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Bioconjugate Chem. 2002, 13, 453−461
453
Polyester Dendritic Systems for Drug Delivery Applications: In
Vitro and In Vivo Evaluation
Omayra L. Padilla De Jesús,† Henrik R. Ihre,† Lucie Gagne,‡ Jean M. J. Fréchet,*,† and
Francis C. Szoka, Jr.*,‡
Department of Chemistry, University of California, Berkeley, California 94720-1460 and School of Pharmacy,
University of California, San Francisco, California 94143-0446. Received October 22, 2001
High molecular weight polymers (> 20 000 Da) have been widely used as soluble drug carriers to
improve drug targeting and therapeutic efficacy. Dendritic polymers are exceptional candidates for
the preparation of near monodisperse drug carriers due to their well-defined structure, multivalency,
and flexibility for tailored functionalization. We evaluated various dendritic architectures composed
of a polyester dendritic scaffold based on the monomer unit 2,2-bis(hydroxymethyl)propanoic acid for
their suitability as drug carriers both in vitro and in vivo. These systems are both water soluble and
nontoxic. In addition, the potent anticancer drug, doxorubicin, was covalently bound via a hydrazone
linkage to a high molecular weight 3-arm poly(ethylene oxide)-dendrimer hybrid. Drug release was
a function of pH, and the release rate was more rapid at pH < 6. The cytotoxicity of the DOX-polymer
conjugate measured on multiple cancer lines in vitro was reduced but not eliminated, indicating that
some active doxorubicin was released from the drug polymer conjugate under physiological conditions.
Furthermore, biodistribution experiments show little accumulation of the DOX-polymer conjugate
in vital organs, and the serum half-life of doxorubicin attached to an appropriate high molecular weight
polymer has been significantly increased when compared to the free drug. Thus, this new macromolecular system exhibits promising characteristics for the development of new polymeric drug carriers.
INTRODUCTION
A variety of approaches have been implemented for the
discovery of medicinal compounds, including the isolation
of naturally occurring bioactive substances and the
design and synthesis of new molecules with biological
activity. Many compounds have been identified by these
means, but the lack of selectivity of the majority of
current highly toxic drugs used for chemotherapy has
motivated the search for other means to improve their
therapeutic efficacy and increase their selectivity (1).
In the past few decades, the use of polymers as carriers
of both covalently bound (2-5) and physically entrapped
drug molecules (6-8) has been widely explored. When a
drug is linked to a polymer, it loses activity because it
cannot interact with the drug target either due to steric
constraints or the inability of the drug to reach the target
site (9). Furthermore, the drug-polymer conjugate can
no longer cross the cellular membrane by simple diffusion, but must do so by endocytosis. The resulting
endosomal compartment containing the polymeric conjugate fuses with the more acidic lysosomal compartment
(pH 4-5), whose hydrolytic enzymes then degrade the
conjugate. This mechanism allows a more selective
uptake of therapeutic agents, in contrast to small drugs
that can easily diffuse into and out of the cell (2, 10). In
certain cells, the endocytotic uptake enables drugpolymer conjugates to bypass multidrug resistance (MDR)
mediated efflux mechanisms hence to overcome drug
resistance in the cancer cells (11, 12). Furthermore, the
anatomical differences between normal and tumor tissue
allows for preferential accumulation of a polymeric drug
in neoplastic tissue. High molecular weight polymers
preferentially accumulate in solid tumor tissue due to a
combination of the leaky character of tumor blood vessels
†
‡
University of California, Berkeley.
University of California, San Francisco.
formed during neo-angiogenesis and to limited lymphatic
drainage. The combination of these two factors is responsible for the enhanced permeability and retention effect
(EPR) observed with tumor tissue, which leads to a
passive targeting of drugs to tumors (13, 14). In addition,
the larger hydrodynamic volume of polymers contributes
to the increased plasma half-life of the drug-polymer
conjugates, increasing the probability of accumulation of
the therapeutic agent in the tumor tissue by means of
the EPR effect (3, 15). Drugs have also been conjugated
to polymers to improve their water solubility properties,
to decrease their toxicity due to local accumulation of the
drug prior to reaching the target tissue, and to protect
them from possible enzymatic degradation or hydrolysis.
Polymers previously evaluated for delivery of covalently attached drugs have primarily been random coil
linear polymers such as poly(ethylene glycol) (PEG) (16),
N-(2-hydroxypropyl)methacrylate (HPMA) (17), and styrene maleic anhydride (SMA) (5). However, the synthesis
of some of these systems results in highly polydisperse
materials with a wide range of molecular weights due to
their mode of preparation (18). Molecules of different
molecular weights found in a polydisperse sample will
behave differently in the body. Therefore, either monodisperse or low-polydisperse (PDI < 1.1) polymers are
required to achieve systems displaying homogeneous and
reproducible pharmacokinetic properties. The intrinsic
properties of dendrimers, with their well-defined structures and their flexibility for tailored functionalization,
classify these molecules as good candidates for the
assembly of multivalent drug carriers (19-25).
The well-designed step-by-step preparation of dendrimers results in a monodisperse system (26-28).
Few dendritic systems have been evaluated previously,
due to their lack of availability. At present, probably the
best-known dendritic system is the polyamidoamine
(PAMAM) Starburst family of dendrimers (29). These are
10.1021/bc010103m CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/24/2002
454 Bioconjugate Chem., Vol. 13, No. 3, 2002
commercially available, but their polycationic character
must be pacified to reduce their in vivo toxicity (30-33).
In addition, their polyamidoamine linkage is resistant
to degradation in the body. Another well-known dendritic
system is based on the polyaryl ether convergent dendrimers (34). Although promising preliminary studies
were performed with the latter by coupling the drug
methotrexate (MTX) (21), these dendrimers also have a
backbone that is inherently resistant to degradation and
thus were thought to be less suitable for drug delivery
purposes than other structures incorporating hydrolyzable linkages.
Thus, it is of interest to develop and evaluate novel
water-soluble, biocompatible, dendritic systems. A polyester dendritic scaffold based on 2,2-bis(hydroxymethyl)propanoic acid has been investigated as a possible drug
carrier. Herein, the biological evaluation of various
polyester dendritic model compounds is described. The
biodistribution of a doxorubicin-polymer conjugate is
also reported, as well as drug release studies. The
synthesis and chemical characterization of the systems
evaluated are reported elsewhere (35).
MATERIALS AND METHODS
Cell Culture. The murine melanoma cell line B16F10
was obtained from the UCSF Cell Culture Facility. The
cells were cultured in prewarmed medium (MEM Eagle’s
with EBSS medium containing 10% fetal bovine serum,
1% MEM nonessential amino acids, 110 mg/L sodium
pyruvate, and 1% penicillin-streptomycin) at 37 °C in a
humidified atmosphere composed of 5% CO2. MDA-MB231 and MDA-MB-435 breast cancer cell lines were
cultured in a medium consisting of Dulbecco’s Modified
Earl’s medium (DME) H-16, containing 10% FBS and 1%
MEM nonessential amino acids. The medium for culturing the monkey kidney fibroblast CV-1 cells consisted of
DME H-21 (high glucose 4.5 g/L) containing 10% fetal
bovine serum, 1% MEM nonessential amino acids, 1%
HEPES buffer, and 1% penicillin-streptomycin. For all
of our experiments, cells were harvested from subconfluent cultures using trypsin-versene (0.05 and 0.02%
respectively) and were suspended in medium. Cell viability was determined using the trypan blue exclusion
method.
Cell Cytotoxicity Assay. The cytotoxicity of the
polymeric carriers, and of the free or conjugated doxorubicin, was determined using a target cell line and the
sulforhodamine B assay (SRB) (36). The target cells used
were murine B16F10 melanoma cells and the human
breast cancer cell lines MDA-MD-231 and MDA-MD-435.
Cells were seeded into a 96-well plate at a density of
3 × 104 cells/mL with 100 µL per well for 19 h (37 °C
and 5% CO2) before the assay. The medium of each well
was then replaced by 100 µL of antibiotic-free medium
containing various concentrations of the polymer with no
drug, drug, or conjugated doxorubicin. The tests were
conducted in replicates of four to eight for each drug dose.
The cells were incubated at 37 °C/5% CO2. Following the
incubation period, the cells were fixed for 1 h at 4 °C by
adding 25 µL of ice-cold 50% trichloroacetic acid (TCA)
to the growth medium of each well. The wells were
washed 5 times with cold water to remove the excess TCA
and then air-dried at room temperature for several
minutes. Once dry, each well received 100 µL of 0.4%
SRB in 1% acetic acid followed with a 30-min incubation
at room temperature. The SRB solution was then aspirated off the cells and the wells were quickly washed with
1% acetic acid. The wells were air-dried at room temperature for several minutes until moisture was no longer
Padilla De Jesús et al.
detected visually. The dye bound to the cells was solubilized by adding 100 µL of 10 mM Tris base, unbuffered,
per well and agitated for 5 min at room temperature. The
optical densities were obtained using an OPTIMAX
microplate reader (Molecular Devices, Sunnyvale, CA) at
a wavelength of 564 nm. OD measured on wells containing cells that did not receive the drug were considered
to represent 100% growth, and OD measured on wells
containing no cells but that received drug are considered
to represent 0% growth. The IC50 is evaluated by
comparing the OD of wells containing cells that were
exposed to at least five doses.
Maximum Tolerated Polymer Intravenous Bolus
Dose. The polymers were dissolved at 162 mg/mL in
HBS. CD-1 female mice, 6-8 week-old, were injected with
increasing volumes of polymer solution via the tail vein
and observed for 24 h postinjection. All mice were then
anesthetized with an intraperitoneal injection of ketamine cocktail (44 mg Ketamine; 2.5 mg Xylazine; 0.75
mg acepromazine in PBS per kg of body mass) and their
liver dissected and observed under a dissecting microscope for signs of toxicity.
Radiolabeling Methods. Compound I and II were
iodinated using carrier-free 125I and the chloramine T
reaction as previously described (37). The radiolabeled
polymer was separated from noncovalently attached
iodine on a Sephadex G-25 column. Compound III
containing the methoxyphenol group was not readily
radiolabeled when using the chloramines T reaction
described in ref 37 but was readily radioiodinated using
noncarrier iodine and an electrophilic protocol based upon
Krummeich et al (38). In brief, 3 mg of III was dissolved
in 0.25 mL of trifluoroacetic acid (TFA), and 0.01 mL of
an aqueous solution of 125I was added containing 1 mCi
125I. Chloramine T (0.54 mg) dissolved in 0.05 mL of TFA
was added to the mixture, and the reaction was heated
at 60 °C for 5 min. The reaction was stopped by the
addition of sodium metabisulfite, 1 mg in 0.01 mL of
water, and the TFA was evaporated under a stream of
nitrogen that was vented through a trap containing
activated carbon (Dupont-New England Nuclear, MA).
The residue was dissolved in 0.1 mL of 0.1 N NaOH
containing 100 mM sodium metabisulfite and chromatographed on a column containing 1.8 mL bed volume of
Dowex 2 anion-exchange resin. The radiolabeled polymer
was eluted in the first 4 mL using water as the mobile
phase. There was about a 20% incorporation of radioiodine into the polymer under these conditions. The
mixture was rechromatographed two additional times,
and greater than 99% of the radioactivity eluted with the
polymer fraction. The radiolabeled material eluted in the
void volume from a Sephadex 25 size exclusion column.
A suitable quantity of radiolabeled polymer was mixed
with the nonradiolabeled polymer in sterile HBS to create
a 10 mg/mL polymer concentration with a specific activity
of 4 µCi/mL for use in the biodistribution studies.
Biodistribution Assays for Compounds I, II, and
III. A known amount of radiolabeled polymer was added
to a stock solution of nonradiolabeled polymer prepared
at a concentration of 10 mg/mL in sterile HBS, and 100
µL was injected intravenously into 6-8 week-old CD-1
female mice. Each mouse was housed separately to collect
the urine and feces. At different times postinjection,
either three or four mice per group were anesthetized
with an intraperitoneal injection of a ketamine cocktail
and sacrificed. The feces and urine were collected. Each
mouse was bled by heart puncture, and the volume of
blood measured and transferred to a scintillation vial.
The circulation system was perfused with PBS via the
Polyester Dendritic Systems for Drug Delivery
right ventricle to reduce blood in the organs. All the
organs were collected separately and transferred to
preweighed scintillation vials. The carcass was sectioned
into three portions and placed into preweighted vials. The
radioactivity contained in each organ was counted on a
Beckman Gamma 8000 Spectrometer (Irvine, CA) and
expressed as counts per minute/gram of tissue. Using this
procedure we were able to account for 95 ( 5% of the
injected radioactivity.
Blood Clearance Assay for Compounds I, II, and
III. Female mice of 6-8 week-old (Charles River) were
injected with 1 mg of radiolabeled polymer in 100 µL of
HBS via the tail vein. At time 3, 10, 30, 60, and 90 min
postinjection, each mouse was anesthetized with isoflurane, and 50 µL of blood was collected from the orbital
sinus using a heparinized capillary. The level of radioactivity (cpm) found in the blood was determined using
a Beckman Gamma 8000 gamma spectrometer (Irvine,
CA) and the elimination rate computed using a loglinear regression of CPM/mL blood versus time after
injection.
Biodistribution Assay for Compound IV. A known
amount of the doxorubicin conjugate was dissolved in a
5 mM HEPES and 5% glucose solution. CD-1 females
mice at 6-8 weeks old were injected i. v. via tail with
200 µL of the conjugate solution. Four mice per group
were anesthetized with a ketamine cocktail by i. p.
injection. All mice were bled by heart puncture, and the
volume of blood was measured and transferred to a
scintillation vial. An injection of 1 mL of PBS to the right
ventricle of the heart was used to flush the circulation
system. The whole organs were dissected and their
weight was recorded. The organs were put into 2 mL
tubes containing 1 mL of acidified alcohol (90% 2-propanol/0.075 HCl) and zirconia beads, followed by homogenization (Bead Beater, Biospec, Bartlesville, OK) for 20
s at 5000 rpm. The blood was allowed to coagulate at 4
°C and then centrifuged for 10 min at 1000 rpm. The
serum (upper phase) was collected, and its volume was
recorded. An aliquot of 100 µL of serum was transferred
to a 2-mL tube containing 1 mL of acidified alcohol. The
homogenized organ samples and the sera were placed in
the refrigerator for 24 h to extract the drug. The cell
debris in the organ homogenates was removed by centrifugation at 14 000 rpm for 5 min at 4 °C. An aliquot
(400 µL) of the supernatants were transferred to tubes
containing 1.6 mL of acidified alcohol. For the quantification of doxorubicin present in the organs, drug-free
homogenates of organs and sera from untreated mice
were spiked with free doxorubicin. Various organs had
different background fluorescence levels, which required
separate calibration curves for each organ. Fluorescence
emission from the drug was measured in fluorescence
intensity units (Ex 490 nm: Em 590 nm) in a Quantech
Turner fluorometer (Barnstead/Thermolyne, Dubuque,
IO). Calibration curves were linear over two orders and
drug levels to 10 ng/g organ could be reliably measured.
Confocal Microscopy. GFP transfected and nontransfected MDA-MB-435 cells were plated on sterilized
4-well slides by the addition of 1 mL of a 1 × 105 cells/
mL solution. After incubation for 24 h, the media was
gently removed and the cells were washed once with 1
mL of PBS, followed by the addition of 0.5 mL of media
containing free doxorubicin and conjugated doxorubicin.
The control cells contained only media. The concentration
of free and conjugated doxorubicin equals the IC50 of free
doxorubicin for this cell line, as determined previously
in vitro experiments (0.62 mg/mL). Cells were incubated
at 37 °C and 5% CO2 for 1 and 24 h. After incubation the
Bioconjugate Chem., Vol. 13, No. 3, 2002 455
Figure 1. Dendritic polyester model compound I with molecular mass of 3790 Da.
cells were fixed for 1 h at 4 °C in 1.0 mL of 4% paraformaldehyde in PBS. The cells were mounted in Vector
Vectrashield (Burlingame, CA) for confocal microscopy.
Confocal images were obtained using LaserSharpe software installed on a Bio-Rad (Hercules, CA) kryptonargon laser scanning confocal microscope (MRC-1024)
outfitted with a Nikon Diaphot 200 microscope using an
×60 objective lens. 512 × 512 pixel sections were taken
every 2 µm using all laser lines, 10% laser power, slow
scan, and Kalman averaging of three scans. For both GFP
transfected and untransfected MDA-MB-5 cells, doxorubicin was imaged by PMT1 using low signal amplification, an iris of 2.5, a gain of 1500, and a black level of
-6. For the GFP transfected MDA-MB-5, GFP was
imaged on PMT2 using an iris of 2.5, a gain of 1500, and
a black level of -3. Images were analyzed using Confocal
Assistant 4.02 software.
Drug Release Studies as a Function of Buffer pH.
A stock solution of conjugate was prepared using distilled
water as a solvent. An aliquot of the stock solution was
added to the respective prewarmed buffer solutions (37
°C), to initiate the reaction. The final concentration of
the conjugate was of 3 mg/mL, 40 mM in buffer ion
and 100 mM NaCl. Solutions at pH 2.4 (HCl), 4.5
(acetate), 5.5 (acetate), 6.5 (MES), and 7.0 (phosphate)
were used to evaluate drug release as a function of pH.
Quantification of drug released was performed by HPLC
analysis on a model 2690 XE Alliance Separations
module (Waters, USA) and a Photodiode Array detector
(Waters 996). Aliquots of 10 µL were injected with builtin autoinjector at different times after the incubation
started. The separation was carried out with a Waters
Symmetry C18 column (2.1 × 150 mm, 3.5 µm) using a
mobile phase consisting of 40% phosphate buffer pH 7.0
and 60% methanol at a flow rate of 0.3 mL/min. The data
acquisition and processing were controlled by Millenium
32 software. In contrast to commonly used mobile phases
for the separation of DOX conjugates from the free DOX,
we used a neutral pH system, to minimize cleavage of
DOX from the polymer during the analysis. A calibration
curve was prepared with fresh doxorubicin and the extent
of release was monitored by peak height based on the
absorbance at 490 nm.
RESULTS AND DISCUSSION
Brief Description of Structure of Model Compounds Evaluated. Model compounds I, II, and III
were evaluated as possible polymeric drug carriers
(Figures 1-3). The common denominator of these systems is the polyester dendritic scaffold derived from the
monomer unit 2,2-bis(hydroxymethyl)propanoic acid. These
456 Bioconjugate Chem., Vol. 13, No. 3, 2002
Padilla De Jesús et al.
Figure 2. Modified dendritic polyester model compound II with molecular mass of 11 500 Da.
Figure 3. Model compound III, composed of 3-arm poly(ethylene oxide) star and [G-2] polyester dendrons (23 500 Da).
three polymers differ in molecular weight and architecture, so we could evaluate these factors on toxicity and
biodistribution. Compounds I and II mainly consist of two
fourth generation [G-4] polyester dendrons attached to
a trisphenolic core. Carrier I has a molecular mass of
3790 Da and contains a fully activated surface with 32
hydroxyl groups, which can be further modified. The
surface of II was modified with methoxy terminated
tri(ethylene glycol) oligomeric units to increase the mass
of the dendritic molecule by a factor of 3 in one single
synthetic step, achieving a mass of 11 500 Da. An
attractive feature of this polyester scaffold is that it is
highly water soluble (at least 200 mg/mL) in an uncharged state, avoiding the necessity of introducing
ionizable functional groups to achieve good solubility
properties. This property decreases the possibilities for
rapid elimination as is observed in the case for cationic
systems, due to electrostatic interactions with the negatively charged cellular membranes and extracellular
matrixes (30-33) or in the case of anionic systems due
to their interaction with the scavenger receptor (39).
Second, the sterically hindered ester bond of the monomer unit makes this backbone more stable toward both
nucleophilic attack and acid-catalyzed hydrolysis avoiding its rapid and premature degradation.
In contrast to I and II, compound III consists of a
hybrid between a 3-arm poly(ethylene oxide) (PEO) star
and three [G-2] polyester dendritic units. This system has
a molecular mass of ca. 23 500 Da, and its molecular
architecture has a more linear character, as compared
to the compact shape of the strictly dendritic systems I
and II. In this instance, the trifunctional PEO core
contributes most of the mass of the system. The advantages of using a star-PEO also include its contribution
to biocompatibility and water solubility (40), without
jeopardizing the polydispersity of the system, since starPEO molecules with low polydispersity indices (PDI <
1.1) are commercially available. On the other hand, the
[G-2] polyester dendrons of III provide the multivalency
necessary for drug attachment. In contrast to linear
polymers containing statistical amounts of reacting sites,
Polyester Dendritic Systems for Drug Delivery
Bioconjugate Chem., Vol. 13, No. 3, 2002 457
Figure 5. Cell viability results upon treatment of B16F10 cells
with polymeric carriers I, II, and III after 48 h of incubation.
Figure 4. Model compound IV. Polymer drug conjugate
consisting of modified III and doxorubicin using a hydrazone
covalent bond as a linker.
this architecture provides a known and reproducible
number of functional groups.
Finally, the drug-polymer conjugate IV (Figure 4)
consists of the potent anticancer drug doxorubicin (DOX)
and a derivative of the polymeric carrier III. DOX was
covalently bound to the polymer by means of an acidlabile hydrazone linkage (41-42). An acid-labile linkage
was selected because it can remain stable under physiological conditions, but once it is internalized in the cell
by endocytosis, the more acidic environment of lysosomal
compartments will trigger the release of the drug. An
additional advantage of a pH-sensitive linker is that the
release rate of the drug attached by an acid-labile linkage
is often less affected by the type of drug than in the case
of enzyme cleavable linkages, which have been difficult
to address (43). Finally, the environment around tumor
tissue is slightly more acidic than normal tissue; therefore, an acid labile linkage might be cleaved more rapidly
in the vicinity of a tumor prior being internalized by the
cancer cell.
Biological Evaluation of Model Compounds. In
Vitro Cytotoxicity of the Carriers. In vitro studies were
performed on the polymeric model compounds I, II, and
III to evaluate the possible toxic effect of the polyester
dendritic scaffold itself. The cell viability studies were
performed on melanoma B16F10 cancer cells and cell
proliferation was quantified by the sulforhodamine B
assay (SRB), which measures the level of protein remaining on a cell culture dish. All of the polymers examined
were well tolerated by cells in culture (Figure 5). For
instance, after incubation for 48 h, cells exposed to I at
a polymer concentration of 10 mg/mL retained 87% of
the growth rate of the control cells. In the case of model
compound II, cell proliferation was slightly suppressed
to 81% of control at 5 mg/mL. Model compound III, the
highest molecular weight carrier evaluated, also was very
well tolerated with 87% of the growth rate of the control
culture at 10 mg/mL of polymer (Figure 5).
An interesting observation on this polymer is that even
though there is an apparent growth inhibition at the
highest concentrations, dead cells were not evident under
microscopic observation even at 40 mg/mL, rather the cell
density was decreased. This suggests a growth inhibition
effect as a result of the very high polymer concentration.
These results are very promising, considering that previously evaluated dendritic systems, such as PAMAM, polyethyleneimine and poly(ethylene oxide) grafted carbosilanes inhibit cell viability and induce hemolysis or
cytolysis at concentrations much lower than 5 mg/mL
(31).
In Vivo Tolerability of the Dendritic Polyesters. Nonionic polymers such as the poly(ethylene glycols) are very
well tolerated upon injection into animals. Given the low
toxicity of the polyester dendrimers in culture, we
thought they would also be well tolerated upon injection
into animals. However, we did not believe it warranted
conducting a complete toxicity profile of the polymers at
this stage of the investigation; rather, we undertook to
learn if the polymers could be administered as an
intravenous bolus at levels greater than 1 g/kg body
weight in mice. If the polymers could be administered at
this dose, a wide variety of drug delivery applications
would be possible. Compound I was injected i.v. over a
10 s period into a number of animals at increasing doses.
At the dose of 1.3 g/kg body weight, 1 of the 2 animals
injected survived. In the animal that died, there was no
evidence for blood hemolysis. The surviving animal was
sacrificed at 24 h postinjection, the organs removed and
visually examined. There was no gross evidence for
toxicity in any organ examined.
Compound III was injected intravenous as a bolus into
two mice at a dose of 1.3 g/kg body weight. Both mice
458 Bioconjugate Chem., Vol. 13, No. 3, 2002
survived with no evidence for any adverse effect of the
polymer. The animals were sacrificed at 24 h, the organs
were removed and visually examined. There was no
evidence for gross pathology in the liver, lungs, heart,
kidney, or intestine.
These preliminary experiments confirmed that the
polymers were very well tolerated in mice when administered by i.v. bolus. Upon the basis of this result, we
decided to undertake biodistribution experiments at
doses of 0.1 g/kg body weight. This is comparable to what
has been used to study biodistribution of dextrans (44)
and HPMA, polymers that have been used as drug
carriers.
Biodistribution Studies. Dendrimer I was radiolabeled
with 125I, and the biodistribution of the radiolabeled
polymer was monitored in CD-1 mice after intravenous
administration. Polymer I was renally excreted and
preferential accumulation in specific organs was not
observed (Figure 6a). At 10 min postinjection, only 14%
of the injected material remained in the serum, and the
polymer was essentially completely excreted after 4 h.
The low circulation time is most likely related to the
compact shape of the branched dendritic structure and
its modest molecular size.
Biodistribution of model compound II was evaluated
and even though the mass of this system was 3 times
larger than I, the polymer was also rapidly excreted in
the urine. At 30 min postinjection, only 4% of the
administered dose remained in serum, and after 5 h the
material was essentially completely excreted from the
body with no evidence for accumulation in any tissue
(Figure 6b).
Model compound III was labeled with 125I as well, but
in contrast to I and II the radioactive label was attached
to a p-methoxyphenyl acetic acid moiety that was statistically coupled to the surface of III. This compound
exhibited a highly significant accumulation in the liver.
At 10 min postinjection, 70% of injected dose is in the
liver and 5% in serum. After 5 h, 53% of the injected dose
was still found in the liver (Figure 6c).
This distribution behavior was not expected. One major
difference between compound III and compounds I and
II is that the labeled moiety was at the periphery of the
PEO chains in III, as opposed to the well protected core
in compounds I and II. This could possibly account for
the liver uptake since it was previously observed that
iodophenol containing PEGs would expose the methoxy
iodophenol to the putative liver receptors whereas the
iodophenol would be sterically hindered in compounds I
and II (37). Another possibility is that the iodinecontaining moiety was hydrolyzed from the polymer and
incorporated into the liver rather than the model compound itself. Given the rapidity of uptake in the liver,
this is unlikely. Moreover, iodinated polymer recovered
from serum eluted in the high molecular weight fraction
on a G-50 spin column. Thus, we attributed the biodistribution profile of compound III to the exposed iodinated
phenol. Despite the uptake of III into the liver, we
decided to prepare a polymer-doxorubicin conjugate with
compound III for further biodistribution studies.
In addition to being a highly potent anticancer agent,
doxorubicin is a fluorescent compound, providing a
convenient analytical tool for monitoring distribution of
the conjugate IV. In contrast to the radiolabeled III, the
polymer-DOX conjugate IV showed no significant accumulation in any vital organ, including the liver, heart,
and lungs (Figure 7). This is a significantly different
distribution pattern than is observed for the free drug,
which partitions into a variety of organs such as the liver
Padilla De Jesús et al.
Figure 6. Biodistribution of model compounds (a) I, (b) II, and
(c) III. The data are expressed in % of dose injected per gram
of organ tissue. The blood is expressed in % of dose injected per
mL, for ca. 2 mL of total blood volume in a CD-1 mouse.
and heart (45). Most importantly, conjugate IV had
circulation half-life of 72 min, which is significantly
longer than both the half-life of the free drug (ca. 8 min)
and the carriers I and II. This demonstrates that the
polymeric form of the carrier alters the pharmacokinetics
and the distribution of the drug.
Drug Release Studies. One of the important features
to take into consideration when designing polymeric
carriers is the means of attachment of the molecule to
be delivered. The nature of the attachment sites will
determine whether selective release of the bioactive
molecule can be attained. A hydrazone linkage was
selected due to its known stability under neutral pH
conditions and its ability to undergo hydrolysis under
acidic conditions. The goal is to have a linkage that will
be sufficiently labile at a pH range of 4-6 to provide a
therapeutic level of the drug. This is the pH found in the
endosomal and lysosomal environments, which are the
Polyester Dendritic Systems for Drug Delivery
Bioconjugate Chem., Vol. 13, No. 3, 2002 459
Figure 7. Biodistribution of IV.
Figure 8. HPLC chromatogram showing increment in free
drug concentration upon release from polymer conjugate IV. The
detection was observed at 490 nm.
Figure 10. Comparative cell viability results upon treatment
of (a) B16F10, (b) MDA-MB-231, and (c) MDA-MB-435 cell lines
with bound DOX (IV) and free DOX. Results are compared in
terms of DOX equivalents.
Figure 9. pH profile for release of doxorubicin from conjugate
IV.
compartments where polymeric drugs are often internalized. The linkage we used and evaluated throughout this
study is a hydrazone linkage.
Drug release was monitored by an HPLC method.
Figure 8 shows the well-resolved peaks corresponding to
the conjugate and free doxorubicin at retention times of
5.4 and 6.3 min, respectively. As shown in Figure 9, the
hydrazone linkage is indeed very stable at neutral pH,
while it is cleaved more rapidly under more acidic
conditions. Due to the highly reversible character of the
hydrolysis of hydrazone linkages, an apparent equilibrium is observed under the experimental nonsink conditions of the release assay. One can estimate the time for
complete release of DOX from the conjugate based on the
computed initial rates of release; a 100% release would
be achieved after 10 min, 3 h, 26.5 h, and 10 days for pH
2.5, 4.5, 5.5, and 6.5, respectively. Thus, the hydrazone
linkage provides a suitable linker for a pH-dependent
release that is compatible with conditions found in
tumors.
Cytotoxicity of Drug-Polymer Conjugate in Vitro. The
cell proliferation results suggest that these systems based
on polyester dendrimers do not exhibit a significant toxic
effect and the drug release rates from the hydrazone
linker encouraged us to further evaluate model compound
IV as a polymeric drug carrier. In vitro evaluation of the
conjugate was performed in various cancer cell lines to
compare the cytotoxic activity of the bound drug in
relation to the free drug (Figure 10).
The three cell lines examined exhibited a range of
sensitivity to the free doxorubicin, from 0.025 µg/mL for
B16F10 to 0.62 µg/mL for the MDA-MB-435 cell lines.
In all three cell lines examined, the free drug was
considerably more potent than the drug-polymer conjugate (Figure 10); 6-fold in the B16F10 cells, 50-fold in
the MDA-MB-231, and 9-fold in the MDA-MB-435 cells.
In the experiments, the conjugated doxorubicin was
chromatographed in a gel permeation column within 1
day of testing the material so that there was less than
460 Bioconjugate Chem., Vol. 13, No. 3, 2002
Padilla De Jesús et al.
Figure 11. Fluorescence confocal microscopy images of localized DOX and DOX-polymer conjugate IV in MDA-MD-435 cells. MDAMD-435 cells were grown to 40% confluence on sterilized coverslips and incubated for 1 and 24 h.
1% free doxorubicin in the conjugate preparation. Moreover, the amount of polymer at the highest dose tested
(100 µg/mL) was substantially below the concentration
where antiproliferation effects of the polymer were
observed. Therefore, the antiproliferation effects of the
conjugate were most likely due to the drug that had been
released from the polymer in the course of the incubation.
These results are encouraging since the doxorubicin
conjugate is less toxic than the free drug yet more toxic
than the polymer alone. The question arises as to where
the doxorubicin is being released from the polymer. At
neutral pH in buffer, drug release is very slow. However,
at low pH drug release is substantially faster. Therefore,
we examined if the drug-polymer conjugate was internalized by cells.
MDA-MB-431 cells were treated for 1 h and 24 h with
the drug-polymer conjugate (IV) or the free doxorubicin.
The fluorescence of doxorubicin provides a means for
monitoring cell uptake of the drug using fluorescence
confocal microscopy. Both the cytoplasm and the nuclei
were highly fluorescent both at 1 h and 24 h after
exposition to free doxorubicin. When cells were exposed
to polymeric drug, fluorescence is observed in the cytosol,
confirming the cell uptake of the conjugate by endocytosis
(Figure 11). However, as also observed with the HPMA
derivative of doxorubicin (46), little fluorescence was
observed in the cell nucleus with the drug-polymer
conjugate. This may be due to the slow release of the drug
from the carrier at the time of evaluation.
CONCLUSIONS
Various polyester dendritic carriers were evaluated as
possible drug carriers. Each of the three drug carriers
(I, II, III) are highly water soluble and nontoxic. Model
compound III exhibited the longest circulatory half-life
(72 min) as compared to the lower molecular weight
systems I and II and was selected for further investigation. A doxorubicin-polymer conjugate was synthesized
using III as the polymeric carrier (IV), and the drug was
attached by means of an acid-labile hydrazone linkage.
The cytotoxicity of the drug was significantly reduced
(80-98%) after attaching it to the polymer. However,
doxorubicin could be rapidly released from the polymer
in an active form at pH values found in the lysosome.
Uptake of IV by cells in culture was also observed by
fluorescence confocal microscopy. In biodistribution experiments, none of these systems accumulated in any
organ examined, including the liver, heart, and lungs.
The results suggest that this polyester dendritic backbone
is a highly water soluble, nontoxic, and biocompatible
polymer. However, a higher molecular weight system
must be prepared to further increase the circulation halflife to effectively exploit the EPR phenomenon. This new
system is a promising polymeric backbone for use as
scaffolds in the development of well-defined macromolecular drug carriers.
ACKNOWLEDGMENT
We thank Dr. Nikolay Vladimirov and Dr. Miroslav
Janco (UCB) for their significant contribution in the
development of the HPLC method for the performance
of the drug release studies. We also thank John Andrew
MacKay (UCSF) for his valuable assistance in the experiments involving confocal microscopy. Financial support
of this research by UC Breast Cancer Research and
Development Program (6JB-0137), the National Science
Foundation (DMR-9816166), the Department of Energy
(Basic Energy Sciences), and the National Institutes of
Health (Grant GM 65361) is acknowledged with thanks.
LITERATURE CITED
(1) Langer, R. (1990) New Methods of Drug Delivery. Science
249, 1527-1533.
(2) Duncan, R., Dimitrijevic, S., and Evagorou, E. G. (1996) The
Role of Polymer Conjugates in the Diagnosis and Treatment
of Cancer. S. T. P. Pharma Sciences 6, 237-263.
(3) Maeda, H., Seymour, L. W., and Miyamoto, Y. (1992)
Conjugates of Anticancer Agents and Polymers: Advantages
of Macromolecular Therapeutics in Vivo. Bioconjugate Chem.
3, 351-362.
(4) Mehvar, Reza (2000) Dextrans for Targeted and Sustained
Delivery of Therapeutic and Imaging Agents. J. Control.
Release 69, 1-25.
(5) Matsumura, R. B., and Maeda, H. (1986) A New Concept
for Macromolecular Therapeutics in Cancer Chemotherapy:
Mechanism of Tumoritropic Accumulation of Proteins and the
Antitumor Agent SMANCS. Cancer Res. 46, 6387-6392.
(6) Ellens, H., Bentz, J., and Szoka, F. C., Jr. (1985) H+-Induced
and Ca-2+-Induced Fusion and Destabilization of Liposomes.
Biochemistry 24, 3099-3106.
(7) Watkins, D. M., Sayed-Sweet, Y., Klimash, J. W., Turro, N.
J., and Tomalia, D. A. (1997) Dendrimers with hydrophobic
Polyester Dendritic Systems for Drug Delivery
cores and the formation of supramolecular dendrimersurfactant assemblies. Langmuir 12, 3136-3141.
(8) Hawker, C. J., Wooley, K. L., and Fréchet, J. M. J. (1993)
Unimolecular Micelles and Globular Amphiphiles - Dendritic
Macromolecules as Novel Recyclable Solubilization Agents.
J. Chem. Soc., Perkin Trans. 1, 12, 1287-1297.
(9) Eliaz, R., and Szoka, F. C., Jr. (2001) Liposome Encapsulated Doxorubicin Targeted to CD44: a Strategy to Kill CD44Overexpressing Tumor Cells. Cancer Res. 61, 2592-2601.
(10) Duncan, R. (1992) Drug-polymer Conjugates: Potential for
Improved Chemotherapy. Anticancer Drugs 3, 175-210.
(11) Cuvier, C., Roblot-Treupel, L., Milliot, J. M., Lizard, G.,
Chevillards, S., Manfait, M., Couvreur, P., and Poupon, M. F.
(1992) Doxorubicin-loaded Nanospheres bypass Tumour Cell
Multidrug Resistance. Biochem. Pharmacol. 44, 509-517.
(12) Omelyanenko, V., Kopečková, P., Gentry, C., and Kopeček,
J. (1998) Targetable HPMA Copolymer-Adriamycin Conjugates: Recognition, Internalization, and Subcellular Fate. J.
Control. Release 53, 25-37.
(13) Maeda, H., Wu, J., Sawa, T., Matsumura, Y., and Hori, K.
(2000) Tumor Vascular Permeability and the EPR Effect in
Macromolecular Therapeutics: A Review. J. Control. Release
65, 271-284.
(14) Seymour, L. W. (1992) Passive Tumor Targeting of Soluble
Macromolecules and Drug Conjugates. Crit. Rev. Ther. Drug
9, 135-187.
(15) Seymour, L. W., Miyamoto, Y., Maeda, H., Brereton, M.,
Strohalm, J., Ulbrich, K., and Duncan, R. (1995) Influence of
Molecular Weight on Passive Tumour Accumulation of a
Soluble Macromolecular Drug Carrier. Eur. J. Cancer 31A,
766-770.
(16) Greenwald, R. B., Conover, C. D., and Choe, Y. H. (2000)
Poly(Ethylene Glycol) Conjugated Drugs and Prodrugs: A
Comprehensive Review. Crit. Rev. Ther. Drug 17, 101-161.
(17) Vasey, P. A., Kaye, S. B., Morrison, R., Twelves, C., Wilson,
P., Duncan, R., Thomson, A. H., Murray, L. S., Hilditch, T.
E., Murray, T., Burtles, S., Fraier, D., Frigerio, E., and
Cassidy, J. (1999) Phase I Clinical and Pharmacokinetic
Study of PK1 [N-(2-Hydroxypropyl)methacrylamide Copolymer Doxorubicin]: First member of a New Class of Chemotherapeutic Agents-Drug-Polymer Conjugates Clin. Cancer
Res. 5, 83-94.
(18) Ulbrich, K., Šubr, V., Strohalm, J., Plocavá, D., Jelı́nkova,
M., and Řı́hová, B. (2000) Polymeric Drugs Based on Conjugates of Synthetic and Natural Macromolecules: I. Synthesis
and Physicochemical Characterization. J. Control. Release 64,
63-79.
(19) Liu, M., and Fréchet, J. M. J. (1999) Designing Dendrimers
for Drug Delivery. Pharm. Sci. Technol. Today 2, 393-401.
(20) Liu, M., Kono, K., and Fréchet, J. M. J. (1999) WaterSoluble Dendrimer-Poly(ethylene glycol) Starlike Conjugates
as Potential Drug Carriers. J. Polym. Sci., Polym. Chem. 37,
3492-3502.
(21) Kono, K., Liu, M., and Fréchet, J. M. J. (1999) Design of
Dendritic Macromolecules Containing Folate or Methotrexate
Residues. Bioconjugate Chem. 10, 1115-1121.
(22) Grayson, S. M., Jayaraman, M., and Fréchet, J. M. J. (1999)
Convergent synthesis and ‘surface’ functionalization of a
dendritic analogue of poly(ethylene glycol). Chem. Commun.
14, 1329-1330.
(23) Liu, M., Kono, K., and Fréchet, J. M. J. (2000) Watersoluble dendritic unimolecular micelles: Their potential as
drug delivery agents. J. Control. Release 65, 12-131
(24) Esfand, R., and Tomalia, D. A. (2001) Poly(Amidoamine)
(PAMAM) Dendrimers: From Biomimicry to Drug Delivery and
Biomedical Applications. Drug Discovery Today 6, 427-436.
(25) Plank, C., Mechtler, K., Szoka, F. C., Jr., and Wagner, E.
(1996) Activation of the Complement System by Synthetic
DNA Complexes: A Potential Barrier for Intravenous Gene
Delivery. Hum. Gene Ther. 7, 1441-1450.
(26) Tomalia, D. A., and Durst, H. D. (1993) Genealogically
Directed Synthesis - Starburst Cascade Dendrimers and
Hyperbranched Structures. Top. Curr. Chem. 165, 193-313.
(27) Fréchet, J. M. J. (1994) Functional Polymers and Dendrimers - Reactivity, Molecular Architecture, and Interfacial
Energy. Science 263, 1710-1715.
Bioconjugate Chem., Vol. 13, No. 3, 2002 461
(28) Newkome, G. R., Moorefield, C. N., and Vögtle, F. (1996)
Dendritic Macromolecules: Concepts, Synthesis, Perspectives,
VCH: Weinheim, Germany.
(29) Tomalia, D. A., Baker, H., Dewald, J., Hall, M., Kallos, G.,
Martin, S., Roeck, J., Ryder, J., and Smith, P. (1985) A New
Class of Polymers-Starburst-Dendritic Macromolecules. Polym.
J. 17, 117-132.
(30) Haensler, J., and Szoka, F. C., Jr. (1993) Poliamidoamine
Cascade Polymers Mediate Efficient Transfection of Cells in
Culture. Bioconjugate Chem. 4, 372-379.
(31) Malik, N., Wiwattanapatapee, R., Klopsch, R., Lorenz, K.,
Frey, H., Weener, J. W., Meijer, E. W., Paulus, W., and
Duncan, R. (2000) Dendrimers: Relationship Between Structure and Biocompatibility in Vitro, and Preliminary Studies
on the Biodistribution of 125I-Labeled Polyamidoamine Dendrimers in Vivo. J. Control. Release 65, 133-148.
(32) Supattapone, S., Wille, H., Uyechi, L., Safar, J., Tremblay,
P., Szoka, F. C., Jr., Cohen, F. E., Prusiner, S. B., and Scott,
M. R. (2001) Branched Polyamines Cure Prion-Infected
Neuroblastoma Cells. J. Virol. 75, 3453-3461.
(33) Duncan, R., and Malik, N. (1996) Dendrimers: Biocompatibility and Potential for Delivery of Anticancer Agents.
Proc. Int. Symp. Controlled Release Bioact. Mater. 23, 105-106.
(34) Hawker, C. J., and Fréchet, J. M. J. (1990) Preparation of
Polymers with Controlled Molecular Architecture - A New
Convergent Approach to Dendritic Macromolecules. J. Am.
Chem. Soc. 112, 7638-7647.
(35) Ihre, H. R., Padilla De Jesús, O. L., Szoka, F. C., Jr., and
Fréchet, J. M. J. (2002) Polyester Dendritic Systems for Drug
Delivery Applications: Design, Synthesis, and Characterization. Bioconjugate Chem. 13, 443-452.
(36) Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon,
J., Vistica, D., Qarren, J. T., Bokesch, H., Kenney, S., and
Boyd, M. R. (1990) New colorimetric cytotoxicity assay for
anticancer drug screening. J. Natl. Cancer Inst. (Bethesda)
82, 1107-1112.
(37) Larwood, D. J., and Szoka, F. C., Jr. (1984) Synthesis,
Characterization, and In Vivo Disposition of Iodinatable
Polyetylene Glycol Derivatives: Differences In Vivo as a
Function of Chain Length. J. Label. Comput. Radiopharmaceut. 21, 603-614.
(38) Krummeich, C., Holschbach, M., and Stöcklin, G. (1994)
Direct nca electrophilic radioiodination of tyrosine analogues;
their in vivo stability and brain-uptake in mice. Appl. Radiat.
Isotopes 45, 929-935.
(39) Meijer, D. K., and Molema, G. (1995) Targeting of drugs
to the liver. Semin. Liver Dis. 15, 202-56.
(40) Francis, G. E., Delgado, C., Fisher, D, Malik, F., and
Agrawal, A. K. (1996) Polyethylene Glycol Modification:
Relevance of Improved Methodology to Tumour Targeting.
J. Drug Target. 3, 321-340.
(41) Kaneko, T., Willner, D., Monkovı́c, I., Knipe, J. O., Braslawsky, G. R., Greenfield, R. S., and Vyas, D. M. (1991) New
Hydrazone Derivatives of Adriamycin and Their Immunoconjugates- a Correlation between Acid Stability and Cytotoxicity. Bioconjugate Chem. 2, 133-141.
(42) Lemieux, G. A., and Bertozzi, C. R. (1998) Chemoselective
ligation reactions with proteins, oligosaccharides and cells.
TIBTECH 16, 506-511.
(43) Melton, R. G., Knox, R. J., Eds. (1999) Enzyme Prodrug
Strategies for Cancer Therapy, Kluwer Academic/Plenum
Publishers, New York.
(44) Takakura, Y., Takagai, A., Hashida, M., and Sezaki, H.
(1987) Disposition and tumor localization of mitomycin CDextran Conjugates in Mice. Pharm. Res. 4, 293-300.
(45) Krishna, R., and Mayer, L. D. (1997) Liposomal doxorubicin
circumvents PSC 833-free drug interactions, resulting in
effective therapy of multidrug-resistant solid tumors. Cancer
Res. 57, 5246-5253.
(46) Hovorka, O., Št’astný, M., Etrych, T., Šubr, V., Strohalm,
J., Ulbrich, K.; Řı́hová, B. (2002) Differences in the intracellular fate of free and polymer-bound doxorubicin. J.
Controlled Release 80, 101-117.
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