Dendrimer Versus Linear Conjugate: Influence of Polymeric

Bioconjugate Chem. 2006, 17, 1464−1472

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Dendrimer Versus Linear Conjugate: Influence of Polymeric Architecture on the Delivery and Anticancer Effect of Paclitaxel Jayant J. Khandare,† Sreeja Jayant,† Ajay Singh,‡ Pooja Chandna,† Yang Wang,† Nicholi Vorsa,‡ and Tamara Minko*,† Department of Pharmaceutics, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, and Department of Plant Biology and Pathology, The State University of New Jersey, New Brunswick, New Jersey 08854. Received August 1, 2006; Revised Manuscript Received September 25, 2006

The relative difference in polymeric architectures of dendrimer and linear bis(poly(ethylene glycol)) (PEG) polymer in conjugation with paclitaxel has been described. Paclitaxel, a poorly soluble anticancer drug, was covalently conjugated with PAMAM G4 hydroxyl-terminated dendrimer and bis(PEG) polymer for the potential enhancement of drug solubility and cytotoxicity. Both conjugates were characterized by 1NMR, HPLC, and MALDI/TOF. In addition, molecular conformations of dendrimer, bis(PEG), paclitaxel, and its polymeric conjugates were studied by molecular modeling. Hydrolysis of the ester bond in the conjugate was analyzed by HPLC using esterase hydrolyzing enzyme. In vitro cytotoxicity of dendrimer, bis(PEG), paclitaxel, and polymeric conjugates containing paclitaxel was evaluated using A2780 human ovarian carcinoma cells. Cytotoxicity increased by 10-fold with PAMAM dendrimer-succinic acid-paclitaxel conjugate when compared with free nonconjugated drug. Data obtained indicate that the nanosized dendritic polymer conjugates can be used with good success as anticancer drug carriers.

INTRODUCTION Paclitaxel (Taxol) is a diterpenoid obtained from the bark of the pacific yew tree Taxus breVifolia (1) and is one of the most promising chemotherapeutic agents acclaimed by the National Cancer Institute (2). The drug promotes tubulin polymerization by forming a hyperstabilized structure, disrupting the normal tubule dynamics essential in cellular division, thereby inducting cell death (3). Due to low aqueous solubility of paclitaxel (about 0.3 µg/mL), numerous attempts are being made to increase solubility of the drug using various formulations and prodrug conjugates (4-8). Among the latter, poly(ethylene glycol) (PEG) prodrugs have been the most promising, providing for an increased aqueous solubility, enhanced pharmacological activity, lowered systemic toxicity, reduced immunogenicity, and prolonged plasma lifetime. Greenwald et al. proposed several relatively high molecular weight PEG prodrugs to render paclitaxel more soluble and increase its systemic circulation time (9). Various polymers with different architectures have been used for the delivery of therapeutic agents (10-19). Only a few of these polymers, such as N-(2-hydroxypropylmethacrylamide) (HPMA), PEG, and poly(glutamic acid) (PGA), and their polymeric prodrug conjugates have shown promise in clinical applications for the delivery of anticancer drugs. Among these, only PGA is a biodegradable polymer. In the case of PEG and HPMA copolymers, molecular weights have been limited to 40 kDa to ensure their renal elimination (20). Recently, novel branched polymers (e.g., dendrimers) have been introduced as carriers of therapeutic agents. Such branched * To whom correspondence should be addressed: Tamara Minko, Ph.D., Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854-8020. Phone: 732-445-3831 x 214. Fax: 732445-3134. Email: [email protected]. † Department of Pharmaceutics. ‡ Department of Plant Biology and Pathology.

carriers are characterized by nanosized, monodispersed peripheral multiend sites that allows for conjugation of multiple drug molecules per one molecule of the carrier. In addition, dendrimers possess unique characteristics including specific shape, branch length-to-density ratio, and surface functionality, which distinguish them from conventional linear polymers (21-23). Moreover, these polymers have entered into therapeutics with successful human clinical (Phase I) trials, namely, dendrimerbased nanopharmaceuticals (VivaGel), nanodiagnostics (Stratus), DNA delivery vectors (Superfect), and other drug delivery/ polyvalency prototypes (24). Many attempts have been made to improve the solubility of the highly water insoluble paclitaxel using formulations, surfactants, PEGylated polymers, Cremophor, solid dispersions, micro-nanoparticles, and complexation with cyclodextrins (25). Another strategy involves prodrug conjugation using small molecules or polymers as solubilizing entities at either C2′primary or C7 secondary hydroxyl positions in paclitaxel (26-30). Such conjugates can potentially improve aqueous solubility of the drug and thereby enhance its antitumor activity. However, polymeric carriers with different architectures have not been compared on the similar experimental conditions in order to assess the effectiveness of the various architectural elements in the delivery of anticancer drug by the carrier (i.e., by linear or branched). The present work is aimed at comparing the ability of carriers with two different polymeric architectures, i.e., (a) polyamidoamine (PAMAM) G4 hydroxyl-terminated dendrimer and (b) conventional linear PEG polymer to deliver anticancer drug paclitaxel. Paclitaxel was covalently conjugated with bis(carboxymethyl) PEG and dendrimer by the condensation method. To study the cellular entry dynamics by confocal microscopy, fluorescent probe fluoroisothiocynate (FITC) was covalently conjugated with paclitaxel, polymers, and polymeric conjugates containing paclitaxel. Conjugates were characterized by 1HNMR, MALDI/TOF, and HPLC methods. The rate of hydrolysis of the ester bond in the conjugates was analyzed by

10.1021/bc060240p CCC: $33.50 © 2006 American Chemical Society Published on Web 10/20/2006

Dendrimer vs Linear Conjugate

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Figure 1. Synthesis of conjugates. (A) Paclitaxel (1) was first bound to succinic acid (2, SA) forming paclitaxel-SA conjugate (3). (B) PAMAM G4 hydroxyl-terminal dendrimer (4) was further coupled with paclitaxel-SA conjugate (3) to form paclitaxel-dendrimer conjugate (5). (C) bis(PEG) (6) was joined with paclitaxel (1) to yield PEG-paclitaxel conjugate (7).

HPLC using esterase enzyme. In addition, polymers and conjugates with paclitaxel were studied for molecular conformations by the molecular modeling method. In vitro cytotoxicity (IC50) of the conjugates was evaluated.

EXPERIMENTAL SECTION Materials. Paclitaxel (Mw ∼853.9 Da) was obtained from Sigma Chemical Co. (Atlanta, GA); R,ω-bis(2-carboxyethyl)poly(ethylene glycol) (Mw ∼10 000 Da) and fluorescein isothiocyanate (FITC) were obtained from Fluka (Allentown, PA). PAMAM-G4-OH (Mw ∼14 279 Da, 64 end groups) dendrimers, N,N-diisopropylethylamine (DIEA), 4-(methylamino)pyridine (DMAP), and succinic acid (SA) were purchased from Sigma-Aldrich (St. Louis, MO). N-(3-dimethylaminopropyl)N-ethylcarbodiimide HCl (EDC.HCl) was purchased from Fluka. Dialysis membrane of the molecular weight cutoff of 3500 Da (Spectra Pore), dimethyl sulfoxide (DMSO), dimethyl forma-

mide (DMF), dichloromethane (DCM) acetone, ethanol, and diethyl ether were purchased from Fischer Scientific. Jupiter C5 column was purchased from Phenomenex (Torrance, CA). Cell Line. The human ovarian carcinoma A2780 cell line was obtained from Dr. T. C. Hamilton (Fox Chase Cancer Center, PA). Cells were cultured in RPMI 1640 medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT). Cells were grown at 37 °C in a humidified atmosphere of 5% CO2 (v/v) in air. All experiments were performed on cells in the exponential growth phase. Synthesis of Paclitaxel-Succinic acid (SA) Conjugate (Figure 1A). SA (2) is a bis(carboxylic acid) moiety and is reacted on an equimolar basis with the hydroxyl group in paclitaxel (1) to form a paclitaxel-SA conjugate (3), leaving one free carboxyl group for further conjugation with hydroxylterminal dendrimer. Briefly, paclitaxel (10.0 mg, 0.0118 mM)

1466 Bioconjugate Chem., Vol. 17, No. 6, 2006

Figure 2. Proton nuclear magnetic resonance (1H NMR) spectra of bis(PEG)-paclitaxel conjugate (A) and paclitaxel-SA-PAMAM dendrimer conjugate (B). 1H NMR spectra were recorded on 400 MHz spectrophotometer using DMSO-d6 as a solvent.

and succinic acid (1.4 mg, 0.0118 mM) were dissolved in 3.0 mL of anhydrous DMSO and 10.0 mL of anhydrous DCM. To the resulting mixture, N-(3- dimethylaminopropyl)-N-ethylcarbodiimide HCl (EDC.HCl, 2.30 mg, 0.0118 mM) and DMAP (1.0 mg) were used as a catalyst. The reaction was carried out with continuous stirring for 24 h at room temperature. The resulting solution was filtered to remove dicyclohexilurea (DCU) obtained as a byproduct during the reaction. Paclitaxel-SA conjugate (3) was precipitated using diethyl ether and dried under vacuum. Synthesis of PAMAM-G4-Succinic Acid-Paclitaxel Conjugate (Figure 1B). Conjugation of succinic acid to paclitaxel resulted in the formation of monocarboxylic acid conjugate (3), which was further conjugated with hydroxyl groups in PAMAMG4-OH dendrimer (4). Briefly, dendrimer (88 mg, 0.0061 mM) and paclitaxel-succinic acid conjugate (3) (6.0 mg, 0.0061 mM) were dissolved in 4.0 mL of anhydrous DMSO and 10.0 mL of anhydrous DCM. The mole ratio of dendrimer to paclitaxelSA conjugate was maintained at 1:1. EDC.HCl (1.2 mg, 0.0062 mM) was added as a coupling agent, and DMAP (1.0 mg) was used as a catalyst. The reaction was stirred continuously for 24 h at room temperature. The resulting solution was filtered to remove DCU, and the filtrate was dialyzed extensively with anhydrous DMSO using dialysis membrane of molecular weight cutoff of 2000 Da for 24 h to remove unreacted paclitaxel-SA conjugate and EDC.HCl. Further, the conjugate was purified by size exclusion chromatography to remove unreacted paclitaxel-SA conjugate and excess EDC.HCl. Conjugate was dried under vacuum at room temperature. 1H NMR was recorded in DMSO-d6 on a 400 MHz NMR spectrophotometer and characterized for the peaks of dendrimer and paclitaxel (Figure 2B). Dendrimer, bis(PEG), and paclitaxel conjugates were analyzed for molecular mass by MALDI/TOF (Figure 3A,B) and HPLC (Figure 3D-F). Synthesis of r,ω-Bis((2-carboxyethyl)PEG)-Paclitaxel Conjugates (Figure 1C). R,ω-Bis(PEG10 000)-citric acid (6) containing carboxyl end groups at the terminals was coupled

Khandare et al.

with hydroxyl groups in paclitaxel to form an ester conjugate (1). Briefly, bis((2-carboxyethyl)PEG) (50 mg, 0.005 mM) and paclitaxel (4.2 mg, 0.005 mM) were dissolved in 5.0 mL of anhydrous dimethylformamide (DMSO) and 15 mL of anhydrous dichloromethane (DCM). To this solution, 1-[3-(dimethylaminopropyl]-3-ethylcarbodiimide HCl (EDC.HCl, 1.0 mg, 0.0052 mM) was added as a coupling agent and 4-(methylamino)pyridine (DMAP, 0.5 mg, 0.004 mM) was used as a catalyst. The reaction solution was stirred continuously for 24 h at room temperature. The reaction yields formation of carbodiimide urea, which was removed by filtration. The conjugate was purified to remove unreacted paclitaxel and EDC.HCl by the dialysis method using a Spectra/Por membrane (Mw cutoff ∼2000 Da) in DMSO as a solvent. After the additional purification by size exclusion chromatography, R,ω-bis((2-carboxyethyl)PEG-paclitaxel conjugate was dried under vacuum at room temperature. 1H NMR spectra were recorded in DMSO-d on a 400 MHz 6 NMR spectrophotometer and characterized for the peaks of bis(PEG) polymer and paclitaxel (Figure 2A). In addition, the molecular mass was measured by MALDI/TOF (Figure 3C) and characterized by HPLC (Figure 3F). FITC Labeling of Bis(PEG), PAMAM Dendrimer, Free Paclitaxel, PEG-Paclitaxel, and PAMAM Dendrimer-SAPaclitaxel Conjugates. FITC possesses carboxyl as well as hydroxyl groups for chemical conjugation with either hydroxyl or carboxyl group-possessing compounds. The reacting components were used in the following proportions: carboxylterminated bis(PEG) (60.0 mg, 0.006 nM) and FITC-OH (2.33 mg, 0.006 nM); PAMAM dendrimer (45 mg, 0.0032 nM) and FITC (5.0 mg, 0.0128 nM); paclitaxel (5.0 mg, 0.0058 nM) and FITC (2.28 mg, 0.0058 nM); PEG-paclitaxel conjugate (27 mg, 0.0025 nM) and FITC (1.0 mg, 0.0025 nM); PAMAM dendrimer-SA-paclitaxel conjugate (25 mg, 0.00163 nM) and FITC (2.5 mg, 0.0064 nM). All the components were dissolved separately in a round-bottom flask, and anhydrous DMSO and dichloromethane (DCM) were added. EDC.HCl was added with equivalent moles of FITC to be conjugated, and the reaction was carried out for 24 h at room temperature. Urea salt was precipitated and removed by filtration. All the FITC-labeled components (except paclitaxel-FITC conjugate) were dialyzed extensively using anhydrous DMSO (dialysis membrane of molecular weight cutoff of 2000 Da) for 24 h to remove unreacted FITC and EDC.HCl. Further, the conjugates were purified using size exclusion chromatography to remove unreacted FITC and excess EDC.HCl. All the conjugates were precipitated three times in acetone to remove free FITC. HPLC. Quantitative evaluations of individual samples were obtained using Water Empower HPLC consisting of an in-line degasser, a 600E multisolvent delivery pump, a 717plus autosampler, and a Photodiode array detector 996. The separations were performed using a Jupiter column (C5, 4.6 × 250, 5-300 A) from Phenomenex (Torrance, CA). Two solvent systems were used. Solvent system A consisted of 99.9% water with pH 3.5 (adjusted with formic acid) and 10% methanol. Solvent B consisted of 20% water with pH 3.5 (adjusted with formic acid), 20% methanol, and 60% acetonitrile. The flow rate was maintained at 1 mL/min using step gradients of 0 min solvent A 100%; 10 min solvent A 100%; 20 min solvent A 0%, and 30 min solvent A 0%, 32 min solvent A 100%. The spectra were recorded ranging from 210 to 600 nm at 1.5 nm step. The spectra of each peak were superimposed with corresponding spectra and retention times. Only peaks which showed no more than 1.0% difference were used for integration and evaluation. Twenty microliters of each sample and standard were injected. The stock solutions (1 mg/mL) of analyzed substances were prepared using methanol as a solvent. The calibration standards of paclitaxel (0.24 mg/mL to 0.000296 mg/



Figure 3. Characterization of synthesized polymer conjugates. Mass of PAMAM G4 OH dendrimer (A, 15.22 KDa), paclitaxel-SA-PAMAM dendrimer (B, 16, 18 KDa) and paclitaxel-bis(PEG) conjugates (C, 12.90 KDa) were measured by MALDI/TOF (Voyager De-Pro) system using sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid) as a matrix. The total mass of the components indicate the appropriate formation of the paclitaxel conjugates. Calibration curve for paclitaxel (D) and typical HPLC chromatograms (E,F) of paclitaxel-SA-PAMAM dendrimer conjugate and PEG-paclitaxel conjugate, respectively. Peaks from 3.5 to 5 min retention time indicate the formation of high molecular weight conjugates.

mL) were prepared by serial dilution to achieve the nominal concentrations in reaction mass and hydrolyzed reaction mass. Release of Paclitaxel from the Conjugates. The release of drug from the conjugates was assessed by HPLC method using esterase as hydrolyzing enzyme at pH 7.4. The standard curve of paclitaxel (Figure 3D) was obtained in a special series of HPLC experiments and used for calculating drug release from the conjugates. The data are presented as a percentage of total bound of paclitaxel, which is released from the conjugates (Figure 5). Molecular Modeling. The studies were focused on analysis of conformational structures, energy minima, and molecular dynamics of bis(PEG) polymer, PAMAM dendrimer, free paclitaxel, and paclitaxel containing polymeric conjugates. The chemical structures were built using ChemDraw 9.0 Pro (Cambrigesoft Corp., Cambridge, MA). Free PEG and its bioconjugate with paclitaxel were built to represent seven

ethylene repeat (-CH2-O-CH2-) units. The settings for energy minima and molecular dynamics were of the order reported by us earlier: 1.000 step interval, 2.0 fs; frame interval, 10 fs; terminated after 10 000 steps; heating/cooling rate, 1.000 kcal/atom/ps; target temperature, 300 K. Distance between the first and last carbon atom was measured for the molecular dynamics structures of bis(PEG) and its conjugation with paclitaxel. RasTop molecular visualization software (Philippe Valadon, San Diego, CA) was used for obtaining lengths of free drug, polymer, and its conjugates. Cellular Internalization (Confocal Microscopy). Cellular internalization of FITC-labeled PEG-polymer, dendrimer, paclitaxel, PEG-paclitaxel, and PAMAM-dendrimer-paclitaxel conjugates was studied by confocal microscopy. To assess intracellular distribution of the substances, six optical sections, known as a z-series, were scanned sequentially along the vertical (z) axis from the top to the bottom of the cell.


Khandare et al.

Figure 5. A release profile of paclitaxel from the conjugates determined by HPLC after enzymatic hydrolysis using esterase as the enzyme at pH 7.4. After 24 h, the release of paclitaxel in the PEG conjugate was observed to be 30%. In the case of paclitaxel-SA-PAMAM dendrimer conjugate, the 30% release of the drug was achieved after 48 h, indicating slower release.

growth by 50%) were calculated. A decrease in the IC50 dose indicates an increase in drug toxicity. Statistics. All in vitro experiments were performed in quadruplicate. The results are expressed as mean ( SD from 4-8 independent measurements. Statistical analysis was performed as a one-way analysis of variance (ANOVA) and comparisons among groups were performed by independent sample t-tests.

RESULTS

Figure 4. Molecular modeling studies of paclitaxel-SA-PAMAM dendrimer (A) and PEG-paclitaxel conjugate (B). Polymeric structures were built in Chemdraw software and allowed to undergo conformational stabilization for energy minima. The stabilized structure of PEGpaclitaxel conjugate was exported to RasTop software to estimate the distances between the first and last carbon atoms (here, C1-C71). The distance was measured to be 11.32 Å for energy-minimized molecule.

In Vitro Cytotoxicity. The cytotoxicity of paclitaxel conjugates and free paclitaxel was assessed by using a modified MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as previously described (31, 32). To measure cytotoxicity, cells were separately incubated in a microtiter plate with different concentrations of free paclitaxel, PEG-paclitaxel, and PAMAM dendrimer-paclitaxel conjugates. Control cells received an equivalent volume of fresh medium. The duration of incubation was 24 h. On the basis of these measurements, IC50 doses of free paclitaxel and its polymeric conjugates (the concentrations of active ingredients necessary to inhibit the cell

Synthesis and Characterization of Paclitaxel Conjugates. R,ω-Bis(PEG)-paclitaxel and dendrimer-SA-paclitaxel conjugates were prepared by using EDC.HCl as a coupling agent. Conjugates were purified by membrane dialysis using the molecular weight cutoff method and by size exclusion chromatography. Each of the conjugates was characterized by 1H NMR on 400 MHz spectrophotometer. Proton peaks arising from paclitaxel, bis(PEG), and PAMAM dendrimer were analyzed (Figure 2A,B), and the peak area was integrated and compared for the conjugation ratio (integration values not shown in proton spectra). Both the conjugates and free paclitaxel were further analyzed and characterized by HPLC. In addition, the masses of the PAMAM dendrimer, bis(PEG) polymer, and their conjugates having paclitaxel were characterized by MALDI/ TOF. FITC was covalently labeled with paclitaxel, polymers, and their paclitaxel conjugates, for evaluating cellular dynamics using confocal microscopy. NMR Peaks Characterization and Estimation of Conjugation Ratios. PEG-Paclitaxel Conjugate. 1H NMR spectrum (Figure 2A) showed the broad peaks from 3.2 to 3.9 ppm indicating protons for -CH2CH2O- in bis(PEG) polymer. A peak at 8.85 ppm shows the presence of two hydrogen atoms arising from the phenyl ring in paclitaxel at the C2′ position (33). Proton peaks from δ 3.2 to 3.9 ppm corresponded to PEG and have the integrated peak area of 639 arbitrary units (integration values not shown in proton spectra). Bis(PEG) polymer with molecular weight 10 000 Da has approximately 257 protons in this region. So, each proton corresponds to a value of 2.49 units (639/257). On the other hand, a peak at δ 8.85 ppm corresponds to a value of 0.025 units for a single proton in the phenyl ring of paclitaxel (integration for value ∼0.05/2 ) 0.025). Therefore, the ratio of protons of PEG to paclitaxel is inferred to be around 0.99. Dendrimer-SA-Paclitaxel Conjugate. 1H NMR spectrum (Figure 2B) showed the multiplets between δ 2.0 and 3.6 ppm


corresponding to the presence of 985 protons of CH2 of PAMAM-G4-OH (integration values not shown in the proton NMR spectra) (34). On the other hand, a peak at 8.85 ppm showed the presence of two hydrogen atoms on the phenyl ring in paclitaxel at the C2′ position. The integrated peak areas for protons in dendrimer and paclitaxel are 104.17 and 0.18, respectively. Since the value for each proton in dendrimer and paclitaxel corresponds to 0.105 and 0.09, respectively (i.e., 104.17/985 ) 0.105 and 0.18/2 ) 0.09), the ratio of paclitaxel molecules per dendrimer is ∼0.857 (0.09/0.105 ) 0.857). The proton NMR provides evidence for the formation of PEGpaclitaxel and PAMAM dendrimer-paclitaxel conjugates. Characterization of Conjugates. MALDI/TOF (Figure 3AC). Masses of PAMAM-G4-OH dendrimer, paclitaxel-SAPAMAM dendrimer, and paclitaxel-bis(PEG) conjugates were measured by MALDI/TOF Voyager DE-Pro system (Applied Biosystems, Foster City, CA) using sinapinic acid (3,5-dimethoxy4-hydroxycinnamic acid) as a matrix. The molecular masses were found to be 15.22 KDa for PAMAM-G4-OH dendrimer (Figure 3A), 16, 18 KDa for paclitaxel-SA-PAMAM dendrimer (Figure 3B), and 12.90 KDa for paclitaxel-bis(PEG) conjugate (Figure 3C). The total mass of the compounds indicates formation of the paclitaxel conjugates. In addition, HPLC data confirmed the peaks between 3.5 and 5 min (Figure 3E,F) indicating high molecular weight conjugate formation for both PAMAM-SA-paclitaxel and PEG-paclitaxel. HPLC. In this study, we used HPLC for analysis of paclitaxel in conjugates and free form. Quantification of free and polymerbound paclitaxel was achieved using the reverse-phase HPLC method preceded by C5 column (4.6 × 250, 5-300 A, Phenomenex, Torrance, CA). Paclitaxel has an absorbance spectrum with the maximum at 280 nm allowing quantitative determination of polymer-drug conjugates. The use of the C5 column improved the retention time of polymer-bound paclitaxel when compared with C18 and C8 columns (Figure 3E,F). The chromatogram represents the broad peak which may arise from conjugation formed at primary as well as secondary hydroxyl positions. The stability of the bond in paclitaxel-polymer conjugates was investigated by measuring the release of free paclitaxel in stock solution. The conjugates were stored at 4 and -20 °C for 1 month, and no significant release of free drug was observed. In addition, the release of paclitaxel from the conjugates after enzymatic hydrolysis was determined using esterase as enzyme at pH 7.4 (Figure 5). Data obtained showed that about 30% of paclitaxel were released from PEG-paclitaxel conjugate after 24 h of incubation. In contrast, less than 20% of the drug was released after 24 h of incubation from PAMAM-SA-paclitaxel dendrimer, reaching 30% only after 48 h of the experiment. Although dendrimer-SA-paclitaxel conjugate has two ester sites for hydrolysis (Figure 1A,B), its enzymatic degradation was slower when compared with PEG conjugate, which has only one ester site (Figure 1C). This may be a result of specific nanosized dendrimer architecture which exhibits higher steric hindrance for esterase degradation at macromolecular ester sites when compared with linear PEG conjugate. Enhanced Aqueous Solubility of Paclitaxel Using Dendritic and PEG Conjugates. Both of these polymers increased aqueous solubility of the drug. Solubility was higher for dendrimer-paclitaxel conjugate when compared to PEGpaclitaxel conjugates. While the aqueous solubility of paclitaxel is limited to 0.3µg/mL, the solubility of paclitaxel-bis(PEG) conjugate was observed to be 2.5 mg/mL. Moreover, the solubility for PAMAM-paclitaxel conjugate was further improved and was found to be 3.2 mg/mL. Molecular Modeling Studies. We compared the structural stability of the linear PEG polymer with the well-defined


Figure 6. Confocal microscopy fluorescent images of human A2780 ovarian carcinoma cells incubated for 24 h with FITC-labeled substances (z-series, from the top of the cell to the bottom).

molecular structure of PAMAM dendrimer, built structures, and evaluated the energy minima and molecular dynamics of paclitaxel, bis(PEG), PAMAM dendrimer, and polymeric conjugates containing paclitaxel. An example of molecular modeling of PAMAM dendrimer is shown in Figure 4. The energy minima conformational distance for paclitaxel was observed to be 8.24 Å (C1 to C47, the figure not shown). On the other hand, the molecular dynamics structure showed a distance of 7.99 Å (figure not shown). Energy minima conformation for paclitaxelSA-PEG conjugate (Figure 4B) was observed to be 11.32 Å (C1 to C71). On the other hand, the molecular conformation of PAMAM dendrimer was found to be more stable than that of its paclitaxel conjugate, indicating conformational bulkiness of the resulting conjugate due to addition of the drug (Figure 4A). Cellular Localization of Labeled FITC Having Bis(PEG) and Dendrimer with Paclitaxel. Theoretically, high molecular weight drug conjugates could adhere to the surface of cancer cells and erroneously be visualized on microscopic images as internalized within cells. To exclude such errors, we analyzed the distribution of FITC-labeled polymers, polymer-drug conjugates, and free paclitaxel in different cellular layers from the upper to the lower of the fixed cell using confocal fluorescent microscopy (z-sections, Figure 6). In these experiments, the studied components were labeled with FITC and incubated with A2780 human ovarian carcinoma cells. The cells were fixed and subjected to confocal microscopy. The z-sections of single cells showed the homogeneous and uniform distribution of labeled polymers with or without paclitaxel. In contrast, the distribution of free paclitaxel inside the cancer cell was highly inhomogeneous both within the one layer and through different layers of a single cell. Free paclitaxel accumulated mainly in the cytoplasm near the plasma membrane and predominantly on the top of the cell in the place of cellular contact with the medium containing the drug (the bottom surface of the cell was attached to the flask wall). These data suggest that both linear PEG polymer and PAMAM dendrimer enhance the penetration of paclitaxel into cancer cells, resulting in a more homogeneous drug distribution inside the single cells. In Vitro Cytotoxicity. Conjugation of paclitaxel to linear PEG polymer and PAMAM dendrimer substantially influenced its cytotoxicity (Figure 7). While linear PEG-paclitaxel conjugate showed more than 25-fold lower toxicity (IC50 dose increased), PAMAM-dendrimer-paclitaxel conjugate showed


Figure 7. In vitro cytotoxicity of free paclitaxel, PEG-paclitaxel, and paclitaxel-SA-PAMAM dendrimer conjugates toward A2780 human ovarian carcinoma cells. PAMAM-SA dendrimer substantially enhances toxicity of paclitaxel by decreasing the dose of the drug required to kill 50% of cells (IC50 dose). Means ( SD are shown. *P < 0.05 when compared with free paclitaxel.

significantly (P < 0.05) higher toxicity (IC50 dose was more them 10 times lower) when compared with free drug.

DISCUSSION Polymeric architecture, molecular weight, and molecular charge play important roles in delivering drugs (35). This is especially true when the polymeric materials have linear, branched, and graft architectures. In addition, such polymers exhibit high steric hindrance for cellular internalization and might often result in delayed cellular entry. For instance, PEGylated adenovirus vectors exhibit longer plasma half-life when compared with free adenovirus, and their entry into cells has been prevented by steric hindrance by poly(ethylene glycol) (PEG) chains (36). Moreover, linear PEG polymer may possess higher polydispersity, and theoretical size may range greater than 300-500 nm even for lower molecular weight polymer. In contrast, hyperbranched polymers, e.g., dendrimers, due to their well-defined monodispersity (PI ≈ 1.0), controlled architecture, nanosize (20-40 nm), and shape, are often considered most promising vehicles for the delivery of drugs, proteins, and other therapeutic agents (21, 37-39). Dendrimers in conjugation with FITC have been reported to achieve rapid cellular entry even in 5 min (34). Dendrimers have unique architecture, as they are prepared through controlled synthetic methods: either by divergent or convergent method having control over the branching architecture. Therefore, in a short time, they have emerged as the most promising drug delivery carriers. In this work, we compared the difference in delivery ability of these polymers with the conventional linear PEG polymers. Conjugation of a low-solubility drug with high-solubility polymers bears an additional advantage of forming a prodrug with enhanced aqueous solubility. Such soluble prodrugs further increase the bioavailability of the drug and therefore have a potential to enhance its activity (cytotoxicity in the case of an anticancer drug). Recently, polyglycerol dendrimers (PGDs) with 4-5 generations were used to investigate the effect of dendritic architecture and its generation on the aqueous solubilization of paclitaxel (7). It was shown that the increase in the paclitaxel solubility by PGDs was dependent on the dendrimer generation. In the present study, both polymers increased the aqueous solubility of the drug. However, the increase in the solubility was higher for the dendrimerpaclitaxel conjugate when compared with the PEG-paclitaxel conjugate. The aqueous solubility of paclitaxel-bis(PEG) was equal to 2.5 mg/mL, while that of PAMAM-SA-paclitaxel conjugate was 3.2 mg/mL. The aqueous solubility of free

Khandare et al.

paclitaxel was about 0.3 µg/mL. Such a dramatic enhancement in paclitaxel solubility allowed dissolving of conjugates in buffer for our in vitro cytotoxicity studies, while the free drug was dissolved in ethanol. Besides solubility and size, the bioavailability and anticancer activity of conjugated drug substantially depend on the type of bond between the drug and a carrier. Specific spacers are often used to control the rate of drug release from the conjugate. We used succinic acid as a spacer to bind paclitaxel to PAMAM dendrimer. SA is a bis functional carboxylic acid and therefore can be conjugated with hydroxyl groups in paclitaxel, thereby resulting in forming an ester bond with the drug, and yet leaves one free carboxyl group for further conjugation with another component. Paclitaxel-SA conjugate was coupled with hydroxyl-terminal PAMAM dendrimer to obtain dendrimer-SApaclitaxel conjugate. In the past, the role of bis functional spacers (e.g., glutaric acid) has been established in increasing the reactivity of the low-reactivity drugs with the hyperbranched polymers (34). Additionally, bis functional spacers such as succinic acid may decrease the steric hindrance for conjugation with polymers, especially with the hyperbranched polymers, due to crowded functional groups (34). This is especially important when the dendrimer conjugate is expected to obtain a high payload of drug in the conjugate. It should be noted that, in the present conjugation reaction, two ester bonds are formed, i.e., one with the drug and another with the dendrimer, thereby providing two sites for hydrolysis to release the free drug. It is expected that free paclitaxel will be released from the conjugated drug following hydrolysis of this bond after internalization of the whole complex into the cancer cell. On the other hand, R,ωbis(PEG10 000) polymer contains carboxyl end groups at the terminals and therefore can be coupled directly with 2′ hydroxyl groups in paclitaxel. Although the bis(PEG) polymer consists of two reactive sites, only one molecule of paclitaxel was coupled using equivalent moles of drug and EDC.HCl as a condensing agent. It has been reported in the past that the most suitable position for structural conjugation in paclitaxel is either C2′ or C7′ hydroxyl (33). It has been well-realized that usually the primary hydroxyl C2′ site is a more active site than the secondary hydroxyl C7′ site due to less steric hindrance, and esterification is preferentially formed at C2′. However, it is possible to form more than one conformation of the conjugates reacting either at C2′ or at C7′. The second free carboxyl group available on the bis(PEG)-paclitaxel conjugate was utilized for coupling with the hydroxyl group in FITC, allowing for visualization of the conjugate by fluorescent/confocal microscopy. A polymer and its reaction with bulkier drug molecules undergo steric hindrance due to higher molecular mass and unstable conformations (35). The net result may lead to lower yields or may even form by-products through incomplete conjugation reactions with free and unreacted drug and polymer chains. Prodrug conjugation reactions may be studied using modeling through the conformations and minima energies. In addition, the reactivity of both polymers as well as drugs could be increased by incorporating spacer molecules. One of the objectives of the present study was to compare the structural stability of the linear PEG polymer with well-defined, monodispersed PAMAM dendrimers. In the past, polymeric conformations with a glycine spacer have been evaluated using modeling studies to estimate the spacer length and molecule stability (40, 41). More recently, few studies suggest applications of modeling in prodrug bioconjugates, involving incorporation of spacers and strategies to reduce the steric hindrance in prodrug synthesis (42). The author has demonstrated steric hindrance associated with conjugation of an anticancer drug with PEG polymer by a molecular modeling method to determine


the most suitable bicarboxylic amino acid that possesses the least steric hindrance. Recently, we reported molecular structures of bis(PEG) polymer with multiple copies of the drug camptothecin (CPT) using molecular modeling tools (40). The minima represented the minimum energies (kcal) indicating the degree of conformational freedom for (a) seven repeating units of bis(PEG) polymeric chain conjugate, (b) generation 2 hydroxyl-terminated PAMAM dendrimer, and (c) its conjugate with paclitaxel. The minimum-energy conformational distance for paclitaxel was observed to be 8.24 Å (C1 to C47). On the other hand, the molecularly dynamic structure showed a distance of 7.99 Å. The minimum-energy conformation for paclitaxel-SA-PEG conjugate was observed to be 11.32 Å (C1 to C71). On the other hand, molecular confirmation of PAMAM dendrimer was found to be more stable than its paclitaxel conjugate, indicating conformational bulkiness of the resulting conjugate due to addition of the drug. Previously, we reported the results of molecular modeling studies which support our synthetic strategies in selecting reactive peptides, polymers, drugs, and spacers to reduce steric hindrance, especially prodrug conjugates involving multiple components (35, 40). Although conjugation of paclitaxel to both traditional PEG polymer and PAMAM G4-SA dendrimer improve the bioavailability of the drug, the influence of the conjugation on anticancer activity of paclitaxel depended on the type of drug carrier. Conjugation to PEG polymer significantly decreased the toxicity of paclitaxel. In fact, the IC50 dose of PEG-paclitaxel conjugate was more than 25 higher when compared with free drug. Such a decrease in anticancer activity might be explained by the increase in molecular mass of the whole complex, which in turn changed the mechanism of the cellular internalization of PEG-paclitaxel polymer from diffusion (free drug) to endocytosis. In contrast, conjugation of paclitaxel to PAMAM G4-SA dendrimer substantially enhanced cytotoxicity of the drug leading to the decrease in the IC50 dose more than 10-fold when compared with free drug.

CONCLUSIONS Taken together, the data obtained suggest that PAMAM G4SA dendrimer represents a promising vehicle for intracellular delivery of low-solubility drugs such as paclitaxel. The developed dendrimer provides both cytoplasmic and nuclear delivery of therapeutics and enhances anticancer activity of paclitaxel.

ACKNOWLEDGMENT This work was supported by National Institutes of Health grant CA100098 from the National Cancer Institute.

LITERATURE CITED (1) Wani, M. C., Taylor, H. L., Wall, M. E., Coggon, P., and McPhail, A. T. (1971) Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J. Am. Chem. Soc. 93, 2325-2327. (2) Singla, A. K., Garg, A., and Aggarwal, D. (2002) Paclitaxel and its formulations. Int. J. Pharm. 235, 179-192. (3) Wall, M. E., and Wani, M. C. (1996) Camptothecin and taxol: from discovery to clinic. J. Ethnopharmacol. 51, 239-253; discussion 253-254. (4) Dorr, R. T. (1994) Pharmacology and toxicology of Cremophor EL diluent. Ann. Pharmacother. 28, S11-14. (5) Feng, X., Yuan, Y. J., and Wu, J. C. (2002) Synthesis and evaluation of water-soluble paclitaxel prodrugs. Bioorg. Med. Chem. Lett. 12, 3301-3. (6) Fjallskog, M. L., Frii, L., and Bergh, J. (1993) Is Cremophor EL, solvent for paclitaxel, cytotoxic? Lancet 342, 873.

Bioconjugate Chem., Vol. 17, No. 6, 2006 1471 (7) Ooya, T., Lee, J., and Park, K. (2004) Hydrotropic dendrimers of generations 4 and 5: synthesis, characterization, and hydrotropic solubilization of paclitaxel. Bioconjugate Chem. 15, 1221-1229. (8) Rowinsky, E. K., Eisenhauer, E. A., Chaudhry, V., Arbuck, S. G., and Donehower, R. C. (1993) Clinical toxicities encountered with paclitaxel (Taxol). Semin. Oncol. 20, 1-15. (9) Greenwald, R. B., Gilbert, C. W., Pendri, A., Conover, C. D., Xia, J., and Martinez, A. (1996) Drug delivery systems: water soluble taxol 2′-poly(ethylene glycol) ester prodrugs-design and in vivo effectiveness. J. Med. Chem. 39, 424-431. (10) Allen, T. M., and Cullis, P. R. (2004) Drug delivery systems: entering the mainstream. Science 303, 1818-1822. (11) Choi, Y., Thomas, T., Kotlyar, A., Islam, M. T., and Baker, J. R., Jr. (2005) Synthesis and functional evaluation of DNA-assembled polyamidoamine dendrimer clusters for cancer cell-specific targeting. Chem. Biol. 12, 35-43. (12) Conover, C. D., Linberg, R., Gilbert, C. W., Shum, K. L., and Shorr, R. G. (1997) Effect of polyethylene glycol conjugated bovine hemoglobin in both top-load and exchange transfusion rat models. Artif. Organs 21, 1066-1075. (13) Dharap, S. S., Wang, Y., Chandna, P., Khandare, J. J., Qiu, B., Gunaseelan, S., Sinko, P. J., Stein, S., Farmanfarmaian, A., and Minko, T. (2005) Tumor-specific targeting of an anticancer drug delivery system by LHRH peptide. Proc. Natl. Acad. Sci. U.S.A. 102, 12962-12967. (14) Duncan, R. (2003) The dawning era of polymer therapeutics. Nat. ReV. Drug DiscoVery 2, 347-360. (15) Kitchens, K. M., El-Sayed, M. E., and Ghandehari, H. (2005) Transepithelial and endothelial transport of poly (amidoamine) dendrimers. AdV. Drug DeliVery ReV. 57, 2163-2176. (16) Kopecek, J., Kopeckova, P., Minko, T., Lu, Z. R., and Peterson, C. M. (2001) Water soluble polymers in tumor targeted delivery. J. Controlled Release 74, 147-158. (17) Maeda, H. (2001) The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. AdV. Enzyme Regul. 41, 189-207. (18) Putnam, D. (2006) Polymers for gene delivery across length scales. Nat. Mater. 5, 439-451. (19) Wang, C., Ge, Q., Ting, D., Nguyen, D., Shen, H. R., Chen, J., Eisen, H. N., Heller, J., Langer, R., and Putnam, D. (2004) Molecularly engineered poly(ortho ester) microspheres for enhanced delivery of DNA vaccines. Nat. Mater. 3, 190-196. (20) Vicent, M. J., and Duncan, R. (2006) Polymer conjugates: nanosized medicines for treating cancer. Trends Biotechnol. 24, 3947. (21) Gurdag, S., Khandare, J., Stapels, S., Matherly, L. H., and Kannan, R. M. (2006) Activity of dendrimer-methotrexate conjugates on methotrexate-sensitive and -resistant cell lines. Bioconjugate Chem. 17, 275-283. (22) Patri, A. K., Myc, A., Beals, J., Thomas, T. P., Bander, N. H., and Baker, J. R., Jr. (2004) Synthesis and in vitro testing of J591 antibody-dendrimer conjugates for targeted prostate cancer therapy. Bioconjugate Chem. 15, 1174-1181. (23) Svenson, S., and Tomalia, D. A. (2005) Dendrimers in biomedical applications-reflections on the field. AdV. Drug DeliVery ReV. 57, 2106-2129. (24) Tomalia, D. A., and Frechet, J. M. (2005) Introduction to “Dendrimers and dendritic polymers”. Progr. Polym. Sci. 30, 217219. (25) Sweetana, S., and Akers, M. J. (1996) Solubility principles and practices for parenteral drug dosage form development. PDA J. Pharm. Sci. Technol. 50, 330-342. (26) Deutsch, H. M., Glinski, J. A., Hernandez, M., Haugwitz, R. D., Narayanan, V. L., Suffness, M., and Zalkow, L. H. (1989) Synthesis of congeners and prodrugs. 3. Water-soluble prodrugs of taxol with potent antitumor activity. J. Med. Chem. 32, 788-792. (27) Li, C., Yu, D. F., Newman, R. A., Cabral, F., Stephens, L. C., Hunter, N., Milas, L., and Wallace, S. (1998) Complete regression of well-established tumors using a novel water-soluble poly(Lglutamic acid)-paclitaxel conjugate. Cancer Res. 58, 2404-2409. (28) Vyas, D. M., Ueda, Y., Wong, H., Matiskella, J. D., Hauck, S., Mikkilineni, A. B., Farina, V., Rose, W. C., and Casazza, A. M. (1995) Phosphate-activated prodrugs of paclitaxel. In Taxane anticancer agents: Basic science and current status (Chen, T. T.,

1472 Bioconjugate Chem., Vol. 17, No. 6, 2006 Ojima, I., and Vyas, D. M., Eds.) pp 124-137, American Chemical Society, Washington, DC. (29) Zhang, X., Li, Y., Chen, X., Wang, X., Xu, X., Liang, Q., Hu, J., and Jing, X. (2005) Synthesis and characterization of the paclitaxel/ MPEG-PLA block copolymer conjugate. Biomaterials 26, 21212128. (30) Zhao, Z., Kingston, D. G. I., and Crosswell, A. R. (1991) Modified taxols, 6. Preparation of water-soluble prodrugs of taxol. J. Nat. Prod. 54, 1607-1611. (31) Minko, T., Kopeckova, P., and Kopecek, J. (1999) Chronic exposure to HPMA copolymer-bound adriamycin does not induce multidrug resistance in a human ovarian carcinoma cell line. J. Controlled Release 59, 133-148. (32) Pakunlu, R. I., Wang, Y., Tsao, W., Pozharov, V., Cook, T. J., and Minko, T. (2004) Enhancement of the efficacy of chemotherapy for lung cancer by simultaneous suppression of multidrug resistance and antiapoptotic cellular defense: novel multicomponent delivery system. Cancer Res. 64, 6214-6224. (33) Shi, Q., Wang, H. K., Bastow, K. F., Tachibana, Y., Chen, K., Lee, F. Y., and Lee, K. H. (2001) Antitumor agents 210. Synthesis and evaluation of taxoid-epipodophyllotoxin conjugates as novel cytotoxic agents. Bioorg. Med. Chem. 9, 2999-3004. (34) Khandare, J., Kolhe, P., Pillai, O., Kannan, S., Lieh-Lai, M., and Kannan, R. M. (2005) Synthesis, cellular transport, and activity of polyamidoamine dendrimer-methylprednisolone conjugates. Bioconjugate Chem. 16, 330-337. (35) Khandare, J. J., and Minko, T. (2006) Polymer drug conjugates: Progress in polymeric prodrugs. Prog. Polym. Sci. 31, 359-397.

Khandare et al. (36) Eto, Y., Gao, J. Q., Sekiguchi, F., Kurachi, S., Katayama, K., Maeda, M., Kawasaki, K., Mizuguchi, H., Hayakawa, T., Tsutsumi, Y., Mayumi, T., and Nakagawa, S. (2005) PEGylated adenovirus vectors containing RGD peptides on the tip of PEG show high transduction efficiency and antibody evasion ability. J. Gene Med. 7, 604-612. (37) Grainger, D., and Okano, T. (2003) Biomedical micro- and nanotechnology. AdV. Drug DeliVery ReV. 55, 311-313. (38) Kono, K., Liu, M., and Frechet, J. M. (1999) Design of dendritic macromolecules containing folate or methotrexate residues. Bioconjugate Chem. 10, 1115-1121. (39) Morgan, M. T., Carnahan, M. A., Immoos, C. E., Ribeiro, A. A., Finkelstein, S., Lee, S. J., and Grinstaff, M. W. (2003) Dendritic molecular capsules for hydrophobic compounds. J. Am. Chem. Soc. 125, 15485-15489. (40) Khandare, J. J., Chandna, P., Wang, Y., Pozharov, V. P., and Minko, T. (2006) Novel polymeric prodrug with multivalent components for cancer therapy. J. Pharmacol. Exp. Ther. 317, 929937. (41) Paranjpe, P. V., Chen, Y., Kholodovych, V., Welsh, W., Stein, S., and Sinko, P. J. (2004) Tumor-targeted bioconjugate based delivery of camptothecin: design, synthesis and in vitro evaluation. J. Controlled Release 100, 275-292. (42) Schiavon, O., Pasut, G., Moro, S., Orsolini, P., Guiotto, A., and Veronese, F. M. (2004) PEG-Ara-C conjugates for controlled release. Eur. J. Med. Chem. 39, 123-133. BC060240P

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