumor growth more significantly than free DOX and DOX micelles. This could be attributed to the ‘‘enhanced permeation and retention’’ (EPR) effect of nano-sized micellar delivery systems [15,20,24]. Fast growing tumor tissues need a tremendous amount of oxygen and nutrients supplied by blood vessels. They release special growth factors including vascular endothelial cell growth factor (VEGF) to facilitate neo-vascularization. As a result, many new vessels are formed, but their cell junctions are not as tight as those of normal tissues. DOX/FOL micelles having a size of about 110
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Fig. 6. Tumor volume growth for various formulations of free DOX, DOX micelles, and DOX/FOL micelles as a function of time.
nm were likely to freely pass through the endothelial junctions of the capillaries in tumor tissue, but not in normal tissue. In addition, passively targeted DOX/ FOL micelles that were accumulated in the solid tumor region might be more readily taken up by tumor cells by a receptor-mediated endocytosis process. A combined effect of the passive targeting and enhanced cellular uptake would be the main reason for the suppression of tumor growth. It should be mentioned that the mice were allowed to eat diet including folate throughout the experiments. A previous study reported that folate-restricted diet can lower the concentration of folate in the bloodstream, resulting in enhancing the extent of cellular uptake into tumor cells over-expressing folate receptors [25]. If folate-
Fig. 7. DOX concentration in the solid tumor for free DOX, DOX/ FOL micelles, and DOX micelles.
Fig. 8. Biodistribution profiles of (A) free DOX, (B) DOX/FOL micelles, and (C) DOX micelles.
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free diet were used for the current animal experiment, the tumor suppression effect of DOX/FOL micelles could have been more prominent. In order to examine passive targeting effect of DOX and DOX/FOL micelles, the amount of DOX in tumor tissue was determined (Fig. 7). For free DOX, the DOX level in the tumor reached a peak level of 3.5 Ag/g at 2 h, and it quickly dropped to 0.2 Ag/g at 12 h and to an imperceptible level after 24 h. In contrast, when DOX/ FOL micelles or DOX micelles were administered, it maintained at a high level up to 96 h. For DOX/FOL micelles, it increased to the highest level, 8.7 Ag/g at 24 h and slowly decreased to 8.3 and 4.4 Ag/g at 48 and 96 h, respectively. The change of the DOX level showed a similar pattern when DOX micelles were administered, while the values were lower than those of DOX/FOL micelles. For DOX micelles, DOX level reached a peak level of 6.3 Ag/g at 24 h and slowly decreased to 6.1 and 3.6 Ag/g at 48 and 96 h, respectively. These results are in good agreement with the previous studies employing polymer –DOX conjugates [20,24,26]. Drug– polymer conjugates tend to accumulate at solid tumors by the aforementioned EPR effect, resulting from enhanced permeability of blood vessels on the sites. Kopecek et al. showed that N-(2-hydroxypropyl)methacrylate copolymer – DOX conjugates accumulated in solid tumors, thereby exerting their therapeutic effects on the tumor [20,24]. DOX/FOL micelles exhibited a greater extent of DOX accumulation in the tumor than DOX micelles, suggesting that DOX/FOL micelles were more effectively transported within KB cells than DOX micelles due to receptor-mediated endocytosis. This enhanced DOX accumulation profile in the tumor is most likely to be responsible for the tumor regression result shown in Fig. 6. Biodistribution profiles of DOX in other tissues including heart, liver, lung, spleen, and kidney were measured after administrating DOX/FOL micelles, DOX micelles, and free DOX to KB tumor xenograft mice (Fig. 8). For free DOX, the experiment was done within 24 h because the DOX level was not detected by a fluorescence detector after 24 h postadministration (Fig. 8A). In contrast, DOX was detectable in all organs up to 96 h for DOX/FOL micelles (Fig. 8B) and DOX micelles (Fig. 8C). For DOX/FOL micelles and DOX micelles, a significantly lower amount of DOX was detected in the heart than for free DOX at 24 h, indicating that the cardiac toxicity of DOX might be substantially reduced by the formula-
tion of DOX micelles. This result is encouraging since the cardiac toxicity is one of the leading side-effects of DOX chemotherapies. The reduced extent of accumulation in the heart can be attributed to selective localization of DOX within the tumor by the EPR effect, resulting in lowering the concentration of DOX in the blood stream while maintaining a higher concentration of DOX in the tumor. For both DOX/FOL and DOX micelles, the accumulation amount in the liver was comparatively higher than in any other organs. The liver accumulation of drug –polymer conjugated has been reported in the several publications [20,24,26].
4. Conclusions Folate receptor targeted PLGA – PEG micelles entrapping a high loading amount of DOX demonstrated superior cellular uptake over DOX and DOX micelles against a folate-receptor positive cell line. The enhanced cellular uptake was caused by a folatereceptor mediated endocytosis process, which also resulted in increased cytotoxicity. In vitro and in vivo studies confirmed that selective passive targeting of DOX into the tumor occurred in a site specific manner.
Acknowledgements This work was supported by the Ministry of Science and Technology (M1021400017-02b150002110), Republic of Korea.
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Folate receptor targeted biodegradable polymeric doxorubicin micelles Hyuk Sang Yoo, Tae Gwan Park * Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea Received 12 November 2003; accepted 3 February 2004
Abstract Biodegradable polymeric micelles, self-assembled from a di-block copolymer of poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG), were prepared to achieve folate receptor targeted delivery of doxorubicin (DOX). In the diblock copolymer structure of PLGA – b-PEG, DOX was chemically conjugated to a terminal end of PLGA to produce DOX – PLGA – mPEG, and folate was separately conjugated to a terminal end of PEG to produce PLGA – PEG – FOL. The two diblock copolymers with different functional moieties at their chains ends were physically mixed with free base DOX in an aqueous solution to form mixed micelles. It was expected that folate moieties were exposed on the micellar surface, while DOX was physically and chemically entrapped in the core of micelles. Flow cytometry and confocal image analysis revealed that folate conjugated mixed micelles exhibited far greater extent of cellular uptake than folate unconjugated micelles against KB cells over-expressing folate receptors on the surface. They also showed higher cytotoxicity than DOX, suggesting that folate receptor medicated endocytosis of the micelles played an important role in transporting an increased amount of DOX within cells. In vivo animal experiments, using a nude mice xenograft model, demonstrated that when systemically administered, tumor volume was significantly regressed. Biodistribution studies also indicated that an increased amount of DOX was accumulated in the tumor tissue. D 2004 Elsevier B.V. All rights reserved. Keywords: Folate; Micelle; Doxorubicin; Targeting; PLGA
1. Introduction Folate receptor has been known to be vastly overexpressed in several human tumors [1 –3]. Folate has been popularly employed as a targeting moiety of various anti-cancer agents to avoid their non-specific attacks on normal tissues as well as to increase their cellular uptake within target cells, as studied in several * Corresponding author. Tel.: +82-42-869-2621; fax: +82-42869-2610. E-mail address: [email protected] (T.G. Park). 0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2004.02.003
previous studies [4– 7]. Folate was often covalently attached to a wide array of drug delivery carriers such as liposomes, polymer conjugates, and nano-particulates [2,6,8 –10]. Among them, anti-cancer liposomes with having surface exposed folate moieties have been extensively studied for passively targeting cancer cells. A folate group covalently attached to phospholipid or cholesterol was used to form doxorubicin (DOX) encapsulated liposomes [7,9,10]. This liposomal DOX formulation exhibited prolonged systemic circulation time and much lower cardiotoxicity than free DOX [10]. The folate-targeted liposomal DOX
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showed superior cytotoxicity to liposomal DOX formulation in cultured folate receptor (+) tumor cells in vitro. Furthermore, in a folate receptor (+) carcinoma murine xenograft model, the folate-targeted liposomal DOX demonstrated greater tumor inhibition effect with increasing the life-span of mice compared to the liposomal DOX [11,12]. Over the past decade, polymeric micelles have received much attention as an alternative delivery carrier for various drugs including anti-cancer agents [13 – 15]. Amphiphatic di-block copolymers self-assembled into polymeric micelles in an aqueous solution. The size of polymeric micelles is approximately less than 100 nm, which not only makes them ideal drug delivery carriers for escaping from renal exclusion and the reticulo-endothelial system, but also gives them an enhanced vascular permeability. Polymeric micelles are composed of a hydrophilic outer shell exposed to the aqueous phase and a hydrophobic inner core encapsulating drug molecules. For anticancer drugs such as DOX, biodegradable polymeric micelles were extensively utilized for passive targeting to solid tumors [16 – 18]. Very hydrophobic drugs such as paclitaxel were also successfully solubilized within the core of micelles to enhance their water solubility for the intravenous injection [19]. In this study, biodegradable DOX polymeric micelles having a targeting ability of folate receptor were prepared. DOX was chemically conjugated to the PLGA terminal end of a di-block copolymer composed of poly(D,L-lactic-co-glycolic acid) (PLGA)
and methoxy-poly(ethylene glycol) (mPEG). Folate was separately conjugated at the PEG terminal end of PEG –PLGA di-block copolymer. DOX conjugated PLGA – mPEG copolymer, folate conjugated PLGA – PEG copolymer, and unprotonated DOX were blended to form self-assembled micelles in aqueous solution. It was postulated that PLGA segments with terminally conjugated DOX were buried, along with physically entrapped free DOX, in the core, and PEG segments with terminally conjugated folate were oriented outside towards aqueous medium. This is schematically shown in Fig. 1. The formation of polymeric micelles was characterized and their cytotoxic effects against KB cells (folate receptor positive) were investigated in vitro. A flow cytometry study was also carried out to elucidate selective targeting capability of folate DOX micelles. The tumor suppression efficacy and biodistribution of the micelles were also evaluated on human tumor xenografted nude mice in vivo.
2. Materials and method 2.1. Materials PLGA (RG502H) (weight average MW: 8000) was purchased from Boehringer Ingelheim (Germany). Methoxy-poly(ethylene glycol)-amine (mPEG amine, CH 3 O-PEG-NH 2 ) and poly(ethylene glycol)-bisamine with MW 3400, N-hydroxysuccinimide (NHS)
Fig. 1. Schematic representation of DOX – PLGA – mPEG/PLGA – PEG – FOL mixed micelles that physically entrap free base DOX in the core while exposing folate on the surface.
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and dicyclohexylcarbodiimde (DCC), folate, DOX, and 3-(4,5-dimethylthiaol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO). Human squamous cell carcinoma cell line of the oral cavity (KB cell) was obtained from the Korean Cell Line Bank (KCLB). All other chemicals were of analytical grade.
gation percentage was calculated by determining the amount of DOX conjugated in DOX – PLGA –mPEG by a spectroscopic method. The conjugation process was monitored by a gel permeation chromatography and no detectable unreacted mPEG peak and PLGA peaks were found in the chromatogram. A synthetic scheme is shown in Fig. 2A.
2.2. Preparation of doxorubicin conjugated di-block copolymers
2.3. Preparation of folate-conjugated di-block copolymer
DOX was conjugated to a terminal hydroxyl group of PLGA in PLGA –mPEG di-block copolymer as described in the previous study with a minor modification [17]. Briefly, 5 g of PLGA dissolved in methylene chloride was activated by 206 mg of DCC and 115 mg of NHS at room temperature under nitrogen atmosphere for 24 h (PLGA/NHS/DCC stoichiometric molar ratio: 1/2/2). The resultant solution was filtered and precipitated by dropping into ice-cold diethyl ether and the activated PLGA was completely dried under vacuum. The activated PLGA (1 g) and mPEG amine (0.75 g) dissolved in 10 ml of methylene chloride were reacted at room temperature under nitrogen atmosphere for 3 h. The resultant solution was precipitated by dropping into ice-cold diethyl ether. The precipitated product, PLGA – mPEG was filtered and dried. One gram of PLGA – mPEG dissolved in 10 ml of methylene chloride was activated by adding 43 mg of p-nitrophenyl chloroformate and 29 mg of pyridine (PLGA – mPEG/p-nitrophenyl chloroformate/pyridine stoichiometric molar ratio: 1/ 2.8/4.8) at 0 jC. The reaction was carried out for 3 h at room temperature under nitrogen atmosphere. The activated PLGA –mPEG (0.1 g) dissolved in 3 ml of dimethylformamide (DMF) was reacted with 5.3 mg of DOX in the presence of 3.1 mg of triethylamine (TEA) for 24 h at room temperature under nitrogen atmosphere (stoichiometric molar ratio of activated PLGA – mPEG/DOX/TEA: 1/1.2/4). Unreacted DOX and other chemicals were removed by transfer of the organic phase into deionized water, which was followed by three subsequent dialyzing procedures against deionized water for 4 h (Spectra/Por 7, MWCO 10,000). The purified DOX – PLGA – mPEG was freeze-dried and stored at 20 jC for further use. The conjugation percentage of DOX to PLLA – PEG was 67.1% on a molar ratio basis. This conju-
Five gram of PLGA dissolved in methylene chloride was activated by 206 mg of DCC and 115 mg of NHS at room temperature under nitrogen atmosphere for 24 h (PLGA/NHS/DCC stoichiometric molar ratio: 1/2/2). The resultant solution was filtered and precipitated by dropping into ice-cold diethyl ether and the activated PLGA was completely dried under vacuum. The activated PLGA (1 g) dissolved in 8 ml of methylene chloride was added slowly to 2.1 g of PEG – bis-amine dissolved in 2 ml of methylene chloride in a drop-wise manner with gentle stirring. The reaction was carried out for 6 h under nitrogen atmosphere (PLGA/PEG – bis-amine stoichiometric molar ratio: 1/5) and the resultant solution was precipitated by addition of ice-cold diethyl ether. The precipitated product, amine-terminated di-block copolymer, PLGA – PEG –NH2 was filtered and dried. Folate-conjugated di-block copolymer was synthesized by coupling the PLGA – PEG – NH2 di-block copolymer to an activated folic acid as described in the previous studies with a minor modification [9,10]. Briefly, 500 mg of the di-block copolymer dissolved in 5 ml of DMSO was mixed with 13 mg of folic acid and 13 mg of DCC. The reaction was performed at room temperature for 7 h and then mixed with 50 ml of distilled water and centrifuged at 3000 rpm. After discarding the pellet, the supernatant was dialyzed and dried. A synthetic scheme is shown in Fig. 2B. The formation of folate-conjugated di-block copolymer, PLGA – PEG – FOL, was monitored and confirmed by a HPLC. The conjugation percentage of folate to PLGA –PEG was 44.8% on a molar ratio basis. The conjugation percentage was calculated by determining the amount of folic acid conjugated in PLGA – PEG – FOL. A known amount of dried PLGA – PEG –FOL was dissolved in dimethysulfoxide (DMSO) and an UV absorbance value at 365 nm was measured to
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determine the concentration of conjugated folic acid. Serially diluted concentrations of folic acid in DMSO were used to construct a calibration curve. 2.4. Preparation and size distribution of doxorubicin micelles Folate-targeted DOX micelles (DOX/FOL micelles) and DOX micelles (DOX micelles) were prepared by combining DOX – PLGA – mPEG, PLGA – PEG – FOL, and free DOX. For DOX/FOL micelles, 70 mg of DOX –PLGA –mPEG, 10 mg of PLGA – PEG –FOL and 20 mg of free DOX were dissolved in acetone in the presence of 6.8 mg of TEA. The organic phase was directly dispersed into aqueous phase with gentle stirring for 24 h to produce mixed micelles. Untrapped DOX and TEA were removed by extensive dialysis
against distilled water (SpectraPor 6, MWCO 10,000). The loading amount of DOX within the micelles was calculated by combining the amount of conjugated DOX plus the amount of physically entrapped DOX in the core. Size distribution of the DOX micelles was measured using a laser light scattering technique (ZetaPlus, Brookhaven Instrument, USA). 2.5. Flow cytometry, confocal image analysis, and cytotoxicity assay The extent of cellular uptake was compared for different micellar formulations using a flow cytometry (FACSCalibur, NJ). The cellular uptake was visualized by a confocal microscopy (Carl Zeiss LSM5100, Germany). MTT-based assay was performed to compare the cytotoxic effects of DOX/FOL and DOX micelles
Fig. 2. Synthetic schemes of (A) DOX – PLGA – mPEG and (B) PLGA – PEG – FOL.
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Fig. 2 (continued).
against KB cells in vitro according to the previously established method [16,17]. KB cells were maintained in RPMI1640 medium without folic acid, and with 10% fetal bovine serum (FBS) at 37 jC in 5% CO2 atmosphere. Cells harvested in a logarithmic growth phase were seeded on 96 wells at a cell density of 5 104 cells per ml. After incubating the cells in a logarithmic phase with various concentrations of DOX/FOL micelles, DOX micelles, or free DOX for 48 h, the MTT assay was performed and the percentage of cell viability was then determined.
micelles, or free DOX. DOX/FOL micelles, DOX micelles, or free DOX (an equivalent dose of DOX = 5 mg/kg) suspended in PBS were administered through the tail vein of animals at day 0 and 7. At predetermined time points, a major axis and a minor axis of tumors were measured using a caliper. Tumor volume was then calculated using the formula: (3/ 4)pa2b, where a and b are the length of the minor and major axis of tumor, respectively. All animals were accommodated in a pathogen-free laboratory environment throughout the experiments.
2.6. Animal experiments
2.7. Biodistribution of doxorubicin and doxorubicin micelles
Female athymic nude mice (nu/nu, body weight = 20 – 25 g) were subcutaneously implanted with a human epidermal carcinoma xenograft cell line, KB cells (1 106 cells per animal). After the implantation, tumors were allowed to grow for 21 days, followed by dosing with DOX/FOL micelles, DOX
Animals received a single intravenous (i.v.) injection of DOX/FOL micelles, DOX micelles, or free DOX at an equivalent DOX dose of 5 mg/kg. At the desired time, the animals were sacrificed and then liver, heart, spleen, lung, kidney, and tumor were removed
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without perfusion. The organs were freeze-dried and weighed. DOX distributed in different organs was extracted using chloroform and isopropanol as described in literature with a minor modification [19,20]. Briefly, lyophilized tissue was homogenized in 200 mM sodium phosphate buffer at pH 7.4, which was followed by an acidic hydrolysis in 2.0 M HCl solution at 80 jC for 10 min. Then, 0.9 ml of chloroform:isopropanol (3:1, v/v) was added to 0.7 ml of homogenized tissue in PBS (20 mg/ml). After vigorous vortex-mixing, an organic layer was collected by centrifugation at 16,000 g for 15 min at room temperature. The solution was completely dried under vacuum and re-dissolved in DDW. The recovery efficiency was >80% and could be corrected by using an internal standard. The recovered solution was analyzed by a HPLC system equipped with a fluorescence detector and a reversed phase C18 column. The mobile phase was composed of 0.01% trifluoroacetic acid aqueous solution and acetonitrile; increasing the percentage of acetonitrile from 5% to 45% in 40 min. A single DOX peak was detected at an excitation wavelength of 480 nm and an emission wavelength of 580 nm.
environment for physical entrapment of free base DOX within the core of micelles. It is also reported that DOX molecules are easily stacked together due to p – p interaction between aromatic multi-ring structures [21]. The size of mixed micelles containing DOX – PLGA – mPEG/PLGA – PEG – FOL was 104.9 F 11.5 nm and the loading amount of DOX was 19.6% (w/w). To comparatively estimate the targeting capacity of mixed micelles to folate receptor over-expressed cells, folate-deficient mixed micelles (DOX – PLGA – mPEG) were also prepared without mixing PLGA – PEG – FOL in the formulation. The
3. Results and discussion As shown in Fig. 2, DOX – PLGA – mPEG and PLGA – PEG – FOL were separately synthesized. The two di-block copolymers, at a weight ratio of DOX – PLGA – mPEG/PLGA – PEG –FOL = 7/1, were co-dissolved in acetone along with DOXHCl and TEA. The solution was dialyzed in aqueous solution to form mixed PLGA – PEG micelles entrapping free base DOX. As shown in Fig. 1, a folate moiety at the PEG end of PLGA – PEG – FOL is expected to be oriented outside, while a DOX moiety at the PLGA end of DOX – PLGA – mPEG is buried in the core. To increase the loading amount of DOX in the internal core of micelles, the mixed micelles were prepared with DOX and an excess amount of TEA in the acetone solution. In the presence of TEA, DOX was deprotonated and became hydrophobic. Free base DOX was likely to be partitioned to a greater extent into the hydrophobic core of micelles than its hydrophilic salt form of DOXHCl. Terminally conjugated DOX at the PLGA end, enriched in the core of micelles, might provide a more favorable partitioning
Fig. 3. Flow cytometry results of (A) DOX micelles and (B) DOX/ FOL micelles in the presence and absence of folate in the medium.
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size of DOX – PLGA – mPEG micelles was 111.7 F 17.2 nm and the loading amount of DOX was 19.2% (w/w). When DOX – PLGA –mPEG without incorporating the unprotonated DOX was used for the formation of micelles, the loading amount (chemically conjugated DOX) was 2.18% (w/w). When DOX hydrochloride was encapsulated within PLGA – mPEG micelles without using TEA, the loading amount (physically entrapped DOX) was merely about 0.5% (w/w) [17]. Thus it is obvious that DOX molecules loaded within the core of micelles were mostly in a physically entrapped state and they were aggregated in a nano-scale size. DOX – PLGA – mPEG micelles containing DOX nano-aggregates exhibited far improved physical stability compared to PLGA – mPEG micelles, suggesting that terminally conjugated
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DOX in the di-block copolymer was likely to stabilize the DOX aggregates. The p –p interaction occurring at the interface is expected to play a significant role in stabilizing hydrophobically aggregated DOX molecules in the core. Flow cytometry analysis was performed to compare endocytosis of folate-targeted DOX micelles (DOX – PLGA – mPEG/PLGA – PEG – FOL:DOX/ FOL micelles) and DOX micelles (DOX – PLGA – mPEG:DOX micelles) using a folate receptor positive cancer cell line, KB cell, in the presence and absence of folate in the incubation medium. This is shown in Fig. 3. When KB cells were incubated with DOX micelles, there was little difference in cellular uptake of DOX regardless of the addition of folate in the medium (A). However, when KB cells were incubated
Fig. 4. Confocal microscopic images of KB cells incubated with (A, B) DOX micelles and (C, D) DOX/FOL micelles in the presence and absence of folate in the medium.
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with DOX/FOL micelles, much greater cellular uptake of DOX was observed in the folate-free medium than in the folate medium. This suggests that free folate in the medium prevented DOX/FOL micelles from transporting into KB cells by competitive binding to folate receptors on the cell surface. The flow cytometry results directly indicate that the DOX/FOL micelles were transported within cells by a folate receptormediated endocytosis process. These results are consistent with those reported previously on the folate receptor-dependent cellular uptake of folate-coated liposomes for anti-cancer therapy [12,13]. Cellular uptake extents of DOX/FOL micelles were also evaluated by using confocal microscopy. For DOX micelles, there was no detectable change in fluorescent intensity of KB cells cultured in the presence and absence of folate in the medium. In contrast, for DOX/FOL micelles, more fluorescently labeled cells can be clearly visualized in the absence of folate in the medium, suggesting DOX/FOL micelles were endocytosed in a receptor-mediated manner. In accordance with the result from Fig. 3, Fig. 4 results confirmed that DOX/FOL micelles could be targeted to cancer cells over-expressing folate receptors on their surface. Employing folate as a targeting moiety was widely studied in various delivery vehicles, especially liposomes for anti-cancer therapy. In the present study, polymeric micelles with a targeting moiety were utilized as an alternative DOX delivery vehicle circumventing the instability concern occurring in the most cases of liposomes. The polymeric micelles not only provided a relatively large drug reservoir for the hydrophobic active ingredient, DOX, but also provided a specificity against certain types of cancer cells. Fig. 5 compared cytotoxic effects of free DOX, DOX micelles, and DOX/FOL micelles against KB cells. DOX micelles show a comparable cytotoxicity to free DOX. However, DOX/FOL micelles exhibit higher cytotoxicity than DOX micelles or free DOX. The IC 50 values of DOX/FOL micelles, DOX micelles, and free DOX were about 50, 70, and 75 AM, respectively. These IC50 values for KB cells are comparatively higher than the reported values of 1 – 10 AM for other cell lines, indicating that KB cells over-expressing folate receptors are more resistant to the treatment of DOX [22,23]. The results in Fig. 5 are consistent with those of Figs. 3 and 4. As a greater
Fig. 5. Cell viability results for free DOX, DOX micelles, and DOX/ FOL micelles.
amount of DOX could be intra-cellularly delivered into cells in the form of nano-sized micelles by endocytosis, the cells were more vulnerable to the cytotoxic effect of DOX. For free DOX, a multi-drug resistant effect, out-fluxing DOX through the p-glycoprotein pump, might play an additional role in decreasing the intracellular concentration of DOX. Thus, the higher cytotoxic result for DOX/FOL micelles can be attributed to the increased cellular uptake of DOX. In vivo animal studies were carried out to examine targeting and anti-tumor effects of DOX/FOL micelles using a KB cell xenografted nude mouse model. Fig. 6 shows the progress of tumor volume growth observed for 24 days with different formulations, free DOX, DOX micelles, and DOX/FOL micelles. It can be seen that DOX/FOL micelles suppressed the tumor growth more significantly than free DOX and DOX micelles. This could be attributed to the ‘‘enhanced permeation and retention’’ (EPR) effect of nano-sized micellar delivery systems [15,20,24]. Fast growing tumor tissues need a tremendous amount of oxygen and nutrients supplied by blood vessels. They release special growth factors including vascular endothelial cell growth factor (VEGF) to facilitate neo-vascularization. As a result, many new vessels are formed, but their cell junctions are not as tight as those of normal tissues. DOX/FOL micelles having a size of about 110
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Fig. 6. Tumor volume growth for various formulations of free DOX, DOX micelles, and DOX/FOL micelles as a function of time.
nm were likely to freely pass through the endothelial junctions of the capillaries in tumor tissue, but not in normal tissue. In addition, passively targeted DOX/ FOL micelles that were accumulated in the solid tumor region might be more readily taken up by tumor cells by a receptor-mediated endocytosis process. A combined effect of the passive targeting and enhanced cellular uptake would be the main reason for the suppression of tumor growth. It should be mentioned that the mice were allowed to eat diet including folate throughout the experiments. A previous study reported that folate-restricted diet can lower the concentration of folate in the bloodstream, resulting in enhancing the extent of cellular uptake into tumor cells over-expressing folate receptors [25]. If folate-
Fig. 7. DOX concentration in the solid tumor for free DOX, DOX/ FOL micelles, and DOX micelles.
Fig. 8. Biodistribution profiles of (A) free DOX, (B) DOX/FOL micelles, and (C) DOX micelles.
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free diet were used for the current animal experiment, the tumor suppression effect of DOX/FOL micelles could have been more prominent. In order to examine passive targeting effect of DOX and DOX/FOL micelles, the amount of DOX in tumor tissue was determined (Fig. 7). For free DOX, the DOX level in the tumor reached a peak level of 3.5 Ag/g at 2 h, and it quickly dropped to 0.2 Ag/g at 12 h and to an imperceptible level after 24 h. In contrast, when DOX/ FOL micelles or DOX micelles were administered, it maintained at a high level up to 96 h. For DOX/FOL micelles, it increased to the highest level, 8.7 Ag/g at 24 h and slowly decreased to 8.3 and 4.4 Ag/g at 48 and 96 h, respectively. The change of the DOX level showed a similar pattern when DOX micelles were administered, while the values were lower than those of DOX/FOL micelles. For DOX micelles, DOX level reached a peak level of 6.3 Ag/g at 24 h and slowly decreased to 6.1 and 3.6 Ag/g at 48 and 96 h, respectively. These results are in good agreement with the previous studies employing polymer –DOX conjugates [20,24,26]. Drug– polymer conjugates tend to accumulate at solid tumors by the aforementioned EPR effect, resulting from enhanced permeability of blood vessels on the sites. Kopecek et al. showed that N-(2-hydroxypropyl)methacrylate copolymer – DOX conjugates accumulated in solid tumors, thereby exerting their therapeutic effects on the tumor [20,24]. DOX/FOL micelles exhibited a greater extent of DOX accumulation in the tumor than DOX micelles, suggesting that DOX/FOL micelles were more effectively transported within KB cells than DOX micelles due to receptor-mediated endocytosis. This enhanced DOX accumulation profile in the tumor is most likely to be responsible for the tumor regression result shown in Fig. 6. Biodistribution profiles of DOX in other tissues including heart, liver, lung, spleen, and kidney were measured after administrating DOX/FOL micelles, DOX micelles, and free DOX to KB tumor xenograft mice (Fig. 8). For free DOX, the experiment was done within 24 h because the DOX level was not detected by a fluorescence detector after 24 h postadministration (Fig. 8A). In contrast, DOX was detectable in all organs up to 96 h for DOX/FOL micelles (Fig. 8B) and DOX micelles (Fig. 8C). For DOX/FOL micelles and DOX micelles, a significantly lower amount of DOX was detected in the heart than for free DOX at 24 h, indicating that the cardiac toxicity of DOX might be substantially reduced by the formula-
tion of DOX micelles. This result is encouraging since the cardiac toxicity is one of the leading side-effects of DOX chemotherapies. The reduced extent of accumulation in the heart can be attributed to selective localization of DOX within the tumor by the EPR effect, resulting in lowering the concentration of DOX in the blood stream while maintaining a higher concentration of DOX in the tumor. For both DOX/FOL and DOX micelles, the accumulation amount in the liver was comparatively higher than in any other organs. The liver accumulation of drug –polymer conjugated has been reported in the several publications [20,24,26].
4. Conclusions Folate receptor targeted PLGA – PEG micelles entrapping a high loading amount of DOX demonstrated superior cellular uptake over DOX and DOX micelles against a folate-receptor positive cell line. The enhanced cellular uptake was caused by a folatereceptor mediated endocytosis process, which also resulted in increased cytotoxicity. In vitro and in vivo studies confirmed that selective passive targeting of DOX into the tumor occurred in a site specific manner.
Acknowledgements This work was supported by the Ministry of Science and Technology (M1021400017-02b150002110), Republic of Korea.
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