All-trans retinoic acid loaded block copolymer nanoparticles efficiently induce cellular differentiation in HL-60 cells

European Journal of Pharmaceutical Sciences 44 (2011) 643–652

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All-trans retinoic acid loaded block copolymer nanoparticles efficiently induce cellular differentiation in HL-60 cells Manu D. Tiwari a,c, Sarika Mehra b, Sameer Jadhav b, Jayesh R. Bellare a,b,c,⇑ a

Department of Biosciences and Bioengineering, IIT Bombay, Mumbai 400076, India Department of Chemical Engineering, IIT Bombay, Mumbai 400076, India c Centre for Research in Nanotechnology and Science, IIT Bombay, Mumbai 400076, India b

a r t i c l e

i n f o

Article history: Received 23 August 2011 Received in revised form 18 October 2011 Accepted 21 October 2011 Available online 28 October 2011 Keywords: Acute Promyelocytic Leukemia All-trans retinoic acid Differentiation therapy Polymer nanoparticles PEG-PLA PEG-PC

a b s t r a c t All-trans retinoic acid (atRA) is used in the differentiation therapy of Acute Promyelocytic Leukemia. However, its therapeutic success is limited by the appearance of relapse and recalcitrant cases, poor aqueous solubility and high degradability. In the current work, we prepared two types of atRA-loaded copolymer nanoparticles – PL1RA and PC1RA, based on poly(ethyleneglycol) (PEG)-poly(L-lactide) and PEG-poly(e-caprolactone), respectively. We then evaluated their physico-chemical properties and compared their differentiation-inducing potential of HL-60 cells with free atRA. These nanoparticles were in the size range 100–150 nm, possessed moderate colloidal stability and exhibited around 30% encapsulation efficiencies. In vitro release studies indicated pseudo-zero order release of a sustained nature, with PL1RA showing 71% and PC1RA exhibiting 84% drug release over a period of two weeks. Photostability measurements exhibited considerable increase in atRA photostability in the nanoparticle forms: 25% of the drug in PL1RA and 19% in PC1RA was intact as compared to only 5% for free atRA after 8 h of light exposure. PL1RA and PC1RA exhibited efficacies comparable to free atRA in inducing HL-60 respiratory burst as assessed by nitroblue tetrazolium and 20 ,70 -dichlorodihydro fluorescein diacteate assays. The average CD11b expressions for atRA, PL1RA and PC1RA on day5 of treatment were 58%, 49% and 60%, respectively. Post-differentiation apoptosis (40%) and reduction in mitochondrial transmembrane potentials (60–70%) were also comparable across all treatment groups. Therefore, our block copolymer nanoparticles, PL1RA and PC1RA, are attractive and effective vehicles for atRA delivery which maintain its activity and enhance its stability resulting in efficient induction of HL-60 differentiation. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction Retinoids, the naturally occurring or synthetic metabolites and analogs of Vitamin A, are general teratogens which are known to exert anti-cancer activities against an array of cancer types (Freemantle et al., 2003; Lotan, 1996). All-trans retinoic acid (atRA; the acid form of Vitamin A) plays indispensable roles in a variety of growth, morphogenetic and developmental processes such as the differentiation of adipocytes and epithelial cells, maturation of hematopoietic cells and embryonic development (De Luca, 1991; Ross et al., 2000). It has also been shown to be effective in the treatment of breast and lung cancers, head and neck cancer, adenocarcinomas and Acute Promyelocytic Leukemia (APL) (De Luca, 1991; Miller and Waxman, 2002; Warrell et al., 1993).

⇑ Corresponding author at: Silicate Technology Lab, Department of Chemical Engineering, IIT Bombay, Mumbai 400076, India. Tel.: +91 22 25764217; fax: +91 22 25726895. E-mail address: [email protected] (J.R. Bellare). 0928-0987/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2011.10.014

Cellular differentiation is at the center of all developmental paradigms. Understandably, any abnormality in this process has the potential to beget serious pathological consequences. APL, a subtype of myelogenous leukemia, is characterized by a block in granulocytic differentiation which leads to the accumulation of promyelocytes (Warrell et al., 1993). atRA induces immature promyelocytes to differentiate along the granulocytic lineage and is used clinically in the differentiation therapy of APL (Breitman et al., 1980; Warrell et al., 1993). The HL-60 promyelocytic cell line, derived from an APL patient, is a continuous cell line which has been utilized extensively to probe its mechanism and test the efficacy of potential therapies (Fleck et al., 2005). Although atRA differentiation therapy, when used together with conventional chemotherapy, induces complete remission in a large proportion of APL patients, yet there are several challenges to be met (Tallman et al., 1997). Primary amongst them are relapse cases and cases exhibiting the retinoid syndrome in which relapsed patients become clinically ‘resistant’ to further atRA treatment (Degos et al., 1995). These are believed to occur because of a reduction in the plasma half-life of atRA due to its catabolism by

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cytochrome P450s (Leo et al., 1989). Muindi et al. (1992) showed that continuous atRA treatment reduces its plasma concentrations to levels below those which sustain differentiation of leukemic cells in vivo. Additionally, atRA is extremely hydrophobic, sensitive to oxidation and has poor aqueous solubility which compounds the problem of its efficient delivery. Several strategies have been attempted to overcome the acquired retinoid resistance; these include use of cytochrome P450 enzyme inhibitors (ketoconazole, liarozole), low-dose retinoid treatment and intermittent dosing (Warrell, 1993). The limited success associated with these, however, stems from the fact that retinoid resistance develops due to pharmacological factors because atRA plasma concentrations at the time of relapse are markedly lower than concentrations on the first day of treatment (Guiso et al., 1994). Hence, pharmacological avenues which result in delivery of atRA at a sustained rate while maintaining its activity and stability are needed. Many nano- and micro-structure based formulations for atRA delivery have been described including liposomes and solid-lipid nanoparticles (Ioele et al., 2005; Lim et al., 2004). Polymer based biodegradable nanoparticles offer exciting opportunities for delivery of anticancer drugs since they can be synthesized in a variety of types and can be functionalized. In particular, poly(ethyeleneglycol) (PEG) based copolymers have emerged as vehicles of choice for hydrophobic anticancer therapeutics since they result in water-dispersible core–shell nanoparticles (Soppimath et al., 2001). Choi et al. (2001) developed atRA-loaded PEG-poly(lactide) (PLA) microspheres and achieved sustained drug release for over 5weeks. Jeong et al. (2004) reported increase in cytotoxicity of atRA upon encapsulation in PEG-poly(caprolactone) (PC) nanoparticles. Despite these reports of atRA encapsulation, considerably less attention has been given in the literature to evaluate the differentiation-inducing activity of nano-encapsulated atRA. Such an analysis is essential as it can serve as an unfailing indicator of atRA stability and activity in the encapsulated form. In this study, we synthesized PEGylated block copolymers and their atRA-loaded nanoparticles with an aim to investigate the differentiation of HL-60 cells as induced by free atRA and the nano-encapsulated forms. We performed physico-chemical evaluations of nanoparticle characteristics and evaluated HL-60 differentiation by employing different biochemical and immunological assays. This study can serve as the basis for using nano-encapsulated atRA for differentiation therapy of APL. 2. Materials All-trans retinoic acid (atRA), 20 ,70 -dichlorodihydro fluorescein diacteate (DCHF-DA), dimethyl sulfoxide (DMSO), L-lactide ((3S)cis-3,6-dimethyl-1,4-dioxane-2,5-dione), e-caprolactone, methoxy-poly(ethylene glycol) (mPEG; mwt 5000), PEG (mwt 6000), stannous octoate (SnO2), propidium iodide (PI), ribonucleaseA, nitroblue tetrazolium (NBT) and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma–Aldrich, St. Louis, MO, USA. RPMI-1640, fetal calf serum (FCS) and antibiotics (penicillin/streptomycin) were procured from HiMedia Labs, Mumbai, India. Phycoerythrin (PE)-conjugated CD11b antibody (CD11b-PE; isotype IgG2a, j) was obtained from BD Biosciences, NJ, USA. Rhodamine B was procured from SD Fine Chem, Mumbai, India. 3. Methods

amounts of lactone (L-lactide or e-caprolactone) (5 g each) and PEG (mPEG-5000 or PEG-6000, respectively) (1 g each) were dried under reduced pressure at 70 °C for 2 h. To these, 0.75wt% SnO2 (37.5 mg in 30 ml toluene) was added as an initiator under N2 atmosphere. After refluxing for 24 h at 150 °C, the reaction mixtures were cooled and solvents were removed under reduced pressure. The products (PL1 and PC1) were dried overnight in vacuum, dissolved in minimal amount of dichloromethane and precipitated using acetone:diethyl ether (cold) (1:4) followed by methanol:hexane (4:1). Finally, they were dried overnight at room temperature under vacuum. 1H NMR (400 MHz; Mercury Plus, Varian Inc., CA, USA) was used to probe the chemical structures and environments of the synthesized copolymers using CDCl3 as the solvent. 3.2. Synthesis of atRA-loaded block copolymer nanoparticles In a typical nanoparticle preparation procedure, 50 mg of the copolymer (PL1 or PC1) was dissolved in acetone, with or without atRA (5 mg), and this solution was added at a constant rate onto an aqueous solution under moderate stirring. After bath sonication for 3 min, the organic solvent was removed by rotary vacuum evaporation. The nanoparticle suspension (PL1RA or PC1RA) so obtained was washed twice with distilled water, lyophilized and stored at 4 °C until further use. All the procedures involving atRA were performed under dark conditions. 3.3. Characterization of atRA-loaded block copolymer nanoparticles The mean hydrodynamic diameters and size distributions of nanoparticles were measured by Dynamic Light Scattering (DLS; BI-9000AT, 90Plus Particle Sizer, Brookhaven Instruments Corporation, NY, USA) at a scattering angle of 90° at 27 °C. Particle suspensions were filtered with 0.45 lm filters and appropriately diluted before measurements. Zeta potential measurements were performed by laser Doppler anemometry (Zeta Potential analyzer; Brookhaven Instruments Corporation, NY, USA). Nanoparticle yield was calculated as the ratio of masses of nanoparticle obtained after lyophilization to that of the ingredients (copolymer/drug) used during preparation. The morphological examination of nanoparticles was conducted by Transmission Electron Microscopy (TEM; Tecnai 12, FEI Company, OR, USA). Nanoparticle suspensions were negatively stained using phosphotungstic acid solution (1% w/v, pH 7.2) and then transferred onto carbon-coated copper grids. Images were observed on the microscope after drying at room temperature. To measure the amount of drug in the nanoparticles, lyophilized atRA-loaded nanoparticles were dissolved in acetone:PBS (1:3, v/v) and the amount of atRA was determined by UV spectroscopy (Nicolet Evolution 300, Thermo Electron Corporation, MA, USA) at 337 nm after ruling out absorbance interference from the copolymer under similar conditions. Encapsulation efficiency (EE) was calculated as the ratio of amounts of drug present in nanoparticles to that used during formulation. For assessing in vitro drug release, 20 mg of lyophilized atRAloaded nanoparticles were dispersed in 2 ml phosphate-buffered saline (PBS, pH 7.4) and introduced in a dialysis bag immersed in 98 ml of PBS (pH 7.4) solution. The system was maintained at 37 °C and shaken at 80 rpm. Three milliliters of PBS solution was taken out at pre-determined time intervals and evaluated for atRA content by UV spectroscopy. Cumulative release (%) was calculated according to the calibration curve.

3.1. Synthesis and characterization of block copolymers 3.4. Culture of HL-60 cells Block copolymers were synthesized by a solution polymerization method based on ring-opening polymerization of the corresponding lactones (Riley et al., 1999). Briefly, predetermined

The HL-60 cell line was obtained from NCCS, Pune and cultured on RPMI-1640 medium supplemented with 10% heat-inactivated

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FCS and penicillin (100U/ml)/streptomycin (100 lg/ml) and incubated under standardized conditions (37 °C, 5%CO2). Cells were passaged by regular dilution in fresh media to a density of 1  106 cells/ml. 3.5. Induction of HL-60 differentiation For induction of differentiation, exponentially growing HL-60 cells were seeded at 2  105 cells/ml in complete RPMI-1640 media. Following treatments were carried out for a period of 5 days – cells treated with (free) atRA only, cells treated with two types of atRA-loaded nanoparticles (PL1RA and PC1RA); cells treated with blank nanoparticles at concentrations corresponding to those used for PL1RA and PC1RA showed no cytotoxicity. atRA was added at a final concentration of 1 lM by dilution from a 10 mM stock solution prepared in DMSO; PL1RA and PC1RA nanoparticles were seeded corresponding to 1 lM atRA. Vehicle control cells were treated with a similar dilution of DMSO which was found to have no effect on the differentiation or the rate of cell division. At the end of the designated culture periods, cells were harvested and processed further. 3.6. Cellular assays 3.6.1. Nitroblue tetrazolium (NBT) assay Differentiation of HL-60 cells was measured by adding 1 ml of cell suspension (1  106 cells) to a solution containing 2 mg/ml of NBT and 20 ng/ml of PMA in phosphate-buffered saline (PBS, pH 7.4) followed by incubation for 1 h at 37 °C. Incubation was stopped by addition of 0.4 ml of cold 2 M HCl and the formazan products were obtained by centrifugation at 700g for 10 min. Finally, the formazan was dissolved in 1 ml of DMSO and absorbance was measured at 560 nm on a UV spectrophotometer. 3.6.2. Determination of Reactive Oxygen Species (ROS) ROS levels were measured by using DCFH-DA as a probe. Following induction treatments, 1–2  106 cells were washed and re-suspended in PBS containing 5 lM DCFH-DA. After 1 h incubation in dark at 37 °C, cells were analyzed by flow cytometry (BD FACS Aria).

3.6.3. CD11b expression Expression of CD11b was measured to assess the induced differentiation at indicated time periods. Following induction treatments, 1–2  106 cells were pelleted by centrifugation at 700g for 5 min, re-suspended in blocking solution (0.05% BSA) for 1 h and washed once by centrifugation at 14000g for 1 min. The cells were then exposed to anti-CD11b-PE antibody (1:100 dilution in blocking solution) for 2 h at room temperature. At the end of this incubation, cells were pelleted by centrifugation at 700g for 5 min, re-suspended in blocking solution, washed twice and finally suspended in PBS. The antibody stained samples were then examined by flow cytometry (BD FACSAria, BD Biosciences, NJ, USA) and analyzed by FCS Express 4 (De Novo software, CA, USA) software. 3.6.4. Mitochondrial trans-membrane (Wm) potential Changes in rhodamine B fluorescence were used to assess mitochondrial trans-membrane (Wm) potential in the treated cells. At the end of indicated time periods, 1–2  106 cells in growth media were incubated at 37 °C for 10 min with 10 lg/ml rhodamine B, washed twice, re-suspended in PBS and assessed for fluorescence intensity on a flow cytometer. 3.6.5. Determination of cellular apoptosis Cells were analyzed for apoptosis by PI staining. At the end of their respective treatment periods, cells were washed with PBS and incubated in 1 ml of staining solution (50 lg/ml PI, 10 lg/ml ribonuclease A, 0.1% sodium citrate and 0.1% Triton X-100) at room temperature for 30 min and analyzed by flow cytometry. 3.7. Data and statistical analysis All data indicates values from at least three independent determinations and is expressed as mean ± SD. The t-test and the oneTable 1 Characteristics of blank and atRA-loaded PL1 and PC1 nanoparticles. Sample

Size (nm)

Polydispersity

f (mV)

Yield (%)

EE (%)

PL1 PC1 PL1RA PC1RA

121 ± 7 90 ± 2 149 ± 11 101 ± 4

0.08 ± 0.03 0.11 ± 0.03 0.12 ± 0.01 0.17 ± 0.01

8 ± 1 7 ± 3 13 ± 2 17 ± 1

72 ± 3 84 ± 2 69 ± 3 80 ± 4

35 ± 2 30 ± 1

Fig. 1. 1H NMR spectra and spectral assignments of (a) PL1 and, (b) PC1 copolymers.

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Fig. 2. Representative TEM micrographs of (a) PL1RA and, (b) PC1RA nanoparticles.

Fig. 3. (a) atRA release from core–shell type PL1RA and PC1RA nanoparticles. (b) Photostabilities of atRA in free, PL1RA and PC1RA forms.

way analysis of variance (ANOVA) were performed to compare two or multiple groups, respectively. Difference between treatments was considered to be significant at a level of at least p < 0.05. For all flow cytometry data, at least 10,000 events were recorded.

3.65 ppm arose due to the methylene protons of homo-sequences of the PEG oxyethylene units (Govender et al., 2000).

4. Results and discussion 4.1. PEGylated block copolymers for core–shell nanoparticles Amphiphilic block copolymers offer improved avenues for encapsulation of hydrophobic drugs since they result in core–shell type of nanoparticle systems (Soppimath et al., 2001; Veronese and Pasut, 2005). PEG, a non-immunogenic and non-toxic polymer, is most commonly used to impart hydrophilic character to lactone based polymers via copolymerization (Soppimath et al., 2001). In the current study, two block copolymers-PL1 and PC1, were synthesized by the anionic ring opening polymerization of L-lactide and e-caprolactone in the presence of methoxyPEG (Mn = 5 kDa) and PEG (Mn = 6 kDa), respectively. Copolymer compositions and structures were confirmed by 1H NMR as shown in Fig. 1. For PL1, peaks at 1.56 and 5.17 ppm were attributable to the methyl (–CH3) and methine (–CH–) protons of the PLA block, respectively. Peaks at 1.38, 1.63, 2.33 and 4.09 ppm in the 1H NMR spectrum of PC1 copolymer arose due to the methylene (–CH2–) protons of – (CH2)3–, –OCCH2– and –CH2OOC– units, respectively; the very weak peak at 4.32 ppm originated due to methylene proton of the PEG end unit. In both the spectra, a sharp single peak at

Fig. 4. Induction of respiratory burst in HL-60 cells as determined by NBT reduction assay. Data represent mean ± SD, n = 3. ⁄Statistically different from control, p < 0.05 (one-way ANOVA).

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4.2. PL1RA and PC1RA exhibit sustained atRA release and improve its stability PEG-based copolymeric systems have been widely utilized for delivering hydrophobic anticancer drugs to different tumor sites (Kwon, 2003). In the present work, atRA-loaded copolymeric nanoparticles were prepared by a nano-precipitation method (Jeong et al., 2004). They exhibited mean hydrodynamic diameters between 100 nm and 150 nm and low polydispersities (Table 1), indicating narrow size distributions, as measured by dynamic light scattering. All the nanoparticles exhibited negative zeta potentials (17 to 7 mV) indicative of moderate colloidal stability. TEM

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observations showed well-dispersed, spherically-shaped nanoparticles without any agglomeration (Fig. 2). Due to its hydrophobic nature, atRA was physically encapsulated in the hydrophobic cores of the nanoparticles. The atRA encapsulation efficiencies for PL1RA and PC1RA were determined by UV absorbance and were found to be 35 ± 2% and 30 ± 1%, respectively. The in vitro atRA release profiles of PL1RA and PC1RA nanoparticles are shown in Fig. 3(a). Both types of nanoparticles exhibited constant atRA release; PL1RA showed 71% and PC1RA exhibited 84% drug release over a period of two weeks indicative of pseudo-zero order profiles. It has been reported earlier that for biodegradable polymers such as PEG-PLA and PEG-PC, partial drug

Fig. 5. (a) Histograms showing increases in cellular ROS levels upon treatment with free atRA, PL1RA and PC1RA for 5 days. (b) Relative gains in ROS levels over control expressed as the percentage of M2. Data represent mean ± SD, n = 3.

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crystallization in the nanoparticles contributes to its diffusioncontrolled slow release (Gref et al., 1994). Choi et al., 2001, showed that increasing the atRA loading in PEG-PLA blended microspheres shifts the in vitro drug release profile from pseudo-first order to pseudo-zero order. Similarly, Jeong et al. (2004) showed that atRA release from PEG-PC nanoparticles is slower at higher drug loading. Due to its extreme sensitivity to air, heat and light, maintaining the stability of atRA is a cardinal aspect of any formulation (Brisaert et al., 1995). As shown in Fig. 3(b), light exposure at room temperature rapidly decreased the content of intact atRA to 5% within 8 h. In contrast, PL1RA and PC1RA exhibited improved atRA stabilities and the corresponding values of intact atRA were found to be 25% and 19%, respectively, after termination of light exposure. This indicates that atRA encapsulation in PC1RA and PL1RA significantly protects it against light-induced degradation. Similar improvements in atRA stability upon incorporation in lipid nanoparticles, liposomes and niosomes have also been reported earlier (Ioele et al., 2005; Lim et al., 2004; Manconi et al., 2002). 4.3. PL1RA and PC1RA induce respiratory burst in differentiating HL-60 cells The terminal differentiation of promyelocytic HL-60 cells by atRA results in acquisition of granulocytic and neutrophilic characters like phagocytosis and chemotaxis (Collins, 1987; Fleck et al., 2005). This forms the basis of its clinical usage in the differentiation therapy of APL wherein immature promylelocytes are induced to differentiate into an array of mature types (metamyelocytes, banded and segmented neutrophils) (Castaigne et al.,

1990; Degos et al., 1995). We examined and compared HL-60 differentiation by free (atRA) and nanoparticle-loaded forms (PL1RA and PC1RA) by several cellular and biochemical markers. Such an evaluation of activities is essential since it has been reported earlier that the clinical efficacy of atRA can be significantly improved in nanoparticle-based formulations (Lim et al., 2004). It has been shown earlier that HL-60 differentiation is accompanied by a gradual acquisition of the functional capacity to produce a respiratory burst (Levy et al., 1990). The NBT reduction assay is commonly used to assess this phagocytosis-associated cellular respiratory burst by colorimetrically measuring the amount of formazan produced in mature HL-60 cells (Breitman et al., 1980). Fig. 4 shows the NBT reduction activities of free atRA, PL1RA and PC1RA after treatment for 5 days. Absorbance profiles, as measured by UV spectroscopy, across all the three treatment groups were similar and maximum absorbance was observed on day 4 in all the groups. These values were significant at p < 0.05 between control cells and atRA-, PL1RA- and PC1RA-treated cells. No statistical differences, however, were observed between the treatment groups indicating that they all achieved similar inductions. Additionally, terminal differentiation associated cessation of proliferative capacity was also observed in all the treated samples. It is well known that the cellular redox status is a key determinant of a cell’s proliferative, maturational and developmental potential (Hitchler and Domann, 2007; Yanes et al., 2010). In case of bipotent HL-60 cells, there was a marked increase in cellular ROS levels with time upon induction by atRA and stimulation by PMA as shown in Fig. 5. Maximum increase was observed on day 5 in the PC1RA-treated group which exhibited an increase of around 150%. Furthermore, these values were significant at

Fig. 6. (a) Day 4 confocal micrograph of HL-60 cells treated with PL1RA showing surface expression of CD11b; scale bar represents 10 lm. (b) Levels of CD11b expression upon treatment with free atRA, PL1RA and PC1RA for 5 days as determined by flow cytometry analysis. Data represent mean ± SD, n = 3. ⁄Statistically different from control, p < 0.05 (one-way ANOVA).

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p < 0.05 between control cells and atRA-, PL1RA- and PC1RA-treated cells. No statistical differences, however, were observed between the treatment groups indicating that they all achieved similar increases in ROS levels. It has been shown earlier that granulocytic phagocytosis involves activation of a membrane-associated NADPH-oxidase enzyme which generates superoxide (O 2 ) and other ROS (singlet oxygen, hypochlorous acid and hydroxyl radicals). Upon being induced to differentiate by atRA, HL-60 cells begin to acquire granu-

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locytic characters which contribute to the observed increase in cellular ROS levels (Trayner et al., 1995). Newburger et al., 1984, showed that the NADPH-oxidase activity increases during HL-60 differentiation and causes over 10-fold increase in PMA-stimulated rate of O 2 production. Interestingly, increase in cellular ROS levels have also been demonstrated with another APL cell line (NB4) and other polar compounds which act as inducers of HL-60 differentiation (dimethyl sulfoxide and dimethylformamide) (Collins et al., 1980; Miyoshi et al., 2010).

Fig. 7. (a) Histograms showing HL-60 cell death upon treatment with free atRA, PL1RA and PC1RA for 5 days. (b) The extents of HL-60 cell death as quantified by the population M2. Data represent mean ± SD, n = 3. ⁄Statistically different from control, p < 0.05 (one-way ANOVA).

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4.4. Differentiating HL-60 cells express CD11b It has recently been shown that oxidative stress markedly enhances the granulocytic differentiation of HL-60 cells (Ogino et al., 2010). Expression of the CD11b surface antigen, an integrin, is considered as a reliable marker of HL-60 differentiation into granulocytes (Brackman et al., 1995). CD11b is upregulated during inflammation and complexes with CD18 to recognize the C3bi complement receptor (CR3). In vivo, it is presented on the surfaces

of granulocytes and NK cells (Fleck et al., 2005). Drayson et al. (2001) showed that CD11b expression is an integral feature of HL-60 differentiation by atRA and is controlled independently of such changes in cell cycle which direct proliferation. Also, its mode of expression seems to be directed by the transcription factor NFjB which upregulates CD11b transcription in HL-60 cells and is activated by atRA (Sokoloski et al., 1993). Fig. 6 shows the extents of CD11b expression upon treatment of HL-60 cells with atRA, PL1RA and PC1RA for 5 days. Being a cluster

Fig. 8. (a) Histograms showing reductions in rhodamine MFI upon treatment with free atRA, PL1RA and PC1RA for 5 days. (b) The levels of reduction in rhodamine MFI. Data represent mean ± SD, n = 3.

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of differentiation (CD) molecule, CD11b expression was limited to the cellular surface only as seen in the confocal micrograph (Fig. 6(a)). Expression was almost 20% for atRA on day 1 and it increased with time. By day 5, atRA- and PC1RA-treated groups exhibited 58% and 60% expression levels, respectively, and PL1RA-treated group showed 49% expression indicating successful induction of differentiation. These expression values were found to be significant at p < 0.05 between control cells and atRA-, PL1RAand PC1RA-treated cells. No statistical differences, however, were observed between the treatment groups indicating similarity in treatments.

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were found to be significant at p < 0.05 between control cells and atRA-, PL1RA- and PC1RA-treated cells. No statistical differences, however, were observed between the treatment groups indicating similarity in treatments. Similar reductions in DWm of HL-60 cells have also been reported earlier upon their differentiation to neutrophils by DMSO and arsenic trioxide (Cai et al., 2000; Collins and Foster, 1983). It is relevant to note here that neutrophils possess an intricate mitochondrial network and the disruption of DWm is a known early marker of commitment of neutrophils into apoptosis (Fossati et al., 2003). 5. Conclusions

4.5. Differentiating HL-60 cells undergo cell death atRA has been shown to produce a concomitant effect in HL-60 cells –induction of differentiation which is followed by cell death. Watson et al. (1997) showed that HL-60 cells induced to morphologically and functionally differentiate towards the granulocytic lineage subsequently undergo spontaneous programmed cell death, apoptosis. Also, the appearance of apoptotic characters is correlated with the predominance of mature neutrophils (Martin et al., 1990). It is pertinent to note here that, in order to maintain cellular homeostasis, normal human peripheral blood neutrophils also exhibit apoptosis within a definite time period (Simon, 2003). Such apoptosis is presumably due to the increase in reactive oxygen content of the cells and, both, NADPH-oxidase and mitochondria-derived oxidants have been postulated to be involved in the process (Fossati et al., 2003). The frequencies and extents of cell death at different time points observed in HL-60 cells induced to differentiate by atRA, PL1RA and PC1RA are shown in Fig. 7. PI staining was used to evaluate apoptosis by using flow cytometry dual parameter analysis based on side scatter vs PI and data were expressed as histograms. As observed, there was an increase in the apoptotic population with time with around 10% cell death on day 1 incrementing to almost 40% by day 5 across all treatment groups. These values were found to be significant at p < 0.05 between control cells and atRA-, PL1RA- and PC1RA-treated cells. No statistical differences, however, were observed between the treatment groups indicating similarity in treatments. It has been demonstrated earlier that a reduction in the mitochondrial transmembrane potential (DWm), which occurs due to the opening of mitochondrial permeability transition pores, is a key event preceding apoptotic cell death (Zamzami et al., 1996). This depolarization of the mitochondrial membrane is aided by the release of cytochrome c into cytosol leading to activation of the caspase cascade (Rosse et al., 1998). Both these events are blocked by the expression of the proto-oncogene bcl-2 which is regulated by atRA (Niizuma et al., 2006; Yang et al., 1997). Niizuma et al. (2006) further showed that atRA upregulates bcl-2 in LA-N-5 and RTBM1 cell lines, in which it induces neuronal differentiation and downregulates it in CHP134 and NB-39-nu cell lines, in which it induces apoptosis. For HL-60 cells, it has been shown that Bcl2 inhibits apoptosis associated with their terminal differentiation and atRA treatment results in destabilization of the Bcl2 mRNA (Naumovski and Cleary, 1994; Otake et al., 2005). The observed reductions in DWm of HL-60 cells upon treatments with atRA, PL1RA and PC1RA were measured by rhodamine B staining and evaluated on a flow cytometer. Rhodamine B is a sensitive probe for assessing DWm since it distributes across the membrane in response to the electrical transmembrane potential and is largely irresponsive to P-glycoprotein- and MRP1-proteinmediated efflux (Reungpatthanaphong et al., 2003). Fig. 8 shows the corresponding decreases in rhodamine mean fluorescence intensities (MFI) with respect to control cells. Reductions across groups were comparable and were 60–70% by day 5. These values

With an aim to evaluate the differentiation-inducing potential of nano-encapsulated atRA, two (PL1 and PC1) biodegradable PEGylated copolymers based on L-lactide and e-caprolactone, respectively, were synthesized and used for preparing atRA-loaded nanoparticles. 1H NMR analysis indicated successful copolymer synthesis. The sizes and polydispersities of nanoparticles were found to slightly increase upon atRA loading. In vitro release studies exhibited pseudo-zero order release of the drug indicating diffusion-controlled slow release. atRA photostability was considerably improved upon encapsulation. The NBT reduction assay and DCHF-DA staining for ROS indicated successful induction of respiratory burst in HL-60 cells upon treatment with PL1RA and PC1RA. These formulations were also able to stimulate CD11b expression and post-differentiation apoptosis in HL-60 cells at levels comparable to free atRA. In conclusion, PL1RA and PC1RA are attractive and efficient vehicles for atRA delivery. Acknowledgements The authors thankfully acknowledge Flow Cytometry and Dynamic Light Scattering Facilities at SAIF, IIT Bombay and are grateful to the Department of Science and Technology, Govt of India, for financial support through Nano Mission, SERC and IRPHA. References Brackman, D., Lund-Johansen, F., Aarskog, D., 1995. Expression of cell surface antigens during the differentiation of HL-60 cells induced by 1,25dihydroxyvitamin D3, retinoic acid and DMSO. Leuk. Res. 19, 57–64. Breitman, T.R., Selonick, S.E., Collins, S.J., 1980. Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc. Nat. Acad. Sci. 77, 2936–2940. Brisaert, M.G., Everaerts, I., Plaizier-Vercammen, J.A., 1995. Chemical stability of tretinoin in dermatological preparations. Pharm. Acta Helv. 70, 161–166. Cai, X., Shen, Y.-L., Zhu, Q., Jia, P.-M., Yu, Y., Zhou, L., Huang, Y., Zhang, J.-W., Xiong, S.-M., Chen, S.-J., Wang, Z.-Y., Chen, Z., Chen, G.-Q., 2000. Arsenic trioxideinduced apoptosis and differentiation are associated, respectively, with mitochondrial transmembrane potential collapse and retinoic acid signaling pathways in acute promyelocytic leukemia. Leukemia 14, 262–270. Castaigne, S., Chomienne, C., Daniel, M.T., Ballerini, P., Berger, R., Fenaux, P., Degos, L., 1990. All-trans retinoic acid as a differentiation therapy for acute promyelocytic leukemia. I. Clinical results. Blood 76, 1704–1709. Choi, Y., Kim, S.Y., Kim, S.H., Lee, K.-S., Kim, C., Byun, Y., 2001. Long-term delivery of all-trans-retinoic acid using biodegradable PLLA/PEG-PLLA blended microspheres. Int. J. Pharm. 215, 67–81. Collins, J.M., Foster, K.A., 1983. Differentiation of promyelocytic (HL-60) cells into mature granulocytes: mitochondrial-specific rhodamine 123 fluorescence. J. Cell Biol. 96, 94–99. Collins, S., 1987. The HL-60 promyelocytic leukemia cell line: proliferation, differentiation, and cellular oncogene expression. Blood 70, 1233–1244. Collins, S.J., Bodner, A., Ting, R., Gallo, R.C., 1980. Induction of morphological and functional differentiation of human promyelocytic leukemia cells (HL-60) by compounds which induce differentiation of murine leukemia cells. Int. J. Cancer 25, 213–218. De Luca, L.M., 1991. Retinoids and their receptors in differentiation, embryogenesis, and neoplasia. FASEB J. 5, 2924–2933. Degos, L., Dombret, H., Chomienne, C., Daniel, M.-T., Miclea, J.-M., Chastang, C., Castaigne, S., Fenaux, P., 1995. All-trans-retinoic acid as a differentiating agent in the treatment of acute promyelocytic leukemia. Blood 85, 2643–2653. Drayson, M.T., Michell, R.H., Durham, J., Brown, G., 2001. Cell proliferation and CD11b expression are controlled independently during HL60 cell differentiation

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