Development of elastin-like polypeptide for thermally targeted delivery of doxorubicin

Article abstract of DOI:10.1016/j.bcp.2006.10.028

The chemotherapeutic drug doxorubicin (Dox) is widely used as an antitumor agent in hematological malignancies and solid tumors. However, one of the limitations of its clinical use is that systemic administration of an effective dose of Dox results in nonselective cardiac toxicity and myelosuppression. In order to minimize this nonspecific toxicity, Elastin-like polypeptide (ELP) was examined for its ability to serve as a macromolecular carrier for thermally targeted delivery of Dox. The ELP-based doxorubicin delivery vehicle (Tat-ELP-GFLG-Dox) consists of: (1) a peptide derived from the HIV-1 Tat protein to facilitate its cellular uptake, (2) ELP to allow thermal targeting, and (3) the lysosomally degradable glycylphenylalanylleucylglycine (GFLG) spacer and a cysteine residue conjugated to a thiol reactive doxorubicin derivative. Cytotoxicity of Tat-ELP-GFLG-Dox in MES-SA uterine sarcoma cells was enhanced 20-fold when aggregation of ELP was induced with hyperthermia. The ELP delivered doxorubicin displayed a cytoplasmic distribution and induced temperature dependent caspase activation.

Full text of DOI:10.1016/j.bcp.2006.10.028

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Development of elastin-like polypeptide for thermally
targeted delivery of doxorubicin
Gene L. Bidwell IIIa, Izabela Fokt b, Waldemar Priebe b, Drazen Raucher a,
*
a The University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, United States
b The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The chemotherapeutic drug doxorubicin (Dox) is widely used as an antitumor agent in
hematological malignancies and solid tumors. However, one of the limitations of its clinical
use is that systemic administration of an effective dose of Dox results in nonselective
cardiac toxicity and myelosuppression. In order to minimize this nonspecific toxicity,
Elastin-like polypeptide (ELP) was examined for its ability to serve as a macromolecular
carrier for thermally targeted delivery of Dox. The ELP-based doxorubicin delivery vehicle
(Tat-ELP-GFLG-Dox) consists of: (1) a peptide derived from the HIV-1 Tat protein to facilitate
its cellular uptake, (2) ELP to allow thermal targeting, and (3) the lysosomally degradable
glycylphenylalanylleucylglycine (GFLG) spacer and a cysteine residue conjugated to a thiol
reactive doxorubicin derivative. Cytotoxicity of Tat-ELP-GFLG-Dox in MES-SA uterine sar-
coma cells was enhanced 20-fold when aggregation of ELP was induced with hyperthermia.
The ELP delivered doxorubicin displayed a cytoplasmic distribution and induced tempera-
ture dependent caspase activation.
Received 6 August 2006
Accepted 20 October 2006
Keywords:
Elastin-like polypeptide
Doxorubicin
Thermal targeting
Macromolecule
Drug delivery
# 2006 Elsevier Inc. All rights reserved.
1.
Introduction
more effective and less toxic by increasing the amount of drug
reaching the intended target.
The efficacy of traditional cytotoxic chemotherapy drugs is
limited by their adverse effects on non-cancerous tissue. Only
a small fraction of the administered dose of drug reaches the
tumor site, while the rest of the drug is distributed throughout
the body. This causes undesirable damage to normal tissue
when used in doses required to eradicate cancer cells,
resulting in a limited therapeutic index. Use of the topoi-
somerase II poison doxorubicin (Dox) is limited by the
induction of myelosuppression and cardiotoxicity [1]. Site-
specific drug delivery vehicles would make chemotherapy
A commonly used approach to address the issue of drug
delivery to solid tumors is to attach the drug to a macro-
molecular carrier. Soluble polymeric carriers are attractive for
systemic drug delivery because polymer–drug conjugates
preferentially accumulate in tumors due to their enhanced
microvascular permeability and retention [2–5] and exhibit
significantly lower systemic toxicity compared to free drug [6–
8]. Studies have shown that water-soluble polymer carriers can
overcome multidrug resistance [9–12]. The most compelling
evidence for the advantages of using polymer–drug conjugates
* Corresponding author at: Department of Biochemistry, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS
39216, United States. Tel.: +1 601 984 1510; fax: +1 601 984 1501.
E-mail address: draucher@biochem.umsmed.edu (D. Raucher).
Abbreviations: aa, amino acid; BSA, bovine serum albumin; DIC, differential interference contrast; Dox, doxorubicin; ELP, elastin-like
polypeptide; FBS, fetal bovine serum; HPMA, N-(2-hydroxypropyl)methacrylamide; MW, molecular weight; PBS, phosphate-buffered
physiological saline; Tat, cell penetrating peptide derived from HIV-1 Tat protein; Tt, transition temperature
0006-2952/$ – see front matter # 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2006.10.028

b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 6 2 0 – 6 3 1
621
over free chemotherapeutic agents for the treatment of cancer
comes from extensive preclinical and clinical studies by
Kopecek and colleagues on the use of N-(2-hydroxypropyl)-
methacrylamide (HPMA) copolymers as drug carriers [13,14].
Elastin-like polypeptide (ELP) is a protein comprised of a
five amino acid repeat (XGVPG, where X is any amino acid
except proline). ELPs are attractive as polymeric carriers for
drug delivery because they undergo an inverse temperature
phase transition [15,16]. Below a characteristic transition
temperature (Tt), ELPs are structurally disordered and highly
solvated. But, when the temperature is raised above their Tt,
they undergo a sharp (2–3 8C range) phase transition, leading
to desolvation and aggregation of the biopolymer [15,16]. This
process is fully reversible when the temperature is lowered
below Tt.
accumulated in the cell cytoplasm. The ELP-delivered Dox was
cytotoxic to MES-SA uterine sarcoma cells, and the toxicity
was enhanced 20-fold by the application of hyperthermia. The
ELP-delivered Dox induced apoptosis by caspase activation in
a temperature dependent manner.
2.
Materials and methods
2.1.
Synthesis of WP936, the thiol reactive doxorubicin
derivative
N-succinimidyl ester of 6-maleimidocaproic acid was pre-
pared as shown in Fig. 1A. The mixture of 6-maleimidocaproic
acid (211 mg, 1 mmol) and sodium carbonate (50.3 mg,
0.5 mmol) in water (10 ml) was prepared and stirred at room
temperature until all acid was dissolved. Water was evapo-
rated to dryness. Residue was dissolved in methanol (10 ml),
toluene (25 ml) was added, and solvents were evaporated to
dryness. Addition and evaporation of the mixture of methanol
and toluene was repeated three times. The obtained white
powder of sodium salt (Fig. 1, (1)) was dissolved in ice-cold DMF
(2 ml). N,N-dissucinimidyl carbonate (281.8 mg, 1.1 mmol) was
added and the reaction mixture was stirred at 0 8C for 1 h. The
reaction mixture was diluted with dichloromethane (25 ml),
washed with water (3 ꢀ 15 ml), and dried over sodium sulfate.
Inorganic salts were filtered off, solvents were evaporated to
dryness, and residue was purified by column chromatography
(SilicaGel 60, Merck) using dichloromethane as eluent, to give
184 mg of 2, yield 60%.
The phase transition of these polypeptides may be
exploited for use in drug delivery by applying focused, mild
hyperthermia to the tumor site. Systemically injected ELP will
remain soluble and freely circulate at normal body tempera-
ture. However, at localized sites where hyperthermia is
applied to raise the tissue above the ELP’s Tt, the polypeptide
will aggregate and accumulate [17]. Attachment of drugs to ELP
offers the capability to specifically deliver these drugs to the
desired tissue by focused application of externally applied
hyperthermia. The use of hyperthermia has an added
advantage of increasing vessel permeability [18–20]. The
ELP-based drug delivery system described here combines
the advantages of macromolecular delivery, hyperthermia,
and thermal targeting.
A previous study demonstrated the ability of ELP to deliver
doxorubicin into the cell cytoplasm and induce cytotoxicity,
but with no significant enhancement in cell toxicity in
response to heat [21]. Since the ultimate goal of drug delivery
by ELP is to thermally target the chemotherapeutic, it is
imperative that cytotoxicity be enhanced in response to
temperature increase. In this study, a thermally responsive
drug carrier was generated by modifying the sequence of ELP
to include additional targeting features, and the result was a
drug delivery vector that achieved a 20-fold enhancement of
cell killing in response to hyperthermia.
The mixture of doxorubicin (hydrochloride salt) (38.4 mg,
0.065 mmol), (2) (30 mg, 0.08 mmol), diisopropylethylamine
(28 ml, 0.16 mmol) and DMF (1 ml) was prepared and stirred at
room temperature (Fig. 1B), while progress of the reaction was
monitored by TLC (chloroform:methanol:ammonia = 85:15:2).
After 40 min, the reaction was completed. The reaction
mixture was diluted with dichloromethane (2 ml) and pre-
cipitated with hexanes (50 ml). The obtained solid was
separated from solvents by centrifugation. The product was
separated by column chromatography (SilicaGel 60, Merck)
using chloroform, chloroform:methanol 98:2, 95:5 as eluents.
Fractions containing WP936 were pooled together, evaporated
to dryness, dissolved in chloroform (1 ml) and precipitated
with hexanes (25 ml). The obtained solid was dried under
vacuum to give 24 mg of WP936 (yield 60%). The 1H NMR
spectrum was obtained for WP936 and was in agreement with
the proposed structure.
1H NMR (CDCl3, d) ppm: 14.0, 13.25 (2s, 1H ea, 6,11-OH), 8.04
(dd, 1H, J = 7.6 Hz, J = 0.7 Hz, H-1), 7.72 (dd, 1H, J = J = 8.4 Hz, H-
2), 7.4 (d, 1H, J = 8.1 Hz, H-3), 6.67 (s, 2H, CH maleimid), 5.82 (d,
1H, J = 8.6 Hz, NH), 5.50 (d, 1H, J = 3.5 Hz, H-10), 5.38 (bs, 1H, H-7),
4.76 (s, 2H, 14-CH2), 4.56 (s, 1H, 9-OH), 4.16 (q, 1H, J = 6.1 Hz, H-
50), 4.08 (s, 3H, OMe), 3.63 (bs, 1H, H-4), 3.49 (t, 2H, J = 7.1 Hz,
CH2-linker), 3.28 (dd, 1H, J = 18.8 Hz, J = 1.8 Hz, H-10), 3.24 (d,
1H, J = 18.8 Hz, H-10), 3.02 (m, 1H, H-30), 2.34 (d, 1H, J = 14.7 Hz,
H-8), 2.17 (dd, 1H, J = 14.7 Hz, J = 4.1 Hz, H-8), 2.12 (dd, 2H,
J = 7.2 Hz, J = 2.4 Hz, CH2 from linker), 1.83 (dd, 1H, J = 12.7 Hz,
J = 5.5 Hz, H-20e), 1.78 (ddd, 1H, J = J = 12.7 Hz, J = 4.1 Hz, H-20a),
1.63–1.55 (m, 4H, CH2 from linker), 1.29 (d, 3H, J = 6.1 Hz, H-6’),
1,30–1.25 (m, 2H, CH2 from linker).
The thermally responsive ELP polypeptide was modified
with the addition of a cell penetrating peptide derived from the
HIV-1 Tat protein (Tat) and by employing
a cleavable
tetrapeptide linker for attachment of the drug to the
macromolecule. The Tat peptide is known to facilitate
transport of large molecules across the plasma membrane
[22], and a previous study demonstrated its ability to enhance
ELP uptake by an endocytic mechanism [23]. In addition, the
ELP contains a tetrapeptide glycylphenylalanylleucylglycyl
(GFLG) linker and a C-terminal cysteine residue. The thiol of
the terminal cysteine is used for attachment of drugs, and the
GFLG linker can be cleaved by lysosomal proteases of the
cathepsin family [24], resulting in intracellular drug release.
For this study, a thiol reactive derivative of doxorubicin
(WP936) containing a maleimido moiety linked to the C-30
amino group was designed and synthesized. WP936 was
attached to the C-terminal cysteine residue of the ELP carrier
(Tat-ELP-GFLG-Dox). The Dox delivery construct exhibited a Tt
of 40 8C, and its cellular uptake was enhanced by both the Tat
peptide and hyperthermia. Dox delivered by the ELP vector

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b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 6 2 0 – 6 3 1
Fig. 1 – Synthesis of N-succinimidyl ester of 6-maleimidocaproic acid (A). Synthesis of WP936, a thiol reactive derivative of
doxorubicin (B). Schematic of the doxorubicin carrier polypeptide (C). The amino acid sequence of the Tat cell penetrating
peptide and the ELP polypeptide are shown in single letter amino acid codes, and the chemical structure of the GFLG linker
with the cysteine-conjugated WP936 is shown.
Anal. Elem. For C37H40N2O14ꢁ2H2O Calcd. C, 57.51; H, 5.74; N,
were lysed by sonication and, after precipitation of nucleic
acids, Tat-ELP-GFLG was precipitated from the soluble lysate
by increasing the sodium chloride concentration and raising
the temperature above the ELP Tt. The protein was collected by
centrifugation, and this process was repeated until the desired
purity was obtained as assessed by a single band on an SDS-
PAGE gel.
3.63, Found: C, 57.90; H, 5.42; N, 3.60.
2.2.
Expression and purification of ELP
The ELP1 and ELP2 gene sequences were graciously supplied
by Dr. Chilkoti. Tat-ELP-GFLG and other constructs used in this
study were generated as described previously [25]. All ELP
constructs were expressed in E. coli using the hyperexpression
system [26] and purified by inverse transition cycling [25].
Briefly, the E. coli strain BLR(DE3) containing the Tat-ELP-GFLG
construct in the pET 25b expression vector was grown for 24 h
at 37 8C in Circle Grow media (Q-Biogene, Irvine, CA). The cells
2.3.
Conjugation of ELP with WP936
A solution of ELP (ELP-GFLG or Tat-ELP-GFLG) was diluted to
100 mM in 50 mM Na2HPO4. Tris-(2-carboxyethyl)phosphine
(TCEP; Molecular Probes, Eugene, OR) was added to a 10-fold

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623
molar excess. WP936 was slowly added while mixing to a final
concentration of 100 mM and incubated with continuous
stirring overnight at 4 8C. Unreacted label was removed by
three inverse transition cycles [25] into PBS. Efficiency of
labeling on the single cysteine residue was assessed by UV–vis
spectrophotometry (modified from Ref. [21]). The typical molar
label to protein ratio was 0.83 ꢂ 0.15.
histogram was a unimodal peak (n = 5000 cells), and the peak
mean was normalized to propidium standard beads.
2.7.
Laser scanning confocal fluorescence microscopy
MES-SA cells were plated on 22 mm2 cover slips at approxi-
mately 50% confluence and treated as described above with
Tat-ELP-GFLG-Dox. Cells were rinsed with PBS at the indicated
times, fixed with paraformaldehyde (PFA, 2%, v/v) and
visualized using a TCS SP2 laser scanning confocal microscope
with a 100ꢀ oil immersion objective (Leica, Wetzlar, Germany).
PMT voltages were lowered for imaging of cells treated at 42 8C
during image acquisition to maximize image resolution and
intensity. Therefore, the difference in image intensity between
cells treated at 37 8C and 42 8C does not represent the actual
difference in the amount of polypeptide in the cells.
2.4.
Characterization of the transition temperature
The temperature induced aggregation of the proteins was
characterized by monitoring absorbance at 350 nm while
increasing the temperature. For initial analysis of Dox-labeled
ELPs, solutions containing 10 mM protein in PBS were heated or
cooled at a constant rate of 1 8C/min in a temperature-
controlled multicell holder in a UV–vis spectrophotometer
(Cary 100, Varian instruments). The analysis was repeated
with Tat-ELP1-GFLG-Dox at concentrations ranging from 1 mM
to 30 mM in cell culture media in order to determine the
concentration dependence of the phase transition under
experimental conditions. Absorbance data was converted to
percentage of the maximal absorbance for each curve, and the
Tt was defined as the temperature at which the OD350 reached
50% of the maximum turbidity. The concentration depen-
dence of the Tt was fit using a logarithmic equation.
2.8.
Cytotoxicity assay
Cells were plated and treated for 1 h at 37 8C and 42 8C as
described above with Tat-ELP1-GFLG-Dox, Tat-ELP2-GFLG-
Dox, or control polypeptides lacking the Tat sequence or
unlabeled with Dox. Cells were rinsed and harvested 72 h after
treatment by trypsinization, collected by centrifugation, and
resuspended in isotonic buffer. Cell number was determined
using a Coulter counter and expressed as a percentage of
untreated cells.
2.5.
Cell culture and polypeptide treatment
MES-SA uterine sarcoma cells (ATCC, Manassas, VA) were
grown as a monolayer in 75 cm2 tissue culture flasks and
passaged every 3–5 days. MES-SA cells were grown in McCoy’s
5A medium supplemented with 10% fetal bovine serum (FBS),
100 U/ml penicillin, 100 mg/ml streptomycin, and 25 mg/ml
amphotericin B (Invitrogen, Carlsbad, CA). Cultures were
maintained at 37 8C in a humidified atmosphere + 5% CO2.
For experiments, cells were removed from tissue culture flasks
by brief treatment with 0.05%, v/v trypsin-EDTA (Invitrogen),
plated in six well plates (300,000 cells/well for flow cytometry,
25,000 cells/well for proliferation) and allowed to grow for
24 h. Cells were treated with media containing polypeptides
for 1 h at the indicated temperature, rinsed, and replaced with
fresh media. In order to eliminate variability due to variation
in labeling levels among different proteins or labeling batches,
concentrations for cell treatments were always based on the
Dox concentration as judged by the absorbance peak at
495 nm.
2.9.
Apoptosis assays
For differential interference contrast (DIC) microscopy, MES-
SA cells were plated onto 22 mm2 coverslips at 50% con-
fluence. Cells were treated as described above with the
indicated protein, rinsed with PBS, and mounted onto glass
slides. The unfixed cells were imaged immediately using a
Zeiss Axiovert 200 DIC microscope with a 40ꢀ oil immersion
objective and a Coolsnap HQ camera.
Caspase activation was measured using a carboxyfluor-
escein FLICA polycaspase assay (Immunochemistry Technol-
ogies, Bloomington, MN). Cells were treated as described
above with the indicated protein, collected by trypsinization,
and stained for 2 h at 37 8C with the carboxyfluorescein FLICA
reagent as described by the manufacturer. Cells were rinsed
twice and analyzed for caspase activation by flow cytometry
using a Cytomics FC 500 flow cytometer (n = 5000 cells).
Forward versus side scatter gating was used to eliminate cell
debris from the analysis, and a histogram of fluorescein
fluorescence (channel FL1) was bimodal with peaks for
caspase positive and caspase negative cells. The percentage
of caspase positive cells was determined from the histograms
and expressed as an average of three experiments.
2.6.
Polypeptide uptake
Cells were treated with 20 mM Tat-ELP1-GFLG-Dox, Tat-ELP2-
GFLG-Dox, or control polypeptides lacking the Tat sequence as
described above. Duplicate plates were treated for 1 h at 37 8C
and 42 8C. Cells were rinsed with PBS and harvested using
nonenzymatic cell dissociation buffer (Invitrogen), centri-
fuged for 2 min, and resuspended in 0.5 ml PBS. Total uptake of
the doxorubicin labeled polypeptides was measured using the
2.10.
Data fitting and statistical analysis
The concentration dependence of the Tt was fit with a
logarithmic equation using Microsoft Excel. Dose–response
curves were fit using exponential or sigmoid equations as
applicable using Microcal Origin in order to determine the 50%
inhibitory concentration (IC50). The thermal targeting index
was calculated for each construct by dividing the IC50 at 37 8C
intrinsic Dox fluorescence by
a Cytomics FC 500 flow
cytometer (Beckman Coulter, Fullerton, CA). Forward versus
side scatter gating was used to remove cell debris from the
analysis, and Dox fluorescence was measured using FL3. Each

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b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 6 2 0 – 6 3 1
Fig. 2 – Thermal properties of Dox delivery constructs. The turbidity of a 10 mM solution of labeled and unlabeled Tat-ELP-
GFLG in PBS was monitored while the temperature was increased at a rate of 1 8C/min (A). The effect of ELP concentration on
the Tt under cell culture conditions was determined by repeating the turbidity assay using concentrations of Tat-ELP1-
GFLG-Dox ranging from 1 to 30 mM in cell culture media (B). Optical density (OD) data is converted to a percentage of the OD
at 60 8C for each curve in order to view all concentrations on the same scale. The midpoint of the phase transitions in (B)
were plotted versus the protein concentration and fit using a logarithmic equation (R2 = 0.9942) in order to determine the
concentration range in which the Tt lies between 37 8C and 42 8C (C).
by the IC50 at 42 8C. The significance of the thermal targeting
index was assessed by comparing the normothermia group
with the hyperthermia group using a paired Student’s t-test in
Microsoft Excel. p-Values of less than 0.05 were considered
statistically significant. Polypeptide uptake and caspase
activation were analyzed for statistical differences using
one-way ANOVA analyses with post hoc Scheffe’s tests for
pair-wise comparisons of treatment groups. The statistical
significance level was p < 0.05.
was constructed including ELP2, which contains 160 XGVPG
repeats (MW = 61 kDa), where X is represented by V, G, and A in
a 1:7:8 ratio. ELP2 has a similar molecular weight to ELP1, but it
does not undergo its phase transition during the mild
hyperthermia used in this study. Thus it serves as a non-
thermally responsive control for the effects of hyperthermia
[17]. Tat-ELP-GFLG was designed to include the Tat cell
penetrating peptide [22] to allow cellular entry of the
macromolecular construct, and the GFLG tetrapeptide linker
was included to allow intracellular drug release by lysosomal
proteases [24]. The C-terminal amino acid of the vector is a
cysteine, which provides a chemically reactive thiol group for
drug conjugation. WP936 is a thiol reactive Dox derivative
obtained by conjugating a maleimido moiety via a linker with
the amino group at the C-30 position (Fig. 1A and B). WP936 can
be covalently attached to the cysteine sulfur by nucleophilic
addition. The labeling reaction resulted in an average molar
ratio of Dox:protein of 0.83 ꢂ 0.15. The amino acid sequence
and chemical structure of WP936 covalently bound to the
sulfur atom of the C-terminal cysteine is shown in Fig. 1C.
3.
Results
3.1.
Design, synthesis, and conjugation of Tat-ELP-GFLG
and WP936
The ELP drug delivery vector is composed of the pentapeptide
repeat XGVPG, and it is designed to undergo a phase transition
when the temperature is raised above its characteristic Tt. Two
versions of the Dox delivery vector were made using two
different ELP repeats. The ELP1-based construct contains 150
pentapeptide repeats (MW = 59.1 kDa) with the guest position
comprised of the amino acids V, G, and A in a 5:3:2 ratio. ELP1
was designed to have a Tt just above physiologic temperature
for use as a thermally targeted vector. A second polypeptide
3.2.
Thermal properties of the Dox delivery vector
In order to apply ELP-based polypeptides for drug delivery,
a suitable temperature transition must be attained. The

b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 6 2 0 – 6 3 1
625
drug-labeled ELP should undergo its phase transition between
39 8C and 42 8C, a temperature range sufficiently above normal
body temperature to prevent unwanted systemic aggregation.
This temperature range is preferred for clinical applications of
hyperthermia because it minimizes the incidence of edema
and necrosis in healthy tissue surrounding a heated tumor
[27]. Elastin-like polypeptide is ideally suited as a thermally
targeted drug carrier because the Tt can be easily manipulated
by varying the molecular weight or the amino acid composi-
tion of the guest residue X in the XGVPG sequence using
simple molecular biology techniques [28]. The Tt of Tat-ELP-
GFLG-Dox was assessed by monitoring the turbidity of a 10 mM
solution of the polypeptide while heating in PBS. The Tt of Tat-
ELP1-GFLG was found to be 46 8C. However, labeling the
polypeptide with Dox caused a significant downshift in the Tt
(Fig. 2A), and as a result, Tat-ELP1-GFLG-Dox had an ideal
phase transition for drug delivery (Tt = 40 8C). The polypeptide
was soluble and the solution was clear at physiological
temperature (37 8C), but when the temperature was raised
to the hyperthermia temperature (42 8C), the polypeptide
aggregated and the solution approached its maximum
turbidity. Tat-ELP2-GFLG-Dox is a polypeptide similar in size
to Tat-ELP1-GFLG-Dox, but a different ELP moiety is incorpo-
rated. Tat-ELP2-GFLG-Dox aggregates at a temperature sig-
nificantly above the hyperthermia temperature (Tt = 65 8C),
and it serves as a control to distinguish effects of the ELP phase
transition from nonspecific hyperthermia-induced effects.
The phase transition of ELP is inversely related to the
concentration of ELP and is also influenced by the concentra-
tion of other co-solutes. Therefore, in order to test the
concentration range in which the Tat-ELP1-GFLG-Dox Tt is
in the desired temperature range of 37–42 8C, the turbidity
assay was repeated with various concentrations of Tat-ELP1-
GFLG-Dox in cell culture media containing 10% FBS (Fig. 2B).
The Tt was determined for each curve, and a plot of Tt versus
polypeptide concentration revealed an inverse relationship
which was best fit using a logarithmic function (Fig. 2C). This
analysis showed that the midpoint of the phase transition was
in the desired temperature range for concentrations between
10 mM and 39 mM. It should be noted, however, that some
aggregation does occur at 42 8C for concentrations lower than
10 mM. Therefore, some thermal effect may be seen for cells
treated under these conditions. The Tat-ELP-GFLG-Dox con-
centration is an important characteristic for thermal targeting
because if the concentration of the ELP carrier is too high, it
will undergo its phase transition at normal physiological
temperature.
Fig. 3 – Cellular uptake of Dox labeled ELP. MES-SA uterine
sarcoma cells were incubated with Dox labeled constructs
(20 mM in cell culture media) for 1 h at 37 8C or 42 8C. The
Dox fluorescence intensity was determined using flow
cytometry and is expressed in relative fluorescence units
(RFU). The data represents the mean of at least three
z
experiments (error bars, S.E.M.). Significant difference
y
compared with ELP1-GFLG-Dox at 37 8C. Significant
difference compared with Tat-ELP1-GFLG-Dox at 37 8C
( p < 0.0001, one way ANOVA, p < 0.05, Scheffe’s test).
both internalized and bound to the cell membrane. Fig. 3
shows that in the absence of the Tat peptide, low levels of
ELP were associated with the cells at either temperature.
However, cell association and uptake of Tat-ELP1-GFLG-Dox
was increased over three-fold as compared to ELP1-GFLG-
Dox when cells were treated at 37 8C ( p < 0.05). In addition,
hyperthermia further enhanced the association and uptake
of Tat-ELP1-GFLG-Dox. Cellular fluorescence was increased
over two-fold when the MES-SA cells were treated with
Tat-ELP1-GFLG-Dox at 42 8C rather than 37 8C ( p < 0.05).
Levels of Tat-ELP2-GFLG-Dox, the non-thermally responsive
control polypeptide, were unaffected by treatment
with hyperthermia. This supports the hypothesis that
the additional cellular association observed with Tat-
ELP1-GFLG-Dox and hyperthermia is due to the polypeptide
phase transition and not to nonspecific effects of
hyperthermia.
3.4.
Cellular distribution
To confirm that Tat-ELP-GFLG-Dox was internalized and not
merely attached to the cell exterior, the cellular distribution of
the ELP-delivered drug was examined by confocal fluores-
cence microscopy. MES-SA cells were treated for 1 h at 37 8C
and 42 8C with 10 mM Tat-ELP1-GFLG-Dox. The cells were
rinsed and analyzed immediately after treatment and 24 h
later using the intrinsic Dox fluorescence. Taking a confocal
section through the center of the cells confirmed internaliza-
tion of Tat-ELP1-GFLG-Dox immediately after treatment, with
the drug accumulating in the plasma and nuclear membranes
as well as in a punctate pattern in the cytoplasm (Fig. 4A, top,
left). When Tat-ELP1-GFLG-Dox treatment was combined with
hyperthermia, the membrane and cytoplasmic distribution
was still present, but the protein was present in large
3.3.
Cellular uptake of ELP-delivered doxorubicin
MES-SA uterine sarcoma cells were used in this study to
evaluate the efficiency of ELP-mediated cellular delivery of
doxorubicin. MES-SA cells were incubated with 20 mM Tat-
ELP-GFLG-Dox or a control polypeptide which lacks the Tat
sequence for 1 h at either 37 8C or 42 8C. After harvesting the
cells with non-enzymatic cell dissociation buffer to prevent
degradation of polypeptide attached to the cell surface, the
non-fixed cells were analyzed by flow cytometry to measure
the Dox fluorescence. This assay provides information
about the total amount of Dox associated with the cells,

626
b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 6 2 0 – 6 3 1
Fig. 4 – Intracellular localization. Confocal fluorescence images of MES-SA cells were collected 1 h and 24 h after a 1 h exposure
to Tat-ELP1-GFLG-Dox (10 mM in cell culture media) at 37 8C or 42 8C (A). PMT voltages were decreased for collection of 42 8C
images. Therefore, the image intensity does not reflect the actual level of Dox in the cells. Control cells were treated with free
Dox (10 mM in cell culture media) (left panel) or untreated to check for autofluorescence (right panel) (B).
aggregates and the cells had begun to show hallmarks of
apoptosis including rounding and membrane blebbing
(Fig. 4A, bottom, left). Twenty-four hours after treatment,
the ELP-delivered Dox still remained in the cell cytoplasm, and
it displayed a more diffuse distribution (Fig. 4A, top, right). The
cells observed 24 h after Tat-ELP1-GFLG-Dox treatment com-
bined with hyperthermia again showed accumulation of the
large ELP-drug aggregates in a diffuse cytoplasmic distribution
and extensive apoptosis (Fig. 4A, bottom, right). In contrast to
the ELP-delivered doxorubicin, the free drug was found
exclusively in the cell nucleus (Fig. 4B, left panel). Untreated
cells had no signal under the imaging settings used, ruling out
any contribution to the images from autofluorescence (Fig. 4B,
right panel).
3.5.
Cytotoxicity of ELP-delivered Dox
The growth-inhibitory activity of Tat-ELP-GFLG-Dox was
evaluated in the MES-SA uterine sarcoma cell line. MES-SA
cells were treated for 1 h at 37 8C or 42 8C with various
concentrations of Tat-ELP-GFLG-Dox; ELP-GFLG-Dox, which
lacks the Tat cell penetrating peptide, the Tat-ELP-GFLG
polypeptide without Dox; or free Dox. After the 1 h treatment,
the cells were rinsed and fresh media was replaced for 72 h.
Cells remaining after 72 h were rinsed, collected by trypsini-
zation, and counted using a Coulter counter. Fig. 5A shows
that Tat-ELP1-GFLG-Dox was cytotoxic at 37 8C, but high
concentrations were needed. However, when Tat-ELP1-GFLG-
Dox was applied in combination with hyperthermia, the

b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 6 2 0 – 6 3 1
627
Fig. 5 – Cell proliferation. MES-SA cells were exposed to varying concentrations of Tat-ELP1-GFLG-Dox (A), Tat-ELP2-GFLG-
Dox (B), Tat-ELP1-GFLG (C), ELP1-GFLG-Dox (D), or free Dox (E) for 1 h at 37 8C or 42 8C, and cells were counted after 72 h. The
data represent an average of at least three experiments (bars, S.E.M.).
cytotoxicity was greatly enhanced. Tat-ELP2-GFLG-Dox was
also cytotoxic (Fig. 5B), but the toxicity was not affected by
hyperthermia and was similar to Tat-ELP1-GFLG-Dox at 37 8C.
The toxicity of the unlabeled Tat-ELP-GFLG protein was
negligible when treated below the Tt. However, Tat-ELP1-
GFLG did show some toxicity at high concentrations when
treated at 42 8C, but this nonspecific toxicity did not approach
the toxic levels observed with the Dox labeled protein (Fig. 5C).
ELP1-GFLG Dox exhibited very poor potency at either treat-
ment temperature, which is consistent with its poor cellular
uptake in the absence of the Tat peptide (Fig. 5D). Free Dox was
more cytotoxic than the polymer-delivered drug (Fig. 5E),
which reflects its passive cellular entry and is consistent with
other studies of Dox bound to synthetic polymers [29,30]. The
Dox toxicity was also affected by hyperthermia, which is likely
due to enhanced diffusion of the drug into the cells.
in Table 1. This calculation allows analysis of the enhance-
ment of polypeptide toxicity by its hyperthermia induced
phase transition. The IC50 of free Dox was about 150 times
lower than the Tat-ELP-GFLG delivered Dox. The Dox IC50 was
enhanced by heat treatment, but the temperature enhance-
ment was only 2.3-fold ( p = 0.02). Some toxicity was noted
with the Tat-ELP1-GFLG protein that does not contain Dox,
especially when aggregation was induced with heat. However,
the level of cell killing was ten-fold less potent than Tat-ELP1-
GFLG-Dox. ELP1-GFLG-Dox, which is thermally responsive but
lacks the Tat cell penetrating peptide, showed a slight thermal
effect, but the potency was two-fold lower at 37 8C and 15-fold
lower at 42 8C than Tat-ELP1-GFLG-Dox. In fact, under
hyperthermia conditions, Tat-ELP1-GFLG is more toxic than
ELP1-GFLG-Dox. Tat-ELP1-GFLG-Dox was cytotoxic at 37 8C,
but relatively high concentrations were needed to achieve
significant cell killing. However, the IC50 was reduced 20-fold
when Tat-ELP1-GFLG-Dox treatment was combined with
hyperthermia ( p = 0.04). The 20-fold enhancement of cyto-
toxicity induced by hyperthermia and Tat-ELP1-GFLG-Dox
treatment results from a combination of the toxicities of the
For direct comparison of the cytotoxic effects of the Dox
delivery constructs, each data set was fitted to an exponential
or sigmoid equation to determine the IC50 value. Additionally,
the thermal targeting index was calculated by dividing the IC50
of each polypeptide at 37 8C by its IC50 at 42 8C, and presented

628
b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 6 2 0 – 6 3 1
Table 1 – IC50 values for all ELP-doxorubicin constructs
ꢃ
ꢃ
C
37
42
50
Thermal targeting index, ðIC C=IC
Þ
Construct
IC50 (37 8C) (mM ꢂ S.E.M.)
IC50 (42 8C) (mM ꢂ S.E.M.)
50
Dox
0.130 ꢂ 0.01
45.1 ꢂ 12*
44 ꢂ 8*
0.058 ꢂ 0.02
10.1 ꢂ 0.36
15.4 ꢂ 3.9
1.1 ꢂ 0.1
2.3y
4.47y
2.85y
19.9y
1.1
Tat-ELP1-GFLG
ELP1-GFLG-Dox
Tat-ELP1-GFLG-Dox
Tat-ELP2-GFLG-Dox
21.9 ꢂ 8.2
18.3 ꢂ 11.4
17.3 ꢂ 12.8
IC50 values for each construct. Data from Fig. 5 was fit to calculate the IC50 value for each construct with treatment at 37 8C or 42 8C. The
thermal targeting index was calculated by dividing the IC50 value for each construct at 37 8C by its IC50 value with 42 8C treatment. ySignificant
difference between the 37 8C and 42 8C IC50 ( p < 0.05, Student’s t-test). *IC50 extrapolated from curve fitting because the required concentration
was unreachable.
Tat peptide and Dox. The Tat-ELP2-GFLG-Dox IC50 was
unaffected by the hyperthermia treatment, indicating that
the thermal enhancement of toxicity observed with the ELP1-
based construct was due to enhanced cellular delivery by
aggregation of the polymer.
averaged to determine the percentage of the cell population
in which caspases were activated under each treatment
condition. Caspase activity induced by hyperthermia alone,
by Tat-ELP1-GFLG, or by ELP-GFLG-Dox was similar to
control levels (Fig. 6C). When cells were treated at 37 8C,
Tat-ELP1-GFLG-Dox induced apoptosis slightly above control
levels. However, when the treatment temperature was
raised to 42 8C, Tat-ELP1-GFLG-Dox induced caspase activa-
tion in a significantly higher proportion of the population. In
fact, the percent of caspase positive cells was enhanced
almost 4 fold when cells were treated with Tat-ELP1-GFLG-
Dox at 42 8C versus 37 8C ( p < 0.05). Apoptosis induction by
Tat-ELP2-GFLG-Dox was not enhanced by hyperthermia,
supporting the hypothesis that the higher levels of apoptosis
seen with Tat-ELP1-GFLG-Dox and hyperthermia were due
to accumulation of the polypeptide as a result of the ELP
phase transition. Free Dox induced apoptosis under the
conditions tested ( p < 0.05), but the levels were not affected
by hyperthermia.
3.6.
Induction of apoptosis
As mentioned above, hallmarks of apoptosis were noted in
the cell morphology after treatment with Tat-ELP1-GFLG-
Dox, especially in combination with hyperthermia. This
effect was documented by treating MES-SA cells adhered to
glass cover slips with each construct and imaging them by DIC
microscopy. Cells were treated for 1 h as described above,
then immediately mounted on slides and imaged without
fixation. Fig. 6A shows representative images of each
treatment population. Untreated cells were spread onto the
glass, and morphological features such as nuclei and
endocytic vesicles were readily visible. This morphology
was not altered by simply exposing the cells to hyperthermia.
When the cells were treated with Tat-ELP1-GFLG, their
morphology was unaltered with treatment below the Tt,
and treatment above the Tt resulted in slight rounding and
minor blebbing in only a small portion of the population. Cells
treated with ELP1-GFLG-Dox showed some hallmarks of
apoptosis, including rounding and membrane blebbing.
However, such effects were minor and limited to a fraction
of the cells. Treatment with Tat-ELP1-GFLG-Dox at 37 8C
caused more extensive rounding and blebbing. When cells
were treated with Tat-ELP1-GFLG-Dox at 42 8C, normal cell
morphology was completely destroyed. All cells examined
were rounded with no discernable nucleus and fragmented
chromosomes. Tat-ELP2-GFLG-Dox induced minor blebbing
in a proportion of the population with no heat effect, similar
to Tat-ELP1-GFLG-Dox at 37 8C. Free Dox also caused notice-
able rounding and membrane blebbing, but not to the extent
of Tat-ELP1-GFLG-Dox at 42 8C.
4.
Discussion
It has been shown previously that the endocytic uptake of a
thermally responsive ELP is significantly enhanced by the
thermally triggered phase transition of the polypeptide [31].
This observation was confirmed in this study where it was
shown that cellular uptake of the ELP-delivered Dox was
enhanced by both the Tat peptide and hyperthermia. Inter-
estingly, cellular uptake of ELP1-GFLG-Dox was not signifi-
cantly enhanced by heat treatment in the absence of the Tat
peptide, even though both constructs undergo their phase
transition in the 37–42 8C window. This result differs from
previous reports of ELP1 labeled with fluorescein [25,31], in
which as much as a two-fold enhancement of cellular uptake
with hyperthermia was seen in several different cell lines. A
possible explanation is that the fluorescein dye is less
hydrophobic than Dox, and therefore the large aggregates of
ELP1-GFLG-Dox may have a reduced affinity for the charged
cell surface. Clearly, however, the addition of the Tat peptide
increases the affinity of the large ELP aggregates for the cell
membrane, as the uptake of Tat-ELP1-GFLG-Dox was
increased three-fold at 37 8C and six-fold at 42 8C as compared
to ELP1-GFLG-Dox.
The mechanism by which Tat-ELP1-GFLG-Dox induced
apoptosis was investigated by determination of caspase
activity. MES-SA cells were treated as described above, cells
were harvested after the 1 h exposure, and caspase activity
was detected using a fluorescent caspase inhibitor and flow
cytometric analysis. A histogram of fluorescence versus cell
number revealed two peaks, one with bright fluorescence
which includes cells with positive caspase activity and one
with lower fluorescence comprised of caspase negative cells
(Fig. 6B). Cell numbers under each peak were quantified and
The cellular Dox levels resulting from Tat-ELP-GFLG-Dox
treatment were much lower than what results from treatment
with free doxorubicin under similar conditions (data not

b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 6 2 0 – 6 3 1
629
Fig. 6 – Induction of apoptosis by ELP-delivered Dox. DIC images of MES-SA cells were collected after treatment for 1 h with
each construct (10 mM) at 37 8C or 42 8C (A). Histograms of caspase activation in MES-SA cells were obtained after the 1 h
treatment (B). The percentage of cells with active caspases was measured from the histograms and averaged (C). Data
represent the average of at least 3 experiments (bars, S.E.M.). zSignificant difference compared with untreated controls at 37 8C.
ySignificant difference compared with Tat-ELP1-GFLG-Dox at 37 8C ( p < 0.0001, one was ANOVA, p < 0.05, Scheffe’s test).
shown). This reflects the disparate mechanisms by which the
two molecules gain cellular entry. Free Dox is sufficiently small
and hydrophobic to gain access to the intracellular environ-
ment by passive diffusion [32], while Tat-ELP-GFLG-Dox relies
on an active transport mechanism for cellular entry [23]. This
contrast in cellular uptake mechanisms of free and polymer
bound Dox has been observed previously with HPMA delivered
Dox [33]. In the case of HPMA–Dox, the endocytic uptake is an
advantage, leading to an alternative toxicity mechanism
involving lipid peroxidation in cell membranes [29], enhanced
tumor activity in vivo [34], lowered systemic toxicity [34], and
the ability to overcome multidrug resistance [12].

630
b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 6 2 0 – 6 3 1
The data presented here demonstrate that the Tat-ELP-
penetrating peptide. In the Dreher et al. study, cells were
continuously exposed for 24 h or 72 h to ELP-Dox with
hyperthermia applied only during the first hour, which would
diminish the influence of hyperthermia. In the study
presented here, the cells were exposed to both drug and
hyperthermia for only 1 h in order to highlight the influence of
hyperthermia treatment. During this time, the hyperthermia
causes the protein to aggregate, and the polypeptide aggre-
gates accumulate on the surface of the cell. As a result, there is
an increase in the amount of polypeptide bound to the cells
and eventually internalized by the cells when compared to
soluble polypeptides at the normothermic temperature. The
thermal enhancement observed in this study was also aided
by the use of the Tat peptide. Some cytotoxicity was observed
with the unlabeled Tat-ELP-GFLG polypeptide, which is
possibly due to the highly charged polymer’s interaction with
and disruption of the plasma membrane [38]. The exact
mechanism for this toxicity is under further evaluation.
However, cell killing by Tat-ELP-GFLG-Dox is not due simply
to the toxicity of Tat-ELP-GFLG, as the unlabeled protein was
an order of magnitude less potent than the Dox labeled
construct and induced no caspase activation in the absence of
Dox in MES-SA cell line. The Tat peptide enhanced the cellular
delivery of the large protein aggregates, as ELP1-GFLG-Dox
only showed a thermal targeting index of 2.85 compared to an
index of 19.9 for Tat-ELP1-GFLG-Dox.
GFLG delivered Dox does not localize to the nucleus. This is in
contrast to the free drug, which localizes entirely in the
nucleus where it serves as an intercalator and topoisomerase
II poison. Similar to HPMA delivered Dox, Tat-ELP-GFLG
delivered Dox displayed a cytoplasmic distribution. The cause
of this altered distribution may be the endocytic mode of
cellular entry used by Tat-ELP-GFLG [23], the failure of Dox to
be released from the ELP carrier, or the modifications made to
the Dox structure. Interestingly, the Tat-ELP-GFLG-delivered
Dox is still cytotoxic regardless of its lack of nuclear
localization, suggesting an alternate mechanism of toxicity
for free and Tat-ELP-GFLG delivered Dox [21]. Tat-ELP-GFLG-
Dox also induced caspase activation more effectively than free
Dox in spite of its less potent cytotoxicity, adding additional
support to the hypothesis that the two forms of the drug are
acting through different mechanisms. It has been shown that
HPMA conjugated Dox causes alterations in plasma mem-
brane permeability [30], induces lipid peroxidation [29], and
induces both caspase dependent apoptosis [35] and necrosis
[32]. Experiments are underway to determine the mechanism
of cytotoxicity by Tat-ELP-GFLG delivered Dox.
In the in vitro assay used in this study, free Dox was found
to be much more potent than the ELP-delivered Dox. However,
the cytotoxicity of the two agents cannot be compared directly
because of the different levels of drug in the cells. In fact, a
previous study using HPMA delivered Dox found that, when
corrected for intracellular drug levels, the cytotoxicity of
polymer-delivered Dox was comparable to free Dox [29].
Because of the complexities of drug pharmacokinetics and
tissue distribution in vivo, the in vitro potency is a poor
predictor of a drug’s therapeutic value, especially when
comparing a polymer-bound drug to the free drug. HPMA–
Dox conjugates have been shown to have in vitro IC50s in the
This study demonstrates the use of ELP as a thermally
targeted drug carrier for the intracellular delivery of doxor-
ubicin. The Dox delivery construct underwent its phase
transition at 40 8C, and its internalization by cells was
enhanced by both the use of a cell penetrating peptide and
by its hyperthermia-induced phase transition. The ELP vector
delivered Dox to the cell cytoplasm and killed cells by
induction of apoptosis through a caspase dependent mechan-
ism. The cytotoxicity was enhanced 20-fold when treatment
was combined with hyperthermia. This work provides initial
proof of principle for the use of ELP as a thermally targeted
delivery system for doxorubicin in vitro, and it opens the door
for the future evaluation of the efficacy of ELP as a potential
drug carrier in vivo.
micromolar range [12,30], much higher than the free Dox IC50
,
but these HPMA–Dox conjugates have shown promise in
preliminary clinical evaluations [36]. The IC50 values of HPMA–
Dox and Tat-ELP-GFLG-Dox are comparable, indicating that a
therapeutic concentration of Tat-ELP-GFLG-Dox is achievable.
A previous study by Dreher et al. reported cytotoxicity with
ELP delivered Dox in a squamous cell carcinoma line (FaDu)
[21]. However, they did not observe enhancement of the
cytotoxic effect with hyperthermia. Several differences
between the Dreher et al. work and this exist. First, Dreher
et al. used an acid-labile hydrazone linker for conjugation of
the drug to ELP, which contrasts with the protease-cleavable
linker used here. Second, the site of modification of Dox with
the reactive moiety was different. Dreher et al. used a
multistep synthesis process to first introduce a reactive
moiety into ELP, then attached Dox via the C-13 carbonyl
group [37]. In this study, doxorubicin was modified by addition
of a maleimide group via a linker to the amine nitrogen at C-30,
allowing conjugation to a unique cysteine residue in a single
step. Third, Dreher et al. used the squamous cell carcinoma
line (FaDu), while in this study, MES-SA uterine sarcoma cells
were used. However, it is unlikely that these differences led to
the enhancement of cytotoxicity by hyperthermia observed in
this study. It is more likely that the thermally induced
cytotoxicity reported here is due to the experimental condi-
tions and to modification of ELP by addition of the cell
Acknowledgements
This work was partially supported by the American Cancer
Society Institutional Research Grant to the University of
Mississippi Medical Center, the Wendy Will Case Cancer Fund,
and The Welch Foundation (Houston, TX).
r e f e r e n c e s
[1] Denny WA. DNA-intercalating ligands as anti-cancer drugs:
prospects for future design. Anticancer Drug Des
1989;4(4):241–63.
[2] Cassidy J, Duncan R, Morrison GJ, Strohalm J, Plocova D,
Kopecek J, et al. Activity of N-(2-
hydroxypropyl)methacrylamide copolymers containing
daunomycin against a rat tumour model. Biochem
Pharmacol 1989;38(6):875–9.

b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 6 2 0 – 6 3 1
631
[3] Maeda H, Seymour LW, Miyamoto Y. Conjugates of
doxorubicin conjugate for cancer therapy. J Control Release
2003;91(1–2):31–43.
[22] Vives E, Brodin P, Lebleu B. A truncated HIV-1 Tat protein
basic domain rapidly translocates through the plasma
membrane and accumulates in the cell nucleus. J Biol
Chem 1997;272(25):16010–7.
[23] Massodi I, Bidwell III GL, Raucher D. Evaluation of cell
penetrating peptides fused to elastin-like polypeptide for
drug delivery. J Control Release 2005;108(2–3):396–408.
[24] Duncan R, Cable HC, Lloyd JB, Rejmanova P, Kopecek J.
Degradation of side-chains of N-(2-
anticancer agents and polymers: advantages of
macromolecular therapeutics in vivo. Bioconjug Chem
1992;3(5):351–62.
[4] Takakura Y, Fujita T, Hashida M, Sezaki H. Disposition
characteristics of macromolecules in tumor-bearing mice.
Pharm Res 1990;7(4):339–46.
[5] Yamaoka T, Tabata Y, Ikada Y. Distribution and tissue
uptake of poly(ethylene glycol) with different molecular
weights after intravenous administration to mice. J Pharm
Sci 1994;83(4):601–6.
[6] Duncan R, Coatsworth JK, Burtles S. Preclinical toxicology
of a novel polymeric antitumour agent: HPMA copolymer-
doxorubicin (PK1). Hum Exp Toxicol 1998;17(2):93–104.
[7] Seymour LW, Duncan R, Kopeckova P, Kopecek J.
Daunomycin- and adriamycin-N-(2-
hydroxypropyl)methacrylamide copolymers by lysosomal
thiol-proteinases. Biosci Rep 1982;2(12):1041–6.
[25] Bidwell III GL, Raucher D. Application of thermally
responsive polypeptides directed against c-Myc
transcriptional function for cancer therapy. Mol Cancer
Ther 2005;4(7):1076–85.
[26] Daniell H, Guda C, McPherson DT, Zhang X, Xu J, Urry DW.
Hyperexpression of a synthetic protein-based polymer
gene. Methods Mol Biol 1997;63:359–71.
[27] Dewhirst MW, Prosnitz L, Thrall D, Prescott D, Clegg S,
Charles C, et al. Hyperthermic treatment of malignant
diseases: current status and a view toward the future.
Semin Oncol 1997;24(6):616–25.
[28] Meyer DE, Chilkoti A. Genetically encoded synthesis of
protein-based polymers with precisely specified molecular
weight and sequence by recursive directional ligation:
examples from the elastin-like polypeptide system.
Biomacromolecules 2002;3(2):357–67.
[29] Minko T, Kopeckova P, Kopecek J. Comparison of the
anticancer effect of free and HPMA copolymer-bound
adriamycin in human ovarian carcinoma cells. Pharm Res
1999;16(7):986–96.
[30] Demoy M, Minko T, Kopeckova P, Kopecek J. Time- and
concentration-dependent apoptosis and necrosis induced
by free and HPMA copolymer-bound doxorubicin in human
ovarian carcinoma cells. J Control Release 2000;69(1):185–
96.
hydroxypropyl)methacrylamide copolymer conjugates;
toxicity reduction by improved drug-delivery. Cancer Treat
Rev 1987;14(3–4):319–27.
[8] Yeung TK, Hopewell JW, Simmonds RH, Seymour LW,
Duncan R, Bellini O, et al. Reduced cardiotoxicity of
doxorubicin given in the form of N-(2-
hydroxypropyl)methacrylamide conjugates: and
experimental study in the rat. Cancer Chemother
Pharmacol 1991;29(2):105–11.
[9] St’astny M, Strohalm J, Plocova D, Ulbrich K, Rihova B. A
possibility to overcome P-glycoprotein (PGP)-mediated
multidrug resistance by antibody-targeted drugs
conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA)
copolymer carrier. Eur J Cancer 1999;35(3):459–66.
[10] Ryser HJ, Shen WC. Conjugation of methotrexate to poly(L-
lysine) increases drug transport and overcomes drug
resistance in cultured cells. Proc Natl Acad Sci USA
1978;75(8):3867–70.
[11] Ohkawa K, Hatano T, Yamada K, Joh K, Takada K, Tsukada
Y, et al. Bovine serum albumin–doxorubicin conjugate
overcomes multidrug resistance in a rat hepatoma. Cancer
Res 1993;53(18):4238–42.
[12] Minko T, Kopeckova P, Pozharov V, Kopecek J. HPMA
copolymer bound adriamycin overcomes MDR1 gene
encoded resistance in a human ovarian carcinoma cell line.
J Control Release 1998;54(2):223–33.
[31] Raucher D, Chilkoti A. Enhanced uptake of a thermally
responsive polypeptide by tumor cells in response to its
hyperthermia-mediated phase transition. Cancer Res
2001;61(19):7163–70.
[13] Kopecek J, Kopeckova P, Minko T, Lu Z. HPMA copolymer–
anticancer drug conjugates: design, activity, and mechanism
of action. Eur J Pharm Biopharm 2000;50(1):61–81.
[14] Kopecek J. Smart and genetically engineered biomaterials
and drug delivery systems. Eur J Pharm Sci 2003;20(1):1–16.
[15] Urry DW, Luan C-H, Parker TM, Gowda DC, Prasad KU, Reid
MC, et al. Temperature of polypeptide inverse temperature
transition depends on mean residue hydrophobicity. J Am
Chem Soc 1991;113:4346–8.
[16] Urry DW. Free energy transduction in polypeptides and
proteins based on inverse temperature transitions. Prog
Biophys Mol Bio 1992;57(1):23–57.
[17] Meyer DE, Kong GA, Dewhirst MW, Zalutsky MR, Chilkoti A.
Targeting a genetically engineered elastin-like polypeptide
to solid tumors by local hyperthermia. Cancer Res
2001;61(4):1548–54.
[18] Fujiwara K, Watanabe T. Effects of hyperthermia,
radiotherapy and thermoradiotherapy on tumor
microvascular permeability. Acta Pathol Jpn 1990;40(2):79–
84.
[19] Gerlowski LE, Jain RK. Effect of hyperthermia on
microvascular permeability to macromolecules in normal
and tumor tissues. Int J Microcirc Clin Exp 1985;4(4):363–72.
[20] Jain RK. Transport of molecules across tumor vasculature.
Cancer Metastasis Rev 1987;6(4):559–93.
[32] Hovorka O, St’astny M, Etrych T, Subr V, Strohalm J, Ulbrich
K, et al. Differences in the intracellular fate of free and
polymer-bound doxorubicin. J Control Release 2002;80(1–
3):101–17.
[33] Putnam D, Kopecek J. Polymer conjugates with anticancer
activity. Adv Polym Sci 1995;122:55–123.
[34] Minko T, Kopeckova P, Kopecek J. Efficacy of the
chemotherapeutic action of HPMA copolymer-bound
doxorubicin in a solid tumor model of ovarian carcinoma.
Int J Cancer 2000;86(1):108–17.
[35] Minko T, Kopeckova P, Kopecek J. Preliminary evaluation of
caspases-dependent apoptosis signaling pathways of free
and HPMA copolymer-bound doxorubicin in human ovarian
carcinoma cells. J Control Release 2001;71(3):227–37.
[36] Duncan R, Vicent MJ, Greco F, Nicholson RI. Polymer–drug
conjugates: towards a novel approach for the treatment of
endrocine-related cancer. Endocrinol Relat Cancer
2005;12(Suppl 1):S189–99.
[37] Kaneko T, Willner D, Monkovic I, Knipe JO, Braslawsky GR,
Greenfield RS, Vyas DM. New hydrazone derivatives of
adriamycin and their immunoconjugates—a correlation
between acid stability and cytotoxicity. Bioconjug Chem
1991;2(3):133–41.
[38] Choksakulnimitr S, Masuda S, Tokuda H, Takakura Y,
Hashida M. In vitro cytotoxicity of macromolecules in
different cell culture systems. J Control Release
1995;34(3):233–41.
[21] Dreher MR, Raucher D, Balu N, Michael Colvin O, Ludeman
SM, Chilkoti A. Evaluation of an elastin-like polypeptide–