Pharmaceutical Research, Vol. 19, No. 4, April 2002 (© 2002)
Research Paper
camptothecin was also pre-clinically evaluated and is now in
Phase I trials (3).
Targeted Delivery of Doxorubicin
by HPMA Copolymer-
Hyaluronan Bioconjugates
Anti-cancer polymer-drug conjugates can be divided into
two targeting modalities: passive and active. The biologic ac-
tivity of the passive targeting is based on the anatomic char-
acteristics of tumor tissue, and allows polymer-drug conjugate
to more easily permeate tumor tissues and accumulate over
time. This is one of the principal reasons for the success of
polymeric drugs, and it is often referred to as the enhanced
permeability and retention (EPR) effect (1). Active targeting
in drug delivery systems can be achieved by exploiting specific
interactions between receptors on the cell surface and target-
ing moieties conjugated to the polymer backbone. The active
approach therefore takes advantage of the EPR effect, but
further increases the therapeutic index through receptor-
mediated uptake by target cancer cells. Previous studies
showed that N-acylated galactosamine (4) and monoclonal
antibody fragments (5) were valuable targeting moieties for
HPMA-DOX conjugates, selectively increasing the cytotox-
icity of the polymer-drug conjugates to tumor cells.
1
2
3
Yi Luo, Nicole J. Bernshaw, Zheng-Rong Lu,
3
1,2,4
Jindrich Kopecek, and Glenn D. Prestwich
Received December 13, 2001; accepted January 4, 2002
Purpose. Overexpression of hyaluronan (HA) receptors on cancer
cells results in enhanced endocytotic uptake of the drug conjugate.
An N-(2-hydroxypropyl)methacrylamide (HPMA)-HA polymeric
drug delivery system was used for targeted delivery of doxorubicin to
cancer cells.
Methods. HA-doxorubicin (DOX) bioconjugates (HA-DOX), and
HPMA copolymer-DOX conjugates containing HA as a side chain
(HPMA-HA-DOX) were synthesized. The cytotoxicity of the poly-
mer-drug conjugate was evaluated via in vitro cell culture. The inter-
Hyaluronic acid (HA), a linear polysaccharide of alter-
nalization of the conjugate was visualized by fluorescence micros- nating D-glucuronic acid (GlcUA) and N-acetyl-D-glucosa-
copy.
mine (GlcNAc) units, serves a variety of functions within
the extracellular matrix (6). These include direct receptor-
mediated effects on cell adhesion, growth and migration (7)
and as a signaling molecule in cell motility, inflammation,
wound healing, and cancer metastasis (8). These effects occur
via intracellular signaling pathways in which HA binds to, and
is internalized by, cell-surface receptors. Most malignant solid
tumors and their surrounding stromal tissue contain elevated
Results. Cytotoxicity of HPMA-HA-DOX targeted bioconjugate was
higher against human breast cancer (HBL-100), ovarian cancer
(SKOV-3), and colon cancer (HCT-116) cells when compared to the
non-targeted HPMA-DOX conjugate. Fluorescence confocal micros-
copy revealed that the targeted HPMA-HA-DOX conjugates were
internalized more efficiently by cancer cells relative to the non-
targeted HPMA-DOX conjugate. Both HPMA-DOX and HPMA-
HA-DOX showed minimal cytotoxicity toward mouse fibroblast NIH
3
T3 cells. The internalization of polymer conjugates was correlated levels of HA (9), and these high levels of HA production
with their cytotoxicity.
provide a matrix that facilitates invasion (10). In addition to
Conclusions. Selective delivery of anti-cancer agents to cancer cells elevated HA in the environment surrounding tumors, most
was achieved by biochemical targeting. The HA-modified HPMA
malignant cell-types overexpress the HA receptors CD44 and
copolymer showed improved toxicity due to receptor-mediated up-
take of the macromolecular drug.
RHAMM. Isoforms of HA receptors, CD44 and RHAMM
are overexpressed in transformed human breast epithelial
KEY WORDS: polymer-drug conjugates; HPMA copolymer; hyal- cells (11), ovarian tumor cells (12), colon cancer (13), lung
uronan; receptor-mediated targeting; doxorubicin; cancer.
cancer (14), stomach cancer (15), acute leukemia (16), and
other cancers (17). As a result, malignant cells with the high-
est metastatic potential often show enhanced binding and in-
ternalization of HA (18).
INTRODUCTION
Targeting of anti-cancer agents to tumor cells and tumor
A major challenge in cancer therapy is the selective de-
metastases can be accomplished by receptor-mediated uptake
livery of small molecule anti-cancer agents to tumor cells.
of bioconjugates of anticancer agents conjugated to HA
Water-soluble polymer-anti-cancer drug conjugates offer
(
19,20), followed by the release of free drugs through the
great potential and have demonstrated good aqueous solubil-
ity, increased half-life in the body, and high anti-tumor ef-
fects. Poly(styrene-co-maleic acid)-neocarzinostatin conju-
gate (SMANCS) was approved for the treatment of liver can-
cer in Japan (1). The conjugates of doxorubicin (DOX) to
N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers
degradation of HA in cell compartments. In this study, cell-
targeted HA-DOX bioconjuagtes and HPMA copolymer-
DOX conjugates containing HA as a side chain (HPMA-HA-
DOX) were synthesized based on the specific interaction be-
tween HA and its receptors overexpressed on the cancer cell-
surface. Selective in vitro cell cytotoxicity was studied using
three human cancer cell lines (HCT-116 colon tumor, HBL-
(
HPMA-DOX conjugate, PK1) have passed Phase I clinical
trials and are currently in Phase II trials (2). HPMA-
1
00 breast cancer, and SK-OV-3 ovarian cancer), and non-
cancerous mouse fibroblast NIH 3T3 cells as a negative con-
trol. In addition, enhanced uptake of the HPMA-HA-DOX
conjugate into cancer cells was observed relative to the non-
targeted HPMA-DOX, as visualized using the intrinsic fluo-
rescence of the DOX. This direct observation of the intracel-
lular drug-HA conjugate provides additional evidence for
1
Department of Medicinal Chemistry.
Center for Cell Signaling.
Department of Pharmaceutics and Pharmaceutical Chemistry, The
University of Utah, Salt Lake City, Utah.
2
3
4
To whom correspondence should be addressed. (e-mail: gprestwich@ the uptake of the targeted conjugates through a receptor-
deans.pharm.utah.edu)
mediated pathway.
0
724-8741/02/0400-0396/0 © 2002 Plenum Publishing Corporation
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