Histone Deacetylase Inhibitor (HDACi) Conjugated Polycaprolactone for Combination Cancer Therapy

Article abstract of DOI:10.1021/acs.biomac.8b00221

The short chain fatty acid, 4-phenylbutyric acid (PBA), is used for the treatment of urea cycle disorders and sickle cell disease as an endoplasmic reticulum stress inhibitor. PBA is also known as a histone deacetylase inhibitor (HDACi). We report here the effect of combination therapy on HeLa cancer cells using PBA as the HDACi together with the anticancer drug, doxorubicin (DOX). We synthesized γ-4-phenylbutyrate-?-caprolactone monomer which was polymerized to form poly(γ-4-phenylbutyrate-?-caprolactone) (PPBCL) homopolymer using NdCl₃·3TEP/TIBA (TEP = triethyl phosphate, TIBA = triisobutylaluminum) catalytic system. DOX-loaded nanoparticles were prepared from the PPBCL homopolymer using poly(ethylene glycol) as a surfactant. An encapsulation efficiency as high as 88% was obtained for these nanoparticles. The DOX-loaded nanoparticles showed a cumulative release of >95% of DOX at pH 5 and 37 °C within 12 h, and PBA release was monitored by ¹H NMR spectroscopy. The efficiency of the combination therapy can notably be seen in the cytotoxicity study carried out on HeLa cells, where only ～20% of cell viability was observed after treatment with the DOX-loaded nanoparticles. This drastic cytotoxic effect on HeLa cells is the result of the dual action of DOX and PBA on the DNA strands and the HDAC enzymes, respectively. Overall, this study shows the potential of combination treatment with HDACi and DOX anticancer drug as compared to the treatment with an anticancer drug alone.

Full text of DOI:10.1021/acs.biomac.8b00221

Article
pubs.acs.org/Biomac
Cite This: Biomacromolecules XXXX, XXX, XXX−XXX
Histone Deacetylase Inhibitor (HDACi) Conjugated Polycaprolactone
for Combination Cancer Therapy
Ruvanthi N. Kularatne,† Katherine E. Washington,† Chandima Bulumulla,† Erika L. Calubaquib,†
Michael C. Biewer,† David Oupicky,‡ and Mihaela C. Stefan*,†
†Department of Chemistry and Biochemistry, University of Texas at Dallas, Richardson, Texas 75080, United States
‡Center for Drug Delivery and Nanomedicine, Department of Pharmaceutical Sciences, University of Nebraska Medical Center,
Omaha, Nebraska 68198, United States
S
* Supporting Information
ABSTRACT: The short chain fatty acid, 4-phenylbutyric acid (PBA), is used for the treatment of urea cycle disorders and sickle
cell disease as an endoplasmic reticulum stress inhibitor. PBA is also known as a histone deacetylase inhibitor (HDACi). We
report here the effect of combination therapy on HeLa cancer cells using PBA as the HDACi together with the anticancer drug,
doxorubicin (DOX). We synthesized γ-4-phenylbutyrate−ε-caprolactone monomer which was polymerized to form poly(γ-4-
phenylbutyrate−ε-caprolactone) (PPBCL) homopolymer using NdCl3·3TEP/TIBA (TEP = triethyl phosphate, TIBA =
triisobutylaluminum) catalytic system. DOX-loaded nanoparticles were prepared from the PPBCL homopolymer using
poly(ethylene glycol) as a surfactant. An encapsulation efficiency as high as 88% was obtained for these nanoparticles. The DOX-
loaded nanoparticles showed a cumulative release of >95% of DOX at pH 5 and 37 °C within 12 h, and PBA release was
1
monitored by H NMR spectroscopy. The efficiency of the combination therapy can notably be seen in the cytotoxicity study
carried out on HeLa cells, where only ∼20% of cell viability was observed after treatment with the DOX-loaded nanoparticles.
This drastic cytotoxic effect on HeLa cells is the result of the dual action of DOX and PBA on the DNA strands and the HDAC
enzymes, respectively. Overall, this study shows the potential of combination treatment with HDACi and DOX anticancer drug
as compared to the treatment with an anticancer drug alone.
an anticancer drug would be more efficient in terms of potency
to kill cancer cells.
INTRODUCTION
■
4-Phenylbutyric acid (PBA) is a short chain fatty acid used
mainly for the treatment of urea cycle disorders as an ammonia
scavenger1−3 and as endoplasmic reticulum stress inhibitor.4−6
Its ability to activate β-globin transcription allowed it to be used
for the treatment of sickle cell disease and thalassemia.7,8 In
addition, PBA has been shown to have neuroprotective effects9
and is known as a histone deacetylase inhibitor (HDACi).3,10,11
The HDAC inhibitory effect of PBA is attributed to its
anticancer activity. During gene transcription and upon HDAC
inhibition, hyperacetylation of the histone residues in
nucleosomes leads to enhanced differentiation, inhibits
proliferation, and eventually causes cell death.3,10 As such,
PBA is used in clinical trials as an anticancer agent.12,13 Yet, the
effectiveness of HDACi as anticancer agents is limited due to
their dose-limiting toxicity14 and due to acquired resistance.15
Therefore, combination therapy using an HDACi together with
The anthracycline, doxorubicin (DOX), is a widely used
chemotherapeutic agent for the treatment of a wide variety of
cancers. These include breast, lung, ovarian, Hodgkin’s and
non-Hodgkin’s lymphoma, lung, gastric, sarcoma, and pediatric
cancers.16,17 The side effects, such as cardiotoxicity caused by
DOX, however, limits the use of an anticancer drug.17 One
solution to circumvent this issue is the use of nanocarriers to
encapsulate the aforementioned drug. Thereby, the drug will
accumulate specifically in the tumor site either by the enhanced
permeation and retention (EPR) effect or by active targeting
which will reduce the side effects to some extent.
Received: February 8, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.biomac.8b00221
Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
Scheme 1. Schematic Diagram of (a) Synthesis of PPBCL Polymer with NdCl3·3TEP/TIBA Catalytic System, (b) Formation of
DOX-Loaded NP Using PEG and Release of DOX and PBA upon the Introduction to Acidic Medium, and (c) Endocytosis and
Subsequent Release of DOX and PBA in the Cancer Cell
Analysis. Proton and carbon nuclear magnetic resonance (1H and
13C NMR, respectively) spectra were recorded in CDCl3 on a Bruker
AVANCE III 500 spectrometer (500 MHz) at 25 °C unless otherwise
mentioned. Multiplicities were given as s (singlet), d (doublet), t
(triplet), q (quartet), and br (broad). Size exclusion chromatography
(SEC) was carried out on a Viscotek VE 3580 system equipped with
Viscotek columns (T6000M), connected to the refractive index (RI),
low angle light scattering (LALS), right angle light scattering (RALS),
and viscosity detectors. An SEC solvent/sample module (GPCmax)
was used with HPLC grade THF as the eluent, and calibration was
based on polystyrene standards. A PerkinElmer LS 50 BL
luminescence spectrometer was used to collect fluorescence spectra
at 25 °C. Dynamic light scattering (DLS) measurements were
performed using a Malvern Zetasizer Nano ZS instrument equipped
with a He−Ne laser (633 nm) and 173° backscatter detector. The
morphology of empty and loaded nanoparticles was visualized by
transmission electron microscopy (TEM) imaging performed on a
Tecnai G2 Spirit Biotwin microscope operated at 120 keV, and images
were analyzed using Gatan Digital Micrograph software. An Agilent
UV/vis spectrophotometer was used to obtain absorbance spectra.
BioTek Cytation 3 cell imaging multimode reader was used for
cytotoxicity and cellular uptake measurements.
Among the different types of HDACis, hydroxamic acids,18,19
benzamides,20 and aliphatic acids21,22 have been conjugated to
polymer backbones. However, these only focused on the
delivery of the HDACi. The use of nanocarriers made from
HDACi conjugated polymeric material will allow for the
codelivery of the HDACi together with a more potent
anticancer drug, such as DOX. The dual delivery of the
HDACi and the anticancer drug is expected to have enhanced
cytotoxicity to the cancer cells. Sustained release of the HDACi
as well as the anticancer drug at the tumor site can be obtained
by polymeric micelles/nanoparticles. Recently, we reported
PBA conjugated poly(ethylene glycol)-b-poly(γ-4-phenylbutyr-
ate−ε-caprolactone-ran-ε-caprolactone) (PEG-b-(PCL-ran-
PPBCL)) copolymer for the delivery of DOX.23 The γ-4-
phenylbutyrate−ε-caprolactone monomer was copolymerized
with ε-caprolactone in the presence of PEG as the macro-
initiator with tin(II) 2-ethylhexanoate (Sn(Oct)2) as the
catalyst. The low incorporation of the PBA, into the polymeric
backbone, resulted in reduced cytotoxicity to the human
cervical cancer cells (HeLa).
Herein, we report the synthesis of poly(γ-4-phenylbutyra-
te−ε-caprolactone) (PPBCL) with a newly developed catalytic
system, NdCl3·3TEP/TIBA (TEP = triethyl phosphate, TIBA =
triisobutylaluminum) (Scheme 1).24 The synthesized homo-
polymer was employed to prepare nanoparticles (NP) with
PEG surfactant to encapsulate DOX. The formed empty and
DOX-loaded NPs were then analyzed to determine the size,
morphology, drug release behavior, cytotoxicity, and cellular
uptake in HeLa cells (Scheme 1).
Synthesis of 4-Oxocyclohexanoyl Phenylbutanoate (2). 4-
Hydroxycyclohexanone (2.00 g, 0.017 mol), phenylbutyric acid (3.17
g, 0.019 mol), 4-(dimethylamino)pyridine (DMAP) (0.21 g, 1.7
mmol), and dichloromethane (DCM) (60 mL) were cooled to 0 °C in
a round-bottom flask. A solution of 1-ethyl-3-(3-(dimethylamino)-
propyl)carbodiimide hydrochloride (EDCl) (4.04 g, 0.21 mol) in
DCM (40 mL) was added dropwise to the cooled solution. The
reaction was stirred overnight and was washed with a saturated
solution of potassium carbonate and several times with water to
remove any excess DMAP, EDCl, and other water-soluble products.
The organic layer was dried over anhydrous magnesium sulfate, and
the solvent was evaporated under reduced pressure to obtain the pure
MATERIALS AND METHODS
■
1
product as a pale yellow liquid (4.50 g, 97%). H NMR (δ = 7.26,
Materials. All commercially available chemicals and solvents were
purchased from Sigma-Aldrich or Fisher Scientific. 4-Hydroxy-
cyclohexanone and NdCl3·3TEP catalyst were synthesized according
to previously published procedures.24,25
CDCl3): δ = 1.98 (q, 2H), 2.04−2.08 (m, 4H), 2.33−2.39 (m, 4H),
2.5−2.56 (m, 2H), 2.67 (t, 2H), 5.15−5.19 (q, 1H), 7.17−7.21 (m,
3H), 7.27−7.31 (m, 2H).
B
DOI: 10.1021/acs.biomac.8b00221
Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
Synthesis of γ-4-Phenylbutyrate−ε-caprolactone (3). To a
stirred solution of 4-oxocyclohexanoyl phenylbutanoate (4.5 g, 0.017
mol) in DCM (100 mL) at 0 °C was added a solution of 77% m-
chloroperoxybenzoic acid (6.6 g, 0.029 mol) in DCM (100 mL). The
reaction mixture was stirred overnight, and potassium carbonate (10.0
g in 20 mL of water) was added to the reaction mixture and further
stirred vigorously for 4 h. The organic layer was separated, and the
aqueous layer was washed with DCM (2 × 50 mL). The combined
organic layers were dried over anhydrous magnesium sulfate and
concentrated in vacuo. The product was isolated by flash
chromatography with gradient elution using hexanes and ethyl acetate
to yield a pale yellow oil (4.0 g, 83%). 1H NMR (δ = 7.26, CDCl3): δ
= 1.94−2.13 (m, 6H), 2.35 (t, 2H), 2.5−2.55 (m, 1H), 2.66 (t, 2H),
2.85−2.91 (m, 1H), 4.11−4.16 (m, 1H), 4.39−4.44 (m, 1H), 5.09−
5.14 (m, 1H), 7.16−7.23 (m, 3H), 7.27−7.31 (t, 2H).
hydrodynamic size. Size measurements were obtained in triplicate at
25 °C. Morphological studies were carried out by TEM where the
samples were prepared by treating the copper mesh grid with 1 mg/
mL aqueous nanoparticle solution for 2 min, followed by staining with
2% phosphotungstic acid for 30 s.
Release Studies for DOX. A 1 mg/mL solution of DOX-loaded
nanoparticles in deionized water was prepared as stated before. A
volume of 4 mL from the nanoparticle solution was transferred to
Snakeskin dialysis tubing with 3500 Da molecular weight cutoff and
was dialyzed against 15 mL of buffer (pH 7.4, 6.0, and 5.0). Aliquots of
2 mL were withdrawn at various time intervals and were replaced by
the same volume. The samples taken were diluted with DMSO in a
ratio of 1:1, and UV/vis spectra were recorded. DOX release was then
determined by a previously established calibration plot for DOX in
DMSO/DI water.
Synthesis of PPBCL. A 10 mL Schlenk flask was flushed with
nitrogen for 10 min. Triisobutylaluminum (25% w/w) (0.02 mL, 0.014
mmol) and NdCl3·3TEP (0.01 mL, 0.0014 mmol) were added to the
Schlenk flask and stirred for 1 min under a nitrogen environment.
Monomer γ-4-phenylbutyrate−ε-caprolactone (0.2 g, 0.7 mmol) was
added to the catalyst mixture, and the flask was immersed in an oil
bath at 40 °C. The polymer was precipitated out in methanol after
overnight stirring and further purified by washing the polymer with
Release Studies for PBA. Empty nanoparticles were prepared in
deionized water with a concentration of 2 mg/mL. A volume of 4 mL
of the nanoparticle solution was transferred to Snakeskin dialysis
tubing with 3500 Da molecular weight cutoff and was dialyzed against
15 mL of buffer (pH 6.0). Three samples were taken at the initial time,
12 h, and 24 h, and the buffer solution was allowed to evaporate. After
complete evaporation of the buffer solution, D2O (0.5 mL) was added,
1
methanol a few times. Mn = 13 000 Da, PDI = 1.51. H NMR (δ =
1
and H NMR spectra were recorded at different time intervals.
7.26, CDCl3): δ = 1.79−2.01 (m, 6H), 2.29 (br s, 4H), 2.63 (t, 2H),
4.05 (t, 2H), 5.00 (m, 1H), 7.16−7.23 (m, 3H), 7.26−7.32 (m, 2H).
Preparation of Nanoparticles. A solvent evaporation method
was employed for the preparation of nanoparticles with PPBCL:PEG
in a 1:1 weight ratio. A stock solution of PPBCL (10 mg) and PEG
(10 mg) in THF (5 mL) was prepared at a concentration of PPBCL of
2 mg/mL. A volume of 3 mL of this stock solution was added
dropwise to deionized water (6 mL) under vigorous stirring. The
solution was kept under stirring until THF was completely evaporated.
The concentration of the final nanoparticles solution was 1 mg/mL
with respect to PPBCL. The resulting solution was filtered through a
0.22 μm polyamide (PA) syringe filter to remove any polymer that has
precipitated out to obtain the nanoparticles in DI water.
Determination of Critical Aggregation Concentration (CAC).
CAC was determined with the aid of pyrene, a hydrophobic
fluorescent probe. For all polymer samples, PPBCL and PEG were
used in a weight ratio of 1:1. A volume of 200 μL of pyrene in THF
(6.0 × 10−5 M) was added to polymer solutions of various
concentrations. This solution was added dropwise to deionized
water (10 mL) under stirring. The solutions were stirred for 4 h for
complete evaporation of THF and for the formation of nanoparticles.
The concentrations of the resulting solutions were in the range of 1 g/
L to 4 × 10−5 g/L. Fluorescence spectra of the solutions were recorded
with the emission wavelength set at 390 nm. The intensity ratios of
pyrene at the wavelengths of 337.5 and 334.5 nm were plotted against
the logarithm of polymer concentration. CAC was determined as the
value at the x coordinate where the two trend lines intersected.
Preparation of DOX-Loaded Nanoparticles. DOX-loaded
nanoparticles were prepared in a similar manner as the empty
nanoparticles. Briefly, 4 mg of DOX·HCl was neutralized with 3 equiv
of triethylamine (2.9 μL), and a stock solution was prepared in THF
(4 mL). A stock solution of PPBCL (10 mg) and PEG (10 mg) in
THF (5 mL) was made so that the concentration of PPBCL was 2
mg/mL. A volume of 3 mL of this stock solution and 0.6 mL of the
DOX stock solution were added dropwise to deionized water (6 mL)
under vigorous stirring. The weight ratio between PPBCL and DOX
was kept at 10:1. The resulting solution was filtered through a 0.22 μm
PA syringe filter in order to remove any nonencapsulated aggregated
drug molecules to obtain DOX-loaded nanoparticles in deionized
water. Drug loading capacity (DLC) and encapsulation efficiency (EE)
were determined using UV/vis absorbance spectra by diluting the
nanoparticle solutions with DMSO in a 1:1 ratio. The absorbance at
485 nm was fitted to a pre-established calibration curve for DOX in
DMSO/deionized water.
Biological Studies. All cell culture experiments were carried out
using RPMI-1640 medium with L-glutamine and sodium bicarbonate
supplemented with 10% fetal bovine serum and 1% penicillin−
streptomycin. Cells were incubated in a humidified environment with
5% CO2 at 37 °C.
Cytotoxicity Studies for Empty Nanoparticles. HeLa cells were
grown in a 96-well plate at a cell density of 5000 cells per well with 100
μL of growth medium. After the cells were incubated for 24 h for cell
adhesion, the growth medium was removed and washed with 100 μL
of PBS. Volumes of 100 μL of empty nanoparticles prepared in PBS in
concentrations of 0.2, 0.1, 0.06, 0.04, and 0.02 mg/mL were added to
the cells with growth media (100 μL) and were incubated at 37 °C for
24 h. Cytotoxicity studies were performed using CellTiter Blue reagent
using manufacturer recommended protocol (N = 4).
Cytotoxicity Studies for DOX Loaded Nanoparticles. HeLa
cells were grown in a 96-well plate at a cell density of 5000 cells per
well with 100 μL of growth medium. After the cells were incubated for
24 h for cell adhesion, the growth medium was removed and washed
with 100 μL of PBS. 100 μL of DOX-loaded nanoparticles, free DOX,
free PBA, and a combination of DOX and PBA, all prepared in PBS in
concentrations of 0.2, 0.06, and 0.02 mg/mL, were added to the cells
with growth media (100 μL) and were incubated at 37 °C for 24 h.
The amount of free DOX to be added was calculated by DLC, and the
amount of free PBA was calculated by the concentration of PPBCL
used. Cytotoxicity studies were performed using CellTiter Blue
reagent using manufacturer recommended protocol (N = 4). To
determine IC50, 100 μL volumes of DOX-loaded NPs in
concentrations of 1, 0.5, 0.25, 0.2, 0.1, 0.06, 0.02, 0.001, and 0.0001
mg/mL were added with growth media (100 μL) into 96-well plates
with HeLa cells already incubated for 24 h. Cytotoxicity studies were
performed using CellTiter Blue reagent using manufacturer recom-
mended protocol (N = 5).
Cellular Uptake Studies. HeLa cells were grown in 35 mm clear
bottom dishes at a cell density of 250 000 cells per dish with 2 mL
growth media. DOX-loaded micelles in PBS at the concentration of 0.1
mg/mL (1 mL) were added to the cells along with growth media (1
mL). After an incubation period of 4 and 24 h, the media was
removed, washed with PBS (2 mL × 3), fixed with 4%
paraformaldehyde (1 mL, incubated for 10 min at room temperature),
washed with PBS (2 mL × 2), treated with Triton X-100 (1 mL,
incubated for 2 min at room temperature), washed with PBS (2 mL ×
4), and counterstained the nucleus with 4′,6-diamidino-2-phenylindole
(DAPI) according to the manufacturer’s recommended protocol.
DLS and TEM Analysis. DOX-loaded and empty nanoparticles
were prepared as stated above and were analyzed by DLS to obtain the
C
DOI: 10.1021/acs.biomac.8b00221
Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
Scheme 2. Synthesis of γ-4-Phenylbutyrate−ε-Caprolactone
Scheme 3. Polymerization of γ-4-Phenylbutyrate−ε-Caprolactone Using Sn(Oct)2 and Zn(Undec)2 and NdCl3·3TEP/TIBA
good yields. By use of Zn(Undec)2 as the catalyst, the majority
of the caprolactone monomer was converted into the
butyrolactone analogue (Scheme 3 and Table 1). DBU, on
RESULTS AND DISCUSSION
■
Synthesis and Characterization of PPBCL Homopol-
ymer. Monomer γ-4-phenylbutyrate−ε-caprolactone was
synthesized previously by our group with an overall yield of
∼40%.23 In this report, we employed a new synthesis that
increased the overall yield to ∼60%. First, 4-hydroxycylohex-
anone was synthesized from 1,4-cyclohexanediol according to
previous reports,25 and then a Steglich esterification was carried
out followed by Baeyer−Villiger oxidation to form γ-4-
phenylbutyrate−ε-caprolactone (Scheme 2). The previously
reported methodology to synthesize γ-substituted-ε-caprolac-
tone monomers involved the conjugation of the substituent
first, followed by oxidation26−29 which could be a disadvantage
in conjugating substituents that are prone to oxidation or in
some cases that are hydrophilic.
In our previous report, homopolymerization of γ-4-phenyl-
butyrate−ε-caprolactone was unachievable; thus, copolymeriza-
tion with ε-caprolactone was carried out with PEG-OH as the
hydrophilic block to synthesize PEG-b-(PCL-ran-PPBCL).
Only 30 mol % of PBA was incorporated in the polymer;
hence, only minimal activity of HDAC inhibition was observed
in vitro studies.23 In an attempt to further increase the content
of HDACi conjugated to the polymer backbone, exhaustive
trials were carried out to obtain the homopolymer of γ-4-
phenylbutyrate−ε-caprolactone. With the use of the commonly
used catalyst, Sn(Oct)2, for ring-opening polymerizations,30−32
we observed that the major product obtained was not the
homopolymer but the more thermodynamically stable butyr-
olactone analogue (Scheme 3). Therefore, further attempts
were carried out with zinc undecylenate (Zn(Undec)2) and 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU), since both of these
catalysts have shown the ability to polymerize substituted or
unsubstituted ε-caprolactones.33−35 Unfortunately, neither of
these catalytic systems provided the desired homopolymer in
Table 1. Polymerization of γ-4-Phenylbutyrate−ε-
Caprolactone with Different Catalytic Systems
% conv to
polymer
% conv to
Mn
catalyst
Sn(Oct)2
transesterified product (g mol−1
)
PDI
32
73
68
27
6000
1.52
1.51
NdCl3·3TEP/
TIBA
13000
Zn(Undec)2
DBU
yield of the polymer was extremely low; higher
transesterification
no polymer or trans esterified product formed
the other hand, showed no polymerization of the caprolactone
monomer. To confirm the structure of the formed trans-
esterified product, we carried out the synthesis of the
butyrolactone monomer (Scheme S1, Figures S1 and S2, see
1
Supporting Information for details). By comparison of the H
NMR spectra, we confirmed the formation of the butyrolactone
analogue during the polymerization (Figure 1). With the goal
of obtaining the homopolymer in high yields we carried out the
polymerization with a catalytic system introduced by our group,
NdCl3·3TEP/TIBA,24 which produced the homopolymer with
only <20 mol % transesterified product (Table 1 and Figure 1).
With Sn(Oct)2 and Zn(Undec)2, transesterification is more
facilitated due to the higher temperatures employed (110 °C).
The mild polymerization temperatures used with our catalytic
system of NdCl3·3TEP/TIBA (40 °C) favored the formation of
the desired homopolymer. Unfortunately, a major limitation of
this catalytic system is the inability to use a macroinitiator such
as PEG to synthesize amphiphilic block copolymers. Therefore,
nanoparticles of PPBCL were formed using PEG as a
surfactant. The synthesized PPBCL homopolymer was
D
DOI: 10.1021/acs.biomac.8b00221
Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
1
1
Figure 1. H NMR spectra for (i) γ-4-phenylbutyrate−ε-caprolactone and (ii) 2-(5-oxotetrahydrofuran-2-yl)ethyl 4-phenylbutanoate. H NMR
spectra for the polymerization of γ-4-phenylbutyrate−ε-caprolactone with (iii) Sn(Oct)2 and (iv) NdCl3·3TEP/TIBA. All spectra are shown for the
region 3.5−7.5 ppm.
characterized by 1H NMR, 13C NMR (Figures S3 and S4), and
SEC using triple-point calibration (Table 1).
Preparation and Characterization of Nanoparticles.
Nanoparticles were formed with PEG as the surfactant, using a
solvent evaporation method. THF was used as the organic
solvent due to the miscibility with water and the moderate
evaporation of the solvent that limits the formation of
aggregates. Initially, different weight ratios between PPBCL
and PEG were used to make the nanoparticles, and a study was
performed to determine the size of the nanoparticles (Table 2).
Figure 2. CAC plot for PPBCL obtained using the fluorescent probe
Table 2. Size Distribution of the PPBCL Empty
pyrene.
Nanoparticles with the Variation of PEG Weight Ratio
weight ratios of PEG:PPBCL
size (nm)
dispersity
tion efficiency (EE) were estimated by using the following
equations.
4:1
98.6
105.8
94.0
0.075
0.062
0.068
0.099
0.085
2:1
amount of DOX loaded
amount of polymer
1:1
DLC =
× 100
0.5:1
0.25:1
95.1
96.2
amount of DOX loaded
theoretical amount of DOX
EE =
× 100
It was observed that irrespective of the weight ratios used, the
size remained constant at ∼100 nm. However, during the
formation of the nanoparticles with the increasing weight ratios
of PPBCL, the polymer started to precipitate out. Therefore,
for all experiments in this study a weight ratio of 1:1 between
PPBCL and PEG was employed.
The critical aggregation concentration (CAC), which
demonstrates the thermodynamic stability of the formed
nanoparticles, was found to be 6.40 × 10−4 g/L as determined
by using the fluorescent probe pyrene. Upon increasing the
polymer concentration, the excitation of pyrene changed from
334 to 337 nm when the surrounding environment changed
from hydrophilic to hydrophobic, respectively. This change was
used to generate a plot of the intensity ratio of these two
excitation wavelengths (I337/I334) versus the logarithm of the
concentration of the polymer. CAC was determined at the
intersection of the two slopes (Figure 2). The thermodynamic
stability of the formed nanoparticles is comparable to the
micelles that were reported previously with the terpolymer,
PEG-b-(PCL-ran-PPBCL).23
The calculated DLC was ∼8.8%, while EE was ∼88%. This
represents a significant increase in DLC and EE as compared to
our previously reported value of 5.1% DLC and 52% EE which
was obtained for PEG-b-(PCL-ran-PPBCL).23 The increase in
DOX loading is due to the high number of PBA units
conjugated to the polymer backbone which resulted in
hydrogen bonding and π-stacking interactions between DOX
and the polymer backbone.
Size and morphology of the empty and DOX-loaded NPs
were determined by DLS and TEM analysis. The size of empty
NPs and DOX-loaded NPs as obtained from DLS was 94.0 and
167.2 nm, respectively (Figure 3a,b and Figure S5). An evident
increase in size can be observed with DOX loading, and the size
of the DOX-loaded NPs is similar in size to the DOX-loaded
micelles obtained with PEG-b-(PCL-ran-PPBCL).23 TEM
images showed the formation of spherical nanoparticles (Figure
3c,d), and the size distribution was comparable to that obtained
from DLS analysis.
DOX and PBA Release. Cumulative DOX release studies
were carried out using DOX-loaded NPs at three different pH
conditions (pH = 5, 6, and 7.4) at the physiological
temperature of 37 °C. Samples were withdrawn for 24 h, and
DOX-loaded nanoparticles were prepared similarly to the
empty nanoparticles (Scheme 3), by using a ratio of [polymer]:
[DOX] = 10:1. Drug loading capacity (DLC) and encapsula-
E
DOI: 10.1021/acs.biomac.8b00221
Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
Blue reagent. The empty NPs with a polymer concentration in
the range of 0.02−0.2 mg/mL were shown to be nontoxic to
the HeLa cells (Figure 5a) as all concentrations of NPs the cells
Figure 3. DLS of (a) empty NPs (b) DOX-loaded NPs and TEM
images of (c) empty NPs and (d) DOX-loaded NPs.
each time a sample was taken, equal amounts of fresh buffer
solution were added to maintain the floating conditions.
Because of the increased number of ester linkages, a cumulative
DOX release of ∼90% was obtained for both pH 5 and 6 within
8 h (Figure 4), which is higher than the cumulative release
Figure 5. Cell viability on HeLa cells after 24 h of incubation time for
(a) empty NPs and (b) DOX-loaded NPs, free DOX, free (DOX +
PBA), and free PBA (N = 4).
were >95% viable, which suggests the polymer by itself is
nontoxic. Cytotoxicity of the DOX-loaded NPs was then
compared to that of free DOX, free PBA, and free (DOX +
PBA). Dosing for free DOX, free PBA, and free (DOX + PBA)
was calculated based on the DLC of Dox and the degree of
polymerization for PBA, for each concentration. When
comparing the cytotoxicity of the DOX-loaded NPs to that of
free PBA, and free DOX (Figure 5b), the DOX-loaded NPs
showed a significantly higher cytotoxic effect to the HeLa cells.
The free HDACi by itself did not cause cell death within the 24
h time frame. Enhanced cell death was observed with free
(DOX + PBA) when compared with free DOX, due to the dual
action of DOX and the HDACi. Furthermore, the cytotoxicity
caused by the DOX-loaded NPs was similar to that of the free
(DOX + PBA), confirming the release of the HDACi together
with the DOX from the NPs. These cytotoxicity studies can be
related to the release profiles discussed above, where within 24
h, in acidic environments, the release of PBA and >95% of
DOX was observed. The cell viability of the DOX-loaded NPs
within 24 h was ∼20%, whereas the cell viability for the
previously reported PEG-b-(PCL-ran-PPBCL) polymer was
∼20% within 60 h.23 The enhanced cytotoxicity is most likely
due to the increased amount of PBA and DOX. IC50 values
calculated for the DOX-loaded NPs from the dose−response
curve were found to be 0.045 mg/mL (Figure S7). The
accelerated release of both DOX and PBA at conditions which
mimics the endosomal pH conditions is desirable as these
released molecules will then enter the nucleus where DOX will
interact with the DNA strands while PBA inhibits the HDAC
enzymes, causing an increased cellular death to the cancer cells.
Figure 4. Cumulative DOX release for the DOX-loaded NP at 37 °C
in different pH conditions of pH = 5, 6, and 7.4.
obtained for the previously reported PEG-b-(PCL-ran-PPBCL)
polymer.23 The release obtained at pH 5 and 6 was significantly
higher than that measured at pH 7.4. In an acidic environment,
the ester linkages in the polymer backbone of PPBCL will be
cleaved, and thus the NPs will no longer hold the cargo,
thereby releasing the DOX into the buffer.
1
The release of the HDACi was monitored by H NMR
analysis. The study was carried out using the empty NPs at pH
6 for 24 h. The pH 5 buffer was not used for the study due to
the presence of potassium phthalate, which masks the 1H NMR
peaks in the aromatic region of PBA. Samples withdrawn were
evaporated to remove the buffer solution and were resuspended
1
in D2O to obtain the H NMR spectra (Figure S6). The data
analysis showed that in acidic environment the ester hydrolysis
takes place promoting the release of DOX and PBA HDACi
into the buffer solution (Scheme 1b).
In Vitro Cytotoxicity of the NPs. Cytotoxicity studies for
empty and DOX-loaded NPs were carried out using CellTiter
F
DOI: 10.1021/acs.biomac.8b00221
Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
Figure 6. Cellular uptake of DOX-loaded NPs into HeLa cells. NPs were incubated for 4 and 24 h at a DOX concentration of 8.8 mg/L.
Cellular Uptake of DOX-Loaded NPs. Cellular uptake
studies on HeLa cells were performed using DOX-loaded NPs
with a DOX concentration of 8.8 mg/L. The NPs were
incubated with the cells for 4 and 24 h before fixing with
paraformaldehyde and staining the nuclei with DAPI. The
inherent fluorescent ability of DOX was used to determine the
cellular uptake of DOX, and it was seen that DOX accumulated
in the nucleus. The merged bright field and fluorescence images
clearly showed the cellular uptake of DOX and the subsequent
accumulation of DOX in the nucleus (Figure 6). With
increasing incubation time up to 24 h, the intensity of DOX
inside the nucleus increased as compared to 4 h. As observed
from cytotoxicity studies, ∼80% of HeLa cells were killed
within 24 h of treatment with the DOX-loaded NPs, which can
be related to the reduced cell density in cellular uptake studies
performed at 24 h.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail mihaela@utdlaas.edu (M.C.S.).
ORCID
David Oupicky: 0000-0003-4710-861X
Mihaela C. Stefan: 0000-0003-2475-4635
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Funding from NSF (CHE-1566059 and CHE-1609880), NIH
(1R21EB019175-01A1), and Welch Foundation (AT-1740) is
gratefully acknowledged.
REFERENCES
■
(1) Brusilow, S. W.; Maestri, N. E. Urea cycle disorders: diagnosis,
pathophysiology, and therapy. Adv. Pediatr. 1996, 43, 127−170.
(2) Burrage, L. C.; Jain, M.; Gandolfo, L.; Lee, B. H.; Nagamani, S. C.
S. Sodium phenylbutyrate decreases plasma branched-chain amino
acids in patients with urea cycle disorders. Mol. Genet. Metab. 2014,
113 (1−2), 131−135.
CONCLUSION
■
In this study, a polycaprolactone containing a pendant PBA
HDACi was successfully synthesized with the NdCl3·3TEP/
TIBA catalytic system. Commonly utilized catalysts for the
polymerization of caprolactone failed to produce the
homopolymer of γ-4-phenylbutyrate−ε-caprolactone. The
synthesized PPBCL polymer with PEG formed spherical NPs
and successfully encapsulated DOX with an EE of 88%. These
DOX-loaded NPs released both DOX and PBA at pH
conditions that mimic the endosomal environment and also
exhibited increased cytotoxicity (cell viability reduced to
∼20%) when compared to free DOX due to the dual action
of DOX and HDACi. Overall, this study shows the improve-
ment of cytotoxicity by combination treatment with an HDACi
and an anticancer drug as compared to the treatment with an
anticancer drug alone.
(3) Marks, P. A.; Rifkind, R. A.; Richon, V. M.; Breslow, R.; Miller,
T.; Kelly, W. K. Histone deacetylases and cancer: causes and therapies.
Nat. Rev. Cancer 2001, 1 (3), 194−202.
̌
́
(4) Basseri, S.; Lhotak, S.; Sharma, A. M.; Austin, R. C. The chemical
chaperone 4-phenylbutyrate inhibits adipogenesis by modulating the
unfolded protein response. J. Lipid Res. 2009, 50 (12), 2486−2501.
(5) Yam, G. H.-F.; Gaplovska-Kysela, K.; Zuber, C.; Roth, J. r.
Sodium 4-Phenylbutyrate Acts as a Chemical Chaperone on Misfolded
Myocilin to Rescue Cells from Endoplasmic Reticulum Stress and
Apoptosis. Invest. Ophthalmol. Visual Sci. 2007, 48 (4), 1683−1690.
(6) Kolb, P. S.; Ayaub, E. A.; Zhou, W.; Yum, V.; Dickhout, J. G.;
Ask, K. The therapeutic effects of 4-phenylbutyric acid in maintaining
proteostasis. Int. J. Biochem. Cell Biol. 2015, 61, 45−52.
(7) Collins, A. F.; Pearson, H. A.; Giardina, P.; McDonagh, K. T.;
Brusilow, S. W.; Dover, G. J. Oral sodium phenylbutyrate therapy in
homozygous β thalassemia: A clinical trial. Blood 1995, 85 (1), 43−49.
(8) Dover, G. J.; Brusilow, S.; Charache, S. Induction of fetal
hemoglobin production in subjects with sickle cell anemia by oral
sodium phenylbutyrate. Blood 1994, 84 (1), 339.
(9) Mimori, S.; Ohtaka, H.; Koshikawa, Y.; Kawada, K.; Kaneko, M.;
Okuma, Y.; Nomura, Y.; Murakami, Y.; Hamana, H. 4-Phenylbutyric
acid protects against neuronal cell death by primarily acting as a
chemical chaperone rather than histone deacetylase inhibitor. Bioorg.
Med. Chem. Lett. 2013, 23 (21), 6015−6018.
(10) Mottamal, M.; Zheng, S.; Huang, T. L.; Wang, G. Histone
Deacetylase Inhibitors in Clinical Studies as Templates for New
Anticancer Agents. Molecules 2015, 20 (3), 3898−3941.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
mac.8b00221.
Synthesis and characterization of 2-(5-oxotetrahydrofur-
an-2-yl)ethyl 4-phenylbutanoate and H and 13C NMR
1
of PPBCL, DLS comparison of empty and loaded
nanoparticles, release study of PBA and dose-responsive
curve for DOX-loaded NPs (PDF)
G
DOI: 10.1021/acs.biomac.8b00221
Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
(11) Miller, A. C.; Cohen, S.; Stewart, M.; Rivas, R.; Lison, P.
Radioprotection by the histone deacetylase inhibitor phenylbutyrate.
Radiat. Environ. Biophys. 2011, 50 (4), 585.
(12) Warrell, J. R. P.; He, L.-Z.; Richon, V.; Calleja, E.; Pandolfi, P. P.
Therapeutic Targeting of Transcription in Acute Promyelocytic
Leukemia by Use of an Inhibitor of Histone Deacetylase. J. Natl.
Cancer Inst. 1998, 90 (21), 1621−1625.
(13) Phuphanich, S.; Baker, S. D.; Grossman, S. A.; Carson, K. A.;
Gilbert, M. R.; Fisher, J. D.; Carducci, M. A. Oral sodium
phenylbutyrate in patients with recurrent malignant gliomas: A dose
escalation and pharmacologic study. Neuro Oncol. 2005, 7 (2), 177−
182.
(14) How, J.; Minden, M. D.; Brian, L.; Chen, E. X.; Brandwein, J.;
Schuh, A. C.; Schimmer, A. D.; Gupta, V.; Webster, S.; Degelder, T.;
Haines, P.; Stayner, L.-A.; McGill, S.; Wang, L.; Piekarz, R.; Wong, T.;
Siu, L. L.; Espinoza-Delgado, I.; Holleran, J. L.; Egorin, M. J.; Yee, K.
W. L. A phase I trial of two sequence-specific schedules of decitabine
and vorinostat in patients with acute myeloid leukemia. Leuk.
Lymphoma 2015, 56 (10), 2793−2802.
(27) Rainbolt, E. A.; Washington, K. E.; Biewer, M. C.; Stefan, M. C.
Towards smart polymeric drug carriers: self-assembling [gamma]-
substituted polycaprolactones with highly tunable thermoresponsive
behavior. J. Mater. Chem. B 2013, 1 (47), 6532−6537.
(28) Hao, J.; Servello, J.; Sista, P.; Biewer, M. C.; Stefan, M. C.
Temperature-sensitive aliphatic polyesters: synthesis and character-
ization of [gamma]-substituted caprolactone monomers and polymers.
J. Mater. Chem. 2011, 21 (29), 10623−10628.
(29) Trollsas, M.; Lee, V. Y.; Mecerreyes, D.; Lowenhielm, P.;
̊
̈
Moller, M.; Miller, R. D.; Hedrick, J. L. Hydrophilic Aliphatic
̈
Polyesters: Design, Synthesis, and Ring-Opening Polymerization of
Functional Cyclic Esters. Macromolecules 2000, 33 (13), 4619−4627.
(30) Storey, R. F.; Sherman, J. W. Kinetics and Mechanism of the
Stannous Octoate-Catalyzed Bulk Polymerization of ε-Caprolactone.
Macromolecules 2002, 35 (5), 1504−1512.
(31) Rainbolt, E. A.; Miller, J. B.; Washington, K. E.; Senevirathne, S.
A.; Biewer, M. C.; Siegwart, D. J.; Stefan, M. C. Fine-tuning
thermoresponsive functional poly(?-caprolactone)s to enhance micelle
stability and drug loading. J. Mater. Chem. B 2015, 3 (9), 1779−1787.
(32) Washington, K. E.; Kularatne, R. N.; Du, J.; Ren, Y.; Gillings, M.
J.; Geng, C. X.; Biewer, M. C.; Stefan, M. C. Thermoresponsive star-
like [gamma]-substituted poly(caprolactone)s for micellar drug
delivery. J. Mater. Chem. B 2017, 5 (28), 5632−5640.
(33) Washington, K. E.; Kularatne, R. N.; Du, J.; Gillings, M. J.;
Webb, J. C.; Doan, N. C.; Biewer, M. C.; Stefan, M. C. Synthesis of
linear and star-like poly(ε-caprolactone)-b-poly{γ-2-[2-(2-methoxy-
ethoxy)ethoxy]ethoxy-ε-caprolactone} amphiphilic block copolymers
using zinc undecylenate. J. Polym. Sci., Part A: Polym. Chem. 2016, 54
(22), 3601−3608.
(34) Hao, J.; Granowski, P. C.; Stefan, M. C. Zinc Undecylenate
Catalyst for the Ring-Opening Polymerization of Caprolactone
Monomers. Macromol. Rapid Commun. 2012, 33 (15), 1294−1299.
(35) Kato, K.; Inoue, K.; Kudo, M.; Ito, K. Synthesis of graft
polyrotaxane by simultaneous capping of backbone and grafting from
rings of pseudo-polyrotaxane. Beilstein J. Org. Chem. 2014, 10, 2573−
2579.
(15) Robey, R. W.; Chakraborty, A. R.; Basseville, A.; Luchenko, V.;
Bahr, J.; Zhan, Z.; Bates, S. E. Histone Deacetylase Inhibitors:
Emerging Mechanisms of Resistance. Mol. Pharmaceutics 2011, 8 (6),
2021−2031.
́
(16) Cortes-Funes, H.; Coronado, C. Role of anthracyclines in the
era of targeted therapy. Cardiovasc. Toxicol. 2007, 7 (2), 56−60.
(17) Thorn, C. F.; Oshiro, C.; Marsh, S.; Hernandez-Boussard, T.;
McLeod, H.; Klein, T. E.; Altman, R. B. Doxorubicin pathways:
pharmacodynamics and adverse effects. Pharmacogenet. Genomics 2011,
21 (7), 440−446.
(18) el Bahhaj, F.; Denis, I.; Pichavant, L.; Delatouche, R.; Collette,
F.; Linot, C.; Pouliquen, D.; Gregoire, M.; Heroguez, V.; Blanquart,
́ ́
C.; Bertrand, P. Histone Deacetylase Inhibitors Delivery using
Nanoparticles with Intrinsic Passive Tumor Targeting Properties for
Tumor Therapy. Theranostics 2016, 6 (6), 795−807.
(19) Delatouche, R.; Denis, I.; Grinda, M.; Bahhaj, F. E.; Baucher, E.;
Collette, F.; Heroguez, V.; Gregoire, M.; Blanquart, C.; Bertrand, P.
́ ́
Design of pH-responsive clickable prodrugs applied to histone
deacetylase inhibitors: A new strategy for anticancer therapy. Eur. J.
Pharm. Biopharm. 2013, 85 (3), 862−872.
(20) Denis, I.; el Bahhaj, F.; Collette, F.; Delatouche, R.; Gueugnon,
́ ́
F.; Pouliquen, D.; Pichavant, L.; Heroguez, V.; Gregoire, M.; Bertrand,
P.; Blanquart, C. Histone deacetylase inhibitor-polymer conjugate
nanoparticles for acid-responsive drug delivery. Eur. J. Med. Chem.
2015, 95, 369−376.
(21) Senevirathne, S. A.; Boonsith, S.; Oupicky, D.; Biewer, M. C.;
Stefan, M. C. Synthesis and characterization of valproic acid ester pro-
drug micelles via an amphiphilic polycaprolactone block copolymer
design. Polym. Chem. 2015, 6 (13), 2386−2389.
(22) Alfurhood, J. A.; Sun, H.; Kabb, C. P.; Tucker, B. S.; Matthews,
J. H.; Luesch, H.; Sumerlin, B. S. Poly(N-(2-hydroxypropyl)-
methacrylamide)-valproic acid conjugates as block copolymer nano-
carriers. Polym. Chem. 2017, 8, 4983−4987.
(23) Senevirathne, S. A.; Washington, K. E.; Miller, J. B.; Biewer, M.
C.; Oupicky, D.; Siegwart, D. J.; Stefan, M. C. HDAC inhibitor
conjugated polymeric prodrug micelles for doxorubicin delivery. J.
Mater. Chem. B 2017, 5 (11), 2106−2114.
(24) Kularatne, R. N.; Yang, A.; Nguyen, H. Q.; McCandless, G. T.;
Stefan, M. C. Neodymium catalyst for the polymerization of dienes
and vinyl monomers. Macromol. Rapid Commun. 2017, 38, 1700427.
́
(25) Busto, E.; Martínez-Montero, L.; Gotor, V.; Gotor-Fernandez,
V. Chemoenzymatic Asymmetric Synthesis of Serotonin Receptor
Agonist (R)-Frovatriptan. Eur. J. Org. Chem. 2013, 2013 (19), 4057−
4064.
(26) Hao, J.; Cheng, Y.; Ranatunga, R. J. K. U.; Senevirathne, S.;
Biewer, M. C.; Nielsen, S. O.; Wang, Q.; Stefan, M. C. A Combined
Experimental and Computational Study of the Substituent Effect on
Micellar Behavior of γ-Substituted Thermoresponsive Amphiphilic
Poly(ε-caprolactone)s. Macromolecules 2013, 46 (12), 4829−4838.
H
DOI: 10.1021/acs.biomac.8b00221
Biomacromolecules XXXX, XXX, XXX−XXX