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Cholic Acid-Modified Dendritic Multimolecular Micelles and Enhancement of Anticancer Drug Therapeutic Efficacy Kun Zhang,† Yongjian Wang,*,‡ Ao Yu,§ Yue Zhang,‡ Hao Tang,§ and X. X. Zhu*,†,# Key Laboratory of Functional Polymer Materials (Ministry of Education), Institute of Polymer Chemistry; Key Laboratory of Bioactive Materials (Ministry of Education), College of Life Sciences; and Central Laboratory, College of Chemistry, Nankai University, Tianjin 300071, China and De´partement de chimie, Universite´ de Montre´al, C.P. 6128, Succursale Centre-ville, Montre´al, Quebec H3C 3J7, Canada. Received November 8, 2009; Revised Manuscript Received July 15, 2010
To improve the efficacy and bioacceptability of polyamidoamine (PAMAM) in potential biomedical applications, the PAMAM dendrimers (first generation) were partially modified with cholic acid. 1H NMR studies and acid-base titration show that two cholic acid molecules are linked to one PAMAM. The modified PAMAM dendrimers self-assemble to form dendritic multimolecular micelles in aqueous solutions, with a diameter of 120 nm measured by dynamic light scattering. These micelles can encapsulate hydrophobic drug molecules in aqueous media and exhibit pH sensitivity. The in vitro results demonstrate that the anticancer activity of camptothecin is significantly enhanced at low drug dose after being encapsulated by these micelles in the presence of serum. Therefore, the dendritic multimolecular micelles based on low generation dendrimers may have potential applications in the delivery of drugs.
INTRODUCTION Dendrimers, which are monodisperse macromolecules with welldefined highly branched architecture, have attracted much attention in drug delivery (1-4), gene therapy (5-7), MRI contrast enhancement (8, 9), liquid crystalline materials (10, 11), electronics (12-14), and catalysis (15, 16). Their unique architecture makes them capable of encapsulating, complexing, or conjugating various molecules as unimolecular micelles (17). However, dendrimers as unimolecules are small in size. For example, the hydrodynamic diameters of G0-G7 polyamidoamine (PAMAM) dendrimers with an ethylenediamine core are in the range 1.4-8.8 nm (18) and G1-G5 poly(propylene imine) dendrimers are in the range 0.6-2.0 nm (19). Thus, they accumulate less in tumoral tissues than micelles, because the usual size of pharmaceutical carriers with the enhanced permeation and retention (EPR) effect is between 10 and 100 nm (20). On the other hand, the regulatory hurdles of high generation dendrimers with good quality and purity are difficult to overcome. The self-assembly of dendrimers into larger and more complex forms may resolve these problems and lead to a wider range of potential applications. Fahmy and co-workers showed that the self-assembly of partial fluorinated G3 PAMAM into larger, dense nanoparticulates enabled the detection of their site-specific accumulation in vivo by 19F magnetic resonance imaging (21). Thayumanavan and co-workers used facially amphiphilic dendrons (G1-G3) which exhibited environmentally dependent aggregation to selectively extract peptides from aqueous solutions and found that the organization of the dendrons themselves was crucial for the binding event (22, 23). Therefore, the aggregation may improve the properties of dendrimers, especially low generation ones. However, reports on dendritic multimolecular micelles for drug delivery have been rare to date. * Corresponding author. Y. Wang: Tel./Fax: +86 22 23501393,
[email protected]. X. X. Zhu: Tel.: +1 514 340 5172; Fax: +1 514 340 5290,
[email protected]. † Key Laboratory of Functional Polymer Materials, Nankai University. ‡ Key Laboratory of Bioactive Materials, Nankai University. § Central Laboratory, Nankai University. # Universite´ de Montre´al.
Dendritic multimolecular micelles can be obtained by amphiphilic dendrimers with self-assembling properties (22, 24). The end groups of dendrimers can be modified to vary the property of the dendrimers and improve their performance. PAMAM dendrimers, which are commercially available and have been widely studied in biomedical fields (4), may form multimolecular micelles once they are hydrophobically modified on their terminal groups (21, 25-27). The residual surface groups can be further modified into other functional groups to serve for imaging, targeted release, and drug anchors (28-30). Furthermore, the positive charges of the remaining unmodified surface groups improve drug therapeutic efficiency by enhancing intracellular delivery (31). Cholic acid is a facially amphiphilic natural compound and has been used to construct supramolecular systems (32-35). It has pharmacological potential to act as an adjuvant of liverspecific drugs and absorption enhancers (36, 37). In this work, we prepared dendrimer-based multimolecular micelles by the self-assembly of the first generation PAMAM (G1 PAMAM) dendrimers partially modified with cholic acid and investigated its aggregation behavior and application on improving drug efficacy, where camptothecin (CPT) was used as a hydrophobic anticancer drug model.
EXPERIMENTAL PROCEDURES Materials and General Characterization. G1 PAMAM with an ethylenediamine core, cholic acid, pyrene, Oil Red O, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (USA). CPT was purchased from Mianyang Dongfangyuan Biotechnology Co., Ltd. (Sichuan, China). N-Hydroxysuccinimide and dicyclohexyl carbodiimide (DCC) were obtained from Shanghai Medpep Co., Ltd. (Shanghai, China). Methanol-d4 was provided by J&K Chemical, Ltd. (Beijing, China). Methanol, acetonitrile, and tetrahydrofuran (THF) were supplied from Kermel Chemical Reagent Co., Ltd. (Tianjin, China), and distilled prior to use following standard procedures. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco (Grand Island, USA). Fetal bovine serum (FBS) and penicillin-streptomycin
10.1021/bc900490u 2010 American Chemical Society Published on Web 08/18/2010
Dendrimer Micelles and Drug Efficacy Enhancement
were from Us-bio (Tianjin, China). Unless otherwise noted, all chemicals were used without further purification. 1H and 13C NMR spectra were all recorded on a Varian Mercury 300 NMR spectrometer (1H, 300 MHz; 13C, 75 MHz). Mass spectrum was obtained on a Bruker Biflex III MALDI-TOF spectrometer. Synthesis of N-Succinimidyl Ester of Cholic Acid (CANHS). This compound was synthesized by reacting cholic acid with N-hydroxysuccinimide in dry THF and acetonitrile with DCC according to a procedure published previously (38). The crude product was purified by column chromatography (petroleum ether/acetone ) 1/1.5). Yield: 62.6%; mp: 119-120 °C. Synthesis of PAMAM1-CA2. CA-NHS (0.3727 g, 0.738 mmol) and G1 PAMAM (0.5269 g, 0.369 mmol) were mixed in methanol (15 mL) and stirred at room temperature for 48 h. The product was purified by dialysis (MWCO 2000) against methanol. After dialysis, methanol was evaporated under reduced pressure, and the residue was dried in vacuo to afford a white powder. The yield was 0.73 g (89.6%). 1H NMR (300 MHz, CD3OD) δ: 0.70 (s, 6H, 18-H), 0.91 (s, 6H, 19-H), 1.03 (d, 6H, 21-H), 2.37 (s, 24H, CH2CO), 2.48 (s, 4H, the core CH2N), 2.57 (s, 8H, CH2N), 2.79 (s, 24H, NCH2), 3.60 (m, 2H, 3β-H), 3.79 (s, 2H, 12β-H), 3.95 (s, 2H, 7β-H). 13C NMR (75 MHz, CD3OD) δ: 11.97, 16.75, 22.12, 23.15, 26.76, 27.66, 28.12, 28.52, 30.11, 32.16, 33.65, 34.76, 35.38, 35.85, 37.49, 38.95, 39.88, 40.85, 41.67, 41.90, 42.04, 46.33, 46.82, 47.91, 48.76, 50.02, 67.82, 71.69, 72.79, 174.01, 174.94, 175.43. MALDI-TOF: m/z calcd for C110H204N26O20 [M+H]+ 2211.585, [M+Na]+ 2233.567, found [M+H]+ 2211.586, [M+Na]+ 2233.563. Acid-Base Titration. The titration experiments were performed using a Metrohm model 809 Titrando apparatus (Metrohm, Herisau, Switzerland) controlled by Metrohm Tiamo 1.2 software at room temperature. Approximately 150 mg of G1 PAMAM or PAMAM1-CA2 was dissolved in 50 mL of 0.1 M NaCl solution. These solutions were titrated by aqueous hydrochloric acid to a pH value around 2.0, followed by backtitration to pH 11.0 using 0.0935 M NaOH. The number of primary amines was determined from the back-titration data. Preparation of PAMAM1-CA2 Micelles. PAMAM1-CA2 was dispersed evenly in citrate buffer (0.1 M, pH 5.0), phosphate buffer (0.1 M, pH 5.5, 6.8, 7.4), or borate buffer (0.1 M, pH 9.0) after sonication for 2 min. Then, the mixture was equilibrated by shaking at 65 °C for 3 h. Preparation of CPT-Loaded Micelles. A measured volume of a CPT chloroform solution was added to a vial and the solvent was then evaporated. Ten milliliters of PAMAM1-CA2 solution were then added to this vial and dispersed evenly by sonication for 2 min. The mixture was equilibrated at 65 °C for 3 h. After cooling, the solutions were filtered via 0.22 µm filter. The concentration of CPT in the solution was determined by UV-vis spectrometry (Varian Cary 100 BIO, America) (369 nm). Dye Solubilization. A series of PAMAM1-CA2 solutions with different pH values (5.0, 7.4, and 9.0) were added to the vials containing an excess amount of Oil Red O and were equilibrated at 65 °C for 3 h. After cooling, the solutions were filtered to remove the residual dye. Absorbance data were recorded on a UV-vis spectrophotometer at 518 nm. Fluorescence Spectroscopy. The pyrene fluorescence emission spectra were recorded with a Varian Cary Eclipse fluorescence spectrophotometer. A given amount of pyrene stock solution in acetone was placed in empty vials, and the solvent was then evaporated. Five milliliter aliquots of PAMAM1-CA2 aqueous solutions of various concentrations were then added to these vials. The final pyrene concentration was 6 × 10-7 M. The mixtures were equilibrated to form PAMAM1-CA2 micelles. The fluorescence emission spectra of pyrene were
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recorded at an excitation wavelength of 339 nm, and the scanning wavelength range was from 360 to 460 nm. Transmission Electronic Microscopy (TEM) and Dynamic Light Scattering (DLS). The sample for TEM was prepared at room temperature by placing a drop of PAMAM1CA2 solution on 300 mesh carbon-coated copper grids. The excess fluid was blotted off using filter paper, and then the sample was dried in vacuo. Microscopy was done on a FEI Tecnai G2 20 S-TWIN transmission electron microscope equipped with Gatan 794 CCD camera. The DLS measurement was done with a Malvern Nano ZS instrument with a 633 nm 4 mW He-Ne laser. The sample (2 g/L) was measured at 25 °C and at a backscatter angle of 173°. Cytotoxicity Study. Mouse embryonic fibroblast cells (NIH 3T3) and human hepatocellular liver carcinoma cells (HepG2) were cultured in DMEM containing 10% (v/v) FBS and 0.5% (w/v) penicillin-streptomycin, at 37 °C in a humidified 5% CO2/ 95% air atmosphere. Cells were seeded in 96-well plates at a density of 1 × 104 cells/well and allowed to adhere for 24 h. The culture medium was replaced by fresh DMEM containing various drug and dendrimer samples (CPT, PAMAM1-CA2 and CPT encapsulated in G1 PAMAM or PAMAM1-CA2), and the mixture was further incubated for 24 h. Fresh culture medium without drug or dendrimer was used as control. Each sample was replicated in five wells. The cells were then washed twice with phosphate buffer, and the fresh culture medium was added. Ten microliters of MTT solution (5 g/L in phosphate buffer) were added to each well and the cells were incubated further for 4 h at 37 °C. After the media were removed, the cells were washed with phosphate buffer and dissolved in DMSO. The optical density (OD) was measured at 570 nm with a Multiskan Ascent plate reader (Thermo Labsystems, Helsinki, Finland). Cell viability (%) ) (ODsample/ODcontrol) × 100%, where ODcontrol and ODsample were obtained in the absence or presence of drug and dendrimer samples, respectively. The inhibition of cell growth was calculated from: Inhibition (%) ) 100% - cell viability (%).
RESULTS AND DISCUSSION Characterization of the Dendrimers. The G1 PAMAM dendrimers used in this study are based on an ethylenediamine core and possess 8 amino groups on the surface. Cholic acidfunctionalized PAMAM was synthesized by reacting the primary amine groups of G1 PAMAM with CA-NHS under mild conditions (Scheme 1). The modified PAMAM (PAMAM1CA2) has 2 cholic acid groups per PAMAM molecule as characterized by 1H NMR and acid-base titration (Table 1). The product reserves most of the terminal amino groups maintaining its solubility in aqueous solutions (with a saturation concentration of ca. 8 g/L in 0.1 M phosphate buffer). Aggregation Properties. The formation of micelles was evidenced by dye solubilization (39) with Oil Red O, a diazo dye with a strong hydrophobic character. Large amounts of Oil Red O were added to the vials containing PAMAM1-CA2 solutions of various concentrations. The mixtures were equilibrated by shaking at 65 °C for 3 h and cooling to room temperature. Red solutions were obtained after filtering the mixtures to remove residual insoluble dye (Figure 1). The UV-vis absorption results of the filtrates showed that Oil Red O was transferred into the hydrophobic environment of the micelles formed. The encapsulation of the guest molecules by the micelles is pH-dependent since Oil Red O has a lower solubility in acidic media (pH 5.0) than in neutral or basic media (pH 7.4-9.0) (Figure 1). This may be ascribed to the conformation change of PAMAM along with pH (40).
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Scheme 1
Table 1. Numbers of Terminal Amino Groups and Cholic Acid Groups
sample
number of terminal amino groups measured by titration
G1 PAMAM PAMAM1-CA2
7.53 5.36
number of cholic acid groups titrationa 2.17
H NMRb
1
1.96
Number of cholic acid groups ) Number of terminal amino groups of G1 PAMAM - Number of terminal amino groups of PAMAM1CA2. b Obtained from 1H NMR. a
centration above a certain level, since more pyrene molecules entered the hydrophobic environment upon the aggregation of PAMAM1-CA2. From the plot of fluorescence intensity ratio (I3/I1) versus PAMAM1-CA2 concentration (Figure 2), it can be observed that the I3/I1 value was almost constant at low PAMAM1-CA2 concentrations followed by an abrupt increase at a certain PAMAM1-CA2 concentration, indicating the transfer of pyrene into the hydrophobic environment of the micelles formed. The critical micelle concentration (CMC) of PAMAM1CA2 in an aqueous solution is thus defined by the intersection of the two linear segments of the curve. The CMC value was estimated to be ca. 80 mg/L (3.6 × 10-5 M), a value between the CMC of block polymers (10-6-10-7 M) and that of small molecule surfactants (10-3-10-4 M). These results indicate that the micelles are stable in aqueous solutions. The aggregated PAMAM1-CA2 showed spherical nanoparticles, and the mean diameter was in the range 50-70 nm with
Figure 1. Oil Red O solubilization (absorbance at 518 nm) as a function of PAMAM1-CA2 concentration at different pH values, showing that the dye solubilization by PAMAM1-CA2 is pH-dependent. The insert shows the solubilization of Oil Red O in phosphate buffer at pH 7.4 with PAMAM1-CA2 (left) and G1 PAMAM (right).
The formation of micelles in aqueous solution was further verified by a fluorescence technique with pyrene as a hydrophobic probe (41). In the emission spectra, the fluorescence intensity of pyrene in water was very low since pyrene is sparingly soluble in water. However, its intensity in PAMAM1CA2 solutions increased with increasing PAMAM1-CA2 con-
Figure 2. Ratio of vibronic bands (I3/I1) in the pyrene fluorescence emission spectra as a function of PAMAM1-CA2 concentration (concentration of pyrene was 6 × 10-7 M in phosphate buffer at pH 7.4.), showing that the CMC of PAMAM1-CA2 is 80 mg/L.
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Figure 5. Cytotoxicity assay with 3T3 and HepG2 cells (104 cells/ well) after incubation with dendrimers at 37 °C for 24 h, showing that PAMAM1-CA2 is not cytotoxic (>80% cell viability) below a concentration of 100 mg/L for NIH 3T3 and 125 mg/L for HepG2, respectively. Figure 3. TEM image of the PAMAM1-CA2 micelles (100 mg/L in phosphate buffer, pH 7.4), showing that the average size of the micelles is in the range 50-70 nm.
Figure 4. The solubility of CPT in phosphate buffer solutions with PAMAM1-CA2 micelles, showing the pH-dependence of the drug encapsulation.
a narrow size distribution observed from the TEM images (Figure 3), while the diameter of PAMAM1-CA2 in aqueous solution was about 120 nm which was measured by DLS. The dimension of PAMAM1-CA2 molecules should be approximately 4 nm, based on the diameter of G1 PAMAM and the length of the cholic acid molecule (about 2.2 nm (42) and 1.5 nm, respectively). Therefore, PAMAM1-CA2 forms multimolecular aggregates in aqueous solutions. According to the literature (20), the usual size of a pharmaceutical micelle is between 10 and 100 nm, and its CMC value is expected to be in the low millimolar region. Thus, the PAMAM1-CA2 micelle may be used as a drug carrier. Drug Encapsulation Property. On the basis of the fact that the encapsulation of Oil Red O by the micelles is pH-dependent, we further examined the influences of medium pH values on the solubility property of CPT. Four pH values (5.0, 5.5, 6.8, and 7.4) of dissolution media were utilized. As shown in Figure 4, pH values of medium notably affected CPT solubility. When pH decreased from 7.4 to 5.0, the solubility of CPT was much decreased. With this pH-sensitivity, PAMAM1-CA2 may be used for the controlled release of encapsulated drugs by a pH trigger in physiological environments of tumor tissues or intracellular endosome and lysosome where there is a significant drop in the pH value. Therefore, these micelles may act as smart drug-releasing devices responding to pH change and targeting tumor tissues. Cytotoxicity Studies. The cytotoxicity of the dendrimers was done with a methylthiazoletetrazolium method in NIH 3T3 and HepG2. The IC50 value, a concentration at which 50% cells are killed, of PAMAM1-CA2 was ca. 117 mg/L for NIH 3T3, while that of the molecular umbrella analogues (with a similar mo-
lecular weight) and polyethyleneimine (with a molecular weight of 25 kDa) are 4 mg/L (43) and 6 mg/L (44), respectively. The results indicate that the dendrimer-based multimolecular micelles have a lower cytotoxicity. As indicated in Figure 5, PAMAM1-CA2 was not cytotoxic (>80% cell viability) below a concentration of 100 mg/L for NIH 3T3 and 125 mg/L for HepG2, respectively. On the basis of these data, the investigation of anticancer activity of the micelles was carried out in a dendrimer concentration of 100 mg/L. Under this condition, the micelles are not cytotoxic and the enhancement of drug therapeutic efficacy should be attributed to the contribution of the micelles encapsulated with the drug. Inhibition of Tumor Cell Growth. To investigate the enhancement of therapeutic efficacy by the micelles, the inhibition of tumor cell (HepG2) growth was evaluated. HepG2 cells (104 cells/well) were exposed to an equivalent concentration (4.5 mg/ L, 13 µM) of free CPT or CPT encapsulated in G1 PAMAM or the micelles (PAMAM1-CA2 concentration at 100 mg/L) in the presence of serum for 24 h, and the cell growth inhibition was determined using the methylthiazoletetrazolium assay. The inhibition was significantly enhanced in the case of CPT encapsulated in PAMAM1-CA2 (enhancement of 46.4% over free CPT), while it had no apparent increase in the case of CPT encapsulated in G1 PAMAM (only 1.3% enhancement over free CPT) (Figure 6A). These data reveal that the cholic acidmodified dendrimers (PAMAM1-CA2) can improve the drug efficacy at the same drug dose of free CPT while the nonmodified dendrimers (G1 PAMAM) cannot. We further investigated the effect of PAMAM1-CA2 concentration on drug efficacy. The results show that the cell growth inhibition is close to that by free CPT when the PAMAM1-CA2 concentration is lower than its CMC (80 mg/L), but inhibition increases sharply to over 90% at a higher concentration (above CMC, Figure 6B). These results indicate that the individual PAMAM1-CA2 molecules cannot improve the drug efficacy but its micellar aggregates can. Also, the improvement by these dendrimer-based multimolecular micelles does not require a high drug dose. This micellar drug delivery system can effectively avoid the problems of drug accumulation in other tissues and the effects on metabolism which are caused by higher drug doses in the clinical use of drug delivery systems of polymeric micelles or nanoparticles. To examine the effect of dendritic multimolecular micelles on drug efficacy, we further investigated the cytotoxicity of CPT-encapsulated PAMAM1-CA2 micelles against tumor cells at several CPT doses (Figure 7). The PAMAM1-CA2 concentration was fixed at 100 mg/L at which the micelles are noncytotoxic. In comparison with free CPT, the CPT-encapsulated PAMAM1-CA2 micelles induced a significant cytotoxic effect
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process (45, 46). In addition, the positive charges of the amines on the micelle surface may improve the cellular uptake of micelles, including the drugs which encapsulated in the micelles. Hence, the micelles would greatly enhance the cellular uptake efficiency of the drug so that it is much more potent than the free drug, even at a low drug dose. Although the PAMAM dendrimers themselves also have amines on the surface, they scarcely have the capacities to interact with CPT. Therefore, the unmodified PAMAM cannot enhance the cellular uptake of CPT efficiently.
CONCLUSION Multimolecular micelles were obtained in aqueous media by the self-assembly of G1 PAMAM dendrimers partially modified with cholic acid. In comparison with the dendrimer alone, the modified dendrimers can significantly enhance the anticancer activity of camptothecin after its encapsulation in the micelles under low dose in the presence of serum. The new micelles may improve drug efficiency without increasing the drug dose. These multimolecular micelles based on low generation dendrimers may have important clinical relevance for cancer therapy. Issues regarding the stability of the micelles in biological media and the optimization of the system remain to be investigated.
Figure 6. Comparative tumor cell growth inhibition profiles of free CPT and CPT encapsulated in G1 PAMAM (G1 PAMAM/CPT) and PAMAM1-CA2 (PAMAM1-CA2/CPT) in HepG2 cells (A) and the effect of PAMAM1-CA2 concentration on the tumor cell growth inhibition (B). The amount of CPT is 4.5 mg/L in every experiment. HepG2 cells were incubated for 24 h. Dulbecco’s modified Eagle’s medium (DMEM) was chosen as a control. The cytotoxicity was measured by the methylthiazoletetrazolium assay. Data are shown as mean ( SD (n ) 5). The results showed that the drug efficacy was significantly enhanced in the case of CPT encapsulated in micelles. PAMAM1-CA2 had no cytotoxicity under the experimental conditions.
Figure 7. Cytotoxicity assay with HepG2 cells (104 cells/well) after incubation with free CPT and CPT encapsulated in PAMAM1-CA2 micelles (100 mg/L) at 37 °C for 24 h, showing that the dendritic multimolecular micelles can improve drug efficacy.
to the tumor cells, even at relatively low drug concentration. Therefore, the results indicate that the dendritic multimolecular micelles can improve drug efficacy without increasing the chemotherapeutic drug dose. It is necessary for CPT to be transported into the cells before it takes pharmacological effect. Since the CPT molecules go through the plasma membrane by passive diffusion, a high dose is needed to increase the therapeutic efficacy. Micelles may be internalized by cells mainly through an efficient endocytic
ACKNOWLEDGMENT The financial support from the National Natural Science Foundation of China (Grant no. 20574039), Tianjin Natural Science Foundation (Grant no. 07JCYBJC02700), and the Canada Research Chair Program is acknowledged. X.X.Z. thanks Fonds Barré for travelling support during his sabbatical leave in Nankai University.
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