BASIC SCIENCE

Nanomedicine: Nanotechnology, Biology, and Medicine10 (2014) 721–732

Original Article nanomedjournal.com

Epigenetics targeted protein-vorinostat nanomedicine inducing apoptosisin heterogeneous population of primary acute myeloid leukemia cells

including refractory and relapsed casesParwathy Chandran, MTecha, Anu Kavalakatt, MTecha,

Giridharan Loghanathan Malarvizhi, MTecha,Divya Rani Vikraman Nair Vasanthakumari, PhDa, Archana Payickattu Retnakumari, MTecha,

Neeraj Sidharthan, MD, DMb, Keechilat Pavithran, MD, DMb,Shantikumar Nair, PhDa, Manzoor Koyakutty, PhDa,⁎

aAmrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Kochi, Kerala, IndiabDepartment of Medical Oncology, Amrita Institute of Medical Sciences and Research Centre, Kochi, Kerala, India

Received 29 August 2013; accepted 24 September 2013

Abstract

Aberrant epigenetics play a key role in the onset and progression of acute myeloid leukemia (AML). Herein we report in silico modellingbased development of a novel, protein-vorinostat nanomedicine exhibiting selective and superior anti-leukemic activity againstheterogeneous population of AML patient samples (n = 9), including refractory and relapsed cases, and three representative cell linesexpressing CD34+/CD38− stem cell phenotype (KG-1a), promyelocytic phenotype (HL-60) and FLT3-ITD mutation (MV4-11). Nano-vorinostat having ~100 nm size exhibited enhanced cellular uptake rendering significantly lower IC50 in AML cell lines and patientsamples, and induced enhanced HDAC inhibition, oxidative injury, cell cycle arrest and apoptosis compared to free vorinostat. Mostimportantly, nanomedicine showed exceptional single-agent activity against the clonogenic proliferative capability of bone marrow derivedleukemic progenitors, while remaining non-toxic to healthy bone marrow cells. Collectively, this epigenetics targeted nanomedicine appearsto be a promising therapeutic strategy against various French-American-British (FAB) classes of AML.

From the Clinical Editor: Through the use of a protein-vorinostat agent, exceptional single-agent activity was demonstrated against the clonogenicproliferative capability of bonemarrowderived leukemic progenitors, while remaining non-toxic to healthy bonemarrow cells. The studied epigeneticstargeted nanomedicine approach is a promising therapeutic strategy against various French-American-British classes of acute myeloid leukemia.© 2014 Elsevier Inc. All rights reserved.

Key words: Epigenetics; Nano-vorinostat; Protein nanomedicine; Acute myeloid leukemia

This work was supported by Department of Biotechnology (DBT),Government of India, under the project ‘In silico design, development,nanotoxicology and preclinical evaluation of theragnostic cancer nanome-dicine Phase-II’ (BT/PR14920/NNT/28/503/2010). PC thanks Council ofScientific and Industrial Research (CSIR), Government of India, for SeniorResearch Fellowship. The authors are grateful to Mr. Sarath S, Mr. Sajin PRavi and Mrs. Sreerekha P R, for their technical assistance extended duringthe work.

Disclosures/Conflicts of interest: None.⁎Corresponding author: Amrita Centre for Nanosciences and Molecular

Medicine, Amrita Institute of Medical Sciences and Research Centre, Kochi,Kerala, India.

E-mail address: manzoork@aims.amrita.edu (M. Koyakutty).

1549-9634/$ – see front matter © 2014 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.nano.2013.09.008

Please cite this article as: Chandran P, et al, Epigenetics targeted protein-vorprimary acute myeloid leuke.... Nanomedicine: NBM 2014;10:721-732, http://d

Epigenetic alterations are crucial to the onset and progressionof cancer, where malignant cells present higher levels of histonedeactylase than normal tissues, generating hypoacetylatednucleosomal histones, leading to repression of numerousgenes, including tumor suppressor genes.1,2 Unlike structuralabnormalities (i.e., chromosome deletions or gene mutations)causing irreversible loss of gene function, genomic silencinginduced by histone deacetylation can be pharmacologicallyreversed by histone deacetylase (HDAC) inhibitors (HDACi).These compounds constitute a promising class of agents thatreverse aberrant epigenetic states through suppression of HDACenzymatic activity and promotion of histone hyperacetylation.3

They assist in chromatin relaxation and uncoiling which permitsre-expression of silenced tumor suppressor genes and cyclin

inostat nanomedicine inducing apoptosis in heterogeneous population ofx.doi.org/10.1016/j.nano.2013.09.008

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dependent kinase inhibitors, potentially resulting in cellulardifferentiation, inhibition of proliferation by cell cycle arrest,generation of oxidative stress, and apoptosis.4

Acute myeloid leukemia (AML) is a clonogenic disordercharacterized by the somatic acquisition of genetic andepigenetic alterations in hematopoietic progenitor cells thatperturb normal mechanisms of self-renewal, proliferation, anddifferentiation.5 The disease is highly heterogeneous with regardto clinical features and acquired genetic alterations, both thosedetectable microscopically as structural and numerical chromo-some aberrations, and those detected as sub-microscopic genemutations and changes in gene expression.6 Emerging datasupport the notion that recruitment of aberrant HDAC activity byoncogenic fusionproteins, resulting fromchromosomal translocations,contributes to silencing of vital genes in hematopoiesis and promotionof AML.7 In t(8;21)(q22;q22) AML, the resulting chimeric proteinAML1/ETO silences various AML1 target hematopoietic genesthrough direct recruitment of HDAC complex to their promoters.8

Similar mechanisms of transcriptional disruption via histonedeacetylation have been described in t(15;17)-positive acutepromyelocytic leukemia, where the PML/RARα oncogenerecruits an HDAC repressor complex to the promoter of retinoicacid-target genes perturbing differentiation of myeloid pro-genitors and critical tumor suppressive functions includinginduction of apoptosis, growth arrest, and cellular senescence.9

Pharmacologic reversal of atypical epigenetic silencing of thesehematopoietic genes is assumed to restore normal bone marrowfunction leading to clinical response in AML.

Vorinostat (suberoylanilide hydroxamic acid, SAHA; ZolinzaTM,Merck) is a strong inhibitor of histone deacetylase with direct bindingto the catalytic site, which allows the hydroxamic moiety to chelatezinc ion located in the catalytic pockets of HDAC, thereby inhibitingdeacetylation and leading to an accumulation of both hyperacetylatedhistones and transcription factors.10 Vorinostat was the first FDAapprovedHDACi for treatment of patientswith progressive, persistentor recurrent cutaneous T cell lymphoma (CTCL).11 In addition, thedrug is being actively pursued for the clinical management ofleukemia wherein vorinostat demonstrated remarkable in vitroactivity12,13 and has shown improved survival and anti-tumor effectsin rodentmodels of leukemia.12,14 Pooled data fromvorinostat clinicaltrial programs, both as a single agent and in combination with otheragents in AML, have emphasized on its anti-leukemic effect withacceptable safety and tolerability profiles.12 An important attribute ofHDACi is that they induce cancer cell death at concentrations towhichnormal cells are relatively resistant, possibly due to potentially largedifferences in the acetylome of normal versus tumour cells,15 makingthem well suited for cancer therapy.

However, the drug faces challenges of low aqueous solubilityand low cell permeability hindering development of intravenous(i.v.) formulations of the same.16 Mostly, hydrophobic anti-cancer agents rely on solvent-based (e.g., Cremophor EL)delivery vehicles for i.v. administration, which are associatedwith serious and dose-limiting toxicities.17 With the emergenceof nanotechnology, improvised drug delivery platforms utilizinghuman serum protein, albumin, have facilitated delivery ofsignificant amount of drug to target site while avoiding toxicitiesof solvent-based formulations.18-24 Albumin-bound paclitaxel(130 nm nab™-paclitaxel; Abraxane®) exemplified the first

FDA approved, clinically successful albumin based nanomedi-cine intended for i.v. administration, for treatment of metastaticbreast cancer, currently under clinical trials for numerous othersolid tumors.19 Endogenous proteins like albumin possessadvantages over polymeric nano-carriers such as preferentialuptake in tumor and inflamed tissues, lack of toxicity andimmunogenicity and ready availability.18 In addition, hydro-phobicity, neutral charge and significant protein bindingefficiency of vorinostat (~71%) are assumed to aid reversibleand non-covalent binding of the drugs to distinct hydrophobicpockets, formed by the lipophilic amino acid side chains of thepolypeptide backbone, for efficient cellular uptake and release.

Considering the above points, in the present paper, we reportthe development of a human serum albumin bound vorinostatnanomedicine and its excellent anti-leukemic activity towardsprimary leukemic cells derived from a heterogeneous set of nineAML patients, including refractory and relapsed cases and threeAML cell lines representing different FAB classes. Interest-ingly, irrespective of clinical characteristics, all patient samplesshowed promising sensitivity towards nano-vorinostat.

Methods

See Supplementary Material available online at http://www.nanomedjournal.com.

Results

In silico docking, synthesis and physico-chemical characterizationof nano-vorinostat

With the aim of developing a nanoformulation for vorinostat, wehave employed in silico docking simulations prior to wet chemicalsynthesis of human serum albumin (HSA) bound vorinostatnanomedicine, hereafter termed as nano-vorinostat. Ligand-proteindocking simulations (Auto Dock 4.2) were performed to investigatepossible chemical interactions and associated binding energybetween vorinostat and HSA. Docking vorinostat with Pocket II Aof A-chain of HSA, one of the most promising and experimentallydetermined drug binding pockets25 indicated that the drug couldmake strong hydrophobic interactions with Ser 192, Tyr 150, Lys199, Leu 238 and Leu 219 residues in albumin, resulting in bindingenergy −6.0 kcal/mol (Figure 1). The interaction was furtherstrengthened by the formation of hydrogen bonds by vorinostat withSer 192, Ala 291 and Arg 222 residues of HSA. In addition, thepresence of Vander Waal’s forces and intermolecular interactionsalso strongly contribute towards the stable interaction of vorinostatwith albumin residues. List of complete interactions betweenvorinostat and HSA is given in Supplementary Data S1.

Figure 2, A, illustrates the synthesis steps involved in thepreparation of nano-vorinostat. Coacervation technique wasimplemented for nano-vorinostat synthesis. Ethanol served as thedesolvating agent, which when introduced into human serumalbumin containing vorinostat, yielded protein-vorinostat co-acervates. These coacervates were subsequently subjected toEDC mediated crosslinking to further reinforce drug entrapmentand avoid premature drug release. SEM image of nano-vorinostat (Figure 2, B) indicated formation of well dispersed

Figure 1. In silico molecular modeling of interactions between human serum albumin (HSA) and vorinostat.

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spherical particles measuring ~100 nm in size, which was in linewith the DLS measurement showing mean hydrodynamicdiameter of ~91 ± 14 nm (Figure 2, B, inset). Unbound freedrug was removed from nano-vorinostat suspension by dialysis,and UV–vis absorption studies of the washed precipitateindicated relatively high loading efficiency of 72.4% and drugcontent of 10.77 w/w. The nanoformulation recorded an averagezeta potential of −31.12 mV, suggesting colloidal stability inaqueous medium (Figure 2, C). Drug release studies performedat physiological pH showed that ~85% of encapsulatedvorinostat was released from protein nanoparticles within72 hours, which reached almost 100% by 168 hours (5 days).It was observed that the drug was released in a sustained fashion,without any burst release (Figure 2, D). Figure 2, E shows thephotograph of nano-vorinostat colloidal solution.

In vitro cytotoxicity studies and functional assays in AMLcell lines

With the aim of investigating single agent anti-leukemicactivity of nano-vorinostat in patient samples, initially, we havetested the same in three AML cell lines, KG-1a, HL-60 andMV4-11, representing three different FAB classes, namely FABM0, M3 and M5. The cell lines were characterized for theirmolecular or morphological characteristics (Figure 3, A-D).

Figure 3, A shows immunophenotyping data of KG-1a whichrevealed 76.7% positive staining for hematopoietic stem cellmarkers CD34+ CD38−, suggesting primitive nature of cells.The confocal microscopic image (Figure 3, B) shows that mostof KG-1a cells are stained positive for CD34 alone (CD38−),indicating immature, stem cell-like population and a few cellsstained for CD34 and CD38, representing relatively differenti-ated population. Figure 3, C shows optical micrograph of HL-60cells, exhibiting cytological staining pattern indicative ofpromyelocytic nature, with pale staining areas in the nucleus,basophilic cytoplasm and primary granules. Figure 3, D showsagarose gel electrophoresis of MV4-11 PCR product showingband specific to FLT3-ITD mutation, which represents worseprognosis scenario. We have studied intracellular uptake ofnano-vorinostat in these cell lines and a representative confocalimage (Figure 3, E) shows N90% KG-1a cells showinginternalized nanoparticles. For this imaging, nanoparticles weredoped with fluorescent Au clusters in albumin matrix as reportedearlier.20 Further, cytotoxicity caused by free and nano-vorinostat was estimated in all the above cell lines and also inbone marrow derived mononuclear cells (BMMC) from healthyindividuals (Figure 3, F-I). The tested samples showedconcentration dependent anti-proliferative effects, howevernano-vorinostat showed enhanced cytotoxicity up to N80% at2.5 μM, invariably, in all three cell lines. ~100% cell death was

Figure 2. Synthesis and physico-chemical characterization of nano-vorinostat. (A) Schematic representation of steps involved in synthesis of nano-vorinostat.(B) SEM image showing spherical particles of ~100 nm. Inset: DLS data showing hydrodynamic diameter of ~94 ± 8 nm. (C) Drug content, encapsulationefficiency and zeta potential of nano-vorinostat. (D)UV–VIS data showing drug release profile of nano-vorinostat at pH 7.4. (E) Photograph of nano-vorinostatexhibiting excellent colloidal stability.

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observed at higher concentration of 5 μM. In contrast, free drugor nano-vorinostat did not exert any significant cytotoxicitytowards healthy BMMC as shown in Figure 3, I. This wasfurther substantiated by employing methylcellulose based clonalprogenitor assay capable of supporting myeloid colony forma-tion. Treatment with maximum test concentration of 5 μM nano-vorinostat was found to have no adverse effects on clonogenicgrowth pattern of healthy BMMC (Figure 3, J). Figure 3, Kshows graphical representation of median number of colonyforming units formed in untreated, free and nano-vorinostattreated healthy BMMC, after 14 days, confirming the non-toxicity of vorinostat.

We next proceeded to evaluate the mechanism of action ofnano-vorinostat by studying HDAC inhibition, intracellular ROSgeneration, cell cycle arrest and apoptosis. A representative dataon MV4-11 cell line, harbouring MLL translocation and FLT3-ITD mutation, representing FAB M5 class having dismalprognosis (Figure 4, A) shows that nano-vorinostat exertedincreased HDAC inhibition compared to free drug. More than90% HDAC inhibition was registered by 1 μM nano-vorinostatcompared to ~65% by free drug. Concentrations above 1 μMregistered almost complete inhibition as seen from Figure 4, A.

ROS generation capability of free and nano-vorinostat wasassessed in MV4-11 as shown in Figure 4, B. The flowcytometry data showed that ~93% of cells treated with 2.5 μMnano-vorinostat showed positive staining for ROS compared to

~78% cells treated with free vorinostat, indicating enhancedactivity of nanoformulation, although only ~82% drug isreleased as shown in the drug release data (Figure 2, D). Thisis confirmed by confocal microscopic image where MV4-11cells treated with 2.5 μM nano-vorinostat showed bright greenfluorescence of oxidized DCF, corresponding to high levels ofROS in the cytosol (Figure 4, C).

Cell cycle phase distributions induced by 2.5 μM free andnano-vorinostat is shown in Figure 4, D. The results revealedthat MV4-11 cells after 36 hours showed significant decrease inG0/G1 phase content (24.6%) and increase in G2/M (35.6%),compared to untreated control and free vorinostat, suggestive ofcell cycle arrest at G2/M phase. Free vorinostat also registereddecreased G0/G1 content (32%) and G2/M arrest (35.6%)with cells trailing to sub-G1 phase. However, considering G0/G1cell count into account; nanoformulation showed morepronounced effects on cell cycle progression. At 72 hours,nano-vorinostat treatment increased sub-G1 fraction to69.1% compared to its free drug counterpart showing 55.6%(Supplementary Data S2).

Significant accumulation of cells in sub-G1 phase isindicative of apoptotic mode of cell death, which was verifiedusing Annexin V/PI assay. After 72 hours, 2.5 μM freevorinostat treatment showed 72.3% cells in early stage apoptosis,13.2% in late stage apoptosis and 0.4% in necrosis stage, leaving14.1% cells in the live quadrant. In contrast, same concentration

Figure 3. Molecular characterization of AML cell lines. (A) KG-1a immunophenotyping data showing 76.7% positivity for CD34+ CD38−. (B) Confocal image of CD34+ CD38− fraction of KG-1a. (C) Opticalmicrograph of HL-60 cells exhibiting cytological staining pattern of promyelocytes. (D) Agarose gel electrophoresis of MV4-11 PCR product showing band specific to FLT3-ITD. (E) Confocal image of golddoped nano-vorinostat treated KG-1a showing internalized nanoparticles. Nano-vorinostat sensitivity towards (F) KG-1a (G) HL-60 (H) MV4-11 and (I) healthy BMMC. CFU assay showing formation ofcolonies in (J) control and 5 μMnano-vorinostat treated healthy BMMC. (K)Graphical representation of median number of CFUs formed in untreated control, free and nano-vorinostat treated healthy BMMC after14 days.

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Figure 4. Mechanistic studies of nano-vorinostat in MV4-11. (A)HDAC inhibition profile. (B) Flow cytogram showing enhanced intracellular ROS generation in nano-vorinostat treated cells. (C) Confocal imageshowing bright green fluorescence of oxidized DCF in cytosol of nano-vorinostat treated cells. (D) Flow cytogram showing induction of G2/M arrest by nano-vorinostat (36 hours). (E) Flow cytogram showingincreased apoptosis in nano-vorinostat treated cells. (F) Confocal image showing both early and late apoptotic fraction in nano-vorinostat treated cells.

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Figure 5. Vorinostat sensitivity towards primary leukemic cells derived from patient samples (n = 9).

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of nano-vorinostat showed 88.5% cells in early apoptosis stage,9.2% in late apoptosis, 0.2% in necrosis, leaving 2.1% cells alone inthe live quadrant indicating higher apoptosis rates compared to freevorinostat (Figure 4,E). This was further confirmed using confocalmicroscopywhich clearly showed both early and late apoptotic cellfractions in nano-vorinostat treated cells (Figure 4, F).

Nano-vorinostat sensitivity in patient sample derivedleukemic cells

The anti-leukemic effect of free and nano-vorinostatwas furtherevaluated in leukemic blasts isolated from AML patients, culturedex vivo. Peripheral blood/bone marrow derived mononuclear cellswere isolated from nine AML patients, whose clinical character-istics at the time of sampling are summarized in Table 1. Thesepatients included three refractory and one relapsed cases. Testconcentrations (0.1-5 μM) of free and nano-vorinostat wereselected from initial experience with leukemic cell lines. Figure 5shows nano-vorinostat sensitivity profile of primary leukemiccells from these patient samples. Inset of each graph depictsimmunophenotyping data of corresponding patient samples. FABclass, blast percentage, expression levels of CD33 and CD34 andmutation/translocation abnormalities were taken into account foranalyzing the basis of vorinostat sensitivity. Interestingly, withinall FAB classes, nano-vorinostat exerted augmented cytotoxicitytowards all patient samples, with some variations in the percentageviability over the range of test concentrations. Patient sample 6recorded the highest blast percentage of 96.7 and sample 8, the

least with 33.1%. However, irrespective of blast percentage, allpatient samples responded better to nano-vorinostat in a dosedependent manner. Sample 4 showed highest CD33 levels of86.7% and sample 8 had the least with 0.9%. Similarly, patientsample 9 recorded highest CD34 levels of 97.7% and sample 4 theleast with 11.1% and the rest of samples within this wide range.Regardless of CD33 or CD34 levels, nano-vorinostat induced celldeath in all samples. In spite of such huge variations in phenotypicexpressions, all patient samples showed excellent responsetowards nano-vorinostat. In regard with mutations/chromosomalabnormalities, patient sample 1 showed JAK2-V617F mutationand samples 7 and 9 showedMLL re-arrangements. Samples 1 and7 showed almost similar cytotoxicity patterns but patient sample 9registered ~100% sensitivity above 0.1 μM nano-vorinostat itself.Sample 5 obtained from a relapsed FABM2 patient showed N90%cytotoxicity starting from the lowest test concentration (0.1 μM),which increased to ~100% for concentrations above 0.1 μM.Samples 4 (FABM2), 6 (FABM0) and 9 (FABM5) obtained fromrefractory patients also showed results similar to that observedwiththe relapsed patients, all responding invariably to lowest testconcentrations itself.

Inhibition of HDAC activity and clonogenic proliferationpotential of leukemic bone marrow samples

Subsequently we have assessed the nano-vorinostat treatmenteffect on HDAC activity and colony-forming ability of leukemicBMMC isolated from patients 7, 8 and 9, whose bone marrow

Table 1Clinical characteristics of AML patient samples (n = 9) at the time of sampling.

Patientsample #

Age/gender Sample type Diagnosis with FAB class Blasts⁎ (%) Translocations/molecularmarkers/cytogenetics⁎

Clinical status at the timeof sampling

1 57/M PB AML with antecedentpolycythemia vera

88.2 JAK2 -V617F/46, XY Induction chemotherapy

2 66/F PB M2 AML 82 None detected/45,XX, del(9)(p)

On low dose cytarabine dueto co-morbidities

3 27/M PB Biphenotypic AML(AML with T-lymphoid markers)

58.9 None detected/46, XY Untreated

4 31/M PB M2 AML showing aberrantCD19 expression

69.1 None detected/46, XY Refractory to cytarabine/daunorubicin induction

5 11/M PB M2 AML 81.1 None detected/na First relapse6 49/F PB M1 AML 96.7 None detected/46, XX Refractory to cytarabine/

daunorubicin induction7 60/F BM M4 AML 50 t(4:11)/46, XX On chemotherapy with

decitabine8 61/F BM M1 AML 33.1 None detected/46,XX On chemotherapy with

decitabine9 54/M BM M5 AML 80.4 t(4: 11)/46, XY Refractory to cytarabine/

daunorubicin induction

na: not available.⁎ At the time of sampling.

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samples were available. Figure 6, A shows the level of HDACinhibition after free and nano-vorinostat treatment in patientsample 7, wherein 1 μM nano-vorinostat resulted in enhancedHDAC inhibition of N90%, compared to free drug (68%).Figure 6, B shows the effect of 2.5 μM nano-vorinostat on theclonogenic potential of leukemic BMMC from patient sample 7,wherein ~98% reduction in colony forming ability wasobserved, with formation of only a few macrophage colonies(CFU-M). Although both samples belonged to two differentFAB classes, HDAC inhibition was almost absolute in both freeand nano-vorinostat treated patient samples 8 and 9 (Figure 6, Cand E). Figure 6, D shows the HDAC inhibitory effect of nano-vorinostat on patient sample 8 wherein nano-vorinostat treatmentnearly disrupted the colony forming capability of these leukemiccells. Only few rudimentary CFU-G (colony forming unit-granulocyte) colonies were to be seen. Figure 6, F clearly showsthat nano-vorinostat completely derailed the colony formingcapability of sample 9 showing MLL re-arrangement. Figure 6,G shows graphical representation of median number of colonyforming units formed in untreated, 2.5 μM free and nano-vorinostat treated leukemic BMMC from patients 7, 8 and 9,after 14 days.

Discussion

Epigenetic modifications are increasingly recognized to playsignificant roles in both normal cellular physiology and diseaseprocesses, particularly in cancer where aberrant gene expressionhas long been associated with its pathogenesis. The histoneacetylation status, one of the major groups mediating epigeneticmodifications, is determined by the opposing actions of histoneacetyltransferases (HATs) and histone deacetylases (HDACs).HAT inactivation has been linked to oncogenesis and experi-mental evidence suggests that the aberrant HDAC activity leads

to the transcriptional repression of specific tumour suppressorgenes, thus contributing to tumour formation.26 Mountingevidence emphasizes the critical role of histone deacetylationin the pathophysiology of AML.7 Several chromosomal trans-locations in AML that produce chimeric fusion oncoproteinshave been shown to recruit HDACs to the promoters to repressgenes involved in cell-cycle, growth inhibition, differentiation,and apoptosis.27 Consistent with this, treatment of AML cellswith HDAC inhibitors has demonstrated augmentation ofderegulated gene transcription patterns inducing growth arrest,differentiation, and apoptosis in a relatively selective manner incancer versus normal host cells. Therefore, the development ofHDACi as therapeutic agents for AML treatment has recentlybeen intensified.

Vorinostat, a pan-HDACi is shown to inhibit theenzymatic activity of Class I (HDAC1, HDAC2 andHDAC3) and Class II (HDAC6) HDACs at nanomolarconcentrations (IC50 b86 nm) and induce cytotoxicity atconcentrations ranging to 10 μM. Vorinostat has demonstrat-ed excellent anti-leukemic effect both in vitro and in vivo,exhibiting exceptional single-agent activity in patients withadvanced CTCL11 and AML.12 Although the drug presentsexcellent therapeutic potential, vorinostat faces shortcomingsof poor aqueous solubility (0.2 mg/mL) and low permeability(a log partition coefficient of 1.9), as indicated by its ClassIV designation in Biopharmaceutics Classification System,thus hindering development of i.v. formulations of thesame.16 Vorinostat also possesses sub-optimal pharmacoki-netics including low bioavailability (43% for humans),extensive serum clearance and a short elimination half-lifeof approximately 2 hours in both animal and humanstudies.16 Therefore, it is of remedial importance to developnovel formulations of vorinostat for parenteral administrationsthat improve solubility and the overall disposition profileof vorinostat.

Figure 6. Level of HDAC inhibition (A, C, E) and effect on clonogenic proliferation (B, D, F) of leukemic BMMC after free and nano-vorinostat treatment inpatients 7, 8 and 9. (G) Graphical representation of median number of CFUs formed in untreated, free and nano-vorinostat treated leukemic BMMC frompatients 7, 8 and 9, after 14 days.

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Accordingly we have designed and developed a protein basedvorinostat nanomedicine wherein the drug is encapsulated withinhuman serum albumin matrix. Ligand protein docking simula-tions performed to investigate possible chemical interactions andassociated binding energy between vorinostat and HSA, revealedamino acid residues that interacted with vorinostat via stronghydrophobic interactions resulting in binding energy of −6.0 kcal/mol. Vorinostat was also found to interact with HSAthrough a combination of van der Waals forces, hydrogen bonds,and intermolecular interactions, indicating the possibility ofenhanced solubility of this otherwise poorly soluble hydrophobicdrug in protein medium. Thus formed nano-vorinostat yielded~100 nm sized, well dispersed, spherical nanoparticles with anencapsulation efficiency of 72.4% and sustained drug releasepattern (over 5 days) from the albumin nano-carrier.

Cytotoxicity of free and nano-vorinostat was initiallyassessed in three AML cell lines, namely KG-1a, HL-60 andMV4-11 which represented three ‘tough-to-treat’ AML types.KG-1a cells derived from the parent KG1 cell line (establishedfrom erythroleukemia patient evolving to AML, betweenpassages 15 and 35) are morphologically, cytochemically,

immunologically, and functionally less mature than the latterand belongs to the AML FAB M0 class.28 KG-1a, owing to itsprimitive disposition, by virtue of expression of hematopoieticstem cell markers (CD34+CD38−), is actively pursued as anappropriate cell model for leukemic stem cell research.29 Ourimmunophenotyping results also revealed that ~76% cellsexhibited CD34+CD38− stem cell phenotype. HL-60 cell lineoriginally established from an acute promyelocytic leukemicpatient represents AML FAB M2 with maturation.30 Consistentwith its origin, our cytological studies showed HL-60 to possessmyeloblastic or promyelocytic nature. MV4-11 cell line derivedfrom patient with acute myelomonocytic leukemia (AML FABM5) expresses a typical FLT3/ITD (internal tandem duplication)mutation consisting of a 30-bp insertion at nucleotide 1857 andfurther harbours a t(4;11)(q21;q23) MLL translocation, repre-senting mixed lineage or biphenotypic leukemia having poorprognosis.31,32 The presence of FLT3–ITD mutation in AMLpatients significantly correlates with an aberrant proliferativecapability resulting from constitutive phosphorylation of anti-apoptotic proteins, increased risk of relapse and dismal overallsurvival (b6 months).33 The tested concentrations of free and

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nano-vorinostat showed dose dependent progressive loss of cellviability in all of these cell lines. However, nano-vorinostatshowed enhanced cytotoxicity over the free drug with 2.5 μMnano-vorinostat invariably inducing N80% cytotoxicity towardsall three cell lines, which was increased to ~100% at 5 μM. Thisindicates the efficacy of vorinostat towards different FAB classesof AML, irrespective of their primitiveness, differentiation ormutational status. Interestingly, both free drug and nano-vorinostat hardly induced any cytotoxicity towards bone marrowderived mononuclear cells (BMMC) from healthy individuals.The colony forming unit assay using normal healthy BMMCtreated with 5 μM of nano-vorinostat also displayed no adverseeffects on the clonogenic growth suggesting less toxicity ofvorinostat to healthy stem/progenitor cells. It is believed thatdifferences in the acetylome34 and expression levels ofthioredoxin15 in tumor and normal cells may contribute to therelative resistance of the latter to HDACi induced cytotoxicity.Normal cells accumulate thioredoxin (TXN, natural ROSscavenger) after treatment with HDACi. Therefore, ROSgenerated in normal cells gets neutralized by TXN, whereastransformed cells lacking TXN, succumb to oxidative stress.Moreover, normal cells, possessing intact survival mechanisms,are able to recover from HDAC inhibition. However, cancercells having multiple and collective defects in proliferation,differentiation and senescence cascades, may succumb toHDAC inhibition.

Anti-cancer effects of HDAC inhibition by vorinostat havebeen associated with a multitude of mechanisms includingintracellular generation of ROS and cell-cycle arrest, whichcommits neoplastic cells to apoptosis. Our HDAC inhibitionassays performed on a representative AML cell line revealedenhanced activity of nano-vorinostat over its free drugcounterpart. Nano-vorinostat has shown to inhibit HDAC moreeffectively than free drug, probably due to the enhanced cellularuptake of albumin-bound drug, which retained its chemicalstability as well as activity at the molecular level. Inhibition ofHDACs by vorinostat occurs through direct interaction of drugwith the catalytic site of enzyme, leading to a state of histonehyperacetylation, which assists in chromatin remodelling topermit expression of repressed genes.4

Previous studies have reported that intracellular ROS andoxidative injury are one of the prime mechanisms of HDACi-induced apoptosis. Our results also showed that the nanoformu-lation succeeded in producing high levels of intracellular ROSthan free vorinostat. This correlates with enhanced intracellularuptake and HDAC inhibition by the nanoformulation. An almostuniversal effect of HDACi is cell cycle arrest, due to theupregulation of cell cycle genes like CDKN1A (encodes theproduction of p21 cyclin-dependent kinase inhibitor). Our dataon nano-vorinostat treated cells showed cell cycle arrest in G2/Mphase in a time dependent manner, and subsequent accumulationof cells in sub-G1 phase. This indicates cells committing toapoptosis. This effect was prominent in nano-vorinostat treatedsamples compared to free drug indicating its better efficacy,probably due to improved cellular uptake. Subsequent studies onapoptosis using flow cytometry and confocal microscopyconfirmed this finding. We speculate that the combined effectof enhanced HDAC inhibition, generation of high intracellular

levels of ROS and cell cycle arrest, inflicted by nano-vorinostattreatment, proved traumatic enough to trigger apoptosis in AMLcell lines. Collectively, all functional assays pointed towardsthe efficacy of nano-vorinostat, which exhibited enhancedcytotoxicity towards representative cell line, compared to itsfree drug counterpart.

The clinical validity and relevance of the observed anti-leukemic effect of nano-vorinostat were further evaluated inprimary leukemic cells isolated from peripheral blood/bonemarrow samples of AML patients (n = 9). FAB class, blastpercentage, expression levels of CD33 and CD34 and mutation/cytogenetic abnormalities were taken into account in analyzingtheir respective roles in vorinostat sensitivity. Interestingly, mostof the patient cells showed dose dependent loss of viability, withnano-vorinostat registering enhanced cytotoxicity at lower IC50(0.5 μM), compared to that of free vorinostat (1 μM). Allobtained patient samples belonged to either granulocytic (M0-M4) or monocytic (M5) lineages. Within these lineages, nano-vorinostat exerted cytotoxicity towards all patient samples.Similarly, irrespective of blast (CD45dim/− cells) percentage, allpatient samples responded to nano-vorinostat in a dosedependent manner. CD33 is a myeloid specific antigen whoseexpression is directly associated with adverse disease featuresand inversely associated with low-risk disease. Increased CD33percentage is considered as an independent predictor of inferioroutcome in AML.35 Similarly, high expression levels ofhematopoietic stem cell marker, CD34, is directly related toenhanced resistance to apoptosis and is also observed as anotherindependent sign of poor prognosis.36 Interestingly, patientsamples which showed high expression levels of both CD33 andCD34 showed excellent response towards nano-vorinostat,similar to ones which expressed low levels of these markers.In another aspect, JAK2-V617F mutations lead to moreaggressive growth of leukemia owing to the activation ofJAK2-STAT5 cascade which substantially alters apoptoticresponse, self-renewal and proliferative capacity of myeloidcells.37,38 Nano-vorinostat was found to exert better cytotoxicityto JAK2 mutated cells (patient 1) compared to free drug. In yetanother case, two patient samples which presented with MLLrearrangements at 11q23, considered as a poor predictor ofclinical outcome,39,40 responded with varying sensitivity tonano-vorinostat, one at lower concentration of 0.1 μM (sample 9)and the other at 1 μM (sample 7).

It may be noted that there are a considerable number ofpatients diagnosed with AML who either fail to achieveremission or relapse after induction with cytarabine anddaunorubicin. Despite the development of a variety of newinvestigational therapies,41 relapsed or refractory AML remainsto be a complicated clinical challenge. From our results, allrelapsed and refractory patient samples invariably displayedexcellent response to nano-vorinostat starting at lowest concen-tration (0.1 μM). It is noteworthy that the nanomedicineexercised superior cytotoxic effects in otherwise hard-to-treatrelapsed and refractory patient samples, compared to freedrug. This pointed towards the potential of nanomedicine toevolve as a promising strategy against poor prognosis cases,either as a stand-alone drug or as an adjuvant with the existingchemo-regimen.

731P. Chandran et al / Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 721–732

Since, AML exemplifies derailed myelopoiesis, whichnormally involves the growth and maturation of myeloid lineage,colony forming unit-granulocyte macrophage (CFU-GM) assaywas performed to study clonogenic growth patterns of vorinostattreated leukemic BMMC from AML patients. This assay hasbecome a yardstick, functional in vitro assay, providing analternative to in vivo animal models, for testing the efficacy andgeneral/lineage specific toxicity of candidate drugs on hemato-poietic progenitors. The number of colonies obtained in CFUassays should be linearly proportional to the CFU content of theinput cell suspension provided that a sufficiently low number ofcells are plated (1 × 104 cells). Besides, the relatively longincubation period (14 days) also increases the sensitivity of theassay and allows the detection of more long-term effects than aredetectable in a proliferation assay. From our results, it was clearthat nano-vorinostat has succeeded in inhibiting the clonogenicproliferation capability of leukemic BMMC isolated from allthree patient samples (patients 7, 8 and 9). Nano-vorinostat coulddisrupt both granulocytic and macrophage lineage colonyformation as evident from the virtual absence of functionalCFU-G, CFU-M or CFU-GM colonies in nanomedicine treatedsamples. In tune with these important results and considering thepresence of hematopoietic progenitor cells and stem cells in bonemarrow samples, it is imperative to investigate the extent ofdifferential toxicity that may be exerted by nano-vorinostattowards healthy and leukemic cells. Interestingly, nano-vorino-stat did not affect the colony forming capability of normalBMMC, isolated from healthy individuals. This indicated thatthe nanomedicine exerted its toxicity specifically to leukemiccells where HDAC activity was elevated and sparednormal healthy cells. The selective ablation of clonogenicgrowth in leukemic bone marrow samples suggests a strongpossibility of nanomedicine to strike the leukemic stem cells(LSC) population. However, this needs to be confirmed bystudying the effect of the same on isolated LSCs in NOD-SCIDanimal models.

In conclusion, we have developed a novel albumin boundvorinostat nanomedicine and demonstrated its anti-leukemicactivity against three different AML cell lines, and primaryleukemic cells derived from nine patients, from different FABclasses. Nano-vorinostat having size of ~100 nm and encapsu-lation efficiency of 72.4% displayed excellent inhibition ofHDAC activity, induced high levels of intracellular ROS, growtharrest and apoptosis in cell lines as well as patient samples,irrespective of its FAB classes including refractory and relapsedcases. More importantly, nano-vorinostat demonstrated excep-tional single-agent activity against the clonogenic proliferativecapacity of leukemic BMMC from a heterogeneous set of patientsamples, sparing healthy BMMC suggestive of its chances ofstriking the AMLSC population. In effect, the aberrant histonedeacetylation targeted protein-nanomedicine appears to be apromising therapeutic option against various FAB classes as wellas poor prognosis cases of AML. Considering the toxicitychallenges associated with clinically used DNA intercalatingagents and anthracyclins, we believe that an epigenetic targetednanoformulation showing excellent single agent activity inpatient derived cells, sparing healthy bone marrow cells, holdsgreat potential for clinical translation.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.nano.2013.09.008.

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