Polycationic Nanodrug Covered with Hyaluronic Acid for Treatment of

Aug 5, 2010 - Department of Biotechnology, The Catholic University of Korea, 43-1 Yeokkok2-dong,. Wonmi-gu, Bucheon-si, Gyeonggi-do, 420-743, Korea...
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Polycationic Nanodrug Covered with Hyaluronic Acid for Treatment of P-Glycoprotein Overexpressing Cancer Cells Hyeona Yim and Kun Na* Department of Biotechnology, The Catholic University of Korea, 43-1 Yeokkok2-dong, Wonmi-gu, Bucheon-si, Gyeonggi-do, 420-743, Korea Received May 24, 2010; Revised Manuscript Received July 7, 2010

To treat cancer cells overexpressing P-glycoprotein (P-gp), we propose a new concept using a nanodrug. The nanodrug was prepared from polyethyleneimine (PEI)/all-trans retinoic acid (ATRA) conjugates (PRA) and covered with hyaluronic acid (HA) to control the cytotoxicity of PRA (yielding PRA-H). The size distribution of PRA-H was narrow, with an average particle size of approximately 143 nm. Its superior stability in phosphate-buffered saline (PBS) was verified by monitoring changes in particle size and zeta potential for 24 h, which were negligible. In contrast, PEI-H (not conjugated with ATRA) exhibited a significant change in particle size and zeta potential. Although PRA was highly cytotoxic against HCT-8 and SNU-484 cancer cells, both of which overexpress P-gp, the cytotoxicity was significantly reduced by shielding with HA. The cytotoxicity of PRA-H was recovered by treatment with hyaluronidase (HAase), which degrades HA and is present in tumors at high concentrations. These results were confirmed by optical microscopy, fluorescence-activated cell sorting (FACs) analysis, and confocal microscopy. The cytotoxic mechanism of PRA was revealed as a type of necrotic lysis by FACs analysis with propidium iodide (PI) staining. Furthermore, PRA increased HCT-8 cell (colon cancer) permeability to doxorubicin (DOX). Therefore, we concluded that PRA-H is a promising new candidate for the treatment of cells with multidrug resistance (MDR) induced by overexpression of P-gp and cancer stem cells.

Introduction Multidrug resistance (MDR) induced by the overexpression of the 170 kDa plasma membrane-associated glycoprotein known as P-glycoprotein (P-gp) is a major obstacle for the successful treatment of cancer. P-gp, a member of the superfamily of ATP-binding cassette transporters, pumps a wide range of structurally diverse amphipathic anticancer drugs out of the cell, including anthracyclines, vinca alkaloids, epipodophyllotoxins, and taxanes. This phenomenon results in decreased intracellular accumulation of drugs, thus, lowering their cytotoxicity. P-gp expression is either intrinsic (cells in gastrointestinal tract) or acquired (cancer cells) according to the cell. In clinical specimens, MDR tumors reveal a very low response rate to chemotherapy.1-3 To overcome MDR, various P-gp inhibitors (chemosensitizers) with the ability to suppress drug efflux from cells have been developed and their efficacy in vitro and in vivo has been reported. Although chemosensitizers such as verapamil, tamoxifen, cyclosporin A, and quinine/quinidine enhance the sensitivity of MDR cancer cells to anticancer drugs, they did not demonstrate dramatic efficacy during treatment in clinical studies.4-6 Moreover, side effects such as cardiac, hepatic, and renal toxicities have been associated with their use.4 Due to these disadvantages, the use of chemosensitizers in clinical studies is limited in the treatment of cancer cells overexpressing P-gp. Recently, a nanocarrier system for overcoming MDR has been proposed that relies on an internalization mechanism different from that of free anticancer drugs.7-10 Nanocarriers internalize in cells via endocytosis, thus, avoiding efflux by P-gp. In a practical application, pH-sensitive nanocarriers decorated with a targeting ligand that responds to endosomal pH in cancer * To whom correspondence should be addressed. Tel.: +82-2-2164-4832. Fax: +82-2-2164-4865. E-mail: [email protected].

Figure 1. Difference in shielding mechanism between (a) PEI-H and (b) PRA-H. PRA, before the interaction with HA, is already assembled due to ATRA self-aggregation, resulting in the formation of many hydrophobic cores. Thus, HA interacts only with the surface of PRA. However, in the case of PEI-H, HA locates in the inner core or outer shell or both due to the free interaction between PEI and HA.

cells have been designed. After they are internalized to cancer cells via receptor-mediated endocytosis, they killed MDR cells with fast release kinetics by particle disruption.11-13 We suggest a new concept for conquering MDR using a nanodrug system that consists of polyethyleneimine (PEI) with a biocompatible polymer hyaluronic acid (HA) cover (Figure 1a). PEI is a cationic polymer that has been investigated as a gene delivery carrier. It has high transfection efficiency due to its positive charges, enabling it to be readily internalized in cells with a negative surface charge.14-17 However, the positive charge is highly cytotoxic due to an electrokinetic interaction with the negatively charged cell surface via a polarization effect.17-19 Such an interaction could interfere with enzyme activity involved in electron transport and oxidative phosphorylation in the cell membrane. In addition, it is known to induce membrane damage via the disruption of the phospholipid bilayers, resulting in the formation of holes. Cellular damage of this nature leads to rapid cell death.20,21

10.1021/bm100562z  2010 American Chemical Society Published on Web 08/05/2010

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We reasoned that if the positive charge of PEI could be controlled, its cytotoxicity would become manageable. Indeed, the cytotoxicity of PEI complexed with a gene is lower than that of free PEI.17 Therefore, we covered PEI with HA, which is found in the extracellular matrix of most human tissues22,23 and is one of most useful materials in the fields of biomaterials, tissue engineering, and drug delivery. The cytotoxicity of PEI in normal tissue is dramatically decreased due to reduction of the positive charge by HA. However, at the tumor site, HA is degraded by hyaluronidase (HAase), the concentration of which is more than 20-1000 times higher than that in normal tissues.24-27 The largest concentrations are found in high-grade tumor lesions (191 ( 7.9 mU/mg protein), followed by highgrade tumors (36.6 ( 2.9 mU/mg protein) and low-grade tumors (9.4 ( 1.4 mU/mg protein).27 In this manner, the cytotoxicity of PEI is selectively restored. To create a more ideal PEI-HA complex with higher stability and total shielding of the positive charge, PEI was conjugated with all-trans retinoic acid (ATRA) as a hydrophobic moiety to generate PEI-ATRA (PRA). In previous research, we suggest the possibility of PRA-H as anticancer material.28 Cell lines that have different IC50 values in doxorubicin (DOX) were killed at identical IC50 values. In this study, the cytotoxicity of PRA in the presence and absence of HA and following treatment with HAase was measured by the method of transcriptional and translational (MTT) assay and fluorescence activated cell sorting (FACs) analysis. The morphology of cells was also observed by field emission scanning electron microscopy (FE-SEM) and confocal microscopy. In addition, to determine the feasibility of using this system to kill MDR cells, we targeted SNU-484 (stomach cancer) and HCT-8 (colon cancer) cells, both of which intrinsically overexpress P-gp.29-31

Experimental Section Materials. Branched PEI (MW: 25 kDa), ATRA, 1,3-dicyclohexylcarbodimide (DCC), N-hydroxysuccinimide (HOSu), hyaluronidase (HAase) from bovine testes (type 2; 200–300 U/mg), rhodamine B isothiocyanate (RITC), fluorescein isothiocyanate (FITC), and propidium iodide (PI) were purchased from Sigma Chemical Company (St. Louis, MO). HALM (MW: 5800 kDa) was obtained from Bioland Ltd. (Cheonan-si, South Korea). All of the reagents were of extra reagent grade, thus, requiring no further purification. Synthesis of PEI/ATRA (PRA) Conjugates. The synthesis of PEI/ ATRA (PRA) conjugates via DCC- and HOSU-mediated amide formation between the primary amines of PEI and a carboxyl acid of ATRA has previously been reported by our group.17 In brief, a mixture of ATRA (PRA 1, 0.83 mmol; PRA 2, 1.25 mmol; and PRA 3, 1.6 mmol), 1,3-dicyclohexyl-carbodimide (1.2× the concentration of ATRA in moles), and N-hydroxysuccinimide (1.4× the concentration of ATRA in moles) was dissolved in dimethyl sulfoxide (DMSO; 6 mL). This solution was stirred for 3 h. PEI (0.04 mmol) was completely dissolved in DMSO after which the two reactant solutions were mixed and stirred for 24 h. The resulting solution was filtered to eliminate insoluble dicyclohexylurea, and the filtrate was dialyzed against distilled water (DW) for 3 days using a dialysis membrane (MWCO: 3500). PRAs were lyophilized for 3 days then analyzed by 1H NMR and UV-vis spectroscopy. Preparation and Characterization of HA-Covered PRA (PRAH). To prepare PRA2-H, PRA 2 (0.2 mg/mL) dispersed in 1 mL of DW was added to HA solution (0.2 mg) and allowed to sit for 24 h, during which time the transmittance of the solution changed from clear to opaque. The solution was incubated for 24 h at 37 °C to evaluate its stability, then characterized in terms of size and zeta potential (Zetasizer; Malvern Instruments Ltd., Worcestershire, U.K.). The particle size of the resulting nanogels was determined by dynamic light scattering (DLS;

Yim and Na Malvern Instruments Ltd. Series 4700). The DLS experiment was performed with an argon ion laser system tuned to a wavelength of 488 nm. Each sample was filtered through a 0.45 µm filter directly into a precleaned 10 mm diameter cylindrical cell. Intensity autocorrelation was measured at a scattering angle (θ) of 90° at 25 °C. The sample concentration was maintained at 0.1 mg/mL. The morphology of the nanoparticles was observed by FESEM (S- 4700, Hitachi, Japan). A drop of the nanoparticles was placed on a graphite surface and coated with Pt by sputtering for 4 min at 20 mA. Cytotoxicity of PRA 2, PRA 2-H, and PRA 2-H Treated with HAase. The cytotoxicity of samples against SNU-484 and HCT-8 cells, which intrinsically overexpress P-gp,29-31 was measured via an MTTbased assay. Cells were seeded into 96-well plates at a density of 1.0 × 104 cells/well and cultured for 24 h in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics at 37 °C in humidified air containing 5% CO2. After adding PRA 2 (20 µg/mL), PRA 2-H (20 µg/mL), or PRA 2-H (20 µg/mL) with HAase (50 U/mL), the cells were incubated for 24 h. MTT solution (0.5 mg/ mL) was then added to the cells. After incubation for 2 h, the MTTcontaining medium was removed and formazan crystals in living cells were dissolved with DMSO (100 µL). The absorbance of the formazan crystals was measured at 595 nm. Observation of Cell Morphology via Optical Micrographs. To observe changes in the cell surface after treatment with PRA2, HCT-8 cells were observed by FE-SEM. Cells were seeded onto 24 well plates at a density of 1.0 × 105 cells/well and cultured as described above. After 24 h, the medium was exchanged with serum-free RPMI containing PRA 2 (20 µg/mL), PRA 2-H (20 µg/mL), or PRA 2-H (20 µg/mL) with HAase. Cells were reseeded on 6-well plates (1 × 105 cells/well), covered with a coverglass, incubated for 24 h, then rinsed twice with Dulbecco’s phosphate-buffered saline (DPBS) and fixed in a suspension of 4% paraformaldehyde. Changes in cell morphology over time were visualized at 400×. Cell Permeability Test. The permeability of PRA2 (20 µg/mL)treated cells was investigated by FACs analysis using DOX. HeLa, SNU-484, and HCT-8 cells were seeded in 6-well plates (1 × 106 cells/ well) and incubated for 24 h. PRA20 µg/mL was added and the cells were incubated for 2 h and then rinsed twice with DPBS. DOX (1 µg/mL) in serum-free medium was added, and the cells were incubated for 4 h. This step was also applied to cells treated with DOX only. The negative control group was incubated in serum-free medium without drugs for 4 h. Cells were collected in a tube and the concentration of internalized DOX was determined by flow cytometry using FL 3, as described above.32 Cell-Killing Mechanism of PRA. To obtain information on the cellkilling mechanism of PRA, treated cells were analyzed by PI staining.33,34 HCT-8 cells were seeded in 6-well plates (1.0 × 106 cells/ well) in complete RPMI medium and incubated at 37 °C in 5% CO2 for 24 h. The medium was then exchanged with serum-free RPMI containing PRA2 (20 µg/mL), PRA 2-H (20 µg/mL), and PRA2-H (20 µg/mL) with HAase and the cells were incubated for 4 h (PRA2) or 12 h (PRA2-H, PRA2-H with HAase). The medium was removed and the remaining cells were washed twice with DPBS. Try-EDTA was added to the cells (0.5 mL/well) and incubated for 3 min. Cells were centrifuged at 1500 rpm for 3 min, then resuspended in PI solution (1 µg/mL). After incubation for 15 min, the cells were rinsed twice. The resulting cell pellets were then resuspended in DPBS. The fluorescence signal of individual cells was assessed with a Cytomics FC500 flow cytometer (Beckman Coulter) using FL3, a channel in the FACs system mainly used to detect a red fluorescent intensity emitted from PI and doxorubicin (DOX) at 630 nm.35 Data were analyzed using CXP software (Beckman-Coulter, Miami, FL). Confocal Study. HCT-8 cells were seeded onto 6-well plates, covered with an autoclaved cover-glass and incubated overnight. HA and PRA2 were fluorescently labeled with FITC and RITC, respectively. A 2 µg/mL solution of HA-FITC-covered PRA2-RITC (i.e., fluorescently labeled PRA-H) was added to the cultured cells and incubated

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Table 1. Physicochemical Characterization of Various Conjugates of PEI-Grafted-ATRA

PRA 1 PRA 2 PRA 3 a

ATRA/PRA (mole)a

size (A.V. ( SD nm)b

ζ-potential (A.V. ( SD nm)b

PDIb

3.8 6.9 7.5

827.7 ( 47.31 174.4 ( 30.20 249.24 ( 19.43

42.1 ( 0.62 30.77 ( 0.40 22.47 ( 1.18

0.68 0.23 0.30

Values analyzed by UV-vis spectroscopy at 395 nm.

b

Values analyzed by dynamic light scatter.

for 24 h. The cells were then rinsed twice with DPBS and treated with 2 mL of 4% paraformaldehyde solution for 10 min. The solvent was removed and the cells were rinsed again with DPBS. Uptake of PRA into the cells was monitored with a confocal laser scanning microscope (Karl Zeiss, Oberkochen, Germany). Fluorescent images were obtained with a water-immersion lens (400 ×) at an exposure time of 200 ms. With the exception of resizing and preparation of montages by means of NIH Image J version 1.33u, the images were not altered in any other way.

Result and Discussion In general, the cancer cell-killing mechanisms of conventional anticancer drugs such as DOX, taxol, and cisplatin can be divided into three main categories: those that (1) inhibit the synthesis of DNA precursors, (2) directly damage DNA in the cell nucleus, and (3) suppress the synthesis or breakdown of the mitotic spindles.36,37 Thus, these drugs have to penetrate the membranes of cancer cells to reach the cytosol or the nucleus. However, cancer cells overexpressing P-gp (MDR cells) strongly inhibit the internalization of drugs across the cell membrane, thus, reducing their efficacy. Therefore, to overcome P-gp-mediated MDR and obtain a high response rate, it is necessary to enhance the permeability of the cell membrane to anticancer drugs. Recently, several researchers have attempted to use cationic polymers such as PEI, poly-L-lysine, and poly(amidoamine) dendrimers to achieve this goal.14,38-40 Although these polymers enhance cellular membrane permeability via the generation of holes,15,16,38 they are strongly cytotoxic.17 However, if the cytotoxicity could be controlled, they could be employed as powerful anticancer drugs for the treatment of MDR cancer cells and cancer stem cells. The present study is an attempt to prove this hypothesis. Physicochemical Characterization of PRA and HA-Covered PRA. Three PRAs with different molar ratios of ATRA to PEI were synthesized by the conventional carbodiimide method41 and analyzed by 1H NMR and UV-vis spectrophotometry. The 1 H NMR spectra of PRA showed unique peaks for both PEI and ATRA (Figure S1). The physical state of PRA changed from a sticky solid to a fine powder with increasing ATRA content (Figure S2). These results coincide with those of our previous report.17 The ATRA graft ratios of PRA 1, 2, and 3 obtained by measuring the absorbance of each solution (0.1 mg/ mL) at 395 nm via UV-vis spectrophotometry were 3.8, 6.1, and 7.5 mol per mole of PEI, respectively.17 The particle sizes of PRA 2 and 3 were less than 300 nm with monodispersion, while that of PRA 1 was over 800 nm. Therefore, ATRA is well-suited to its role as a hydrophobic moiety to form the hydrophobic core in PRA conjugates. All samples revealed positive charges due to the presence of PEI (Table 1). To control the cytotoxicity of PRA 2, it was covered with the biocompatible anionic polymer HA to generate PRA 2-H, and its stability in terms of size and zeta potential was evaluated. The size of PRA 2-H (PRA/HA, 1:1 wt %) was less than 150 nm with a narrow size distribution. The change in size and size distribution after one day was negligible (Figure 2a). On the other hand, the size of PEI-H was 274.9 ( 20.77 nm, and it

Figure 2. Change in the particle size and size distributions of (a) PRA 2-H and (b) PEI-H in one day.

increased up to three times after one day by complex dissociation and reaggregation (Figure 2b). These results indicate that the stability of PEI-H was much lower than that of PRA 2-H. This difference in stability was due to the difference in the degree of self-assembly between the two types of nanoparticles. Before PRA interacts with HA during the formation of PRA-H, it is already assembled due to the self-aggregation of ATRA, which induces formation of the hydrophobic core. Thus, HA interacts only with the surface of PRA. However, in the case of PEI-H, HA locates at the inner core or outer shell or both due to the free interaction between PEI and HA (Figure 1), thus, resulting in the difference in surface charge between PEI-H particles. This hybridized surface charge would then lead to low charge repulsion between PEI-H particles and particle aggregation. This hypothesis is supported by the zeta potential results. PRA 2-H nanoparticles showed a high negative

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Figure 3. Change in the zeta potential of PRA 2-H (0.1 mg/mL) and PEI-H (0.1 mg/mL) in 1 day.

surface charge with a narrow standard mean deviation (-38.767 ( 3.04 mV). PEI-H had a low negative surface charge with a wider deviation (Figure 3). After 1 day, the surface charge of PEI-H significantly changed to a positive charge while the change in the surface charge of PRA 2-H was insignificant. The results indicated that the surface charge of PEI-H is more heterogeneous than that of PRA-H. The heterogeneous charge of the PEI-H surface leads to additional interactions, such as Coulombic and van der Waals forces interactions, between the PEI-H particles. These interactions facilitated particle aggregations.42 Observation of morphology by FE-SEM revealed that PRA 2-H was well dispersed, with sizes ranging from about 80 to 170 nm (Figure 4a). However, only PRA 2 showed aggregation due to its sticky nature (Figure 4b). Thus, the data indicate that HA contributed to the stability of PRA. Cytotoxicity of PRA 2-H against Cancer Cells Overexpressing P-gp. Many papers have reported that P-gp is inherently overexpressed in a variety of gastrointestinal (GI) tract cells; such cells have been employed for research on MDR.3,29-31 We chose HCT-8, a human colon cancer cell line, and SNU-484, a human stomach cancer cell line, to prove the potential of PRA in the treatment of MDR cells.29-31 HeLa cells, which do not express P-gp, were used as a control. First, to verify drug resistance of the cells, they were treated with various concentrations of DOX. HeLa cells showed IC50 values in the range of 2-10 ug/mL (DOX). However, the IC50 values of SNU-484 (50-150 ug/mL) and HCT-8 (500-1500 ug/mL) were 20- and 300-fold higher, indicating that P-gp was overexpressed in SNU-484 and HCT-8 cells (Figure 5a). This result corresponds to the analysis of P-glycoprotein (P-gp) RNA (Figure

Figure 5. Cytotoxicity of (a) DOX and (b) PRA 2 at various concentrations over 24 h. (0) HCT-8, (∆) SNU-484, and (b) HeLa cells. (c) Cytotoxicity of PRA 2-H (20 µg/mL) with and without treatment with HAase (50 U/mL). (d) Cytotoxicity of PEI-H (20 µg/ mL) with and without treatment with HAase (50 U/mL).

S4) and another report.43,44 In contrast, PRA (not covered with HA) appeared to be highly cytotoxic, with IC50 values in the range of 2-10 µg/mL against SNU-484 and HCT-8 cells, a result that did not correlate with the presence of P-gp (Figure 5b). Therefore, the cytotoxic mechanism of PRA is different from that of DOX. As mentioned above, cationic polymers such as PEI, poly-L-lysine, diethylaminoethyl-dextran, and poly(amidoamine) dendrimers form holes in the cell membrane via an interaction between their positive charge and the negative charge of the cell membrane.14,38-40 Such holes not only enhance the membrane permeability of the material,15,16,38 but also promote cell death. Thus, we infer that the cytotoxicity of PRA was induced by the hole formation mechanism, which destroyed the balance of osmotic pressure. The viability of cells in the presence of PRA 2-H (20 µg/mL) was also measured to evaluate the ability of HA to inhibit cytotoxicity. The viability of all three cell types tested was above 85% (Figure 5c). This result was likely induced by a change in the positive surface charge of PRA to a relatively high negative charge. The reduced cytotoxicity of PRA-H could be recovered by treatment with HAase. Fortunately, for our purposes, the level of HAase at

Figure 4. FESEM images of PRA 2 (0.1 mg/mL) and PRA 2-H (0.1 mg/mL). Scale bar is 500 nm.

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Figure 6. Optical micrographs (400×) of the surface morphology of HCT-8 cells before (PRE), immediately (POST), and up to 24 h after treatment with PRA 2 (20 µg/mL), PRA 2-H (20 µg/mL), and PRA 2-H (20 µg/mL) with HAase (50 U/mL).

Figure 7. Permeabilities of DOX (a,b) and PI (c,d) in cells were measured by FACs. Cells were exposed to DOX at 1 µg/mL for 4 h or incubated with PRA 2 (10–20 µg/mL) for 2 h before treating with DOX. (a) HCT-8 cells, (b) SNU-484 cells, N.C.: negative control (untreated cells). Cells were stained with PI at 1 µg/mL. Flow cytometry of HCT-8 cells treated with (c) only PRA 2, (d) PRA 2-H, and PRA 2-H plus HAase.

the tumor site in metastatic breast cancer and several carcinoma lines is more than 20-100 times higher than that in normal tissue.24-26 Indeed, many studies have described the elevation of HAase content in the sera of cancer patients,18,19 a phenomenon associated with cancer progression.27,45-47 In this study, we used HAase type 2, which is anchored to glycosylphosphatidylinositol in cancer cell membranes.46,47 This enzyme can cleave the middle size molecular weight of HA (until 20 kDa or about 50 disaccharide units).48 As PRA-H circulates in blood or normal tissue, HA shields the interaction of PRA with normal cells. However, after accumulation at the tumor site via an enhanced permeability and retention (EPR) effect, HA on the surface of PRA-H is attacked by the highly concentrated HAase. The exposed PRA is highly cytotoxic. Thus, to mimic a solid tumor site, PRA-H was cocultured with HAase (50 U/mL), and its cytotoxicity was dramatically increased in all three cell lines tested. This behavior is evidence of the potential of PRA-H for the treatment of MDR cells (Figure 5c). However, the cytotox-

icity of PEI-H against HCT-8 cells was not controllable (Figure 5d) because the positive charge of PEI was not perfectly shielded with HA (Figure 1b), as indicated by the zeta potential result (Figure 3). The surface charge of PEI-H is heterogeneous. The heterogeneous charge of the PEI-H surface leads to interaction Coulombic and van der Waals forces between the PEI-H particles.42 The cytotoxicity of HAase was negligible over the range of concentrations tested (Figure S3). Changes in Morphology over Time of Cells Treated with PRA. To confirm the cytotoxicity results, changes in the morphology of HCT-8 cells treated with PRA 2 or PRA 2-H, with or without HAase were observed over time with an optical microscope (Figure 6). In the presence of PRA 2, significant changes in morphology due to cell death were observed after 4 h and were continuously observed up to 24 h. However, PRA 2-H-treated cells maintained normal morphology over 24 h. When PRA 2-H was coincubated with HAase, the cell death process was similar to that

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Figure 8. Confocal images of HCT-8 cells treated with PRA 2 (2 µg/mL), PRA 2-H (2 µg/mL), and PRA2-H (2 µg/mL) plus HAase (50 U/mL) after 4 h. HA was labeled with FITC and PRA 2 was labeled with RITC. Cell nuclei were stained with DAPI.

observed with PRA 2 at 15 h, while the cell morphology was different from that of PRA 2 at the initial 4 h time point. After 24 h, only cell debris was observed. These results support those of the MTT test with PRA 2 (Figures 5 and 6). We infer that the cytotoxic mechanism of PRA is due to the formation of holes in the cell surface. To demonstrate this hypothesis, the change in permeability of SNU-484 and HCT-8 cells to DOX (1 µg/mL) after treatment with PRA 2 (20 µg/ mL) was monitored by FACs analysis (Figure 7a,b) and compared to the results obtained with HeLa cells (Figure S4). In the absence of PRA 2, efflux by overexpressing P-gp prevented a significant increase in the amount of DOX retained in both HCT-8 (Figure 7a) and SNU-484 cells (Figure 7b), thus, leading to reduced cytotoxicity. However, after treatment with PRA 2, a dramatic change in the permeability of both types of cells to DOX was observed (Figure 7a,b), indicating that new channels for DOX transport (such as holes) on the surface of cells were formed by PRA 2. On the other hand, HeLa cells, which do not overexpress P-gp, showed high membrane permeability to DOX whether or not they were treated with PRA 2 (Figure S4). The effect of HA on suppression of hole formation by PRA 2 was also studied by PI staining as an indicator of diffusion due to necrotic lysis (hole formation).15,38 (Figure 7c). PI is membrane impermeable and generally excluded from viable cells. PI only enters cells with membrane damage. It is used an indicator for necrosis. The high intensity of PRA 2 treated cells indicate that the cells membrane was attacked by the cationic polymer PRA 2 and holes occurred. PI was able to diffuse into cells through these holes (Figure 7c). The intensity of PI fluorescence in HCT-8 cells did not increase following treatment with PRA 2-H, indicating that HA successfully shielded the interaction of the polymer with the cells and prevented membrane hole formation (Figure 7d). The uptake of PI dramatically increased after 12 h (Figure 7d). These results correspond to the patterns of cytotoxicity revealed by the MTT assay (Figure 4) and optical microscopy (Figure 6). All of the results regarding the interaction between PRA and HCT-8 cells were verified by confocal microscopy. The micrographs in Figure 8 illustrate the changes in fluorescence of cells cultured with PRA 2-RITC (2 µg/mL) or HA-FITCcovered PRA 2-RITC (fluorescent labeled PRA-H) for 4 h. While cells treated with PRA 2- RITC accumulated fluorescence to a high degree, those treated with labeled PRA 2-H did not

generate a signal; however, fluorescence was recovered by treatment with HAase. These results strongly support the hypothesis that shielding with HA reduces the cytotoxicity of PRA in the cationic nanoparticle.

Conclusion An HA-shielded PRA nanodrug (PRA-H) was investigated as a new concept in the treatment of cancer cells overexpressing P-gp. This system revealed better physical stability than a PEI-H complex due to homogeneous charge surface. The cytotoxicity of PRA-H against various types of cancer cells was controlled by the presence/ absence of the enzyme that is possible degrade HA, which is highly concentrated at the tumor site. In particular, the greater cytotoxicity of PRA-H via holes formation in SNU-484 and HCT-8, both of which are GI tract cancer cell lines overexpressing P-gp, makes it a promising candidate in the next generation of anticancer therapy with the ability to treat MDR cells. Acknowledgment. This work was financially supported by the Ministry of Health and Welfare, Republic of Korea (A084060). Supporting Information Available. Synthesis and characteristics of PRA; physical morphology of PRA 1, 2 and 3; MTT assay of HAase; membrane permeability of Hela cells measured by FACS analysis; P-gp expression in Hela, SNU-484, and HCT-8 cells; and additional synthesis information. This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes (1) Pauwels, E.; Erba, P.; Mariani, G.; Gomes, C. Multidrug resistance in cancer: its mechanism and its modulation. Drug News Perspect. 2007, 20 (6), 371. (2) Gottesman, M.; Fojo, T.; Bates, S. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat. ReV. Cancer 2002, 2 (1), 48–58. (3) Cordon-Cardo, C.; O’brien, J.; Boccia, J.; Casals, D.; Bertino, J.; Melamed, M. Expression of the multidrug resistance gene product (Pglycoprotein) in human normal and tumor tissues. J. Histochem. Cytochem. 1990, 38 (9), 1277. (4) Figueredo, A.; Arnold, A.; Goodyear, M.; Findlay, B.; Neville, A.; Normandeau, R.; Jones, A. Addition of verapamil and tamoxifen to the initial chemotherapy of small cell lung cancer a phase I/II study. Ca-Cancer J. Clin. , 65 (9), 1895–1902. (5) Verweij, J.; Herweijer, H.; Oosterom, R.; Van der Burg, M.; Planting, A.; Seynaeve, C.; Stoter, G.; Nooter, K. A phase II study of epidoxorubicin

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(25)

(26) (27) (28)

in colorectal cancer and the use of cyclosporin-A in an attempt to reverse multidrug resistance. Br. J. Cancer 1991, 64 (2), 361. Tsuruo, T.; Iida, H.; Tsukagoshi, S.; Sakurai, Y. Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil. Cancer Res. 1981, 41 (5), 1967. Verdiere, A.; Dubernet, C.; Nemati, F.; Poupon, M.; Puisieux, F.; Couvreur, P. Uptake of doxorubicin from loaded nanoparticles in multidrug-resistant leukemic murine cells. Cancer Chemother. Pharmacol. 1994, 33 (6), 504–508. Soma, C.; Dubernet, C.; Barratt, G.; Nemati, F.; Appel, M.; Benita, S.; Couvreur, P. Ability of doxorubicin-loaded nanoparticles to overcome multidrug resistance of tumor cells after their capture by macrophages. Pharm. Res. 1999, 16 (11), 1710–1716. Mohajer, G.; Lee, E.; Bae, Y. Enhanced intercellular retention activity of novel pH-sensitive polymeric micelles in wild and multidrug resistant MCF-7 cells. Pharm. Res. 2007, 24 (9), 1618–1627. Jabr-Milane, L.; van Vlerken, L.; Yadav, S.; Amiji, M. Multifunctional nanocarriers to overcome tumor drug resistance. Cancer Treat. ReV. 2008, 34 (7), 592–602. Brigger, I.; Dubernet, C.; Couvreur, P. Nanoparticles in cancer therapy and diagnosis. AdV. Drug DeliVery ReV. 2002, 54 (5), 631–651. Kukowska-Latallo, J.; Candido, K.; Cao, Z.; Nigavekar, S.; Majoros, I.; Thomas, T.; Balogh, L.; Khan, M.; Baker Jr, J. Nanoparticle targeting of anticancer drug improves therapeutic response in animal model of human epithelial cancer. Cancer Res. 2005, 65 (12), 5317. Wong, H.; Bendayan, R.; Rauth, A.; Xue, H.; Babakhanian, K.; Wu, X. A mechanistic study of enhanced doxorubicin uptake and retention in multidrug resistant breast cancer cells using a polymer-lipid hybrid nanoparticle system. J. Pharmacol. Exp. Ther. 2006, 317 (3), 1372. Sera, T.; Shea, L. Materials for non-viral gene delivery: Biomaterials. Annu. ReV. Mater. Sci. 2001, 31, 25–46. Hong, S.; Bielinska, A.; Mecke, A.; Keszler, B.; Beals, J.; Shi, X.; Balogh, L.; Orr, B.; Baker, J.; Banaszak Holl, M. Interaction of poly (amidoamine) dendrimers with supported lipid bilayers and cells: hole formation and the relation to transport. Bioconjugate Chem. 2004, 15 (4), 774–782. Moroson, H. Polycation-treated tumor cells in vivo and in vitro. Cancer Res. 1971, 31 (3), 373. Park, K.; Kang, H.; Cho, J.; Chung, I.; Cho, S.; Bae, Y.; Na, K. Alltrans-retinoic acid (ATRA)-grafted polymeric gene carriers for nuclear translocation and cell growth control. Biomaterials 2009, 30 (13), 2642–2652. Franzmann, E.; Schroeder, G.; Goodwin, W.; Weed, D.; Fisher, P.; Lokeshwar, V. Expression of tumor markers hyaluronic acid and hyaluronidase (HYAL1) in head and neck tumors. Int. J. Cancer 2003, 106 (3), 438–445. Lokeshwar, V.; Young, M.; Goudarzi, G.; Iida, N.; Yudin, A.; Cherr, G.; Selzer, M. Identification of bladder tumor-derived hyaluronidase: its similarity to HYAL1. Cancer Res. 1999, 59 (17), 4464. Helander, I.; Latva-Kala, K.; Lounatmaa, K. Permeabilizing action of polyethyleneimine on Salmonella typhimurium involves disruption of the outer membrane and interactions with lipopolysaccharide. Microbiology 1998, 144 (2), 385. Lv, H.; Zhang, S.; Wang, B.; Cui, S.; Yan, J. Toxicity of cationic lipids and cationic polymers in gene delivery. J. Controlled Release 2006, 114 (1), 100–109. Kuo, J. Practical aspects of hyaluronan based medical products; CRC Press: New York, 2005. Laurent, T. The chemistry, biology and medical applications of hyaluronan and its deriVatiVes; Portland Press: London, 1998. Liu, D.; Pearlman, E.; Diaconu, E.; Guo, K.; Mori, H.; Haqqi, T.; Markowitz, S.; Willson, J.; Sy, M. Expression of hyaluronidase by tumor cells induces angiogenesis in vivo. Proc. Natl. Acad. Sci. U.S.A. 1996, 93 (15), 7832. Lokeshwar, V.; Rubinowicz, D.; Schroeder, G.; Forgacs, E.; Minna, J.; Block, N.; Nadji, M.; Lokeshwar, B. Stromal and epithelial expression of tumor markers hyaluronic acid and HYAL1 hyaluronidase in prostate cancer. J. Biol. Chem. 2001, 276 (15), 11922. Home, C. Hyaluronidase as a therapeutic target in cancer: a matter of too little or too much. Lokeshwar, V.; Lokeshwar, B.; Pham, H.; Block, N. Association of elevated levels of hyaluronidase, a matrix-degrading enzyme, with prostate cancer progression. Cancer Res. 1996, 56 (3), 651. Yim, H.; Jo, E.; Na, K. Development of a new micellar anticancer drug: cationic polymer/vitamin A conjugate covered with hyaluronic acid. Macromol. Res. 2010.

Biomacromolecules, Vol. 11, No. 9, 2010

2393

(29) Zacherl, J.; Hamilton, G.; Thalhammer, T.; Riegler, M.; Cosentini, E.; Ellinger, A.; Bischof, G.; Schweitzer, M.; Teleky, B.; Koperna, T. Inhibition of P-glycoprotein-mediated vinblastine transport across HCT-8 intestinal carcinoma monolayers by verapamil, cyclosporine A and SDZ PSC 833 in dependence on extracellular pH. Cancer Chemother. Pharmacol. 1994, 34 (2), 125–132. (30) Hunter, J.; Hirst, B.; Simmons, N. Epithelial secretion of vinblastine by human intestinal adenocarcinoma cell (HCT-8 and T84) layers expressing P-glycoprotein. Br. J. Cancer 1991, 64 (3), 437. (31) Lee, T.; Park, J.; Min, Y.; Kim, K.; Choi, C. Epigenetic mechanisms involved in differential MDR1 mRNA expression between gastric and colon cancer cell lines and rationales for clinical chemotherapy. BMC Gastroenterol. 2008, 8 (1), 33. (32) Bidwell, G., III.; Fokt, I.; Priebe, W.; Raucher, D. Development of elastin-like polypeptide for thermally targeted delivery of doxorubicin. Biochem. Pharmacol. 2007, 73 (5), 620–631. (33) Umebayashi, Y.; Miyamoto, Y.; Wakita, M.; Kobayashi, A.; Nishisaka, T. Elevation of plasma membrane permeability on laser irradiation of extracellular latex particles. J. Biochem. 2003, 134 (2), 219. (34) Ha, H.; Snyder, S. Poly (ADP-ribose) polymerase is a mediator of necrotic cell death by ATP depletion. Proc. Natl. Acad. Sci. U.S.A. 1999, 96 (24), 13978. (35) Krysko, D.; Vanden Berghe, T.; D’Herde, K.; Vandenabeele, P. Apoptosis and necrosis: detection, discrimination and phagocytosis. Methods 2008, 44 (3), 205–221. (36) Drabløs, F.; Feyzi, E.; Aas, P.; Vaagbø, C.; Kavli, B.; Bratlie, M.; Pena-Diaz, J.; Otterlei, M.; Slupphaug, G.; Krokan, H. Alkylation damage in DNA and RNAsrepair mechanisms and medical significance. DNA Repair 2004, 3 (11), 1389–1407. (37) Manfredi, J.; Horwitz, S. Taxol: an antimitotic agent with a new mechanism of action. Pharmacol. Ther. 1984, 25 (1), 83. (38) Hong, S.; Leroueil, P.; Janus, E.; Peters, J.; Kober, M.; Islam, M.; Orr, B.; Baker, J., Jr.; Holl, M. Interaction of polycationic polymers with supported lipid bilayers and cells: nanoscale hole formation and enhanced membrane permeability. Bioconjugate Chem. 2006, 17 (3), 728–734. (39) Mecke, A.; Lee, D.; Ramamoorthy, A.; Orr, B.; Holl, M. Synthetic and natural polycationic polymer nanoparticles interact selectively with fluid-phase domains of DMPC lipid bilayers. Langmuir 2005, 21 (19), 8588–8590. (40) Davis, M. Non-viral gene delivery systems. Curr. Opin. Biotechnol. 2002, 13 (2), 128–131. (41) Rich, D.; Singh, J. The carbodiimide method. The Peptides: Major methods of peptide bond formation; 1979; p 241. (42) Bouyer, F.; Robben, A.; Yu, W.; Borkovec, M. Aggregation of colloidal particles in the presence of oppositely charged polyelectrolytes: effect of surface charge heterogeneities. Langmuir 2001, 17 (17), 5225–5231. (43) Chen, C.; Chin, J.; Ueda, K.; Clark, D.; Pastan, I.; Gottesman, M.; Roninson, I. Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells. Cell 1986, 47 (3), 381–389. (44) Lee, T.; Park, J.; Min, Y.; Kim, K.; Choi, C. Epigenetic mechanisms involved in differential MDR 1 mRNA expression between gastric and colon cancer cell lines and rationales for clinical chemotherapy. BMC Gastroenterol. 2008, 8 (1), 33. (45) Pham, H.; Block, N.; Lokeshwar, V. Tumor-derived hyaluronidase: a diagnostic urine marker for high-grade bladder cancer. Cancer Res. 1997, 57 (4), 778. (46) Culty, M.; Miyake, K.; Kincade, P.; Silorski, E.; Butcher, E.; Underhill, C. The hyaluronate receptor is a member of the CD44 (H-CAM) family of cell surface glycoproteins. J. Cell Biol. 1990, 111 (6), 2765–2774. (47) Rai, S.; Duh, F.; Vigdorovich, V.; Danilkovitch-Miagkova, A.; Lerman, M.; Miller, A. Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation. Proc. Natl. Acad. Sci. U.S.A. 2001, 98 (8), 4443. (48) Hua, Q.; Knudson, C.; Knudson, W. Internalization of hyaluronan by chondrocytes occurs via receptor-mediated endocytosis. J. Cell Sci. 1993, 106 (1), 365. (49) Idris, S.; Mkhatresh, O.; Heatley, F. Assignment of 1H NMR spectrum and investigation of oxidative degradation of polyethylenimine using 1 H and 13C 1-D and 2-D NMR. Polym. Int. 2006, 55 (9), 1040–1048. (50) Kang, H.; Lee, M.; Bae, Y. Polymeric gene carriers. Crit. ReV. Eukaryotic Gene Expression 2005, 15 (4), 317.

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