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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Leslie Dan Faculty of Pharmacy, University of Toronto, Ontario, Canada (H.L.W., R.B., H.Y.X., K.B., X.Y.W.); and Ontario Cancer Institute, Toronto, Ontario, Canada (A.M.R.)
Received for publication
January 9, 2006
Accepted
March 15, 2006.
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; Tack et al., 2004). However, suboptimal therapeutic responses associated with multidrug resistance (MDR) frequently occur (Longley and Johnston, 2005). Overexpression of P-glycoprotein (Pgp) is one of the prominent mechanisms that contribute to the MDR phenotype (Endicott and Ling, 1989; Gottesman, 2002). Pgp is a 170-kDa membrane-associated glycoprotein that may actively extrude several substrates, including a variety of cytotoxic drugs such as doxorubicin (Dox), from cell cytoplasm to outside of plasma membrane, thus lowering the effective drug concentrations within the cells (Endicott and Ling, 1989; Gottesman, 2002). Because cytotoxic drugs typically carry numerous dose-limiting normal tissue side effects (Tipton, 2003), it is generally impractical to overcome this form of drug resistance simply by increasing the drug dose. To improve the therapeutic ratio of cancer chemotherapy, it is critical to establish alternative approaches that may improve accumulation and prolong retention of cytotoxic drugs in drug-resistant cancer cells without causing additional normal tissue side effects.
Particulate drug delivery systems such as polymeric microspheres (Liu et al., 2001), nanoparticles (de Verdiere et al. 1997; Moghimi and Hunter, 2000), liposomes (Thierry et al., 1993; Romsicki and Sharom, 1999; Booser et al., 2002), and solid lipid nanoparticles (SLNs) (Wong et al., 2004, 2005) offer great promise to achieve the aforementioned goal. Particulate systems are well known to be able to deliver drugs with higher efficiency with fewer adverse side effects (Booser et al., 2002; Lamprecht et al., 2005). Some encapsulated formulations may be further engineered to deliver substances such as Pgp inhibitors or other therapeutic molecules together with cytotoxic drugs to achieve stronger anticancer activity in drug-resistant cancer cells (Liu et al., 2001; Wong et al., 2004; Veldman et al., 2005). In addition, some formulations also possess intrinsic activities to reduce drug resistance (Thierry et al., 1993; Romsicki and Sharom, 1999; Moghimi and Hunter, 2000; Kabanov et al., 2002; Nori et al., 2003). The mechanisms that underlie these MDR reversal activities are diversified and probably vary from one drug carrier to another. Mechanisms established so far include inhibition of Pgp by the polymers making up the drug carriers (Romsicki and Sharom, 1999; Moghimi and Hunter, 2000; Kabanov et al., 2002), local buildup of drug molecules outside the cell membranes (de Verdiere et al., 1997), and increase of cellular drug uptake by endocytosis of the drug carriers (Lee et al., 1992; Soma et al., 1999; Nori et al., 2003). At present, there are few studies devoted to the investigation of the mechanistic issues of MDR reversal by SLNs.
In our previous studies (Wong et al., 2004, 2005, 2006), we have developed polymer-lipid hybrid nanoparticles (PLNs), a modified form of SLN that incorporates an anionic polymer to facilitate loading of water-soluble cationic drugs that contain lipophilic molecular structures, e.g., Dox and verapamil. Compared with the conventional free Dox solution treatment, the new nanoparticle system with Dox encapsulated (Dox-PLN) resulted in approximately an 8-fold increase in cell kill of Pgp-overexpressing human breast cancer cells in clonogenic assay experiments (Wong et al., 2006). This additional anticancer activity against Pgp-overexpressing cancer cells is not caused by the nonactive ingredients of the nanoparticles because the lipid, polymer, and surfactants used in Dox-PLN preparation were shown inoculous in terms of their effects on cancer cell proliferation and cell membrane integrity (Wong et al., 2006). Furthermore, Dox-polymer complexes not encapsulated in lipids were not more effective than Dox only. Therefore, the enhancement in anticancer activity was likely attributable to the fact that Dox was more efficiently delivered to the cells when encapsulated in PLNs (i.e., Dox-PLN).
In the present work, we examined the mechanism by which the PLN system enhanced the activity of Dox in Pgp-overexpressing cancer cells. Studies were performed to evaluate quantitatively how Dox, when administered in the form of Dox-PLN, accumulated in and was retained by Pgp-overexpressing breast cancer cells. Since endocytosis of lipid formulations by various cell types were previously reported (Lee et al., 1992; Soma et al., 1999), endocytosis inhibition studies were carried out to determine whether this mechanism was also involved in the uptake of Dox-PLN by cancer cells. Intracellular distribution of Dox and fluorescence-labeled lipids was examined by fluorescence microscopy to help further understand the drug uptake processes. Insights gained from these studies may help to optimize the use of nanoparticle systems for improved chemotherapy of MDR cancers.
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). Pluronic F68 (i.e., poloxamer 188, a nonionic block copolymer) was supplied by BASF Corp. (Florham Park, NJ). For the Western blot analysis, protease inhibitor cocktail (P8340) and antiactin antibody AC-40 were purchased from Sigma-Aldrich, and Pgp antibody C219 and IgG2a:HRP were purchased from ID Labs Biotechnology (London, ON, Canada) and Serotec (Raleigh, NC), respectively.
Tumor Cell Lines and Culture. The Pgp-overexpressing human breast carcinoma cell line MDA435/LCC6/MDR1 and parental cell line MDA435/LCC6/WT were generous gifts from Dr. Robert Clarke (Georgetown University, Washington, DC). The Pgp-overexpressing murine breast carcinoma cell line EMT6/AR1 and parental cell line EMT6/WT were kindly provided by Dr. Ian Tannock (Ontario Cancer Institute, Toronto, ON, Canada). Monolayers of all cell types (passages 5–30 in our hands) were cultured on 75-cm2 polystyrene tissue culture flasks at 37°C in 5% CO2/95% air humidified incubator. Cancer cells were maintained in growth medium consisting of 
-minimal essential medium (Ontario Cancer Institute Media Lab), pH 7.2, supplemented with 10% fetal bovine serum. Cells grown to confluence (3–5 days for EMT6 cell lines, 5–7 days for MDA435 cell lines after seeding approximately 0.1 million cells/dish) were subcultured with 0.05% trypsin-EDTA (Invitrogen Inc., Burlington, ON, Canada), diluted (1/10) in fresh growth medium, and reseeded.
Preparation of PLNs Containing Dox and Fluorescence Probe. Dox-PLNs were prepared as described previously (Wong et al., 2005, 2006). Typically, a mixture of 100 mg of stearic acid and 0.9 ml of aqueous solution containing 5 mg of Dox and Pluronic-F68 (2.5% w/v) was warmed to 72 to 75°C. Following the addition of 2.5 mg of HPESO polymer, the mixture was stirred for 10 min and then ultrasonicated for 3 min to form submicron-sized lipid emulsion. The emulsion was dispersed in water at 4°C (one part emulsion to four or nine parts of water) to form PLN.
Blank PLNs were similarly prepared except that Dox was omitted. For PLNs loaded with rhodamine-B, rhodamine-B was used in place of Dox. For microscopic imaging that required coloading of fluoresceinamine-labeled stearic acid (FSA; see the method of preparation described below) in Dox-PLNs, stearic acid was substituted with FSA in the PLN preparation.
Dox-PLNs and blank PLNs used in the present work were characterized for their physicochemical properties such as particle size, surface charge, drug loading properties, and drug release kinetics in a previous study. Their properties are summarized in Table 1.
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Preparation of FSA and Evaluation for Labeling Stability and Fluorescence Properties. FSA was prepared according to Zhang et al. (2004) with a few modifications. In brief, 0.0439 g of fluoresceinamine isomer I and 0.0316 g of 1-ethyl-3–3-(3-dimethylaminopropyl)-carbodiimide hydrochloride were added to 1 ml of dimethyl sulfoxide, and the suspension formed was vortexed until homogeneous. The suspension was added drop-wise to stearic acid solution (1 g of stearic acid in 24 ml of dimethyl sulfoxide). The mixture formed was continuously stirred in darkness for 48 h at room temperature. At the end of the reaction, 100 ml of distilled water was added. The precipitate formed was collected by filtration, grounded into a fine powder, and washed repeatedly with copious amounts of ice-cold water until the washout liquid became colorless. The pale-yellow product containing FSA was dried in vacuum and purified by recrystallization in absolute ethanol. The presence of fluorescence activity in the final product was confirmed by scanning its ethanolic solution at 
ex = 430 nm and em = 498 nm (
-Scan fluorometer; Photon Technology International PTI, Birmingham, NJ).
The stability of the fluoresceinamine labeling on PLNs was evaluated by incubating 0.5 ml of PLN suspension (containing 50 mg of FSA) in 10 ml of growth medium at cell culture conditions for 4 h. The suspension was then centrifuged at 120,000g, and the fluorescence signal in the supernatant was measured to examine possible release of FSA from the FSA-PLN.
Western Blot Analysis. Western blot analysis was performed as described previously by our laboratory (Ronaldson et al., 2004) to allow comparison of the Pgp levels of the cell lines used. MDA435/LCC6/MDR1 and EMT6/AR1 cells were harvested by centrifugation (400g), and the cell pellets formed were lysed for 30 min at 4°C in a 250 mM sucrose buffer containing 1 mM EDTA and 0.1% (v/v) protease inhibitor cocktail. Samples were then homogenized on ice for 2 min with a Dounce homogenizer, and aliquots (25 µg of protein) from the suspension were resolved on a 10% SDS-polyacrylamide gel and electrotransferred onto a polyvinylidene difluoride membrane. The membrane was blocked overnight (4°C) in Tris-buffered saline containing 0.05% Tween 20 and 5% dry skim milk powder. Pgp and 
-actin (42 kDa) were detected by treating the samples with monoclonal Pgp antibody C219 (1:500 dilution) and antiactin antibody AC-40 (1:500 dilution), followed by IgG2a:HRP as the secondary antibody. Proteins were visualized using enhanced chemiluminescence according to the manufacturer's instructions (Pierce Chemical, Rockford, IL). Densitometric analysis of protein bands was performed using Image Quant 5.2 (Molecular Dynamics, Piscataway, NJ). Protein concentration of the cell lysate was determined with Bradford's protein assay.
Dox Cellular Uptake and Retention Studies. To establish cellular drug uptake profiles, cells were plated onto 48-well plates at densities of approximately 40,000 to 100,000 cells/well at 37°C. When cells reached confluence, Dox solution or Dox-PLN suspension was added into each well to initiate cellular drug accumulation. In the experiments with MDA435 cell lines, treatment with Dox solution/blank PLN combination was also included to evaluate the impact of blank PLNs and the surfactant. All treatments were adjusted to 10 µg/ml Dox concentration with Earle's balanced salt solution (EBSS). Blank EBSS was used as the negative control. At predetermined time intervals (0–4 h), supernatant was removed, and cells were washed with ice-cold phosphate-buffered saline (PBS; pH 7.6) and lysed with PBS containing 1% Triton X-100. Dox concentrations in the cell lysates were measured with SpectraMax Gemini XS microplate fluorometer (Molecular Devices, Sunnyvale, CA) at an excitation wavelength of ex = 478 nm and an emission wavelength of em = 594 nm. To adjust for background fluorescence from the cellular components, Dox standardization curves were also prepared using cell lysates. In this case, untreated cells were lysed with PBS/1% Triton X-100 containing Dox or Dox-PLNs at known concentrations. Separate standardizations for Dox solution and Dox-PLNs were prepared in each experiment for more accurate Dox concentration calibrations.
To evaluate cellular Dox accumulation by cancer cells after the drug efflux period, monolayer cells grown in 48-well plates were loaded with Dox in the form of free Dox solution or Dox-PLNs containing 10 µg/ml Dox for 2 h. Supernatant was removed at the end of treatment, and cells were washed with ice-cold PBS. The wells were refilled with fresh drug-free EBSS, and cells were incubated at 37°C to facilitate cellular drug efflux. At predetermined time intervals (0–2 h), supernatant containing the effluxed drug was removed. Cells were washed and lysed, and the amount of Dox retained by the cells was measured with a microplate fluorometer as described above.
Cellular Dox uptake or retention is expressed as nanomoles per milligram of protein. Protein concentrations of the cell lysates were determined by the Bradford colorimetric assay (Bradford, 1976) using reagents from Bio-Rad (Melville, NY) and bovine serum albumin (Sigma-Aldrich Inc.) as the standard. Membrane integrity of the cultured cells in the presence of Dox-PLNs and their various components (e.g., stearic acid, pluronic surfactant, HPESO polymer) was previously verified using trypan blue exclusion assay (Wong et al., 2005). There was no significant loss of cell membrane integrity when the cells were treated with Dox-PLNs or their ingredients.
Endocytosis Inhibition Studies. The effects of endocytosis inhibitors on cellular Dox uptakes were evaluated according to Ramge et al. (2000) with some modifications. Cells were pretreated with 25 µg/ml colchicine or 5 µg/ml cytochalasin B, inhibitors of pinocytosis, and phagocytosis, respectively, in EBSS for 1 h at 37°C. Following the removal of the pretreatment solution, the cells were washed with PBS, treated with Dox solution or Dox-PLNs containing 10 µg/ml Dox, and incubated under the conditions used for cell culture. Cellular Dox accumulation at 2 h was measured, and protein standardization was performed as described above. Membrane integrity of cells treated with the endocytosis inhibitors was confirmed by trypan blue exclusion assays. The capacity of the cell lines tested to perform endocytosis has also been confirmed using Lucifer yellow, a well documented endocytosis or exocytosis substrate (Jayanth and Vinod, 2003). Dox uptakes of cells pretreated with endocytosis inhibitors are compared with those without pretreatment, and the reduction in Dox uptake caused by the inhibitors is expressed as percent inhibition of Dox uptake.
Fluorescence Microscopy of Drug and Lipid Uptake into Breast Tumor Cells. For fluorescence microscopy experiments, MDA435/LCC6 cells at the confluent stage of growth were reseeded (approximately 106 cells per dish) and incubated overnight at cell culture conditions in 10-cm Petri dishes, each containing a duplicate of poly-L-lysine-coated coverslips. Cells were then treated with free Dox solution, Dox-PLNs, or Dox-FSA-PLNs for 2 h. At the end of treatment, cells were washed with ice-cold PBS, and one of the two coverslips in each dish coated with the treated cells was removed for imaging within 5 min. The remaining coverslip in each dish was reimmersed in fresh growth medium for an additional 2 h to allow cellular drug efflux. The coverslips, after removal from the Petri dish, were mounted on glass slides, placed on the platform of a differential interference contrast fluorescence microscope (Axiovert 100TV; Zeiss Inc., Oberkochen, Germany), and viewed with an oil immersion objective (100x magnification). Filter sets (ex = 540 nm and em = 590 nm and ex = 430 nm and em = 500 nm) were used for imaging of Dox and FSA, respectively. "Snap-shot" images were acquired using an intensified CCD camera under computer control. The same camera settings were used for the experiments unless otherwise specified.
For comparison, experiments were also conducted using EMT6/AR1 and PLNs loaded with rhodamine-B as substitutes for MDA435 cells and Dox-PLNs, respectively. ex = 520 nm and em = 570 nm filter sets were used for rhodamine detection.
Nuclear localization by the Dox delivered using PLNs was confirmed by nuclear staining of Dox-PLN-treated cells. MDA435/LCC6/MDR1 cells treated with Dox-PLN suspension containing 10 µg/ml Dox for 2 h and allowing for 2 h drug-free period were fixed by immersing in 4% paraformaldehyde in PBS for 20 min. The fixed cells were treated with DAPI solution (2 µM in PBS) for 5 min and rinsed with PBS twice. Fluorescence microscope images of Dox and DAPI were taken from the same microscope field. For detection of DAPI, ex = 360-nm and em = 450-nm filter sets were used. The same wavelength combination was used for Dox detection.
Data Analysis. Results are presented as mean ± S.D. from a minimum of three separate experiments in cells pertaining to different passages, unless otherwise specified. Statistical analysis for unpaired experimental data was performed using Student's t test. In all analyses, a value of p < 0.05 was considered significant.
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170-kDa product; Evers et al., 1998) in both cancer cell lines known to overexpress Pgp. Densitometric analysis showed that the Pgp expression in EMT6/AR1 cells is 1.8- to 2.1-fold higher than MDA435/LCC6/MDR1 cells. This is consistent with our previous results of cytotoxicity studies, which demonstrated higher resistance to Dox in the Pgp-overexpressing murine breast cancer cell line (LD90 > 150 µg/ml) compared with the human cell line (LD90 = 9 µg/ml) (Cheung, 2005; Wong et al., 2006). The results of Western blot of the wild-type cell lines (EMT6/WT and MDA435/LCC6/WT) (data not shown) indicated that Pgp expression in these cells was undetectable (H. L. Wong, K. Babakhanian, R. Bendayan, and X. Y. Wu, unpublished data) (Cheung et al., 2006).
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To determine whether this effect was simply due to the nondrug ingredients of PLN such as the lipid, polymer, and surfactant, we also treated MDA435 cell lines with blank PLN/Dox solution combination. In both MDA435 cell lines, the addition of blank PLNs to Dox solution did not increase cellular Dox uptake. Modest decreases in Dox accumulation were observed instead. However, the decreases were not statistically significant.
Enhanced Dox Retention by PLNs in Pgp-Overexpressing MDA435/LCC6 Cells and EMT6 Cells. Fig. 3, A to D, demonstrates the effects of using different Dox treatments (Dox solution or Dox-PLN) on cellular Dox retention in tumor cells. The declines in the cellular Dox level as a result of drug efflux into fresh EBSS medium are presented in the form of 2-h time profiles. As expected, after treatment with Dox solution, cellular efflux was faster in Pgp-overexpressing cell lines than in their corresponding wild-type cells. In both wild-type cell lines (Fig. 3, A and C), the use of Dox-PLNs in place of Dox solution did not improve drug retention. In contrast, both Pgp-overexpressing cell lines treated with Dox-PLNs demonstrated enhanced Dox cellular retention. Significantly higher cellular Dox levels were observed 1.5 and 1 h after initiation of the drug efflux phase in MDA435/LCC6/MDR1 cells (Fig. 3B) and EMT6/AR1 cells (Fig. 3D), respectively. The enhancement of cellular Dox retention contributed by Dox-PLNs was more prominent in EMT6 cells than in MDA435 cells. When comparing cells treated with Dox-PLNs with those with Dox solution, over 8-fold enhancement in Dox retention was demonstrated in EMT6/AR1 cells at 2 h, whereas an approximately 90% enhancement was observed in MDA435/LCC6/MDR1 cells at the same time point.
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In both Pgp-overexpressing cell lines treated with Dox solution, pretreatment with colchicine or cytochalasin-B enhanced Dox uptake instead (i.e., negative values obtained). This is understandable because both agents are Pgp substrates, and they probably reduced the cellular Dox efflux by competition (Zilfou and Smith, 1995). In comparison, cytochalasin-B demonstrated net inhibitory effect against Dox uptake in both MDA435/LCC6/MDR1 cells and EMT6/AR1 cells treated with Dox-PLNs. In MDA435/LCC6/MDR1 cells, this inhibition was significant compared with cells treated with Dox solution.
Dox Is Better Retained Intracellularly By Pgp-Overexpressing Cells and Tends to Localize in Cell Nuclei When Delivered as Dox-PLNs. Figures 5, 6, 7 present fluorescence microscopic images of cells treated with Dox (Fig. 5, A–J), Dox and DAPI (Fig. 6, A and B), or rhodamine (Fig. 7, A and B) in free solutions or PLN form. The impact of the formulations is compared in terms of the intensity and cellular distribution of fluorescence. As expected, strong fluorescence intensity was detected in wild-type cells, and it did not substantially fade after 2-h efflux test (Fig. 5, A and B). On the other hand, although MDA435/LCC6/MDR1 cells treated with Dox solution exhibited respectable fluorescence intensity after treatment, their intensity became barely detectable after the 2-h drug efflux period (Fig. 5, C and D). In comparison, the fluorescence in MDA435/LCC6/MDR1 cells treated with Dox-PLNs containing the same Dox concentration (5 µg/ml) retained moderately high intensity (Fig. 5, E versus F). The same trend was observed in MDA435/LCC6/MDR1 cells treated with higher Dox concentration (10 µg/ml) (Fig. 5, G–J), where stronger fluorescence was retained in cells treated with Dox-PLNs. When comparing the fluorescence intensities, it should be noted that the brightness setting was intentionally lowered (to approximately 50% sensitivity) during imaging in Fig. 5, A, B, I, and J, to minimize light saturation for improved image clarity.
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Figure 6, A and B, presents the fluorescence microscope images of MDA435/LCC6/MDR1 cells treated with Dox-PLNs and DAPI, respectively. The similar locations of Dox and DAPI seen in the images confirm the nuclear localization of Dox delivered by the PLN formulation.
To determine whether the above trend would also occur in another cell line with a different Pgp substrate, EMT6/AR1 cells were treated with a solution (Fig. 7A) or PLN form (Fig. 7B) of rhodamine for 2 h followed by efflux experiment for another 2 h. Similar to the results observed with Dox, a marked improvement of rhodamine retention is seen when the dye was loaded and administered in the form of PLN.
Dual Fluorescence-Labeled Dox-FSA-PLNs Showed Similar Location and Retention of Dox and Lipids in Pgp-Overexpressing Cells. To delineate whether the lipid played a role in the drug uptake and retention by the Pgp-overexpressing cancer cells, the lipid molecules (stearic acid) used for PLN preparation were conjugated with fluorescence groups, and their cellular distribution was examined and compared with that of the Dox. Examinations using fluorescence microscopy and fluorescence spectroscopy were performed on PLNs labeled with FSA. No noticeable fluorescence intensity in the incubation medium after 4-h incubation at culture conditions was observed (data not shown), indicating negligible leakage of FSA from FSA-PLNs. Initial studies also confirmed no interference of the fluorescence signals from Dox and FSA when they were in a lipid environment. After having been treated with dual-labeled PLNs, i.e., Dox-FSA-PLNs, for 2 h and reincubated in a fresh medium for an additional 2 h, MDA435/LCC6/MDR1 cells still exhibited fluorescence signals specific for Dox and FSA, respectively. The cellular distribution of fluorescence corresponding to Dox (Fig. 8A) and FSA (Fig. 8B) generally overlapped with each other. The fluorescence of Dox, however, appeared to be slightly more diffusive than FSA.
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). Effective chemotherapy thus requires a reasonably high level of drug molecules to accumulate within the cancer cells. In addition, the effectiveness of chemotherapy is also positively correlated to drug exposure time (Millenbaugh et al., 2000). In Pgp-overexpressing cells, it becomes a difficult task to maintain a high intracellular drug level for a reasonable length of time. In the present study, we found that, in addition to moderate enhancement of cellular Dox accumulation (Fig. 2, B and D), the new nanoparticle formulation resulted in substantial improvement of Dox retention by two drug-resistant breast cancer cell lines (Fig. 3, B and D). This may at least in part account for the enhanced in vitro anticancer activity of Dox-PLNs demonstrated in our previous study (Wong et al., 2006).
There have been studies showing that some of the lipids and surfactants used in lipid-based formulations possess intrinsic Pgp inhibitory activities (Batrakova et al., 1999; Romsicki and Sharom, 1999). However, it does not explain the finding that blank PLNs did not improve Dox accumulation in Dox-treated MDA435/LCC6/MDR1 cells (Fig. 2B). These results indicate that the nonactive ingredients of the tested formulations, at least at the concentrations used, do not suppress Pgp activity by themselves. In fact, this is consistent with our previous study, which showed that the cancer-suppressive activity of Dox treatment was also improved only when Dox was an integral part of the nanoparticles and not when it was separately added in the presence of blank PLNs or polymer (Wong et al., 2006).
The advantages of nanoparticles over solution appear to be augmented by the increasing Pgp level of the treated cells. The enhancement in drug accumulation by Dox-PLNs was larger in EMT6/AR1 than in MDA435/LCC6/MDR1. This trend is even more noticeable in the drug retention data (enhancement by Dox-PLNs over Dox solution after 2 h drug efflux: 90% in Fig. 3B, 900% in Fig. 3D). Because Western blot analysis (Fig. 1) shows that EMT6/AR1 expresses a higher Pgp level than MDA435/LCC6/MDR1 and that these drug accumulation and retention-enhancing effects by Dox-PLNs are practically nonexistent in wild-type cells, it is likely that Dox-PLNs are more useful when the cancer cells express higher levels of Pgp (i.e., more drug-resistant). As discussed before, PLNs do not directly inhibit Pgp. Instead, PLNs may improve the treatment by allowing the drug molecules to bypass the efflux action of Pgp.
In fluorescence microscopy studies, regardless of the dosage form, considerable amounts of Dox could be detected in MDA435/LCC6/MDR1 immediately after drug treatment, with Dox-PLN-treated cells accumulating moderately higher cellular Dox levels. The impact of the dosage form was much more noticeable after the 2-h drug efflux period. Dox was clearly better retained in cells treated with Dox-PLNs (Fig. 5F) than Dox solution (Fig. 5D). A similar trend was observed in cells treated with higher Dox concentration (Fig. 5, G–J). These findings generally support the drug uptake and retention data.
Cell nucleus is the organelle of interest in our study because it is the major target of many cytotoxic drugs (Dorr, 1998). Considering that DNA may quench the fluorescence of Dox and low-level Dox binding in cell nuclei is relatively difficult to be detected (Lam et al., 2000), it is encouraging to observe strong fluorescence in the nuclei of cells treated with Dox-PLNs (Fig. 5G). The images show that Dox-PLN is superior to Dox solution in attaining high nuclear drug concentration in Pgp-overexpressing cells. Since it has been demonstrated that the nuclear membrane also has Pgp to prevent drug penetration into the cell nuclei (Baldini et al., 1995), the images suggest that the lipid nanoparticles may also help overcome this possible source of drug resistance, although the operative mechanism requires further investigations.
Wielinga et al. (2000) demonstrated that passive permeation also plays an important and independent role in Dox cellular efflux in drug-resistant cells. Considering that MDA435/LCC6/MDR1 has only moderately high Pgp level, the role of passive diffusion in drug clearance from these cells should be significant. Therefore, it may be argued that the aforementioned findings are attributable to a diminished net outward drug diffusion rate, for example, as a result of a local buildup of Dox-PLNs outside the cell membrane surfaces. However, had substantial reduction in passive drug diffusion occurred, the cellular Dox accumulations in wild-type cells treated with Dox-PLNs would have been similarly enhanced like in their Pgp-overexpressing sublines (Fig. 2, A and C). The cellular drug efflux rates of the EMT6/AR1 cell line, which expresses a higher level of Pgp and its cellular drug efflux rates, should be less reliant on passive diffusion. Instead, EMT6/AR1 cells responded even to a greater extent to Dox-PLNs in terms of Dox accumulation and retention. Furthermore, the microscopic images of EMT6/AR1 cells treated with rhodamine, an efficient substrate for Pgp, also demonstrated distinctively higher drug retention in cells when rhodamine was administered in nanoparticle form (Fig. 7, A and B). These results all suggest that it is more difficult for Pgp to remove the drug molecules from the cells when these molecules are associated with the nanoparticles. It appears that this is the principal mechanism responsible for the enhanced cellular drug retention and to some extent cellular drug uptake. The result with rhodamine also indicates that this phenomenon is not limited to Dox but to other Pgp substrates.
We further examined the role of lipids of the nanoparticles. The similarity of the cellular distributions of FSA and Dox (Fig. 8, A and B) suggests that it is likely that at least part of the Dox retained in the cells was physically associated with the lipids of nanoparticles. This may also explain the granular appearance of the fluorescence in the images of Dox-PLN-treated cells.
As a modified form of SLN, the nanoparticles used in the present study were prepared with solid lipids. In comparison with liquid lipid-based formulations, such as liposomes, the aforementioned Dox-lipid complexes or aggregates are expected to have relatively good physical integrity after being internalized into the cells. These drug-lipid complexes are likely too large to be handled by Pgp and are consequently "trapped" within the cancer cells. It is possible that other forms of SLN may have similar properties once they gain entrance into their targeted cells.
Endocytosis inhibition studies were conducted to further understand how the drug or drug-loaded nanoparticles enter the cells. Colchicine mainly inhibits pinocytotic uptake of liquid and particles smaller than 50 nm in diameter, whereas cytochalasin-B primarily inhibits phagocytosis of particles greater than 100 nm (Ramge et al., 2000). In Fig. 4, A and B, it is evident that Dox-PLN accumulation was inhibited by colchicine in both wild-type cell lines, and the magnitudes of inhibition were not affected by the type of Dox formulations. On the other hand, both wild-type cells pretreated with cytochalasin-B exhibited stronger inhibition of cellular drug uptake (p < 0.05) when treated with Dox-PLNs, indicating significant phagocytosis of Dox-PLNs or their fragments by the wild-type cancer cells.
The situation is more complicated in Pgp-overexpressing cells because both colchicine and cytochalasin-B have Pgp-inhibitory effects (Zilfou and Smith, 1995; Gottesman, 2002), which may lead to various degrees of Dox accumulation enhancement. Nevertheless, Dox-PLN-treated cells were still more responsive to cytochalasin-B, indicating phagocytosis likely occurred in Pgp-overexpressing cells. Overall, the drug accumulation was only incompletely inhibited, and there was no further increase in inhibition when these experiments were repeated at higher concentrations of inhibitors (data not shown). It is evident that a substantial fraction of drug still enters the cells by simple diffusion. This may be practically needed, particularly in solid tumor therapy, because direct contacts between the nanoparticles with all tumor cells are unlikely.
Endocytosis can be receptor- or adsorption-mediated. Endocytosis of anionic liposomes mediated by scavenger receptors has been demonstrated before (Lee et al., 1992), although this possibility would be scarce. Adsorption-mediated endocytosis occurs at a higher efficiency with cationic nanoparticles (Chenevier et al., 2000; Templeton, 2002), but negatively charged particles can still be captured by cells at a reasonably high rate as shown in a number of liposomal formulations (Lee et al., 1992; Miller et al., 1998). The high lipophilicity of PLN may also improve their adsorption onto the cell membranes. Since this adsorption mechanism is nonspecific, for improved efficiency of endocytosis of Dox-PLNs by cancer cells, ligands that specifically target cancer-related receptors and cationic lipids will be considered in the future PLN formulation designs.
Drug in PLNs probably enters cancer cells by a combination of simple diffusion and phagocytosis, as schematically illustrated in Fig. 9. The phagocytotic uptake (pathway 2) is not necessarily more efficient than simple diffusion (pathway 1) because Dox-PLNs were shown not more effective than Dox solution in wild-type cells. However, part of the drug likely remains physically associated with the solid lipids when internalized by cells. In this form, the drug can neither be easily removed by Pgp efflux nor quickly diffuse out and may continue to build up in the cells to serve as intracellular drug sources, which may lead to chronic suppression of the drug-resistant cancer cell proliferation. All in all, the present study suggests a new mechanism of action for a novel lipid-based formulation for overcoming the drug resistance of Pgp-overexpressing tumor cells.
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ABBREVIATIONS: MDR, multidrug resistance; Pgp, P-glycoprotein; Dox, doxorubicin; SLN, solid lipid nanoparticle; PLN, polymer-lipid hybrid nanoparticle; Dox-PLN, Dox-loaded polymer-lipid hybrid nanoparticle; DAPI, 4'-6-diamidino-2-phenylindole; HPESO, hydrolyzed polymer of epoxidized soybean oil; FSA, fluoresceinamine-labeled stearic acid; EBSS, Earle's balanced salt solution; PBS, phosphate-buffered saline.
Address correspondence to: Dr. Xiao Yu Wu, Graduate Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, 19 Russell Street, University of Toronto, ON, Canada M5S 2S2.
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170 kDa) in cultured MDA435/LCC6/MDR1 and EMT/AR1 cells. Whole-cell lysate preparations (25 µgof protein) from MDA435/LCC6/MDR1 (lane 1) and EMT6/AR1 (lane 2) cells were resolved on a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Pgp was detected using the monoclonal Pgp antibody C219 (1:500 dilution), whereas 
