Prodrug strategy for PSMA-targeted delivery of TGX-221 to prostate cancer cells

Article abstract of DOI:10.1021/mp3000309

TGX-221 is a potent, selective, and cell membrane permeable inhibitor of the PI3K p110β catalytic subunit. Recent studies showed that TGX-221 has antiproliferative activity against PTEN-deficient tumor cell lines including prostate cancers. The objective of this study was to develop an encapsulation system for parenterally delivering TGX-221 to the target tissue through a prostate-specific membrane aptamer (PSMAa10) with little or no side effects. In this study, PEG-PCL micelles were formulated to encapsulate the drug, and a prodrug strategy was pursued to improve the stability of the carrier system. Fluorescence imaging studies demonstrated that the cellular uptake of both drug and nanoparticles was significantly improved by targeted micelles in a PSMA positive cell line. The area under the plasma concentration time curve of the micelle formulation in nude mice was 2.27-fold greater than that of the naked drug, and the drug clearance rate was 6.16-fold slower. These findings suggest a novel formulation approach for improving site-specific drug delivery of a molecular-targeted prostate cancer treatment.

Full text of DOI:10.1021/mp3000309

Article
pubs.acs.org/molecularpharmaceutics
Prodrug Strategy for PSMA-Targeted Delivery of TGX-221 to Prostate
Cancer Cells
Yunqi Zhao,† Shaofeng Duan,† Xing Zeng,‡ Chunjing Liu,§ Neal M. Davies,∥ Benyi Li,‡
and M. Laird Forrest*,†
†Department of Pharmaceutical Chemistry, The University of Kansas, Simons Laboratories, 2095 Constant Ave. Rm. 136B, Lawrence,
Kansas 66047, United States
‡Department of Urology, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160, United
States
§Higuchi Biosciences Center, The University of Kansas, 2034 Becker Drive, Lawrence, Kansas 66047, United States
∥Faculty of Pharmacy, The University of Manitoba, 750 McDermot Road, Winnipeg, Manitoba R3Y 2N2, Canada
ABSTRACT: TGX-221 is a potent, selective, and cell
membrane permeable inhibitor of the PI3K p110β catalytic
subunit. Recent studies showed that TGX-221 has anti-
proliferative activity against PTEN-deficient tumor cell lines
including prostate cancers. The objective of this study was to
develop an encapsulation system for parenterally delivering
TGX-221 to the target tissue through a prostate-specific
membrane aptamer (PSMAa10) with little or no side effects.
In this study, PEG-PCL micelles were formulated to encapsulate the drug, and a prodrug strategy was pursued to improve the
stability of the carrier system. Fluorescence imaging studies demonstrated that the cellular uptake of both drug and nanoparticles
was significantly improved by targeted micelles in a PSMA positive cell line. The area under the plasma concentration time curve
of the micelle formulation in nude mice was 2.27-fold greater than that of the naked drug, and the drug clearance rate was 6.16-
fold slower. These findings suggest a novel formulation approach for improving site-specific drug delivery of a molecular-targeted
prostate cancer treatment.
KEYWORDS: PEG-PCL micelle, TGX-221, PSMA, target delivery
The synthetic small molecule TGX-221 (Figure 1) is a
potent, selective, and cell membrane permeable inhibitor of the
PI3K p110 beta catalytic subunit, which is critical for cell
growth, proliferation, and tumorigenesis of PTEN-deficient
tumor cells including prostate cancers.12,13 Therefore, PI3K
p110 beta inhibitors have a great promise as novel chemo-
therapeutic agents to treat PTEN deficient cancer cells.13
However, TGX-221 is poorly soluble and requires organic
solvents, such as dimethyl sulfoxide (DMSO) or propylene
glycol, for intravenous injection, which may cause cardiac
toxicity, unconsciousness, arrhythmia, and cardiac arrest.14
The therapeutic index of anticancer drugs is often very
narrow, and the cytotoxic dose of the drug in the desired tissues
can be maintained over an extended period of time with
minimal side effects by targeted delivery and controlled drug
release.15,16 Both passive and ligand-targeted nanoparticles have
been developed for targeted delivery of cancer therapies.17,18
Passively targeted nanoparticles can accumulate to a greater
extent in tumors compared to healthy tissues due to the
INTRODUCTION
■
The phosphatidylinositol 3-kinase (PI3K)/phosphatase and
tensin homologue (PTEN)/Akt pathway is highly involved in
different types of cancer.1 PI3Ks are a family of enzymes that
phosphorylate PI(4,5)P2 (PIP2) to PI(3,4,5)P3 (PIP3). PIP3 is
a lipid-signaling second messenger that further activates its
downstream effectors, such as Akt, PDK1, and Rac1/cdc42.2
The activation of Akt simulates cell growth, proliferation, and
survival.3 PTEN is a phosphatase that dephosphorylates PIP3
back to PIP2.4 The missing function of PTEN results in
accumulation of PIP3 that mimics the activation of PI3K and
triggers cell growth. PTEN deficiency is found in many types of
cancers, such as prostate cancer (LNCaP), brain cancer
(U87MG), and breast cancer (BT549).5−7
There are three classes of PI3K isoforms. The most
commonly studied class I PI3Ks are further divided into class
IA and IB. Only class IA enzymes were clearly implicated in
human cancers. Class IA PI3K is consists of a p110 catalytic
subunit and a regulatory subunit. There are three highly
homologous p110 catalytic isoforms: p110alpha, p110beta, and
p110delta.8,9 p110beta is a promising target in cancer
therapy,10,11 and PTEN-deficient tumor cells mainly depend
on p110beta for signaling and growth, not p110alpha.9
Received: January 16, 2012
Revised: March 23, 2012
Accepted: April 11, 2012
Published: April 11, 2012
© 2012 American Chemical Society
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in water after comixing the drug with an amphiphilic polymer.
Both poly(ethylene glycol) (PEG) and polycaprolactone
(PCL) are FDA approved biocompatible and biodegradable
materials. Micelles formed by PEG-PCL block copolymers have
been used as an effective drug delivery system for lipophilic
molecules.21−23 Upon pooling in the tumor, micelles will slowly
release the drug and then dissolve into nontoxic degradation
products.24 Nanoparticles actively targeted via ligand binding
can target cancer cells that overexpress specific receptors or
proteins.16 The ligands, monoclonal antibodies or aptamers,
can recognize and bind to complementary molecules expressed
on the tumor cells. We hypothesized that the delivery of a
TGX-221 analogue to prostate cancer cells may be improved if
the drug is encapsulated in targeted nanoparticles. The cancer-
targeted nanoparticles should be effective in suppressing tumor
growth and metastasis with reduced or lack of side effects
associated with drug toxicities to normal tissues.
Aptamers are single-or double-stranded oligonucleotides that
are modified to have high binding affinity and specificity to the
targets,25 and they have emerged as a novel class of active
targeting moieties for therapeutic and diagnostic applications in
cancer treatment. Prostate specific membrane antigen (PSMA)
expression is confined primarily to prostate tissues.26,27 The
expression of PSMA in other tissues, such as the brain and
small intestines, is approximately 1000-fold less than that in the
prostate.28 Prostate specific membrane aptamer A10
(PSMAa10) has nanomolar affinity to the membrane
Figure 1. Structure of (a) TGX-221; (b) BL05; (c) BL05-HA; and (d)
BL05-PA.
enhanced permeability and retention (EPR) effect. The high
accumulation of nanoparticles in the tumor tissues is a
consequence of the poorly aligned endothelial cells allowing
nanoparticles to escape from the blood circulatory system and
to pool in the tumor where there is a lack of effective lymphatic
drainage.19,20 Micelles are attractive nanoparticles for the
delivery of hydrophobic drugs, since they form spontaneously
Scheme 1. Synthesis of TGX-221 (Compound 5) and BL05 (Compound 8)
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expressing PSMA, and it can be used to achieve specific
targeting of the nanoparticles to prostate cancer cells.29
Due to the high biocompatibility of PEG-PCL block
copolymers and the specific expression of PSMA on prostate
cancer cells, the combination of PEG-PCL and PSMAa10 in a
nanoparticle delivery system is promising for the targeted
therapy of prostate cancer.
Synthesis of Compound 3. Compound 2 (2.53 g, 7.80
mmol) in dry DMF (50 mL) was mixed with N,N-
diisopropylethylamine (4.0 mL), butyl vinyl ether (5.0 mL),
and dichloro-1,1′-bis(diphenylphosphino)ferrocene palladium-
(II) (0.25 g, 0.20 mmol) at ambient temperature under argon
for ca. 30 min until a homogeneous solution was formed. The
solution then was heated to 120 °C and stirred for another 16
h. After the solution cooled, it was poured into 200 mL of 1 M
HCl at 0 °C. The mixture then was stirred at ambient
temperature overnight and extracted with DCM (2 × 100 mL).
The combined organic phases were washed with water and
dried over Na2SO4. Removal of the solvent under reduced
pressure followed by purification of the resulting residue
through a silica flash column using EtOAc/hexanes 3:1 as the
eluting solvent led to the desired compound with a yield of 60%
(1.35 g).
Synthesis of Compound 4. Compound 3 (1.35 g, 4.69
mmol) in DCM and methanol (5: 1, 84 mL) was combined
with NaBH4 (0.70 g, 18.5 mmol) in five portions at 0 °C . After
10 min, the cooling bath was removed and the mixture was
stirred at ambient temperature for 3 h. Then the mixture was
cooled to 0 °C and 20 mL of H2O was added slowly. The
mixture was extracted with DCM (2 × 50 mL). The combined
organic phases were washed with water (2 × 50 mL) and brine
(30 mL) and then dried over Na2SO4. Removal of the solvent
under reduced pressure followed by purification of the resulting
residue through a silica flash column using EtOAc/hexanes 3:1
as the eluting solvent led to the desired compound with a yield
of 86% (1.17 g).
Synthesis of Compound 7. A solution of compound 4 (1.17
g, 4.05 mmol) in 10 mL of dry DCM was cooled to 0 °C, and
MsCl (1.70 mL, 24.3 mmol) was added dropwise followed by
dry TEA (1.35 mL). After 10 min, the cooling bath was
removed, and the reaction proceeded at ambient temperature
for ca. 4 h until TLC showed that the starting material was
consumed completely. The resulting mixture was washed with
water (50 mL), 2 M aqueous NaOH (50 mL) and brine (50
mL), and then dried over Na2SO4. Removal of the solvent
under reduced pressure followed by purification of the resulting
residue through a silica flash column using EtOAc/hexanes 1:2
as the eluting solvent led to the desired compound as a pale
yellow solid with a yield of 93% (1.39 g).
Synthesis of TGX-221 (Compound 5). A solution of
compound 4 (0.70 g, 1.91 mmol) in 10 mL of dry DCM was
cooled to 0 °C, and aniline (0.5 mL, 16.3 mmol) was added
dropwise followed by dry TEA (1.0 mL). The mixture was
refluxed for 4 h and then stirred at ambient temperature
overnight. The mixture was washed with water (2 × 50 mL)
and brine (50 mL), and then dried over Na2SO4. Removal of
the solvent under reduced pressure followed by purification of
the resulting residue through a silica flash column using
EtOAc/hexanes 3:1 as the eluting solvent led to the desired
compound as a pale yellow solid with a yield of 80% (0.56 g).
Synthesis of 2-(Phenylamino)ethanol (Compound 6). We
followed the procedure of Bhanu Prasad and Gilbertson30
Briefly, a mixture of aniline (4.66 g, 50 mmol) and 2-
bromoethanol (4.34 g, 33 mmol) was heated at 90 °C under an
argon atmosphere for 4 h. The resulting solid was dissolved in
ethyl acetate (100 mL), washed with 2 M aqueous NaOH (3 ×
20 mL) followed by brine (50 mL), and then dried over
Na2SO4. Removal of the solvent under reduced pressure
followed by purification of the resulting residue through a silica
MATERIALS AND METHODS
■
1. Materials. Azide poly(ethylene glycol) (MW: 5800) was
purchased from Polymer Source Inc. (Quebec, Canada).
Propargyl-dPEG1-NHS ester was purchased from Quanta
BioDesign Ltd. (Powell, Ohio). PSMAa10 {5′-[NH2-(CH2)6-
PEG18-GGG AGG ACG AUG GGG AUC AGC CAU GUU
UAC GUC ACU CCU UGU CAA UCC UCA UCG GC
inverted T]-3′, 2′F pyrimidines} was custom synthesized by
Integrated DNA Technologies (Coralville, IA). ε-Caprolactone,
IR-820, pyrene, 1-amino-3-butyne, and 1 M HCl in diethyl
ether were purchased from Sigma-Aldrich Co. (St. Louis, MO).
Resazurin blue, silica gel, and the organic solvents were
purchased from Fisher Scientific (Pittsburgh, PA).
2. TGX-221 and Prodrug Synthesis. All chemicals were
used as received unless stated otherwise. NMR spectra were
taken on a 400 MHz Bruker with the solvent peak as an internal
reference. Mass spectra were run in the electrospray ionization
mass spectrometry (ESI-MS) mode or atmospheric pressure
chemical ionization mass spectrometry (APCI-MS) mode.
Reactions that required an inert atmosphere were carried out
under dry argon with flame-dried glassware. Tetrahydrofuran
(THF) was freshly distilled over sodium benzophenone.
Dichloromethane (DCM), N,N-dimethylformaldehyde
(DMF), and triethylamine (TEA) were freshly distilled over
CaH2. The synthetic scheme of TGX-221 and its analogue
BL05 are shown in Scheme 1.
Synthesis of Compound 1. Malonyl dichloride (3.2 mL,
32.0 mmol) was added dropwise to a solution of 2-amino-3-
bromo-5-methylpyridine (5 g, 26.7 mmol) in dry DCM (50
mL) cooled to 0 °C. The mixture was stirred at ambient
temperature (ca. 22 °C) for 48 h. The yellow precipitate was
collected by filtration, washed with DCM (3 × 50 mL), and
dried under reduced pressure. The desired compound was
obtained with a yield of 86% (4.88 g), and identity was
1
confirmed by H NMR. The crude compound was used in the
next step without any further purification. The filtrate was
concentrated by rotovaporation. The resulting residue was
suspended in 100 mL of H2O, and the suspension was stirred at
ambient temperature for 1 h. The suspension was filtered, and
the filtrate was neutralized with solid Na2CO3 to recover the
unreacted 2-amino-3-bromo-5-methylpyridine (0.84 g).
Synthesis of Compound 2. Compound 1 (4.88 g, 19.13
mmol) was suspended in dry DCM (100 mL), and TEA (5.40
mL, 38.3 mmol) was added dropwise at 0 °C followed by
methanesulfonyl chloride (MsCl) (2.90 mL, 26.8 mmol). The
mixture was stirred at ambient temperature for 1 h. Morpholine
(5.0 mL, 57.4 mmol) was added, and the mixture was refluxed
for 24 h. The solvent then was removed under reduced
pressure, and the mixture was diluted with H2O to afford a pale
yellow precipitate. The solid was collected by filtration, washed
with 2 × 50 mL of H2O, and dried under reduced pressure. The
residue was purified through a silica flash column using EtOAc/
hexanes 3:1 as the eluting solvent. The desired compound was
obtained as a yellow solid with a yield of 42% (2.61 g).
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flash column using EtOAc/hexanes 1:2 as the eluting solvent
led to the desired compound with a yield of 89% (4.03 g).
Synthesis of Compound 8 (BL05). A solution of compound
5 (0.47 g, 1.28 mmol) in 10 mL of dry DCM was cooled to 0
°C, and a solution of 2-(phenylamino)ethanol (0.70 g, 5.12
mmol) in 5 mL of dry DCM was added dropwise followed by
dry TEA (1 mL). After 10 min, the mixture was refluxed for ca.
15 h until TLC showed that the starting material was consumed
completely. The resulting mixture was washed with 2 M
aqueous HCl, water, saturated NaHCO3 aqueous solution and
brine, and then dried over Na2SO4. Removal of the solvent
under reduced pressure followed by purification of the resulting
residue through a silica flash column using EtOAc/hexanes 5:1
as eluting solvent led to the desired compound with a yield of
69% (0.36 g).
Synthesis of BL05-Palmitate Derivative (BL05-PA). BL05
(compound 8) (0.10 g, 0.24 mmol) and palmatic anhydride
(0.24 g, 0.48 mmol) were suspended in 10 mL of dry DCM,
and 0.20 mL of dry pyridine was added dropwise. The mixture
was stirred overnight at ambient temperature until TLC
showed that the starting material was consumed completely.
The resulting mixture was washed with 2 M aqueous HCl (30
mL), water, saturated NaHCO3 aqueous solution, and brine.
Removal of the solvent under reduced pressure followed by
purification of the resulting residue through a silica flash
column using EtOAc/hexanes 3:1 as the eluting solvent led to
the desired compound with a yield of 95% (0.15 g).
Synthesis of BL05-Hexanoate Derivative (BL05-HA). A
solution of BL05 (compound 8) (0.18 mg, 0.45 mmol) in 5 mL
of dry DCM was cooled to 0 °C, and hexanoyl chloride (0.26
mL, 1.80 mmol) was added followed by dry pyridine (0.38
mL). After 10 min, the cooling bath was removed, and the
reaction proceeded at ambient temperature overnight until
TLC showed that the starting material was consumed
completely. The resulting mixture was washed with 2 M
aqueous HCl, water, saturated NaHCO3 aqueous solution, and
brine. The organic phase was dried over Na2SO4. Removal of
the solvent under reduced pressure followed by purification of
the resulting residue through a silica flash column using
EtOAc/hexanes 2:1 as the eluting solvent led to the desired
compound as a pale yellow solid with a yield of 91% (0.266 g).
3. Synthesis of Azide Poly(ethylene glycol)-block-
Poly(ε-caprolactone) Copolymers (N3-PEG-PCL). All glass-
ware was flamed dried under vacuum and was handled under a
dry argon stream. The N3-PEG-PCL copolymer was prepared
by acid catalyzed ring-opening polymerization reaction of ε-
caprolactone in the presence of N3-PEG-OH as an initiator.
The typical process is described as follows. N3-PEG-OH was
azeotropically dried using anhydrous toluene under reduced
pressure. ε-Caprolactone was dried over CaH2 overnight and
then distilled under vacuum. N3-PEG-OH (0.375 g, 0.0647
mmol) was dissolved in 8 mL of dry DCM, followed by the
addition of 0.75 mL of ε-caprolactone (100 equiv). The
polymerization was initiated by the addition of 1 M HCl in
diethyl ether (0.8 mL, 3 equiv) at 25 °C. After 24 h, the mixture
was poured into diethyl ether to precipitate the copolymer.
Then, the N3-PEG-PCL copolymer was further purified by
precipitation in cold acetone. The residual solvent was removed
under reduced pressure.
performed on a Shodex GPC LF-804 column thermostatted at
40 °C with DMF and 10 mM LiCl as the mobile phase at a flow
rate of 0.8 mL/min. Peaks were detected using a refractive
index detector (RID-10A, Shimadzu). Narrow molecular weight
distribution poly(ethylene glycols) (Scientific Polymer Prod-
ucts Inc., Ontario, NY) were used as standards for GPC
analysis.
5. Micelle Preparation. The N3-PEG-PCL (15 mg, 1.3
mmol) was dissolved in 0.5 mL of dimethylacetamide (DMAc),
and the organic solution was added dropwise to 2 mL of
ddH2O with mechanical stirring. The organic solvent was
removed by overnight dialysis against phosphate buffered saline
(PBS) using 10 000 molecular weight cutoff (MWCO) dialysis
tubing (Spectrum Laboratories, Rancho Dominguez, CA).
Drug-loaded micelles were prepared by mixing the drugs with
the copolymer in DMAc before adding ddH2O.
The aptamer, PSAMa10, was functionalized by addition of an
alkyne group using the following procedure. PSMAa10 (0.2
μmol, 3.7 mg) was dissolved in 500 μL of 0.1 M carbonate
buffer (pH 9.0). The alkyne-NHS ester (2.3 mg) was dissolved
in 60 μL of DMSO, and 6 μL of the alkyne-modified NHS ester
solution was added to the carbonate buffer containing the
aptamer. After mixing for 4 h at ambient temperature, the
alkyne modified PSMAa10 was purified using a centrifugal
filtration device (Amicon Ultra Centrifugal Filter 10K,
Millipore Corp.).
The near-infrared dye, IR-820, was modified to include an
alkyne by conjugation of 1-amino-3-butyne. Briefly, 150 mg of
IR-820 (0.177 mmol) was dissolved in 10 mL of DMF. Then,
61 mg of 1-amino-3-butyne (5 equiv) and 123 μL of TEA (5
equiv) were added. The solution was stirred at 40 °C for 8 h.
The final product was purified by silica flash chromatography
using EtOAc/methanol 1:2 as the elution solvent.
Both alkyne modified PSMAa10 and IR-820 were conjugated
to the micelle’s surface by azide alkyne Huisgen cycloaddition
(i.e., click reaction). Briefly, 50 μg of alkyne modified PSMAa10
and 100 μg of alkyne modified IR-820 were added to N3-PEG-
PCL micelles (2.5 mg/mL) prepared in ddH2O. Copper sulfate
(0.6 μmol/mL) and sodium ascorbate (3.0 μmol/mL) were
used as a catalyst. The reaction mixture was gently stirred at
ambient temperature overnight. The modified micelles were
purified by dialysis against PBS using 10 000 MWCO dialysis
tubing (SnakeSkin Pleated Dialysis Tubing, Thermo Scientific)
for 24 h. To determine the extent of the reaction, the
supernatant was separated from the micelle solution using a
Microcon YM-100 centrifugal concentrator (0.5 mL capacity,
NMWL 100 000, Millipore Corp.) at 11 000g for 15 min. The
filtrate was analyzed by high-performance liquid chromatog-
raphy (HPLC) (LC-2010CHT, Shimadzu).
6. Micelle Characterization. The critical micelle concen-
tration (CMC) of N3-PEG-PCL micelles was determined by
measuring the excitation ratio of pyrene using a fluoropho-
tometer (RF-5301 PC, Shimadzu). For example, solutions of
N3-PEG-PCL micelles were prepared by serial dilution and
then incubated with 0.6 μM pyrene for 1 h at 65 °C and then
18 h at ambient temperature in the dark. The fluorescent
emission of pyrene was measured at 390 nm. The fluorescent
excitement ratio of pyrene at 339 and 334 nm changes in
response to the probe’s microenvironment polarity. A sharp
increase in the 339/334 excitation ratio indicates the CMC as
the pyrene preferentially partitions into the hydrophobic core
of N3-PEG-PCL micelles.
4. Characterization of the Polymeric Material. The N3-
1
PEG-PCL was dissolved in CDCl3 for H NMR spectroscopy.
Gel permeation chromatography (GPC) was used to determine
the polymer molecular weight distribution. GPC analysis was
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The formation of micelles was determined by GPC (Shodex
OHpak SB-803 HQ, Showa Denko America, Inc.) using a
Shimadzu 2010CHT system. GPC was performed using
ddH2O as the mobile phase with a flow rate of 0.8 mL/min,
and the GPC column was thermostatted at 40 °C. Elution
peaks were detected using an evaporative light scattering
detector (ELSD-LTII, Shimadzu). The size and polydispersity
of micelles were measured with a ZetaPALS (Brookhaven
Instruments Corp.) using the Gaussian distribution.
volume of drug-loaded micelles in the dialysis tubing was 5 mL,
and the sink solution was 4 L. The PBS was changed several
times per day to maintain sink conditions. After predetermined
time intervals, samples were withdrawn from the dialysis tubing
and analyzed by HPLC with a reversed phase column (TSK-
GEL ODS-100Z, Tosoh Bioscience) at 50 °C with UV
detection at 280 nm. The chromatography conditions were a
mobile phase A of 100% ddH2O and B of 100% methanol at a
flow rate of 1 mL/min. The gradient program was (i) a linear
gradient from 50% to 90% solvent B over 5 min; (ii) 3.5 min
with 90% solvent B; (iii) 6.5 min with 100% solvent B; and (iv)
10 min with 50% solvent B. Retention times: TGX-221 6.86
min; BL05 6.93 min; BL05-HA 10.14 min; and BL05-PA 17.04
min.
10. Uptake of PSMA-Targeted Micelles by Prostate
Cancer Cells. Fluorescence microscopy was used to examine
cellular uptake of IR-820-labeled PSMA-targeted micelles.
PSMA negative PC3 cells and PSMA positive LNCaP cells
were seeded at a density of 1 × 106 cells in an 8.6 cm2 chamber
slide system (Thermo Scientific). After cells had attached to the
microscope slide’s surface, the PC3 and LNCaP cells were
incubated with the PSMA-targeted (2.5% weight compared
with polymer concentration) or nontargeted micelles for 6 h at
37 °C. Unbound micelles were removed by washing three times
with PBS. Fluorescence images were acquired using a Cy5 filter
set on a Nikon Eclipse 80i microscope equipped for
epifluorescence and an Orca ER camera (Hamamatsu Inc.,
Bridgewater, NJ).
The cellular uptake and accumulation of PSMA targeted
micelles (2.5% weight compared with polymer concentration)
was quantified in LNCaP and PC3 cells. Cells were seeded in
96-well flat-bottom plates at a concentration of 5000 cells per
well in 90 μL of growth medium. After the cells attached to the
surface, 10 μL of targeted or nontargeted IR-820-modified
micelles was added at different concentrations. After 6 h of
incubation, the growth medium was removed and each well was
washed with PBS. The plate fluorescence was measured using a
fluorophotometer at an excitation wavelength of 675 nm and an
emission wavelength of 750 nm.
11. Pharmacokinetic Evaluation. Male Balb/c mice were
maintained in a temperature-controlled room with a 12 h light/
dark cycle for at least 1 week prior to the study. The animal
protocol was approved by the Institutional Animal Care and
Use Committee (IACUC) at the University of Kansas Medical
Center. Animals were administered BL05-HA loaded PSMA-
targeted micelles or BL05-HA prepared in propylene glycol at a
dose of 30 mg/kg via tail vein injection (100 μL). Blood
samples were withdrawn after 5 min and 1, 2, 4, 6, 12, and 24 h
from the saphenous vein. A 20 μL blood sample was added to
50 μL of Alsever’s solution, which is an anticoagulant. After
gently mixing, the blood sample was centrifuged at 3000 rpm
for 10 min. Plasma was prepared by solid phase extraction
(SPE) prior to the HPLC analysis. Briefly, plasma sample
diluted with 2% NH4OH (1:3) was applied to the SPE column
(Bond Elut-C18, Agilent Technologies, Lake Forest, CA) that
was pretreated with 100% MeOH and equilibrated with 100%
ddH2O. Sample was washed with 5% MeOH in ddH2O and
eluted twice with 150 μL 100% MeOH.
The drug loading efficiency (DL %) and encapsulation
efficiency (EE %) of BL05-HA in PEG-PCL micelles were
calculated according to the following equations:
weight of the drug in micelle
weight of the polymer and drug
DL % =
EE % =
× 100%
weight of the drug in micelle
weight of the feeding drug
× 100%
GPC micelle fraction was collected and dried by speed-vap.
The dried micelle fraction was weighed to determined weight of
the polymer and drug. Then, the dried micelle was redissolved
in methanol and drug weight in micelle was evaluated by HPLC
analysis.
7. Cytotoxicity Assay. The prostate cancer cell lines
DU145 and LNCaP were maintained in RPMI-1640 medium,
and PC3 cells were maintained in F-12K medium (ATCC,
Manassas, VA). LNCaP is a PSMA positive cell line, whereas
DU145 and PC3 are PSMA negative. Both were supplemented
with 10% fetal bovine serum (Hyclone Laboratory Inc., Logan,
Utah). Cells were plated in 96-well flat-bottomed plates at a
concentration of 5000 cells per well in 90 μL of growth
medium. After 12 h, TGX-221, BL05, or BL05-HA loaded
micelles in PBS were added at concentrations of 0, 0.1, 1, 5, 10,
50, or 100 μM. PBS and 10 μL of trichloroacetic acid (TCA)
were added to negative and positive control wells, respectively.
After 72 h, 10 μL of 55 μM resazurin blue was added to each
well and incubated at 37 °C for 4 h. After incubation, the
resorufin product was measured with a fluorophotometer
(SpectraMax Gemini; Molecular Devices, CA) using an
excitation wavelength of 560 nm and an emission wavelength
of 590 nm. The IC50 was determined as the midpoint between
positive and negative control groups for each plate using
GraphPad Prism 5 software (GraphPad Software Inc., San
Diego, CA).
8. Western Blot Assay. After serum starvation for 16 h,
LNCaP cells were treated with 2 μM TGX-221 or BL05 and
100 ng/mL EGF for 30 min. Then, cells were pelleted and
lysed with cell lysis buffer containing protease inhibitors.
Protein concentrations were determined by BCA assays, and
equal amounts of protein extract were separated on a 10% SDS
polyacrylamide gel, transferred to a nitrocellulose membrane,
and immunoblotted with anti-pAkt (Ser-473) and Akt (Cell
Signaling Technology, Inc., Danvers, MA) antibodies followed
by HRP-conjugated secondary antibodies (Santa Cruz Biotech,
Inc., Santa Cruz, CA). The specific bands were detected using a
chemiluminescence luminol reagent (Santa Cruz Biotech, Inc.,
Santa Cruz, CA).
9. In Vitro Drug Release Study. The in vitro release of the
drug from N3-PEG-PCL micelles into PBS (pH 7.4) was
monitored by a dialysis method.31 Dialysis was carried out at 37
°C under sink conditions using 10 000 MWCO dialysis tubing
(SnakeSkin, Thermo Scientific Inc., Rockford, IL). The initial
12. Statistical Analysis. GraphPad Prism 5 software was
used for statistical analysis. A t test was used to test for
differences between two data sets, while a one-way ANOVA
and Tukey post test were used to analyze the differences when
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more than two data sets were compared. In all comparisons,
statistical significance was set at p ≤ 0.05.
8.7 Hz, 1H), 6.81 (d, J = 8.2 Hz, 2H), 6.74 (t, J = 7.2 Hz, 1H),
5.61 (s, 1H), 5.60−5.57 (m, 1H), 4.16−4.09 (m, 1H), 4.00−
3.93 (m, 1H), 3.67−3.59 (m, 5H), 3.58−3.49 (m, 1H), 3.41 (t,
J = 4.9 Hz, 4H), 2.34 (d, J = 0.9 Hz, 3H), 2.28 (t, J = 7.5 Hz,
2H), 1.66 (d, J = 7.2 Hz, 4H), 1.61 (t, J = 7.2 Hz, 2H), 1.35−
1.22 (bs, 23H), 0.90 (t, J = 7.0 Hz, 3H). 13C NMR (CDCl3,
100 MHz): δ = 14.1, 17.5, 18.4, 22.7, 24.9, 29.2, 29.3, 29.4
(×2), 29.5 (×2), 29.6 (×2), 29.7(×2), 31.9, 34.2, 44.0, 44.4
(×2), 51.8, 61.8, 66.5 (×2), 81.3, 113.1 (×2), 117.4, 121.7,
124.2, 129.3 (×2), 135.7, 136.0, 147.7, 147.9, 159.0, 160.1,
173.7. HRMS (ESI) Calcd for C39H59N4O4 (M +H)+:
647.4536. Found: 647.4528.
RESULTS
■
1. Drug Synthesis. We successfully synthesized TGX-221,
BL05, and the fatty acid prodrugs. The synthesis of TGX-221
was accomplished with 2-amino-3-bromo-5-methylpyridine and
malonyl dichloride as the starting materials through six steps
with an overall yield of 14% and BL05 with an overall yield of
1
12%. The structure of each compound was determined by H
NMR, 13C NMR or together with high resolution APCI-MS or
ESI-MS.
1
Compound 1. 1H NMR (DMSO-d6, 400 MHz): δ = 8.74 (s,
1H), 8.29 (s, 1H), 5.55 (s, 1H), 2.34 (s, 3H), which was
consistent with the reported data.
BL05-HA. H NMR (CDCl3, 400 MHz): δ = 8.71 (s, 1H),
7.42 (d, J = 1.8 Hz, 1H), 7.23 (d, J = 8.8 Hz, 1H), 7.22 (d, J =
8.7 Hz, 1H), 7.21 (d, J = 8.7 Hz, 1H), 6.81 (d, J = 8.2 Hz, 2H),
6.74 (t, J = 7.2 Hz, 1H), 5.61 (s, 1H), 5.60−5.57 (m, 1H),
4.17−4.09 (m, 1H), 4.00−3.93 (m, 1H), 3.70−3.58 (m, 5H),
3.41 (t, J = 4.9 Hz, 4H), 2.34 (d, J = 0.9 Hz, 3H), 2.28 (t, J =
7.4 Hz, 2H), 1.66 (d, J = 7.0 Hz, 3H), 1.64−1.58 (m, 1H),
1.38−1.26 (m, 5H), 0.91 (t, J = 6.8 Hz, 3H). 13C NMR
(CDCl3, 100 MHz): δ = 13.9, 17.4, 18.4, 22.3, 24.6, 31.3, 34.1,
44.0, 44.4 (×2), 51.8, 61.8, 66.5 (×2), 81.3, 113.1 (×2), 117.4,
121.7, 124.2, 129.3 (×2), 135.7, 136.0, 147.7, 147.9, 159.0,
160.1, 173.7. HRMS (ESI) Calcd for C29H39N4O4 (M + H)+:
507.2971. Found: 507.2955.
1
Compound 2. H NMR (CDCl3, 400 MHz): δ = 8.72 (s,
1H), 7.87 (d, J = 1.9 Hz, 1H), 5.61 (s, 1H), 3.84−3.80 (m,
4H), 3.74−3.69 (m, 4H), 2.35 (s, 3H), which was consistent
with the reported data.
1
Compound 3. H NMR (CDCl3, 400 MHz): δ = 8.88 (s,
1H), 7.86 (d, J = 2.2 Hz, 1H), 5.66 (s, 1H), 3.84−3.79 (m,
4H), 3.67−3.62 (m, 4H), 2.79 (s, 3H), 2.38 (d, J = 1.0 Hz,
3H), which was consistent with the reported data.
1
Compound 4. H NMR (CDCl3, 400 MHz): δ = 8.67 (s,
1H), 7.50 (d, J = 1.9 Hz, 1H), 5.63 (s, 1H), 5.25−5.18 (m,
1H), 4.63 (d, J = 5.0 Hz, 1H), 3.82 (t, J = 2.9 Hz, 4H), 3.61 (t, J
= 4.2 Hz, 4H), 2.35 (d, J = 1.0 Hz, 3H), 1.63 (d, J = 6.6 Hz,
3H). 13C NMR (CDCl3, 100 MHz): δ = 18.1, 22.1, 44.5 (×2),
49.1, 66.4 (×2), 81.1, 117.4, 122.3, 135.3, 137.3, 147.5, 159.0,
160.2. HRMS (ESI) Calcd for C15H19N3 NaO3 (M + Na)+:
312.1324. Found: 312.1329.
2. PEG-PCL Micelle Preparation and Characterization.
The N3-PEG-PCL block copolymer was synthesized by a ring-
1
opening reaction. H NMR spectra showed two main peaks at
3.67 (−OCH2 CH2 ) and 4.08 (−OCH2 CH2 CH2 -
CH2CH2CH2CO−) that correspond to PEG and PCL,
respectively. The molecular weight of N3-PEG-PCL copolymer
determined by 1H NMR was 5800:5800, and the polydispersity
of this polymer measured by GPC was 1.10. The CMC was
determined to be 300 nM using the pyrene incorporation assay.
Based on Gibb’s free energy of micellization, the nanomolar
CMC of PEG-PCL copolymer indicated that the micelle had
high thermodynamic stability. For 10% (w/w) BL05-HA
loaded PEG-PCL micelles, DL % was 9.5 0.2% and EE %
was 74.5 4.6%.
The N3-PEG-PCL micelles were prepared using a solvent
displacement method. The PCL block served as a hydrophobic
core of the micelle and physically entrapped the poorly water-
soluble anticancer drug into the micelle. GPC was used to
confirm the formation of PEG-PCL micelles. PEG-PCL
micelles are known to be stable when diluted below the
CMC during GPC analysis, which reflects a high kinetic
stability against dissociation.32 The micelles’ hydrodynamic
diameter from DLS with Gaussian volume weighting was
approximately 50 nm (Table 1), which is suitable for
intravenous administration and extravasation in tumors.33
3. Conjugation of PSMAa10 to PEG-PCL Micelles. The
alkyne modified aptamer, PSMAa10, was confirmed by ESI-MS
([M+Na]+ = 18 928.0). The alkyne modified PSMAa10 was
conjugated to the azide end group of the PEG-PCL copolymer
by the Huisgen cycloaddition click reaction. The conjugation
efficiency was determined by quantifying the unconjugated
ligand remaining in the supernatant. The amount of PSMAa10
conjugated to the N3-PEG-PCL micelle surface increased with
increasing reaction time: 1 h, 0.017 0.005 (yield: 14.2%); 3 h,
0.103 0.010 (yield: 20.6%); 5 h, 0.123 0.015 (yield: 24.6%)
mg PSMAa10/mg N3-PEG-PCL. Since precipitation occurred
after 5 h, the Click reaction time was controlled at 3 h. The
conjugation of PSMAa10 to the micelle’s surface was examined
Compound 5 (TGX-221). 1H NMR (CDCl3, 400 MHz): δ =
8.67 (s, 1H), 7.60 (d, J = 2.0 Hz, 1H), 7.13 (d, J = 7.4 Hz, 1H),
7.12 (d, J = 6.6 Hz, 1H), 6.68 (t, J = 7.3 Hz, 1H), 6.48 (d, J =
7.6 Hz, 2H), 5.68 (s, 1H), 5.15 (d, J = 5.6 Hz, 1H), 4.39 (bs,
1H), 3.81 (t, J = 5.0 Hz, 4H), 3.72−3.62 (m, 4H), 2.26 (d, J =
1.0 Hz, 3H), 1.59 (d, J = 6.8 Hz, 3H). 13C NMR (CDCl3, 100
MHz): δ = 18.3, 22.1, 44.6 (×2), 49.1, 66.6 (×2), 81.3, 113.2
(×2), 117.6, 122.5, 123.8, 129.2 (×2), 135.4, 137.3, 146.8,
147.5, 159.1, 160.2. HRMS (ESI) Calcd for C21H25N4O2 (M +
H)+: 365.1978. Found: 365.1975.
Compound 6. 1H NMR (CDCl3, 400 MHz): δ = 7.22 (dd, J
= 7.4, 7.3 Hz, 2H), 6.78 (d, J = 7.3 Hz, 1H), 6.69 (d, J = 7.7 Hz,
2H), 3.84 (t, J = 5.1 Hz, 2H), 3.32 (t, J = 4.9 Hz, 2H), 3.03 (bs,
2H).
Compound 7. 1H NMR (CDCl3, 400 MHz): δ = 8.89 (dd, J
= 2.1, 1.1 Hz, 1H), 7.87 (d, J = 2.2 Hz, 1H), 5.88 (q, J = 6.8 Hz,
1H), 5.68 (s, 1H), 3.82 (t, J = 5.2 Hz, 4H), 3.65 (t, J = 5.1 Hz,
4H), 3.16 (s, 3H), 2.39 (d, J = 0.9 Hz, 3H), 1.88 (d, J = 6.8 Hz,
3H). HRMS (ESI) Calcd for C16H22N3O5 S (M + H)+:
368.1280. Found: 368.1276.
1
Compound 8 (BL05). H NMR (CDCl3, 400 MHz): δ =
8.70 (s, 1H), 7.45 (s, 1H), 7.22 (d, J = 8.6 Hz, 1H), 7.20 (d, J =
8.5 Hz, 1H), 6.86 (d, J = 8.2 Hz, 2H), 6.76 (t, J = 6.9 Hz, 1H),
5.64−5.60 (m, 1H), 5.59 (s, 1H), 3.63−3.57 (m, 4H), 3.54 (t, J
= 6.0 Hz, 2H), 3.44 (t, J = 5.6 Hz, 2H), 3.41−3.35 (m, 4H),
2.34 (s, 3H), 1.62 (d, J = 7.0 Hz, 3H). 13C NMR (CDCl3, 100
MHz): δ = 16.9, 18.4, 44.4 (×2), 47.3, 52.3, 60.0, 66.5 (×2),
81.3, 114.2 (×2), 117.8, 121.7, 124.3, 129.2 (×2), 135.5, 136.5,
148.0, 148.2, 159.0, 160.1. HRMS (ESI) Calcd for C23H29N4O3
(M + H)+: 409.2240. Found: 409.2229.
1
BL05-PA. H NMR (CDCl3, 400 MHz): δ = 8.71 (s, 1H),
7.42 (d, J = 1.8 Hz, 1H), 7.23 (d, J = 8.7 Hz, 1H), 7.21 (d, J =
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Table 1. Characterization of Different PEG-PCL Micelle
Formulations
Table 2. IC50 of TGX-221, BL05, and BL05-HA in Different
Prostate Cancer Cell Lines
a
a
size (nm)
(mean SD)
in vitro release
half-life
TGX-221 (μM)
(mean SE)
BL05 (μM)
(mean SE)
BL05-HA (μM)
(mean SE)
empty micelle
27.6 0.2
40.5 0.8
54.5 0.3
DU145
PC3
35.6 0.12
18.2 0.85
3.98 0.18
32.4 0.67
23.4 0.74
4.036 0.11
52.5 0.15
21.8 0.66
3.19 0.12
PSMA-targeted micelle
TGX-221 loaded micelle
(5% w/w)
<1 h
LNCaP
a
All of the drugs showed selective cytotoxicity to PSMA-positive
LNCaP cells (n = 6).
BL05 loaded micelle (5% w/w)
59.8 2.2
49.9 0.5
<1 h
BL05-HA loaded micelle
(5% w/w)
5 days
BL05-PA loaded micelle
(5% w/w)
29.9 0.1
58.8 0.9
6.5 days
BL05-HA loaded PSMA-targeted
micelle
a
All of the formulations had particle sizes that were smaller than 100
nm. The in vitro release half-life was improved by encapsulating more
hydrophobic prodrugs.
Figure 2. Western blot analysis of Akt phosphorylation on Ser473 and
total Akt protein levels with different treatments. Both TGX-221 and
BL05 showed inhibitory effects on the phosphorylation of Akt.
by agarose gel electrophoresis (data not shown). The near-
infrared fluorophore IR-820 was functionalized with an alkyne
by TEA buffered condensation with 1-amino-3-butyne. The
successful conjugation was confirmed by ESI-MS. The micelle
surface also was modified with IR-820 by click reaction. The IR-
820 conjugated micelles had a maximum absorption wavelength
of 675 nm and an emission wavelength of 745 nm.
The cytotoxicity of empty micelles, PSMA-targeted empty
micelles, IR-820-labeled empty micelles, and IR-820-labeled
PSMA-targeted micelles was examined in DU145 prostate
cancer cells (Figure 3). The drug free targeted micelles did not
inhibit cell growth. However, the dye-labeled micelles did
inhibit cell growth, so they were not characterized further in
vivo.
4. Characterization of Conjugated PEG-PCL Micelles.
Particle sizes of different micelle formulations were determined
by dynamic light scattering (Table 1). The sizes of all of the
nanoparticle formulations were smaller than 100 nm, which is
suitable for intravenous application. The in vitro release half-
lives of TGX-221, BL05, BL05-HA, and BL05-PA were
investigated at simulated in vivo conditions using a bath at
pH 7.4 and 37 °C (Table 1). In addition, the release half-lives
of TGX-221 and its analogue, BL05, from PEG-PCL micelles
were identical, which is likely due to the similar lipophilicity of
the two compounds. TGX-221 had a calculated log Po/w of 2.14
0.68, and BL05 was 1.81 0.67 (ALogPS 2.1, http://www.
vcclab.org). The more hydrophobic ester prodrugs, BL05-HA
(log Po/w 4.15 0.31) and BL05-PA (log Po/w 8.42 0.83),
had much longer release half-lives of 5 and 6.5 days in PEG-
PCL micelles, respectively. The prodrugs tended to stay in the
hydrophobic PCL core of the micelle, and they were protected
from hydrolysis in the hydrophobic core due to limited
penetration of water. The aptamer conjugated micelle was
larger than the nontargeted micelle, and the conjugation of IR-
820 was confirmed by observing the increased Stokes shift of
fluorescent dye. The Stokes shift of IR-820 increased after
conjugation to the micelle surface due to electromagnetic
retardation.34
5. In Vitro Cytotoxicity Studies. The IC50's of TGX-221,
BL05, and BL05-HA were determined in DU145, PC3, and
LNCaP cells (Table 2). BL05-PA was not soluble in PBS with
1% DMSO, and it was not tested further. TGX-221, BL05, and
BL05-HA showed selective cytotoxicity to LNCaP cells, which
may be due to the deficiency of PTEN in this cell line and the
accumulation of PIP3 in the cells. A Western blot was
performed to determine if both TGX-221 and BL05 are
involved in the same cell signaling pathway that contributes to
the selective cytotoxicity. As demonstrated by Western blot,
TGX-221 and BL05 inhibited phosphorylation of Akt (Figure
2), which in turn indicated the inhibitor of PI3K activity.
Figure 3. Cytotoxicity of empty micelles in DU145 cells (mean
SD).
6. Micelle Formulation Effects on Cellular Uptake of
the Drug. The DU145 cells were treated with BL05-HA in
DMSO and BL05-HA loaded in nontargeted micelles. The
solubility of BL05-HA in 5% DMSO was determined to be
32.35 μg/mL (49.86 μM). After a 12 h treatment, the cell
culture medium was analyzed for extracellular drug content by
HPLC, and the drug uptaken by the cells was quantified
(Figure 4). Micelle formulations significantly improved the
cellular uptake at all concentrations, and also DMSO was not
required to solubilize BL05-HA in the micelle formulation.
7. PSMAa10 Targeting Improved Cellular Uptake of
Both Micelles and Drug by PSMA-Positive Cells. LNCaP
cells (PSMA+) were treated with PSMA-targeted and IR-820-
labeled micelles with increasing substitutions of the aptamer.
The micelle uptake was estimated by the relative fluorescence
intensity of the IR-820. The fluorescence increased with
increasing aptamer substitution: 1%, 325
103.410; 2.5%,
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Figure 4. Micelle formulation improved DU145’s uptake of BL05-HA
(mean SD) (*, p < 0.001).
574.554 33.614; 5%, 704.055 73.235 and 10%, 945.311
145.445 RFU.
Confocal microscopy was used to examine if PSMA targeting
can improve the micelle uptake by PSMA presenting cells. The
aptamer did not improve the micelle uptake in the PSMA-
negative PC3 cells. However, in the PSMA-positive LNCaP
cells, we detected more fluorescence signal in cells treated with
PSMA-targeted micelles (Figure 5). The uptake was then
quantified with a fluorescence plate reader. The fluorescence
signal was significantly higher in PSMA-positive cells (LNCaP)
treated with PSMA-targeted micelles. However, in PSMA-
negative PC3 cells, the fluorescence was not enhanced by
addition of PSMA to the micelles (Figure 6).
The effect of PSMA-targeting of micelles on the cellular
uptake of BL05-HA was examined in both DU145 (PSMA−)
and LNCaP (PSMA+) cells (Figure 7). PSMA-targeted
micelles significantly improved the drug uptake in PSMA-
positive LNCaP cells but not PSMA-negative DU145 cells. In
addition, BL05-HA formulated in PSMA-targeted micelles
significantly reduced LNCaP cell viability compared to the
naked drug, whereas BL05-HA in nontargeted micelles was no
more effective than the naked drug. In PSMA negative cells,
this significant effect was not observed (Figure 8) (p > 0.05).
8. Pharmacokinetic Evaluation. The pharmacokinetics of
naked BL05-HA and BL05-HA formulated in PSMA-targeted
micelles were compared in male Balb/c mice (n = 3). The
initial drug plasma concentration (C0) of the micelle
formulation was 1.42-fold greater than that of the naked drug
(Figure 9). The plasma concentration of BL05 delivered by
Figure 6. Uptake of micelles by (a) PC3 cells and (b) DU145 cells
(mean SD) (*, p < 0.05; **, p < 0.01).
PSMA-targeted micelles was higher than that of the naked drug
at all time points. A two-compartment pharmacokinetic model
was selected to describe the biexponential nature of the
pharmacokinetics disposition of PEG-PCL micelle formulation,
whereas one-compartment model was used in BL05-HA naked
drug, and the related pharmacokinetic parameters are listed in
Table 3. The area under the plasma concentration time curve
(AUC0−24h) of the micelle formulation was 2.27-fold greater
than that of the naked drug, and the concomitant total body
clearance was 6.16-fold lower.
DISCUSSION
■
Prostate cancer is by far the most commonly diagnosed cancer
among Western males. Most anticancer drugs are administered
intravenously or orally to achieve systemic distribution, and the
nonspecific uptake of anticancer drugs may cause damage to
Figure 5. Fluorescence imaging study of cell uptake of fluorescent PSMA-targeted micelles in PSMA-positive LNCaP cells. (A) PC3 cells treated
with nontargeted micelles; (B) PC3 cells treated with PSMA-targeted micelles; (C) LNCaP cells treated with nontargeted micelles; (D) LNCaP cells
treated with PSMA-targeted micelles.
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intestinal damage, and neurotoxicity.37 The leaky endothelial
vasculature of tumor tissues and the size properties of
nanoparticles play a significant role in passive targeting,38 due
to the EPR effect. Improved targeting can be achieved by
decorating the nanoparticle surface with a tumor specific
targeting molecule, such as a monoclonal antibody or nucleic
acid.39 The tumor specific targeting molecules can bind to
tumor cells and increase the intracellular concentration of the
drug by receptor-mediated endocytosis. In this study, we
formulated a prostate cancer cell-targeted nanocarrier that can
enhance the efficacy of an encapsulated anticancer drug against
prostate cancer cells and also optimize the pharmacokinetic
disposition of the drug in a mouse model because of the
encapsulated carrier. We targeted the carriers to prostate cells
with the PSMA aptamer, because this small ligand has
nanomolar affinities for the PSMA receptor, which is ubiquitous
to clinical prostate cancers.40 We conjugated the ligand to
preformed micelles. This approach is expected to result in a
more homogeneous distribution of the ligands among the
nanoparticles than other approaches, such as attaching ligands
to the monomers before formation or drug encapsulation. The
resulting triazole linker also is more resistant to hydrolytic and
enzymatic cleavage than esters or amides formed by
carbodiimide chemistries.
Figure 7. PSMA-targeted micelles improved drug uptake by PSMA
positive cells. Drug uptake by (a) PSMA-negative DU145 cells and (b)
PSMA-positive LNCaP cells treated with targeted or nontargeted
formulations (mean SD) (*, p < 0.001).
TGX-221 showed selective cytotoxicity against LNCaP cells,
which was consistent with a previous report.13 TGX-221 and its
analogue, BL05, showed similar toxicity in different prostate
cancer cell lines, and Western blotting confirmed their
inhibitory effects on phosphorylation of Akt in LNCaP cells.
The modification of the aniline nitrogen did not affect the drug
activity significantly, which is possible if only the morpholine-
chromone ring system and aniline structure contribute to the
binding to PI3K beta subunit.41
Drug interaction and association with the hydrophobic core
of the micelle can influence a drug’s release rate.42 Both TGX-
221 and BL05 did not stay in the hydrophobic core of PEG-
PCL micelles, which may be due to the amphiphilic nature of
these molecules; therefore, the hydrophobic character of the
drug was modified. The addition of hydrophobic fatty esters
increased the encapsulation efficiency, so we postulated that the
long carbon chain may decrease the release rate by making the
drug more favorable to the hydrophobic core.43,44 The fatty
acid prodrugs are susceptible to intra- and extracellular
esterases and hydrolysis, so the parent drug is expected to
reform rapidly after the drug is released from micelles.
The in vitro cytotoxicity study showed that both PSMA-
targeted and nontargeted empty micelles were not toxic to
DU145 cells. This was not unexpected, since both PEG and
PCL polymers are already FDA approved for use in humans
and PSMAa10 is made of a single stranded oligonucleotide.
However, inhibitory effects were observed with IR-820 labeled
PEG-PCL micelles. The toxic effect came from the dye, IR-820,
which was reported to significantly inhibit cell growth at
concentrations greater than 5 μM.34 Therefore, the IR-820
labeled nontargeted and targeted micelles were only used for
examining delivery efficiency, and not for further in vivo
characterization.
Figure 8. IC50's of different formulations of BL05-HA in LNCaP and
DU145 cells (mean SE) (*, p < 0.01; , p > 0.05).
∧
healthy rapidly dividing cells, such as bone marrow and
hair.35,36 Nanoparticles can passively or actively target to tumor
tissue and minimize the side effects of conventional chemo-
therapies, such as sexual dysfunction, cardiac toxicity, gastro-
Due to the change of partition and diffusion coefficients,
BL05-HA may have lower membrane permeability compared to
its parent drug, BL05, and TGX-221. Thus, the passive
diffusion rate of the naked drug to the cells could be lower.
The micelle formulation significantly improved cellular uptake
Figure 9. Plasma BL05 concentration versus time disposition (mean
SD) (n = 3).
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Table 3. Pharmacokinetic Parameters after i.v. Administration of BL05-HA Loaded PSMA-Targeted Micelles and Naked BL05-
a
HA (mean SD) (n = 3)
parameters
unit
L·kg−1
μg·mL−1
μg·h·mL−1
h−1
PSMA-targeted micelle
naked BL05-HA
Vd
5.715 1.219*
1.809 0.420*
7.682 0.784***
0.647 0.081***
2.174 0.749
8.004 1.209
1.270 0.207
3.385 0.290
2.847 0.083
C0
AUC0−24h
k
k12
k21
Cl
h−1
h−1
0.698 0.195
mL·min−1·kg−1
61.627 13.148***
379.783 57.352
a
*, p < 0.05; **, p < 0.01; ***, p < 0.001.
of BL05-HA. This is because internalization of PEG-PCL
micelles can enhance cellular uptake of encapsulated drugs.45
We found that encapsulating BL05-HA with PSMA targeted
PEG-PCL micelles increased cytotoxicity in LNCaP cells but
not in PC3 cells. This can be explained by the enhanced cellular
uptake of the carrier by PSMA-positive cells through a receptor-
mediated pathway. Cellular uptake of the naked drug is driven
by a concentration gradient. After equilibrium is established, no
more drug can enter into the cells. The PEGylated surface of
the micelles reduces interaction with the cell surface. Therefore,
the cytotoxicities of naked drug and the untargeted drug-loaded
micelles in LNCaP and PC3 cells were not significantly
different. In PSMA positive cells, the receptor mediated
transport of PSMA-targeted micelles improved drug delivery
into cells. This significantly enhanced cytotoxicity in PSMA-
positive cells. Since the expression of PSMA on PSMA-negative
cells was extremely low, the effect of PSMA-targeting was not
significant (p > 0.05).
A two-compartment model was used to describe the
pharmacokinetic (PK) disposition of the PEG-PCL micelle
formulation. The PK profile showed that the plasma drug
concentration declines biexponentially as the sum of distribu-
tion and elimination processes. The rapid drug concentration
decrease in initial phase was due to the drug’s distribution from
the central compartment into the peripheral compartments.
Drug metabolism and excretion contributed to the gradual
decrease in the plasma concentration during the beta phase. In
the pharmacokinetics study, we have shown that the PEGylated
micelles can increase the plasma AUC and detectable systemic
circulation of the drug by avoiding clearance by the
reticuloendothelial system (RES) and kidneys. The RES
consists primarily of monocytes and macrophages in the spleen
and liver that are responsible for clearing molecules bound with
serum proteins. The kidneys also clear unbound drugs by
glomerular filtration that are below the renal exclusion limit of
ca. 20−60 kDa. Stealth nanoparticles surface-modified with
PEG can avoid clearance by the RES and kidneys; thus, they
have prolonged circulation half-lives in the plasma.46 This
approach has been used to optimize pharmacokinetic
disposition of nanoparticles and enhance drug delivery.46,47 In
this study, the high Vd value of naked BL05-HA may be due to
its high tissue distribution. A rapid total body clearance rate is
also apparent. The micelle formulation significantly reduced the
Vd by minimizing nonspecific tissue distribution, and the drug
was consequently retained within the vascular system. This
could also reduce toxicity to normal tissues by reducing their
exposure to the drug through nonspecific distribution.
CONCLUSIONS
■
The syntheses of TGX-221, the analogue BL05, and the BL05-
HA and BL05-PA prodrugs were successfully accomplished.
Both TGX-221 and its analogue BL05 had an inhibitory effect
on phosphatation of Akt in PTEN-deficient LNCaP cells.
Encapsulation of BL05-HA in PEG-PCL micelles resulted in
sustained release over several days. In vitro, we have shown that
micelles targeted with PSMAa10 and loaded with BL05-HA
significantly inhibit the growth of PSMA-positive prostate
cancer cells but not PSMA-negative cells. In vivo pharmaco-
kinetics demonstrated that PSMA-targeted micelles significantly
increased the AUC and decreased the total body clearance rate
of the drug. These findings suggest that PSMA-targeted
micelles are a promising drug delivery vehicle for developing
PI3-kinase inhibitor treatments of PSMA positive prostate
cancers. A future study will examine the efficacy of this
formulation in xenografts of human prostate cancer.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: mforrest@ku.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was funded by Congressionally Directed Medical
Research Program IDEA Award in Prostate Cancer
(KAN0061569), DOD Grant W81XWH-09-1-0455 (PI:
Benyi Li), and Kansas Academy of Science Graduate Student
Research Grant (PI: Yunqi Zhao). We also thank the CCET
Synthetic Medicinal Chemistry Laboratory core (NIH
P20RR015563, PI: Timmerman) for assistance in the synthesis
of the drugs and Dr. Jeffrey Krise for the use of his microscopy
equipment. YZ and SD contributed equally to this study.
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