Pendant HDAC inhibitor SAHA derivatised polymer as a novel prodrug micellar carrier for anticancer drugs

Article abstract of DOI:10.1080/1061186X.2017.1419355

Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (HDACi) approved by FDA for the treatment of cutaneous T cell lymphoma, is a promising anticancer drug for various cancers with a unique mode of action. However, it demonstrates limited clinical benefits in solid tumours as a single drug. In order to achieve enhanced and synergistic co-delivery of SAHA and doxorubicin (DOX), a cleavable SAHA-based prodrug polymer (POEG-b-PSAHA), consisting of hydrophilic poly(oligo(ethylene glycol) methacrylate) (POEG) blocks and hydrophobic SAHA segments, has been developed. POEG-b-PSAHA prodrug polymer was able to form spherical micelles with a diameter around 60 nm and well retained the pharmacological activity of SAHA in either inhibiting the proliferation of tumour cells or inducing histone acetylation. DOX formulated in POEG-b-PSAHA-based micelles showed a sustained release profile. DOX-loaded POEG-b-PSAHA exhibited more potent cytotoxicity towards tumour cells than free DOX and DOX loaded in a pharmacologically ‘inert’ nanocarrier, POEG-b-POM. Consistently, DOX/POEG-b-PSAHA formulation resulted in an improved therapeutic effect in vivo compared to free DOX, Doxil or DOX formulated in POEG-b-POM micelles. These results suggest that SAHA-based prodrug micelles may serve as a dual functional carrier for combination strategies in epigenetic-oriented anticancer therapy.

Full text of DOI:10.1080/1061186X.2017.1419355

Journal of Drug Targeting
ISSN: 1061-186X (Print) 1029-2330 (Online) Journal homepage: http://www.tandfonline.com/loi/idrt20
Pendant HDAC Inhibitor SAHA Derivatized Polymer
as a Novel Prodrug Micellar Carrier for Anticancer
Drugs
Jieni Xu, Jingjing Sun, Pengcheng Wang, Xiaochao Ma & Song Li
To cite this article: Jieni Xu, Jingjing Sun, Pengcheng Wang, Xiaochao Ma & Song Li (2017):
Pendant HDAC Inhibitor SAHA Derivatized Polymer as a Novel Prodrug Micellar Carrier for
Anticancer Drugs, Journal of Drug Targeting, DOI: 10.1080/1061186X.2017.1419355
To link to this article: https://doi.org/10.1080/1061186X.2017.1419355
Accepted author version posted online: 18
Dec 2017.
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Date: 19 December 2017, At: 23:27

Pendant HDAC Inhibitor SAHA Derivatized Polymer as a
Novel Prodrug Micellar Carrier for Anticancer Drugs
Jieni Xua,b,c, Jingjing Suna,b,c, Pengcheng Wanga,b, Xiaochao Maa,b and Song Lia,b,c,*
aCenter for Pharmacogenetics, University of Pittsburgh, Pittsburgh, PA 15261, USA
bDepartment of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA
cUniversity of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA
Corresponding Author
*Song Li, M.D., Ph.D.; Tel, 412-383-7976; Fax, 412-648-1664; Email: sol4@pitt.edu; Center for Pharmacogenetics, Department of
Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, 313 Salk Pavilion, Pittsburgh, PA 15261.
Key Words: Vorinostat, Doxorubicin, Co-delivery, Prodrug micelles

Abstract:
Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (HDACI)
approved by FDA for the treatment of cutaneous T cell lymphoma, is a promising anticancer
drug for various cancers with a unique mode of action. However, it demonstrates limited
clinical benefits in solid tumors as a single drug. In order to achieve enhanced and synergistic
co-delivery of SAHA and Doxorubicin (DOX), a cleavable SAHA-based prodrug polymer
(POEG-b-PSAHA), consisting of hydrophilic poly(oligo(ethylene glycol) methacrylate)
(POEG) blocks and hydrophobic SAHA segments, has been developed. POEG-b-PSAHA
prodrug polymer was able to form spherical micelles with a diameter around 60 nm, and well
retained the pharmacological activity of SAHA in either inhibiting the proliferation of tumor
cells or inducing histone acetylation. DOX formulated in POEG-b-PSAHA-based micelles
showed a sustained release profile. DOX-loaded POEG-b-PSAHA exhibited more potent
cytotoxicity towards tumor cells than free DOX and DOX loaded in a pharmacologically
“inert” nanocarrier, POEG-b-POM. Consistently, DOX/POEG-b-PSAHA formulation
resulted in an improved therapeutic effect in vivo compared to free DOX, Doxil, or DOX
formulated in POEG-b-POM micelles. These results suggest that SAHA-based prodrug
micelles may serve as a dual functional carrier for combination strategies in epigenetic-
oriented anticancer therapy.

1. Introduction:
Cancer development and progression is not only confined to genetic changes, but also
involves epigenetic changes, leading to alterations in gene expression and cell phenotypes.
Epigenetics is concerned with the heritable phenotypes based on changes in chromosome, as
opposed to genetics, whose realm is on the basis of alterations in the primary DNA sequence
(Avendaño and Menéndez, 2015, Berger et al., 2009). Alterations in the structure and
modification status of chromatin represent powerful regulatory mechanisms for gene
expression and genome stability (Avendaño and Menéndez, 2015). The major epigenetic
modifications in mammals, and particularly in humans, are DNA methylation and post-
translational histone modifications (acetylation, methylation, phosphorylation etc.) (Gibney
and Nolan, 2010). In the context of histone modifications, the acetylation status of histones
plays a crucial role in regulating gene expression by affecting the accessibility of DNA
around them (Kouzarides, 2007). Acetylation levels are controlled by the balance of two
enzymes families: histone acetyltransferase (HAT) and histone deacetylase (HDAC). HAT
facilitates the acetylation of lysine in histone tails, which is associated with a more relaxed
chromatin state, opening up access for transcription factors and polymerases, and therefore
gene-transcription activation. In contrast, HDACs deacetylate the lysine residues thereby
promoting a more condensed chromatin state and hence leading to transcriptional gene
silencing (Grunstein, 1997, Gregory et al., 2001). Specially, the removal of the acetyl groups
by HDACs also exposes the protonated amino groups of lysine units of histones, thereby
increasing ionic interactions between the positively charged histones and negatively charged
phosphate backbone of DNA, which yields a more compact chromatin structure and represses
gene transcription by limiting the accessibility of the transcription machinery (Gregory et al.,
2001).

A growing body of evidence indicates that the silence of tumor suppressor genes caused by
HDAC overexpression could be a common phenomenon in tumor onset and progression (Zhu
et al., 2004, Halkidou et al., 2004, Song et al., 2005). Therefore, there is growing interest in
the development of HDAC inhibitors (HDACi) as anticancer agents and a large number of
studies demonstrated the potential of HDACi in different cancer cell lines and in animal
models of tumors (Marks et al., 2001, Johnstone, 2002, Falkenberg and Johnstone, 2014).
The oral drug Vorinostat, also known as SAHA (from suberoylanilide hydroxamic acid) was
the first HDAC inhibitor approved by the U.S. FDA in 2006 for the treatment of cutaneous T
cell lymphoma in patients who have progressive, persistent or recurrent disease or following
failure of two systemic therapies. It was also approved for multiple myeloma with slight
advantage over Velcade®. SAHA is a pan-HDAC inhibitor and inhibits both classes I and II
enzymes, but does not inhibit HDACs belonging to class III (Marks, 2007, Marks et al.,
2001). SAHA acts as a micromolar inhibitor of HDACs by chelating the zinc atoms in
HDAC catalytic sites via its hydroxamic end, with the phenyl ring protruding out of the
binding pocket and lying on the hydrophobic surface of HDAC (Marks and Breslow, 2007).
SAHA blocks the proliferation of cultured tumor cells and suppresses tumor growth in a
variety of tumor models including prostate cancer (Butler et al., 2000), breast cancer (Cohen
et al., 1999), leukemia (He et al., 2001), glioma (Eyupoglu et al., 2005) and lung cancer
(Desai et al., 2003) with little or no toxicity.
Although SAHA has significant anticancer activity as a monotherapy in hematological
malignancies, it demonstrates limited clinical benefit for patients with solid tumors,
prompting the investigation of novel combination treatments with existing neoplastic
interventions (Thurn et al., 2011). Besides, the acceptable toxicity profile of SAHA permits a
broad integration into currently approved chemotherapy regimens (Munster et al., 2009,

Marchion et al., 2005b, Marchion et al., 2005a). Based on a mechanistic rationale, SAHA has
been shown to be additive or synergistic with a wide array of anticancer drugs, including
conventional chemotherapeutics (doxorubicin, paclitaxel, cisplatin, gemcitabine, 5-
fluorouracil and etoposide) (Arnold et al., 2007, Dowdy et al., 2006, Rikiishi et al., 2007,
Kim et al., 2003), targeted agents (imatinib, bevacizumab and trastuzumab) (Nimmanapalli et
al., 2003, Fuino et al., 2003) and transcriptional modulators (all-trans retinoic acid and the
demethylating agent 5-aza-2′-deoxycytidine) (Cameron et al., 1999). One of the most
significant combinations is with DNA damage-inducing therapies, which occurs, in part,
through a SAHA-mediated increase in chromatin accessibility and downregulation of DNA
repair (Thurn et al., 2011). Doxorubicin induces DNA strand breaks by binding to DNA,
stabilizing the Topo II-DNA complex, and inhibiting the re-ligation of DNA strands during
replication (Thorn et al., 2011). SAHA potentiates DOX-induced Topo II-mediated DNA
damage, growth inhibition and cell death through the chromatin decondensation, facilitating
the access of topo II inhibitors to their DNA substrate (Marchion et al., 2005b, Marchion et
al., 2005a). Moreover, the synergy is further intensified by the effect of SAHA on the
expression, regulation and activation of a variety of DNA repair and DNA damage response
genes, like Ku70 (Chen et al., 2007).
Although the combination of SAHA and other anticancer agents helps, to some extent,
achieve better treatment of cancer, the triumph of simple physical mixture of multiple drugs
is largely hindered as a result of poor solubility, minimal distribution to tumor tissue, and
suboptimal dose ratio (Parhi et al., 2012). Carrier-mediated combination therapy could offer
some advantages to overcome the above challenges, including improved solubility and
bioavailability of each drug, increased accumulation at tumor sites through EPR effect,
synchronized and controlled pharmacokinetics of each drug and ratiometric dosing, that is,

the ability to tailor the relative ratios of each agent based on its pharmacological disposition
(Lee and Nan, 2012).
Among the commonly used drug delivery systems, polymers with diverse structures have
been widely selected as the nanocarriers for hydrophobic anticancer drugs, including SAHA,
DOX, and others. Several nanoscopic formulations of SAHA have been developed for
parenteral administrations to improve the solubility and overall disposition profile. For
example, PEG-b-PLA block copolymer-based micelles have been employed as a carrier to
encapsulate SAHA, and it was found that SAHA loaded in micelles provided sustained
exposure and improved pharmacokinetics characterized by a significant increase in serum t1/2,
AUC and mean residence time (Mohamed et al., 2012). However, the use of a large amount
of pharmacologically inert carriers may not only add to the cost but also imposes safety issue
(Croy and Kwon, 2006). Prodrug polymer nanocarriers represent an effective strategy to
reduce the amount of inert materials. More importantly, amphiphilic prodrug polymers self-
assemble in aqueous solution and serve as dual-functional carriers to achieve the additive or
synergistic effect with co-delivered anticancer drugs.
In this study, we designed and synthesized SAHA-based prodrug polymer, denoted as POEG-
b-PSAHA. These amphiphilic polymers could self-assemble into prodrug micelles and serve
as nanocarriers for DOX delivery. The size and structure of POEG-b-PSAHA were
characterized. Moreover, the in vitro pharmacological activity of POEG-b-PSAHA prodrug
micelles was examined and compared with free SAHA. Finally, the antitumor activity of
DOX-loaded POEG-b-PSAHA micelles was investigated in a syngeneic breast cancer model.

2. Methods
2.1 Materials
Aniline, N,N′-dicyclohexylcarbodiimide, 4-(dimethylamino)pyridine, hydroxylamine
hydrochloride, 1-hydroxybenzotriazole hydrate, potassium carbonate, potassium hydroxide,
vinylbenzyl chloride, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid,
oligo(ethylene glycol) methacrylate OEGMA (average Mn
=
500), 2,2-
Azobis(isobutyronitrile) (AIBN), trypsin-EDTA solution, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) and Dulbecco’s Modified Eagle’s Medium (DMEM)
were all purchased from Sigma-Aldrich (MO, USA). AIBN was purified by recrystallization
in anhydrous ethanol. Monomethyl suberate was purchased from TCI (OR, USA). DOX.HCl
was purchased from LC Laboratories (MA, USA). Fetal bovine serum (FBS) and penicillin-
streptomycin solution were purchased from Invitrogen (NY, USA.). Cell culture and animals
were similarly handled as described before (Zhang et al., 2014b).
2.2 Methods
2.2.1 Synthesis of SAHA-monomer
SAHA was synthesized following a published method (Chung et al., 2013).
Vinylbenzyl chloride (1.0 g, 6.6 mmol), succinic acid (7.7 g, 66.0 mmol, 10 equiv) and
K2CO3 (4.5 g, 32.8 mmol, 5 equiv) were dissolved in 30 mL DMF and immersed into an oil
bath at 90 °C. After 4 h, the mixture was filtered through cottons to remove excess amount of
salt. The reaction was quenched by addition of excess amount of pure water and centrifuged
to remove DMF. The precipitate was dissolved in DCM, and the crude product was purified
by column chromatography on silica gel eluted with ethanol/DCM (v/v, 1: 9) to give the
Compound 1 (1.4 mg, 91%).

For the synthesis of SAHA-monomer, CDI (69 mg, 0.43 mmol, 1.2 equiv) was added in
portions to a stirred solution of Compound 1 (100 mg, 0.43 mmol, 1.2 equiv) in DCM. After
stirring for 10 min, the reaction mixture turned into light yellow color. TLC showed that the
intermediate was formed, and then SAHA (94 mg, 0.36 mmol) was added to the mixture in
portions. After 2 h, the reaction was complete as confirmed by TLC, and the reaction was
quenched by the addition of the saturated sodium bicarbonate. The aqueous layer was
extracted with DCM, washed with brine, and dried over MgSO4. The crude product was
chromatographed on silica gel (ethyl acetate/hexane =2:1) to give SAHA monomer (125 mg,
69%).
2.2.2 Synthesis of POEG macroCTA
POEG macroCTA was synthesized and purified following a published literature (Sun et al.,
2016).
2.2.3 Synthesis of POEG-b-PSAHA
POEG macroCTA (150 mg, 0.02 mmol), SAHA-monomer (125 mg, 0.26 mmol, 13 equiv),
AIBN (1 mg, 0.0061 mmol, 0.3 equiv) and 2 mL dried 1, 4-Dioxane were added into An
Schlenk tube and deoxygenated by free-pump-thawing for three times. The mixture was then
protected under N2 and immersed into an oil bath at 90 °C. After 24 h, the reaction was
quenched by immersing the tube into liquid nitrogen. The mixture was purified through three
cycles of dissolution/precipitation in DCM/hexane, and dried in vacuum to afford POEG-b-
PSAHA (260mg, 96%).

2.2.4 Preparation and characterization of DOX-loaded POEG-b-PSAHA micelles
The blank or DOX-loaded POEG-b-PSAHA micelles were prepared through the film
hydration method. First, DOX.HCl was neutralized with 3 equivalents of triethylamine in a
mixture of DCM/methanol (1:1, v/v). DOX (5 mg/mL) was then mixed with POEG-b-
PSAHA (50 mg/mL in DCM) at various carrier/drug weight ratios. After removing the
solvent by nitrogen flow, the thin film was formed, which was further dried under vacuum for
2 h to remove any trace of remaining solvent. Then the thin film was hydrated with PBS
followed by vortexing. The blank micelles were similarly prepared. The average diameter
and the size distribution of micelles were examined via a Zetasizer (DLS). The morphology
of blank or DOX-loaded POEG-b-PSAHA micelles was observed using transmission electron
microscopy (TEM).
The drug loading efficiency of DOX was examined by Waters Alliance 2695 Separations
Module combined with Waters 2475 Fluorescence Detector (excitation, 490 nm; emission,
590 nm; gain, 3; sensitivity (FUFS), 10 000) and high performance liquid chromatography
(HPLC) respectively as described in previous publications (Zhang et al., 2014a). Drug
loading capacity (DLC) and drug loading efficiency (DLE) were calculated according to the
following equation: DLC %= [weight of drug loaded/(weight of polymer used + weight of
drug used)]×100%, DLE %= (weight of loaded drug/weight of input drug)×100 %. The
colloidal stability of drug-loaded micelles with various carrier/drug molar ratios at 4°C was
evaluated by following the changes in sizes of the particles or visible precipitates every hour
in the first 12 h and daily after 12 h following sample preparation.
The CMC of POEG-b-PSAHA was measured by fluorescence spectrometry using nile red as
a fluorescence probe. Briefly, thirty microliters of nile red (0.05 mg/mL in DCM) were added

to each tube and the solvent was removed by nitrogen flow. POEG-b-PSAHA micelles
ranging from 6.1× 10−5 to 5 ×10−1 mg/mL were prepared with serial dilution as described
above and then added into the tubes with nile red. The final concentration of nile red was
kept at 6.0 × 10−7 M. The micelles were vortexed and kept overnight at room temperature to
reach the equilibrium of solubilization. The samples were excited at an excitation wavelength
of 550 nm and fluorescence intensities were recorded at an emission spectrum from 600 to
700 nm. The peak intensities at 647 nm were plotted versus polymer concentrations. The
CMC value was calculated as the cross-point where a sharp increase in fluorescence intensity
was observed.
The in vitro release of DOX from DOX-loaded POEG-b-PSAHA micelles was examined
using a dialysis method. Briefly, two hundred and fifty microliters of DOX/POEG-b-PSAHA
micelles or free DOX·HCl (0.5 mg DOX/mL) were transferred to clamped dialysis bags
(MWCO 3500 Da) in triplicate and incubated in 50 mL PBS as release medium, with gentle
shaking at 100 rpm at 37 °C. One milliliter of medium was withdraw and same volume of
fresh medium was supplemented at 10 min, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48 and 72 h. DOX
release from micelles was measured by fluorescence spectrometry at the excitation
wavelength of 480 nm and emission wavelength of 590 nm by Synergy H1 Hybrid Multi-
Mode Microplate Reader (Winooski, VT).
2.2.5 In vitro cytotoxicity assay
Cytotoxicity assay was performed with 4T1.2 mouse breast cancer cells, MCF-7 human
breast cancer cells, and HCT-116 human colon cancer cells. 4T1.2 (1×103 cells/well), MCF-7
(5×103 cells/well), or HCT-116 (2×103 cells/well) cells were seeded in 96-well plates
followed by 24 h of incubation in DMEM with 10% FBS and 1% streptomycin/penicillin. To
evaluate the combinational effect of SAHA with other anticancer drugs, various

concentrations of free SAHA, free DOX·HCl, or the combination of both were added in
triplicate to cells and incubated for 48 h. To study the cytotoxicity of blank POEG-b-PSAHA
micelles, cells were challenged with various concentrations of POEG-b-PSAHA micelles or
free SAHA (in DMSO) at equivalent SAHA concentrations for 48 h. The cytotoxicity of
DOX-loaded POEG-b-PSAHA micelles was also examined, in comparison to DOX.HCl and
DOX loaded in a biologically inert polymer POEG-b-POM. Cells were treated with different
DOX formulations of varied concentrations. Blank POEG-b-PSAHA or POEG-b-POM at
concentrations equivalent to those of carriers in the corresponding DOX formulation groups,
was also added into cells. Cells were incubated for 48 h and cell viability was determined by
MTT assay. Briefly, ten microliters of 3-(4, 5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide (MTT) (5mg/mL in PBS) were added to cells. Following
incubation for 4 h, the medium was removed and MTT formazan was solubilized by DMSO.
The absorbance was measured by a microplate reader with wavelength at 590 nm and
reference wavelength at 620 nm. Untreated cells were used as a control. Cell viability was
calculated as [(ODtreat -ODblank )/(ODcontrol -ODblank )×100%].
2.2.6 Western blot
Western immunoblotting was conducted with 4T1.2 cells. Cells were treated with 0.1%
DMSO (control) or indicated concentrations (1 M or 5 M) of SAHA or POEG-b-PSAHA,
respectively. After 24 h treatment, cells were rinsed twice with ice-cold PBS and lysed by
RIPA buffer. The lysates were centrifuged at 12,500 rpm for 10 min. Samples with equal
amounts of total cellular proteins were subjected to sodium dodecylsulfate polyacryl amide
gel electrophoresis (SDS-PAGE), followed by transferring to nitrocellulose membranes. The
membranes were first blocked in 5% nonfat dry milk dissolved in DPBS with 0.1% Tween 20
(PBST) for 1 h at RT, and then incubated with primary antibody at a final dilution of 1:1,000

in 5% BSA in PBST overnight at 4 °C. After washing three times with PBST, the membranes
were incubated with secondary antibody at a final dilution of 1:5,000 in PBST for 1 h at room
temperature. After washing three times with PBST, bound antibodies were detected by
chemiluminescence. Beta-actin was used as the loading control. Primary antibodies for AC-
H3 and AC-H4 were from Cell Signaling Technology (MA, USA) and the antibody for beta-
actin was from Sigma-Aldrich (MO, USA).
2.2.7 Plasma pharmacokinetics
For plasma pharmacokinetics, DOX/POEG-b-PSAHA micelles or free DOX.HCl was
injected intravenously into tumor-free female CD1 mice at a DOX dose of 5 mg/kg, with five
mice in each group. Blood samples were collected in heparinized tubes at designated time
points (3min, 10 min, 30 min, 1 h, 2 h, 4 h, 8 h and 12 h) post injection. The samples were
centrifuged at 12,500 rpm for 10 min at 4 °C and 20 μL of the supernatant were collected.
Then 200 μL methanol, containing 500 ng/mL daunorubicin as the internal standard, was
added and vortexed for 3 min. The samples were centrifuged at 12,500 rpm for 10 min at
4 °C and 150 μL of the clear supernatant was collected and injected into HPLC for DOX
analysis.
2.2.8 In vivo therapeutic study
The in vivo antitumor efficacy of the DOX loaded in POEG-b-PSAHA prodrug nanocarrier
was tested in a syngeneic 4T1.2 mouse breast cancer model. Female BALB/c mice were s.c.
inoculated with 4T1.2 cells at right mammary fat pad with 2×105 cells/mouse. When the
tumor sizes reached around 50 mm3, mice were randomly divided into five groups (n=5), and
treated via tail vein injection with PBS, DOX.HCl, Doxil, DOX/POEG-b-POM or
DOX/POEG-b-PSAHA micelles at a DOX dose of 5 mg/kg on days 1, 4 and 7, respectively.

Tumor sizes were measured with the digital caliper every three days following the initiation
of the treatment and calculated by the formula: (L×W2)/2, in which L is the longest and W is
the shortest in tumor diameters (mm). Data were plotted as the actual tumor volume. Body
weights were also monitored for the indication of toxicity. On day 23 post injection, all mice
were sacrificed and tumor tissues were collected for measurement of weights and
photography.
2.2.8 Statistical analysis
In vitro or in vivo data are presented as mean ± standard deviation (SD) or mean ± standard
error of mean (SEM), respectively. Statistical analysis was performed with two-tailed
Student’s T test or one-way analysis of variance (ANOVA) for two groups or multiple groups,
respectively, followed by Turkey simultaneous post hoc test. In all statistical analyses,
P < 0.05 was considered statistically significant.
3. Results and Discussion:
3.1 Effect of combination of SAHA and DOX on tumor cell proliferation
The proliferation inhibitory effect of SAHA and DOX was examined in 4T1.2 and HCT-116
cell lines. As shown in Fig. 1A, SAHA or DOX inhibited the proliferation of 4T1.2 cells in a
concentration-dependent manner. It was also apparent that combination of the two agents led
to enhanced cytotoxicity. A similar combinational effect was found in HCT-116 cells (Fig.
1B). Combination index (CI) was then calculated to assess a potential synergism between
SAHA and DOX, by the equation CI=(d1/IC501)+ (d2/IC502), with d1 or d2 being the
concentration of SAHA or DOX required to achieve 50% killing effect in co-treatment, while
IC501 or IC502 being IC50 of SAHA or DOX in single treatment, respectively. The CI of

SAHA and DOX combination were 0.67 and 0.84 in 4T1.2 and HCT-116 cells respectively,
suggesting a synergism between the two drugs.
3.2 Synthesis and characterization of the POEG-b-PSAHA polymer
We initially designed and synthesized a SAHA-based monomer by directly conjugating
SAHA with methacryloyl chloride. The macro-chain transfer agent POEG was then
synthesized by RAFT polymerization of hydrophilic OEGMA monomer, which was further
used to initiate the polymerization of hydrophobic SAHA monomer. However, micelles
formed from this polymer had a size around 160 nm (data not shown), which is suboptimal
for EPR effect. In order to decrease the hydrodynamic size, we re-designed a new SAHA
monomer by introducing an additional 4 carbon hydrophobic chain and another benzyl ring to
SAHA (Scheme. 1). Vinylbenzyl chloride was linked to succinic acid, followed by the
condensation reaction with SAHA to form a hydrolysable diester bond. Then RAFT
polymerization was similarly performed as described above, yielding the amphiphilic POEG-
b-PSAHA block copolymer. The structure of POEG-b-PSAHA polymer was confirmed by
1H NMR (Fig. 2), and the average degree of polymerization of the SAHA monomer was
calculated. Each POEG-b-PSAHA molecule contained 12 units of SAHA.
3.3 Characterizations of DOX-loaded POEG-b-PSAHA micelles
Blank or DOX-loaded POEG-b-PSAHA micelles were prepared by a simple film hydration
method. Unlike the initial SAHA polymer that formed relatively large-sized micelles (~160
nm), the size of drug-free POEG-b-PSAHA micelles was significantly smaller (63.7 nm).
This is likely due to more effective carrier-carrier interaction as a result of incorporation of
additional 4 carbon hydrophobic chain and another benzyl ring to SAHA monomer. Loading
of DOX into POEG-b-PSAHA micelles at a carrier/drug weight ratio of 20:1 only led to a

slight increase in the particle size (~70 nm) (Fig. 3B). TEM showed sphere shape with a
uniform size distribution, which was consistent with DLS (Fig. 3C, D). The size, DLC, and
formulation stability of drug-loaded POEG-b-PSAHA micelles were then examined and
listed in Table 1. DOX could be loaded into POEG-b-PSAHA micelles at a carrier/drug ratio
as low as 5/1 (mg/mg), at which ratio DOX loading capacity was 14.9 % and DOX-loaded
micelles were stable for 5 days at 4 °C. With an increase in the carrier/drug ratio, the drug
encapsulation efficiency and colloidal stability were further improved. As shown in Table 1,
at a carrier/drug ratio of 50:1, DOX-loaded micelles were stable for 50 days at 4 °C. A
carrier/drug mass ratio of 20:1 and 5:1 was used for in vitro and in vivo study, respectively.
Fig. 4 shows that the CMC of POEG-b-PSAHA micelles was around 0.004 mg/mL as
determined by a rapid change of fluorescence intensity of loaded nile red probe during serial
dilution. This low CMC of POEG-b-PSAHA shall provide a good stability for the micelles
upon dilution in blood stream after i.v. injection.
3.4 In vitro drug release
The release profile of DOX loaded in POEG-b-PSAHA micelles in PBS was investigated by
a dialysis method and compared to that of free DOX. As shown in Fig. 5, around 50% of
DOX was released from DOX.HCl in the first two hours and 85% of total DOX was released
within 12 h. In contrast, less than 1% of DOX was released from the DOX-loaded POEG-b-
PSAHA micelles in the first hour and a relatively sustained release continued for 72 hours,
with only around 30% of total drug released. The slow release kinetics might be due to strong
carrier-drug interaction, including hydrophobic interaction and π-π stacking, which shall
provide a good stability of DOX/POEG-b-PSAHA in blood stream.

3.5 In vitro cytotoxicity of drug-free or DOX-loaded POEG-b-PSAHA micelles
To evaluate whether POEG-b-PSAHA prodrug micelles maintained the ability of SAHA to
inhibit tumor cell proliferation, 4T1.2, MCF-7 or HCT-116 cells were treated with POEG-b-
PSAHA micelles or free SAHA (in DMSO) for 48 h. As shown in Fig. 6A, free SAHA
inhibited the proliferation of 4T1.2 tumor cells in a dose-dependent manner, while POEG-b-
PSAHA showed a comparable cytotoxicity. Similar results were shown in MCF-7 and HCT-
116 tumor cells (Fig. 6B & C), suggesting that SAHA could be effectively released from
POEG-b-PSAHA via the cleavage of carbamide bond.
The in vitro cytotoxicity of DOX-loaded POEG-b-PSAHA micelles was also tested, in
comparison with DOX.HCl and DOX loaded in pharmacologically “inert” POEG-b-POM
micelles. POEG-b-PSAHA and POEG-b-POM carrier alone were included as controls and
were used at the same concentrations as those used in drug-loaded micelles. As shown in Fig.
7A, POEG-b-POM blank micelles showed minimal cytotoxicity even at the highest
concentration (20 μg/mL). In contrast, blank POEG-b-PSAHA micelles exhibited significant
cytotoxicity, which was comparable to that of DOX.HCl. DOX loaded in POEG-b-PSAHA
was more potent in killing tumor cells than DOX.HCl and DOX/POEG-b-POM formulation.
Similar results were observed in MCF-7 and HCT-116 cells (Fig. 7B & C). It is likely that the
improved cytotoxicity of DOX/POEG-b-PSAHA is attributed to a synergistic action between
DOX and SAHA that was released from the carrier following intracellular delivery.
3.6 Effect of POEG-b-PSAHA on histone acetylation
To investigate if the POEG-b-PSAHA-mediated cytotoxicity is attributed to the specific
action of released SAHA, we examined the efficiency of POEG-b-PSAHA in modulating the
acetylation of histone in 4T1.2 breast cancer cells by Western blot. As shown in Fig. 8, the
basal levels of acetylated H3 and H4 were very low. SAHA treatment at submicromolar

concentrations for 24 h led to significant increases in their levels in a dose-dependent manner.
Interestingly, POEG-b-PSAHA was comparable to or even more potent than free SAHA in
inducing acetylation of H3 or H4 at the same concentrations of SAHA. These data indicated
that POEG-b-PSAHA well retained the bioactivity of SAHA in inhibiting HDAC, resulting in
the hyperacetylation of histone H3 and H4 and subsequently inhibition of cancer cell
proliferation. The possible mechanism for the comparable or even further enhanced efficacy
in histone acetylation is that POEG-b-PSAHA protected the hydroxamic acid of SAHA from
hydrolysis and pharmacologically active SAHA was slowly released from the polymer over a
prolonged period of time. On the other hand, free SAHA with unprotected hydroxamic acid
may be subjected to rapid metabolic conversion to inactive metabolites, SAHA-glucuronide
and 4-anilino-4-oxobutanoic acid (Hattori et al., 2011).
3.7 Plasma pharmacokinetics
The DOX/POEG-b-PSAHA or DOX.HCl was injected into tumor-free mice at a DOX dose
of 5 mg/kg. The plasma concentrations of DOX were examined at different time points. The
initial concentration of POEG-b-PSAHA was around 1 mg/mL, which was 250-fold higher
than its CMC (0.004 mg/mL). The concentrations of DOX in the blood following i.v.
injection of different treatments as a function of time were illustrated in Fig. 9. Compared to
DOX.HCl group, the plasma concentrations of DOX from the group treated with POEG-b-
PSAHA/DOX were significantly higher at early time points and maintained at relatively high
levels until 12 h, which is likely attributed to the surface modification of PEG and stealth-
shielding against RES system. These data demonstrated that DOX formulated in POEG-b-
PSAHA prodrug micelles was able to circulate for a significantly longer period of time in the
blood.

3.8 In vivo therapeutic study
Following the demonstration of a potential synergistic effect between POEG-b-PSAHA-
based carrier and the co-delivered DOX in cultured cancer cells, we then evaluated the tumor
growth inhibitory effect of DOX/POEG-b-PSAHA in a highly metastatic syngeneic murine
breast cancer model (4T1.2, s.c.). As shown in Fig. 10A, free DOX exhibited a modest
antitumor activity at a dose of 5 mg/kg. Doxil and DOX/POEG-b-POM formulations were
more effective than free DOX. At the same dose of DOX, DOX/POEG-b-PSAHA was most
effective in inhibiting the tumor growth (Fig. 10A). Fig. 10 B & D show the weights and
images of tumor tissues collected at the end of the experiment, which were consistent with
the result of tumor growth curves (Fig. 10A). The significantly improved anti-tumor activity
of DOX/POEG-b-PSAHA is likely attributed to the effective accumulation of the nanocarrier
at tumor sites due to its small size (~70 nm). The synergistic effect between the released
SAHA and co-delivered DOX may also play an important role in tumor inhibition. More
studies are needed in the future to further delineate the underlying mechanism. There were
slight increases in body weights in all groups over the period of study, suggesting the
negligible toxicity of DOX/POEG-b-PSAHA micelles in vivo.
4. Conclusion:
We have developed a well-characterized POEG-b-PSAHA prodrug micellar nanocarrier,
which consists of a hydrophobic segment composed of 12 SAHA-based units, and a POEG
hydrophilic segment for efficient entrapment of DOX. Our POEG-b-PSAHA prodrug
micelles well retained the biological activity of SAHA in inhibiting the proliferation of tumor
cells and promoting the acetylation of histone H3 and H4. Besides, POEG-b-PSAHA was
effective in formulating DOX and demonstrated a slow kinetics of drug release. More

importantly, in vivo delivery of DOX via POEG-b-PSAHA led to significant inhibition of
4T1.2 tumor, much more effectively than DOX.HCl, Doxil and DOX/POEG-b-POM.
Acknowledgments
We would like to thank Dr. Ming Sun for her help with negative EM study.
Declaration of interest
This work was supported by NIH grants no. R01CA174305 and no. R01CA219399. The
authors declare no competing financial interest.
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Fig. 1
120
100
80
60
40
20
0
120
100
80
60
40
20
0
A
B
4T1.2
HCT-116
0
0
1 µM SAHA
1 µM SAHA
2.5 µM SAHA
2.5 µM SAHA
5 µM SAHA
10 µM SAHA
5 µM SAHA
10 µM SAHA
0
100
200
300
400
500
0
100
200
300
400
500
DOX Concentration (ng/mL)
DOX Concentration (ng/mL)
CI=0.67
CI=0.84
Fig. 2

Fig. 3
Fig. 4

Fig. 5
100
80
60
40
20
0
***
***
***
***
***
***
***
***
***
POEG-b-PSAHA
DOX×HCl
***
0
10 20 30 40 50 60 70 80
Time (hours)
Fig. 6
A
B
C
120
MCF-7
HCT-116
120
100
80
60
40
20
0
120
100
80
60
40
20
0
4T1.2
100
80
60
40
20
0
SAHA
SAHA
SAHA
POEG-b-PSAHA
POEG-b-PSAHA
***
POEG-b-PSAHA
**
***
***
***
***
0.1
1
10
100
0.1
1
10
100
0.1
1
10
100
SAHA Concentration (mM)
SAHA Concentration (µM)
SAHA Concentration (µM)
Fig. 7
A
B
C
120
120
100
80
60
40
20
0
120
100
80
60
40
20
0
MCF-7
HCT-116
4T1.2
100
80
60
40
20
0
*
***
**
&
*
&&&
***
##
&&&
&
#
***
&&
###
&&&
##
&&&
&&&
&&&
***
POEG-b-POM
#
POEG-b-POM
##
POEG-b-POM
&&&
#
&&&
&&&
POEG-b-PSAHA
DOX.HCl
POEG-b-PSAHA
DOX.HCl
POEG-b-PSAHA
***
&&&
***
###
###
*
DOX.HCl
##
POEG-b-POM/DOX
POEG-b-PSAHA/DOX
&&&
POEG-b-POM/DOX
POEG-b-PSAHA/DOX
POEG-b-POM/DOX
***
&&&
###
***
**
#
POEG-b-PSAHA/DOX
#
0.1
1
10
100
1000
0.1
1
10
100
1000
1
10
100
1000
DOX Concentration (ng/mL)
DOX Concentration (ng/mL)
DOX Concentration (ng/mL)

Fig. 8
Fig. 9
40
35
30
25
20
15
10
5
DOX.HCl
POEG-b-PSAHA/DOX
0
0
2
4
6
8
10
12
Time (hours)

Fig. 10

Fig. 11

Scheme 1.

Table 1.
Micelles
Mass ratio
(mg: mg) a
Size (nm)b
PDIc
DLC(%)d DLE(%)e Stabilityf
63.7 ± 0.7 0.14 ± 0.01
78.4 ± 0.4 0.22 ± 0.01
POEG-b-PSAHA:
5:1
14.9
8.2
4.5
3.0
1.9
89.3
90.6
93.5
92.3
96.7
5 d
9 d
POEG-b-PSAHA:
DOX
10: 1
20:1
30: 1
50:1
75.9 ± 0.6 0.18 ± 0.01
73.9 ± 0.9 0.26 ± 0.01
79.7 ± 0.3 0.23 ± 0.01
POEG-b-PSAHA:
DOX
17 d
32 d
50 d
POEG-b-PSAHA:
DOX
POEG-b-PSAHA:
DOX
86.6 ± 0.2 0.20± 0.01
POEG-b-PSAHA:
DOX
b)
a) DOX concentration in micelles was kept at 0.5 mg/mL. Measured by dynamic light scattering
c)
d)
e)
particle sizer. PDI = polydispersity index. DLC = drug loading capacity. DLE = drug loading
efficiency. f) Data mean there was no noticeable size change and visible precipitates during the
follow-up period at 4 °C.