In Vitro and in Vivo Evaluation of Hydrophilic Dendronized Linear

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In Vitro and in Vivo Evaluation of Hydrophilic Dendronized Linear Polymers Cameron C. Lee,† Masaru Yoshida,†,§ Jean M. J. Fre´chet,† Edward E. Dy,‡ and Francis C. Szoka*,‡ Center for New Directions in Organic Synthesis, Department of Chemistry, University of California, Berkeley, California 94720-1460, and Department of Biopharmaceutical Sciences & Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446. Received September 23, 2004; Revised Manuscript Received April 8, 2005

Rigid-rod dendronized linear polymers consisting of a poly(4-hydroxystyrene) backbone and fourthgeneration polyester dendrons were evaluated in vitro and in vivo to determine their suitability as drug delivery vectors. Cytotoxicity assays indicated that the polymers were well tolerated by cells in vitro. Biodistribution studies of the polymers in both nontumored and tumored mice revealed that as for random coil linear polymers, renal clearance was a function of polymer size, with significant urinary excretion observed for a 67 kDa dendronized polymer. High accumulation in organs of the reticuloendothelial system was exhibited by a dendronized polymer with a very high molecular weight (Mn ) 1740 kDa), but was not as significant for smaller polymers with Mn ) 67 kDa and Mn ) 251 kDa. The rank order for tumor accumulation of the polymers on a percent injected dose per gram tumor basis was 251 kDa ∼ 1740 kDa > 67 kDa. These data will help guide the selection of highly functionalizable rigid-rod dendronized polymers with pharmacokinetic properties appropriate for use as drug carriers.

INTRODUCTION

Polymers, both natural and synthetic in origin, have attracted attention as drug delivery vehicles due to their ability to favorably alter the biological properties of attached therapeutic agents (1). For example, polymerdrug conjugates may have increased blood circulation half-lives, reduced toxicities, and increased solubilities relative to the parent drugs (2). In addition, high molecular weight (MW) polymers can exhibit enhanced accumulation in tumor tissues relative to normal tissues. This phenomenon is termed the enhanced permeation and retention effect (EPR effect), and its occurrence is attributed to the “leaky” blood vessels and poorly developed lymphatic drainage system present within tumor tissues (3). To date, the carriers that have been most extensively investigated for drug delivery applications are linear polymers such as poly(ethylene oxide) (PEO) (4) and poly(N-(2-hydroxypropylmethacrylamide)) (HPMA) (5, 6). However, increasing attention is being given to new, more highly branched structures such as dendrimers and star polymers with the hope that interesting effects of architecture on biological properties may be discovered (714). Dendronized linear polymers, a recent addition to the branched polymer family, are linear polymers that bear dendrons at each repeat unit along their backbones (Figure 1). It is believed that as the sizes of the pendant dendrons increase, so do the interactions between adja* To whom correspondence should be addressed: School of Pharmacy S-926, University of California, San Francisco, CA 94143-0446. Tel (415) 476-3895, Fax (415) 476-0688, E-mail [email protected]. † University of California, Berkeley. ‡ University of California, San Francisco. § Permanent address: Nanotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan.

Figure 1. Schematic representation of a dendronized linear polymer and the molecular structure of generation-four dendronized poly(4-hydroxystyrene).

cent dendrons, and at higher generations these macromolecules attain extended conformations and can be described as somewhat rigid, cylindrical rods (15). Dendronized polymers may be interesting scaffolds for drug delivery as the large number of functionalizable peripheral groups on the dendrons should allow for very high levels of drug loading, and furthermore it has been suggested that the shape and multivalency of a macromolecule can influence its biological properties (16-20). However, in contrast to dendrimers, few biological studies of dendronized linear polymers have been reported to date (21-23). Here we describe the cytotoxicity, biodistribution, and pharmacokinetics of synthetic, watersoluble, rigid-rod dendronized linear polymers consisting

10.1021/bc0497665 CCC: $30.25 © 2005 American Chemical Society Published on Web 04/29/2005

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of a poly(4-hydroxystyrene) backbone and fourth-generation polyester dendrons in normal and tumored mice (Figure 1). To the best of our knowledge, this constitutes the first report on the in vivo biodistribution of dendronized polymers.

Lee et al. Table 1. Molecular Weights, Sizes, and Pharmacokinetic Parameters for 1-3 Measured in Mice. SEC-MALLSa (kDa) 1 2 3

MATERIALS AND METHODS

Materials. Unless otherwise noted, reagents and solvents were obtained from commercial suppliers and used without further purification. Polymers 1-3 were prepared as previously reported (24, 25). Characterization. Analytical size exclusion chromatography (SEC) in N,N-dimethylformamide (DMF) with 0.2% LiBr was performed at 70 °C at a nominal flow rate of 1.0 mL/min on a chromatography line calibrated with linear poly(ethylene oxide) (PEO) standards (6450529500 Da) and fitted with two 7.5 × 300 mm PLgel mixed-bed C columns (5-µm particle size). The SEC system used when determining PEO-equivalent MWs consists of a Waters 510 pump, a Waters U6K injector, and a Waters 410 differential refractive index detector thermostated at 35 °C. The SEC system for determining absolute MWs consists of a Waters 510 pump, a 7125 Reodyne injector, a Wyatt DAWN-EOS multi-angle laser light scattering detector (laser of λ ) 690 nm), and a Wyatt Optilab differential refractive index detector. Light scattering data were analyzed using Astra software from Wyatt, and SEC data using the differential refractive index detector were analyzed using Millennium software from Waters. Volume-average particle diameters were determined using dynamic light scattering. Experiments were performed at least three times at 25 °C in saline solution with a Zetasizer Nano ZS (Malvern Instruments) equipped with a 4 mW He-Ne laser at 633 nm. Radioactivity from 125I was quantified with a 1480 Wallac Wizard 3 Automatic Gamma Counter. Counts emitted from samples contained in screw-cap scintillation vials were measured over the course of one minute and were recorded as counts per minute (cpm). Cytotoxicity of 1-3. Cell toxicity studies were performed at the UC Berkeley Tissue Culture Facility. MDAMB-231 human breast cancer cells were seeded in 96well plates at a density of 1.6 × 104 cells per well in 100 µL of DMEM with 10% FBS (DMEM + FBS). After incubation overnight (37 °C, 5% CO2), the medium was removed by aspiration and replaced with fresh medium (100 µL) containing 0-3.0 mg/mL of polymers 1-3. These solutions also contained antibiotics (1% penicillin-streptomycin). (Each of the polymers used in these experiments was purified prior to use by size exclusion chromatography on a PD-10 column, followed by lyophilization.) Following incubation for 48 h, the medium was aspirated off and replaced with 100 µL of fresh medium and 20 µL of a 5 mg/mL MTT solution. The cells were incubated for 4 h, after which time the medium was removed, leaving behind purple crystals. These crystals were dissolved in 200 µL of DMSO and 25 µL of glycine buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The absorbance values at 520 nm were measured using a SpectraMAX 190 microplate reader. (Molecular Devices). The percent viability of cells in treated wells relative to nontreated cells was calculated and represents the average value over four wells (Figure 2). Synthesis of Tyramine-Functionalized Dendronized Polymers 1-3. 1, 2, or 3 was dissolved in 1 mL of anhydrous pyridine and reacted overnight with a solution containing 5-7 mol % (relative to hydroxyls) of 4-nitrophenyl chloroformate in 1 mL of CH2Cl2. The

c

SECb (kDa)

DLS

Mn

Mw

Mn

Mw

PDI

Dc (nm)

67.0 251 1,740

69.0 260 1,890

13.7 38.2 190.0

16.0 45.2 242.0

1.16 1.18 1.27

5.8 10.0 15.7

a Absolute MWs. b Relative PEO MWs from SEC in DMF. Mean volume-average diameter.

solvent was evaporated, and the polymer was dissolved in 2 mL of anhydrous N,N-dimethylformamide. An excess of 4-(2-aminoethyl)phenol (tyramine) and 1 mol equiv of anhydrous triethylamine was added to the solution and was stirred overnight. The polymers were purified of low MW contaminants by dialysis in Spectra/Por regenerated cellulose membranes (MWCO ) 8000, Spectrum Laboratories) against distilled water. The presence of tyramine groups was confirmed by 1H NMR analysis of a lyophilized aliquot of the solution retained in the dialysis membrane. The aromatic protons appear at 6.6 and 6.9 ppm, and the benzylic protons appear at 2.6 ppm. Radioiodination of Tyramine-Functionalized Polymers 1-3. Tyramine functionalized 1, 2, or 3 were iodinated as previously described (8, 26), resulting in solutions of radiolabeled polymer in HBS (10 mM HEPES/ Cl, 140 mM NaCl, pH 7.4). Non-polymer-bound 125I was removed by ion exchange chromatography on Bio-Rad AG 1-X-2 resin (chloride form), and polymers were separated from residual low MW radioactive contaminants by size exclusion chromatography on Bio-Rad 10DG desalting columns that had been equilibrated with HBS. The initial, high-MW fractions were collected and pooled. The specific activities of the solutions for the biodistribution experiments in nontumored mice were measured to be 23, 3, and 35 µCi/mL for polymers 1, 2, and 3, respectively, and for the biodistribution experiments in tumored mice the activities were 15, 26, and 16 µCi/mL, respectively. The final concentration of polymer in the solutions was 0.8 mg/mL. Biodistribution of 1-3 in Nontumored Mice. Polymer solutions (200 µL) were administered intravenously to 6-8 week-old CD-1 female mice (three mice per experimental group). The mice were sacrificed at six different times following injection for biodistribution analyses: 10, 30, 90, 540, 1440, and 2880 min postinjection. The blood (collected by heart puncture), heart, lungs, liver, stomach, spleen, intestines, kidney, and carcass (divided into three portions) were weighed, and the amount of radioactivity present in each organ was quantified. For the 24- and 48-h time points, mice were housed in metabolic cages to allow for the collection of urine and feces. The amount of radioactivity recovered was on average 86 ( 9% of that injected; we attribute the nonquantitative recoveries to injection variability. The data were corrected for radioactive decay and plotted as % of injected dose per gram of organ versus time and as % of injected dose per organ versus time for each organ (Figure 3). The % of the injected dose per gram (% ID/g) of blood versus time curve was analyzed using a two-compartment model, since ln[% ID/g] versus time curves clearly displayed two different rates of decay for early and late time points. The pharmacokinetic parameters for the twocompartment model were estimated using the residuals method (27). A complete list of pharmacokinetic parameters is presented in Table 2. For urine analysis at 24 h, 1.0 mL of urine was loaded onto a PD-10 column and eluted in 1.0 mL fractions to

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Biological Evaluation of Dendronized Polymers Table 2. Pharmacokinetic Parameters for 1-3a t1/2,β (h) t1/2,R (h) k21 (min-1) k12 (min-1) kel (min-1) Co (% ID/g) V1 (g blood) AUC0-∞ (% ID‚min‚g-1)

1

2

3

14 ( 2 0.9 ( 0.2 0.0040 ( 0.0003 0.007 ( 0.002 0.0025 ( 0.0006 40 ( 3 2.5 ( 0.2 16000 ( 3000

19 ( 2 1.3 ( 0.9 0.0054 ( 0.0005 0.003 ( 0.006 0.0010 ( 0.0007 41 ( 4 2.5 ( 0.2 40000 ( 4000

44 ( 9 1.1 ( 0.6 0.0055 ( 0.0007 0.004 ( 0.006 0.0005 ( 0.0003 38 ( 5 2.6 ( 0.3 80000 ( 20000

a Parameters were extracted from %ID/g blood curves assuming a two-compartment model, where the concentration of radioactivity in the blood is represented by the equation: C ) Ae-Rt+ Be-βt. Values for A, R, B, and β were calculated using the residuals method (27). Definitions: t1/2,β, elimination half-life; t1/2,R, distribution half-life; k21, rate of polymer loss from central to peripheral compartment; k12, rate of polymer loss from peripheral to central compartment; kel, rate of elimination from the central compartment; Co, concentration of polymer in central compartment after injection but before clearance or loss to peripheral compartments; V1, apparent volume of the central compartment; AUC0-∞, area under the % ID/g blood curves from zero to infinity.

separate high MW components from lower ones by size exclusion chromatography. Radioactivity was found to elute from the column in two major fractions. The first fractions (1-6) contained high MW species, and the second fractions (7 and above) contained low MW species. The recovery of radioactivity from the columns was quantitative. The percent of the recovered radioactivity found in the high MW fractions for mice given polymers 1, 2, and 3, were 99%, 85%, and 76%, respectively. For feces analysis at 24 h, ∼400 mg of feces were weighed into a tube containing zirconia beads and 1.0 mL of HBS. The tube was capped, and the feces were homogenized using a Bead Beater (Biospec) for 200 s at 5000 rpm. The solids were centrifuged out, and 50-250 µL of the supernatant was removed, loaded onto a PD10 column, and eluted in 1.0 mL fractions in the same manner as for the urine analysis. The recovery of radioactivity from the column was quantitative. Again, the first fractions (1-8) contained high MW species, and the second fractions (9 and above) contained low MW species. The percent of the recovered radioactivity found in the high MW fractions in the feces for mice given polymers 1, 2, and 3, was 97%, 91%, and 95%, respectively. Biodistribution of 1-3 in Tumored Mice. Female 8-week old BALB/c mice were inoculated with C26 tumor cells via subcutaneous injection in the right hind flank on day zero (1 × 106 cells in 50 µL of cell media). On the twelfth day postinoculation, when the tumors were ∼5 mm in diameter, polymer solutions (200 µL) were administered intravenously to the mice (three mice per experimental group). The mice were housed in metabolic cages to allow for the collection of urine and feces, and at 24 h postinjection the mice were sacrificed and the blood (collected by heart puncture), heart, lungs, liver, stomach, spleen, intestines, kidney, tumor, leg muscle, and carcass (divided into three portions) were weighed, and the amount of radioactivity present in each tissue type was quantified. The amount of radioactivity recovered was on average 80 ( 9% of that injected; we attribute the nonquantitative recoveries to injection variability. The data were corrected for radioactive decay and plotted as % of injected dose per gram of organ and as % of injected dose per organ for each organ (Figure 5).

polyester dendron in each case (Figure 1) (24, 25). The absolute and relative MWs of each polymer, as measured by size exclusion chromatography (SEC), are given in Table 1. The MWs measured by SEC relative to PEO calibration standards are much lower than their absolute MWs measured using an online multiangle laser light scattering (MALLS) detector; PEO-equivalent MWs are reported here because they provide an estimate of the hydrodynamic sizes of the dendronized polymers. The dimensions of the polymers were also measured using dynamic light scattering (DLS) and are presented in Table 1. In Vitro Cytotoxicity Studies. To determine the biocompatibility of the dendronized polymers, toxicity experiments were performed in vitro with MDA-MB-231 human breast cancer cells. After a 48 h incubation period, cell viability was evaluated using the MTT assay (Figure 2). Over the concentration range investigated, the polymers did not exhibit high levels of toxicity, with greater than 85% cell viability at a concentration of 0.25 mg/mL and greater than 70% viability at the highest concentration tested (3.00 mg/mL). In Vivo Biodistribution Studies in Nontumored Mice. After determining that cells were viable in the presence of the dendronized polymers, in vivo experiments were performed to determine their time-dependent biodistribution profiles in mice. To track the polymers in vivo, a small fraction of the peripheral hydroxyl groups of each dendronized polymer were statistically converted to tyramine carbamates, and the polymers were then radiolabeled with 125I as previously described (8, 26). Polymer solutions were administered intravenously to 6-8 week-old CD-1 female mice, and their tissue distribution profiles were monitored over time. Polymer 1 showed little tissue-specific accumulation as a function of time (Figures 3a,b). The kidney was the only organ having elevated polymer levels relative to other den-

RESULTS AND DISCUSSION

Polymer Synthesis and Characterization. Three dendronized polymers were prepared starting with poly(4-hydroxystyrene) having different backbone lengths (5, 17, and 130 kDa) and low polydispersity indices (PDIs < 1.3). Dendronization was carried out using the previously reported divergent route up to the fourth-generation

Figure 2. Toxicity of polymers 1-3 toward MDA-MB-231 cells.

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Lee et al.

Figure 3. Biodistribution of (a-b) 1, (c-d) 2, and (e-f) 3 in CD-1 mice, plotted as % injected dose per gram of tissue versus time and % injected dose per organ versus time. The values at 10 min in (a) and the values at 30 min in (e) are averages for two mice, and the error bars therefore represent the range of two values. In b, d, and f, error bars have been omitted from the head, torso, tail, and blood values because the volume/mass recovered from these tissues can vary widely from mouse to mouse. Error bars have been omitted from the feces and urine values because the respective excrements for each time point were not collected individually but were pooled together.

dronized polymers studied (% injected dose/g (%ID/g) > 10%). This kidney accumulation was accompanied by substantial renal clearance, with 19% and 22% of the injected radioactivity excreted into the urine after 24 and 48 h. The small increase in the amount of radioactivity present in the urine 48 h postinjection when compared to the amount present 24 h postinjection indicates that the majority of the polymer was excreted within the initial 24 h. After determining that the data was best described by a two-compartment distribution model, the elimination half-life of 1 was calculated to be 14 h (Table 2). A notable feature of this experiment is the lack of significant liver accumulation. Previous biodistribution experiments in which the radiolabel was placed at the periphery of a PEO star polymer suffered from rapid accumulation in the liver, a finding that was attributed to the interaction of exposed iodophenols with complementary receptors (26). In the present case, however, the radiolabeled phenols are likely shielded within the dense branches of the dendronized structure. It is also possible

that a small number of defects (lack of dendron growth) along the poly(4-hydroxystyrene) backbone lead to iodination of the highly encapsulated phenolic backbone itself. As shown in Figures 3c,d, polymer 2 showed no unusual tissue accumulation. Compared to polymer 1, the amount of radioactivity lost in the urine was significantly lower for polymer 2, with only 6% of the initial dose present in the urine after 48 h. Consequently, the lack of renal clearance resulted in increased blood concentrations of radioactivity for 2 throughout the experiment, with an elimination half-life of 19 h, and in total only 10% of the initial dose was excreted in the urine and feces combined after 48 h. The biodistribution profile of the largest evaluated dendronized polymer 3 differed significantly from that of the previous two polymers (Figures 3e,f). Like 2, polymer 3 was cleared very slowly from the body, with only 6.5% excreted after 48 h (urine + feces). The elimination half-life for this high MW polymer was 44 h. Most striking was the steady increase in polymer concentra-

Biological Evaluation of Dendronized Polymers

tions in the liver and spleen with time. In these organs, maximum polymer concentrations of 21% ID/g in the liver and 30% ID/g in the spleen were reached 48 h after injection. In comparison, mice given polymers 1 or 2 had 3% or 5%, respectively, in the spleen, and 8% or 9%, respectively, in the liver. Previous studies have shown that as polymer MW increases PEO is taken up by Kupffer cells (28) and poly(vinylpyrrolidone) is taken up by macrophages (29) to a greater extent, and a high level of uptake in reticuloendothelial cell-rich organs is not uncommon for large particles such as liposomes and nano/microparticles (30). In the case of particles, uptake is believed to be caused either by filtration in the spleen if their diameters are greater than 200 nm, or by opsonization (absorption of phagocytosis-stimulating proteins) (31). If we consider that the size of 3 measured by DLS is ∼16 nm, it seems unlikely that splenic filtration is occurring, unless polymer aggregation occurs in vivo. However, filtration should not be ruled out, as this polymer is expected to be much less flexible than a nondendronized polymer of an equivalent hydrodynamic size, and its end-to-end dimensions might not be accurately represented in DLS measurements (25). While opsonization is another possibility, it would be surprising, as this behavior was not observed for the two other polymers that have the same surface functionality. Further examination will be necessary to deduce the source of the high reticuloendothelial system accumulation of 3, and to determine whether this uptake is caused by the molecular size, shape, surface chemistry, or some combination of the three. It is important to note that in the urine, SEC analysis revealed that for mice given polymers 1, 2, and 3, 99%, 85%, and 76% of the total excreted radioactivity was associated with high MW species (Figure 4a), respectively, while in the feces, greater than 90% of watersoluble radioactivity was attributable to high MW compounds (Figure 4b). We were unable to assess the high/ low MW nature of the radioactivity that remained insoluble in the feces by this method; however, we were able to estimate the amount of radioactivity that was made soluble as ∼100%, ∼60%, and ∼20% from the feces of mice given polymers 1, 2, and 3, respectively. Since the water solubilities of the dendronized polymers decrease as the polymer chain length increases, and because we would expect small molecule radioactive components to have good water solubility, we believe that the low solubility of radioactivity found in the feces of mice treated with larger polymers indicates that the insoluble radioactivity is associated with high MW material. The small quantity of low MW species excreted by the mice does not amount to more than a few percent of the total injected dose, and therefore we are confident that our data reflects the biodistribution behavior of the polymers and not that of free iodine. Overall, the results indicate that polymer 1 was small enough to pass through pores of the renal membrane, while polymers 2 and 3 were too large to undergo this process (32). Therefore, the apparent MW cutoff for glomerular filtration is between 67 and 251 kDa (Mn) for these fourth-generation dendronized polymers. If the hydrodynamic sizes of these polymers relative to PEO (as measured by SEC, Table 1) are considered, their blood clearance rates are consistent with the reported nominal MW cutoff for PEO of 30-40 kDa (33). In Vivo Biodistribution Studies in Tumored Mice. Recently, Uzgiris has speculated that due to enhanced reptation, linear polymers with extended backbone conformations may be able to more easily traverse the porous

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Figure 4. (a) Size exclusion chromatography of urine collected from mice 24 h postinjection. Fraction no. 11 is actually the sum of fractions 11-20. (b) Size exclusion chromatography of the soluble materials from homogenized feces collected from mice 24 h postinjection. For polymers 1 and 2, fraction no. 11 is actually the sum of fractions 11-20. For polymer 3, fraction no. 10 is actually the sum of fractions 10-19.

capillary walls found in tumor tissues than would a linear polymer in a predominantly coiled conformation (19, 20). Since the dendronized linear polymers presently under study have been shown to have extended conformations in solution (25), preliminary biodistribution experiments were performed in BALB/c mice bearing subcutaneous C26 tumors at a single 24 h time point to determine their propensity for tumor accumulation. Polymers 1, 2, and 3 were found to be present in the excised tumors at concentrations of 6%, 18%, and 14% ID/g, respectively (Figure 5). The significantly lower concentration of 1 found in the tumor is likely a consequence of its shorter blood circulation half-life. In contrast, 2 and 3 have increased access to the tumor vasculature due to their long-circulating nature and thus have sufficient time for the EPR effect to take place (34), resulting in higher concentrations of polymer in the tumors. Interestingly, the polymer concentration in the tumor for 3 was not statistically different from that of 2, even though it is 7-fold larger in mass and has a 2-fold longer blood elimination half-life. The levels of tumor accumulation found for 2 and 3 are comparable with those of drug carriers based on liposomes (35) and are among the highest published values for this tumor model. At present we are unable to determine whether the conformations of the polymers have any effect on their ability to access the interstitial space of the tumor due to a lack of relevant model polymers. Studies seeking to answer this question are underway. Conclusions. In conclusion, we have shown that highly functionalizable, nontoxic, dendronized polymers represent a promising new scaffold for polymeric drug delivery systems. As with linear polymers, a positive correlation was observed between the size of the dendronized polymer and its blood circulation time. The long-

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Figure 5. Biodistribution of 1, 2, and 3 in BALB/c mice bearing subcutaneous C26 tumors, plotted as (a) % injected dose per gram of tissue versus time and (b) % injected dose per organ versus time. Error bars have been omitted from the head, torso, tail, and blood values because the volume/mass recovered from these tissues can vary widely from mouse to mouse. Error bars have been omitted from the feces and urine values because the respective excrements for each time point were not collected individually but were pooled together. In a, the mean concentrations of radioactivity in the tumor were significantly different between 1 and 2 and 1 and 3 (P < 0.05) but not 2 and 3 (P ) 0.16).

circulating nature of these high MW polymers is advantageous for their development as drug carriers since it has been shown that enhanced tumor accumulation is observed for polymers with long circulation half-lives, a feature that was successfully demonstrated in our preliminary biodistribution experiments. Therefore, future work will focus on the application of these novel polymers as carriers of anticancer drugs. In addition, as total body clearance of the higher MW polymers was low (e10%), future studies will utilize polymers with degradable backbones to prevent long-term accumulation (36). ACKNOWLEDGMENT

Financial support of this research by the National Institutes of Health (GM 65361 and EB 002047) is acknowledged with thanks. Fellowship support for M.Y. from the Japan Society for the Promotion of Science (JSPS) is gratefully acknowledged. We thank De Li for assistance with size exclusion chromatography studies, and we also thank Ann Fischer at the University of California Tissue Culture Facility for assistance with cell studies. LITERATURE CITED (1) Duncan, R. (2003) The dawning era of polymer therapeutics. Nat. Rev. Drug Discovery 2, 347-360. (2) Maeda, H., Seymour, L. W., and Miyamoto, Y. (1992) Conjugates of anticancer agents and polymers: Advantages

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