In Vitro and in Vivo Evaluation of Hydrophilic Dendronized Linear Polymers

In Vitro and in Vivo Evaluation of Hydrophilic Dendronized LinearPolymers

Cameron C. Lee,† Masaru Yoshida,†,§ Jean M. J. Frechet,† 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 fourth-generation polyester dendrons were evaluated in vitro and in vivo to determine their suitability asdrug delivery vectors. Cytotoxicity assays indicated that the polymers were well tolerated by cells invitro. Biodistribution studies of the polymers in both nontumored and tumored mice revealed that asfor random coil linear polymers, renal clearance was a function of polymer size, with significant urinaryexcretion observed for a 67 kDa dendronized polymer. High accumulation in organs of thereticuloendothelial 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 ) 251kDa. The rank order for tumor accumulation of the polymers on a percent injected dose per gramtumor basis was 251 kDa ∼ 1740 kDa > 67 kDa. These data will help guide the selection of highlyfunctionalizable rigid-rod dendronized polymers with pharmacokinetic properties appropriate for useas drug carriers.

INTRODUCTION

Polymers, both natural and synthetic in origin, haveattracted attention as drug delivery vehicles due to theirability to favorably alter the biological properties ofattached therapeutic agents (1). For example, polymer-drug conjugates may have increased blood circulationhalf-lives, reduced toxicities, and increased solubilitiesrelative to the parent drugs (2). In addition, high molec-ular weight (MW) polymers can exhibit enhanced ac-cumulation in tumor tissues relative to normal tissues.This phenomenon is termed the enhanced permeationand retention effect (EPR effect), and its occurrence isattributed to the “leaky” blood vessels and poorly devel-oped lymphatic drainage system present within tumortissues (3).

To date, the carriers that have been most extensivelyinvestigated for drug delivery applications are linearpolymers such as poly(ethylene oxide) (PEO) (4) and poly-(N-(2-hydroxypropylmethacrylamide)) (HPMA) (5, 6).However, increasing attention is being given to new, morehighly branched structures such as dendrimers and starpolymers with the hope that interesting effects of archi-tecture on biological properties may be discovered (7-14). Dendronized linear polymers, a recent addition tothe branched polymer family, are linear polymers thatbear dendrons at each repeat unit along their backbones(Figure 1). It is believed that as the sizes of the pendantdendrons increase, so do the interactions between adja-

cent dendrons, and at higher generations these macro-molecules attain extended conformations and can bedescribed as somewhat rigid, cylindrical rods (15). Den-dronized polymers may be interesting scaffolds for drugdelivery as the large number of functionalizable periph-eral groups on the dendrons should allow for very highlevels of drug loading, and furthermore it has beensuggested that the shape and multivalency of a macro-molecule can influence its biological properties (16-20).However, in contrast to dendrimers, few biological studiesof dendronized linear polymers have been reported todate (21-23). Here we describe the cytotoxicity, biodis-tribution, and pharmacokinetics of synthetic, water-soluble, rigid-rod dendronized linear polymers consisting

* To whom correspondence should be addressed: School ofPharmacy S-926, University of California, San Francisco, CA94143-0446. Tel (415) 476-3895, Fax (415) 476-0688, [email protected].

† University of California, Berkeley.‡ University of California, San Francisco.§ Permanent address: Nanotechnology Research Institute,

National Institute of Advanced Industrial Science and Technol-ogy (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan.

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

535Bioconjugate Chem. 2005, 16, 535−541

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

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

MATERIALS AND METHODSMaterials. Unless otherwise noted, reagents and

solvents were obtained from commercial suppliers andused without further purification. Polymers 1-3 wereprepared as previously reported (24, 25).

Characterization. Analytical size exclusion chroma-tography (SEC) in N,N-dimethylformamide (DMF) with0.2% LiBr was performed at 70 °C at a nominal flow rateof 1.0 mL/min on a chromatography line calibrated withlinear poly(ethylene oxide) (PEO) standards (6450-529500 Da) and fitted with two 7.5 × 300 mm PLgelmixed-bed C columns (5-µm particle size). The SECsystem used when determining PEO-equivalent MWsconsists of a Waters 510 pump, a Waters U6K injector,and a Waters 410 differential refractive index detectorthermostated at 35 °C. The SEC system for determiningabsolute MWs consists of a Waters 510 pump, a 7125Reodyne injector, a Wyatt DAWN-EOS multi-angle laserlight scattering detector (laser of λ ) 690 nm), and aWyatt Optilab differential refractive index detector. Lightscattering data were analyzed using Astra software fromWyatt, and SEC data using the differential refractiveindex detector were analyzed using Millennium softwarefrom Waters.

Volume-average particle diameters were determinedusing dynamic light scattering. Experiments were per-formed at least three times at 25 °C in saline solutionwith a Zetasizer Nano ZS (Malvern Instruments) equippedwith a 4 mW He-Ne laser at 633 nm.

Radioactivity from 125I was quantified with a 1480Wallac Wizard 3 Automatic Gamma Counter. Countsemitted from samples contained in screw-cap scintillationvials were measured over the course of one minute andwere recorded as counts per minute (cpm).

Cytotoxicity of 1-3. Cell toxicity studies were per-formed at the UC Berkeley Tissue Culture Facility. MDA-MB-231 human breast cancer cells were seeded in 96-well plates at a density of 1.6 × 104 cells per well in 100µL of DMEM with 10% FBS (DMEM + FBS). Afterincubation overnight (37 °C, 5% CO2), the medium wasremoved by aspiration and replaced with fresh medium(100 µL) containing 0-3.0 mg/mL of polymers 1-3. Thesesolutions also contained antibiotics (1% penicillin-strep-tomycin). (Each of the polymers used in these experi-ments was purified prior to use by size exclusion chro-matography on a PD-10 column, followed by lyophiliza-tion.) Following incubation for 48 h, the medium wasaspirated off and replaced with 100 µL of fresh mediumand 20 µL of a 5 mg/mL MTT solution. The cells wereincubated for 4 h, after which time the medium wasremoved, leaving behind purple crystals. These crystalswere dissolved in 200 µL of DMSO and 25 µL of glycinebuffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The absor-bance values at 520 nm were measured using a Spectra-MAX 190 microplate reader. (Molecular Devices). Thepercent viability of cells in treated wells relative tonontreated cells was calculated and represents the aver-age value over four wells (Figure 2).

Synthesis of Tyramine-Functionalized Dendro-nized Polymers 1-3. 1, 2, or 3 was dissolved in 1 mLof anhydrous pyridine and reacted overnight with asolution containing 5-7 mol % (relative to hydroxyls) of4-nitrophenyl chloroformate in 1 mL of CH2Cl2. The

solvent was evaporated, and the polymer was dissolvedin 2 mL of anhydrous N,N-dimethylformamide. An excessof 4-(2-aminoethyl)phenol (tyramine) and 1 mol equiv ofanhydrous triethylamine was added to the solution andwas stirred overnight. The polymers were purified of lowMW contaminants by dialysis in Spectra/Por regeneratedcellulose membranes (MWCO ) 8000, Spectrum Labo-ratories) against distilled water. The presence of tyraminegroups was confirmed by 1H NMR analysis of a lyophi-lized aliquot of the solution retained in the dialysismembrane. The aromatic protons appear at 6.6 and 6.9ppm, and the benzylic protons appear at 2.6 ppm.

Radioiodination of Tyramine-Functionalized Poly-mers 1-3. Tyramine functionalized 1, 2, or 3 wereiodinated as previously described (8, 26), resulting insolutions of radiolabeled polymer in HBS (10 mM HEPES/Cl, 140 mM NaCl, pH 7.4). Non-polymer-bound 125I wasremoved by ion exchange chromatography on Bio-Rad AG1-X-2 resin (chloride form), and polymers were separatedfrom residual low MW radioactive contaminants by sizeexclusion chromatography on Bio-Rad 10DG desaltingcolumns that had been equilibrated with HBS. Theinitial, high-MW fractions were collected and pooled. Thespecific activities of the solutions for the biodistributionexperiments in nontumored mice were measured to be23, 3, and 35 µCi/mL for polymers 1, 2, and 3, respec-tively, and for the biodistribution experiments in tumoredmice the activities were 15, 26, and 16 µCi/mL, respec-tively. The final concentration of polymer in the solutionswas 0.8 mg/mL.

Biodistribution of 1-3 in Nontumored Mice.Polymer solutions (200 µL) were administered intrave-nously to 6-8 week-old CD-1 female mice (three mice perexperimental group). The mice were sacrificed at sixdifferent times following injection for biodistributionanalyses: 10, 30, 90, 540, 1440, and 2880 min postinjec-tion. The blood (collected by heart puncture), heart, lungs,liver, stomach, spleen, intestines, kidney, and carcass(divided into three portions) were weighed, and theamount of radioactivity present in each organ wasquantified. For the 24- and 48-h time points, mice werehoused in metabolic cages to allow for the collection ofurine and feces. The amount of radioactivity recoveredwas on average 86 ( 9% of that injected; we attributethe nonquantitative recoveries to injection variability.The data were corrected for radioactive decay and plottedas % of injected dose per gram of organ versus time andas % of injected dose per organ versus time for each organ(Figure 3).

The % of the injected dose per gram (% ID/g) of bloodversus time curve was analyzed using a two-compart-ment model, since ln[% ID/g] versus time curves clearlydisplayed two different rates of decay for early and latetime points. The pharmacokinetic parameters for the two-compartment model were estimated using the residualsmethod (27). A complete list of pharmacokinetic param-eters is presented in Table 2.

For urine analysis at 24 h, 1.0 mL of urine was loadedonto a PD-10 column and eluted in 1.0 mL fractions to

Table 1. Molecular Weights, Sizes, and PharmacokineticParameters for 1-3 Measured in Mice.

SEC-MALLSa (kDa) SECb (kDa) DLS

Mn Mw Mn Mw PDI Dc (nm)

1 67.0 69.0 13.7 16.0 1.16 5.82 251 260 38.2 45.2 1.18 10.03 1,740 1,890 190.0 242.0 1.27 15.7

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

536 Bioconjugate Chem., Vol. 16, No. 3, 2005 Lee et al.

separate high MW components from lower ones by sizeexclusion chromatography. Radioactivity was found toelute from the column in two major fractions. The firstfractions (1-6) contained high MW species, and thesecond fractions (7 and above) contained low MW species.The recovery of radioactivity from the columns wasquantitative. The percent of the recovered radioactivityfound in the high MW fractions for mice given polymers1, 2, and 3, were 99%, 85%, and 76%, respectively.

For feces analysis at 24 h, ∼400 mg of feces wereweighed into a tube containing zirconia beads and 1.0mL of HBS. The tube was capped, and the feces werehomogenized using a Bead Beater (Biospec) for 200 s at5000 rpm. The solids were centrifuged out, and 50-250µL of the supernatant was removed, loaded onto a PD-10 column, and eluted in 1.0 mL fractions in the samemanner as for the urine analysis. The recovery ofradioactivity from the column was quantitative. Again,the first fractions (1-8) contained high MW species, andthe second fractions (9 and above) contained low MWspecies. The percent of the recovered radioactivity foundin the high MW fractions in the feces for mice givenpolymers 1, 2, and 3, was 97%, 91%, and 95%, respec-tively.

Biodistribution of 1-3 in Tumored Mice. Female8-week old BALB/c mice were inoculated with C26 tumorcells via subcutaneous injection in the right hind flankon day zero (1 × 106 cells in 50 µL of cell media). On thetwelfth day postinoculation, when the tumors were ∼5mm in diameter, polymer solutions (200 µL) were ad-ministered intravenously to the mice (three mice perexperimental group). The mice were housed in metaboliccages to allow for the collection of urine and feces, andat 24 h postinjection the mice were sacrificed and theblood (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 tissuetype was quantified. The amount of radioactivity recov-ered was on average 80 ( 9% of that injected; we attrib-ute the nonquantitative recoveries to injection variability.The data were corrected for radioactive decay and plottedas % of injected dose per gram of organ and as % ofinjected dose per organ for each organ (Figure 5).

RESULTS AND DISCUSSIONPolymer 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 previouslyreported divergent route up to the fourth-generation

polyester dendron in each case (Figure 1) (24, 25). The ab-solute and relative MWs of each polymer, as measured bysize exclusion chromatography (SEC), are given in Table1. The MWs measured by SEC relative to PEO calibra-tion standards are much lower than their absolute MWsmeasured using an online multiangle laser light scatter-ing (MALLS) detector; PEO-equivalent MWs are reportedhere because they provide an estimate of the hydrody-namic sizes of the dendronized polymers. The dimensionsof the polymers were also measured using dynamic lightscattering (DLS) and are presented in Table 1.

In Vitro Cytotoxicity Studies. To determine thebiocompatibility of the dendronized polymers, toxicityexperiments were performed in vitro with MDA-MB-231human breast cancer cells. After a 48 h incubation period,cell viability was evaluated using the MTT assay (Figure2). Over the concentration range investigated, the poly-mers did not exhibit high levels of toxicity, with greaterthan 85% cell viability at a concentration of 0.25 mg/mLand greater than 70% viability at the highest concentra-tion tested (3.00 mg/mL).

In Vivo Biodistribution Studies in NontumoredMice. After determining that cells were viable in thepresence of the dendronized polymers, in vivo experi-ments were performed to determine their time-dependentbiodistribution profiles in mice. To track the polymersin vivo, a small fraction of the peripheral hydroxyl groupsof each dendronized polymer were statistically convertedto tyramine carbamates, and the polymers were thenradiolabeled with 125I as previously described (8, 26).

Polymer solutions were administered intravenously to6-8 week-old CD-1 female mice, and their tissue distri-bution profiles were monitored over time. Polymer 1showed little tissue-specific accumulation as a functionof time (Figures 3a,b). The kidney was the only organhaving elevated polymer levels relative to other den-

Table 2. Pharmacokinetic Parameters for 1-3a

1 2 3

t1/2,â (h) 14 ( 2 19 ( 2 44 ( 9t1/2,R (h) 0.9 ( 0.2 1.3 ( 0.9 1.1 ( 0.6k21 (min-1) 0.0040 ( 0.0003 0.0054 ( 0.0005 0.0055 ( 0.0007k12 (min-1) 0.007 ( 0.002 0.003 ( 0.006 0.004 ( 0.006kel (min-1) 0.0025 ( 0.0006 0.0010 ( 0.0007 0.0005 ( 0.0003Co (% ID/g) 40 ( 3 41 ( 4 38 ( 5V1 (g blood) 2.5 ( 0.2 2.5 ( 0.2 2.6 ( 0.3AUC0-∞(% ID‚min‚g-1)

16000 ( 3000 40000 ( 4000 80000 ( 20000

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

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

Biological Evaluation of Dendronized Polymers Bioconjugate Chem., Vol. 16, No. 3, 2005 537

dronized polymers studied (% injected dose/g (%ID/g) >10%). This kidney accumulation was accompanied by sub-stantial renal clearance, with 19% and 22% of the injectedradioactivity excreted into the urine after 24 and 48 h.The small increase in the amount of radioactivity presentin the urine 48 h postinjection when compared to theamount present 24 h postinjection indicates that themajority of the polymer was excreted within the initial24 h. After determining that the data was best describedby a two-compartment distribution model, the elimina-tion half-life of 1 was calculated to be 14 h (Table 2).

A notable feature of this experiment is the lack of sig-nificant liver accumulation. Previous biodistribution ex-periments in which the radiolabel was placed at the peri-phery of a PEO star polymer suffered from rapid accu-mulation in the liver, a finding that was attributed tothe interaction of exposed iodophenols with complemen-tary receptors (26). In the present case, however, the ra-diolabeled phenols are likely shielded within the densebranches 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 iodina-tion of the highly encapsulated phenolic backbone itself.

As shown in Figures 3c,d, polymer 2 showed no un-usual tissue accumulation. Compared to polymer 1, theamount of radioactivity lost in the urine was significantlylower for polymer 2, with only 6% of the initial dose pre-sent in the urine after 48 h. Consequently, the lack ofrenal clearance resulted in increased blood concen-trations of radioactivity for 2 throughout the experiment,with an elimination half-life of 19 h, and in total only10% of the initial dose was excreted in the urine and fecescombined after 48 h.

The biodistribution profile of the largest evaluateddendronized polymer 3 differed significantly from thatof the previous two polymers (Figures 3e,f). Like 2,polymer 3 was cleared very slowly from the body, withonly 6.5% excreted after 48 h (urine + feces). The elim-ination half-life for this high MW polymer was 44 h. Moststriking was the steady increase in polymer concentra-

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 timeand % 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, andthe error bars therefore represent the range of two values. In b, d, and f, error bars have been omitted from the head, torso, tail, andblood values because the volume/mass recovered from these tissues can vary widely from mouse to mouse. Error bars have beenomitted from the feces and urine values because the respective excrements for each time point were not collected individually butwere pooled together.


tions in the liver and spleen with time. In these organs,maximum polymer concentrations of 21% ID/g in the liverand 30% ID/g in the spleen were reached 48 h after injec-tion. In comparison, mice given polymers 1 or 2 had 3%or 5%, respectively, in the spleen, and 8% or 9%, respec-tively, in the liver. Previous studies have shown that aspolymer MW increases PEO is taken up by Kupffer cells(28) and poly(vinylpyrrolidone) is taken up by macroph-ages (29) to a greater extent, and a high level of uptakein reticuloendothelial cell-rich organs is not uncommonfor large particles such as liposomes and nano/micropar-ticles (30).

In the case of particles, uptake is believed to be causedeither by filtration in the spleen if their diameters aregreater than 200 nm, or by opsonization (absorption ofphagocytosis-stimulating proteins) (31). If we considerthat the size of 3 measured by DLS is ∼16 nm, it seemsunlikely that splenic filtration is occurring, unless poly-mer aggregation occurs in vivo. However, filtrationshould not be ruled out, as this polymer is expected tobe much less flexible than a nondendronized polymer ofan equivalent hydrodynamic size, and its end-to-enddimensions might not be accurately represented in DLSmeasurements (25). While opsonization is another pos-sibility, it would be surprising, as this behavior was notobserved for the two other polymers that have the samesurface functionality. Further examination will be neces-sary to deduce the source of the high reticuloendothelialsystem accumulation of 3, and to determine whether thisuptake is caused by the molecular size, shape, surfacechemistry, or some combination of the three.

It is important to note that in the urine, SEC analysisrevealed that for mice given polymers 1, 2, and 3, 99%,85%, and 76% of the total excreted radioactivity wasassociated with high MW species (Figure 4a), respec-tively, while in the feces, greater than 90% of water-soluble radioactivity was attributable to high MW com-pounds (Figure 4b). We were unable to assess the high/low MW nature of the radioactivity that remainedinsoluble in the feces by this method; however, we wereable to estimate the amount of radioactivity that wasmade soluble as ∼100%, ∼60%, and ∼20% from the fecesof mice given polymers 1, 2, and 3, respectively. Sincethe water solubilities of the dendronized polymers de-crease as the polymer chain length increases, and be-cause we would expect small molecule radioactive com-ponents to have good water solubility, we believe thatthe low solubility of radioactivity found in the feces ofmice treated with larger polymers indicates that theinsoluble radioactivity is associated with high MW mate-rial. The small quantity of low MW species excreted bythe mice does not amount to more than a few percent ofthe total injected dose, and therefore we are confidentthat our data reflects the biodistribution behavior of thepolymers and not that of free iodine.

Overall, the results indicate that polymer 1 was smallenough to pass through pores of the renal membrane,while polymers 2 and 3 were too large to undergo thisprocess (32). Therefore, the apparent MW cutoff forglomerular filtration is between 67 and 251 kDa (Mn) forthese fourth-generation dendronized polymers. If thehydrodynamic sizes of these polymers relative to PEO(as measured by SEC, Table 1) are considered, their bloodclearance rates are consistent with the reported nominalMW cutoff for PEO of 30-40 kDa (33).

In Vivo Biodistribution Studies in Tumored Mice.Recently, Uzgiris has speculated that due to enhancedreptation, linear polymers with extended backbone con-formations may be able to more easily traverse the porous

capillary walls found in tumor tissues than would a linearpolymer in a predominantly coiled conformation (19, 20).Since the dendronized linear polymers presently understudy have been shown to have extended conformationsin solution (25), preliminary biodistribution experimentswere performed in BALB/c mice bearing subcutaneousC26 tumors at a single 24 h time point to determine theirpropensity for tumor accumulation. Polymers 1, 2, and3 were found to be present in the excised tumors atconcentrations of 6%, 18%, and 14% ID/g, respectively(Figure 5). The significantly lower concentration of 1found in the tumor is likely a consequence of its shorterblood circulation half-life. In contrast, 2 and 3 haveincreased access to the tumor vasculature due to theirlong-circulating nature and thus have sufficient time forthe EPR effect to take place (34), resulting in higherconcentrations of polymer in the tumors. Interestingly,the polymer concentration in the tumor for 3 was notstatistically different from that of 2, even though it is7-fold larger in mass and has a 2-fold longer bloodelimination half-life. The levels of tumor accumulationfound for 2 and 3 are comparable with those of drugcarriers based on liposomes (35) and are among thehighest published values for this tumor model. At presentwe are unable to determine whether the conformationsof the polymers have any effect on their ability to accessthe interstitial space of the tumor due to a lack ofrelevant model polymers. Studies seeking to answer thisquestion are underway.

Conclusions. In conclusion, we have shown thathighly functionalizable, nontoxic, dendronized polymersrepresent a promising new scaffold for polymeric drugdelivery systems. As with linear polymers, a positivecorrelation was observed between the size of the den-dronized polymer and its blood circulation time. The long-

Figure 4. (a) Size exclusion chromatography of urine collectedfrom mice 24 h postinjection. Fraction no. 11 is actually the sumof fractions 11-20. (b) Size exclusion chromatography of thesoluble materials from homogenized feces collected from mice24 h postinjection. For polymers 1 and 2, fraction no. 11 isactually the sum of fractions 11-20. For polymer 3, fraction no.10 is actually the sum of fractions 10-19.


circulating nature of these high MW polymers is advan-tageous for their development as drug carriers since ithas been shown that enhanced tumor accumulation isobserved for polymers with long circulation half-lives, afeature that was successfully demonstrated in our pre-liminary biodistribution experiments. Therefore, futurework will focus on the application of these novel polymersas carriers of anticancer drugs. In addition, as total bodyclearance of the higher MW polymers was low (e10%),future studies will utilize polymers with degradablebackbones to prevent long-term accumulation (36).

ACKNOWLEDGMENT

Financial support of this research by the NationalInstitutes of Health (GM 65361 and EB 002047) is ac-knowledged with thanks. Fellowship support for M.Y.from the Japan Society for the Promotion of Science(JSPS) is gratefully acknowledged. We thank De Li forassistance with size exclusion chromatography studies,and we also thank Ann Fischer at the University of Cali-fornia Tissue Culture Facility for assistance with cellstudies.

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Figure 5. Biodistribution of 1, 2, and 3 in BALB/c mice bearingsubcutaneous C26 tumors, plotted as (a) % injected dose pergram of tissue versus time and (b) % injected dose per organversus time. Error bars have been omitted from the head, torso,tail, and blood values because the volume/mass recovered fromthese tissues can vary widely from mouse to mouse. Error barshave been omitted from the feces and urine values because therespective excrements for each time point were not collectedindividually but were pooled together. In a, the mean concentra-tions of radioactivity in the tumor were significantly differentbetween 1 and 2 and 1 and 3 (P < 0.05) but not 2 and 3 (P )0.16).


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In Vitro and in Vivo Evaluation of Hydrophilic Dendronized Linear Polymers