Accepted Manuscript Curcumin loaded pH-sensitive hybrid lipid/block copolymer nanosized drug delivery systems Ivelina Jelezova, Elena Drakalska, Denitsa Momekova, Natalia Shalimova, Georgi Momekov, Spiro Konstantinov, Stanislav Rangelov, Stergios Pispas PII: DOI: Reference:

S0928-0987(15)00326-7 http://dx.doi.org/10.1016/j.ejps.2015.07.005 PHASCI 3315

To appear in:

European Journal of Pharmaceutical Sciences

Received Date: Revised Date: Accepted Date:

7 April 2015 2 July 2015 6 July 2015

Please cite this article as: Jelezova, I., Drakalska, E., Momekova, D., Shalimova, N., Momekov, G., Konstantinov, S., Rangelov, S., Pispas, S., Curcumin loaded pH-sensitive hybrid lipid/block copolymer nanosized drug delivery systems, European Journal of Pharmaceutical Sciences (2015), doi: http://dx.doi.org/10.1016/j.ejps.2015.07.005

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Curcumin loaded pH-sensitive hybrid lipid/block copolymer nanosized drug delivery systems

Ivelina Jelezova1, Elena Drakalska2, Denitsa Momekova2, Natalia Shalimova1, Georgi Momekov1, Spiro Konstantinov1, Stanislav Rangelov3, Stergios Pispas4

1

Department of Pharmacology, Pharmacotherapy and Toxicology, Faculty of

Pharmacy, Medical University of Sofia, 2 Dunav Str., 1000 Sofia, Bulgaria 2

Department of Pharmaceutical Technology and Biopharmaceutics, Faculty of

Pharmacy, Medical University of Sofia, 2 Dunav Str., 1000 Sofia, Bulgaria 3

Institute of Polymers, Bulgarian Academy of Sciences, Sofia, Bulgaria,

4

Theoretical and Physical Chemistry Institute, National Hellenic Research

Foundation, 48 Vassileos Constantinou Avenue, 11635 Athens, Greece

Corresponding author: Denitsa Momekova, Department of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy, Medical University-Sofia, 2 Dunav St., 1000 Sofia, Bulgaria; Phone: +3592 9236 528; Fax: +3592 9879 874; email: [email protected]

Abstract Curcumin is a perspective drug candidate with pleiotropic antineoplastic activity, whose exceptionally low aqueous solubility and poor pharmacokinetic properties have hampered its development beyond the preclinical level. A possible approach to overcome these limitations is the encapsulation of curcumin into nano-carriers, incl. 1

liposomes. The present contribution is focused on feasibility of using hybrid pHsensitive liposomes, whereby curcumin is entrapped as a free drug and as a water soluble inclusion complex with PEGylated tert-butylcalix[4]arene, which allows the drug to occupy both the phospholipid membranes and the aqueous core of liposomes. The inclusion complexes were encapsulated in dipalmithoyl phosphathydilcholine:cholesterol liposomes, whose membranes were grafted with a poly(isoprene-b-acrylic acid) diblock copolymer to confer pH-sensitivity. The liposomes were characterized by DLS, ζ-potential measurements, cryo-TEM, curcumin encapsulation efficacy, loading capacity, and in vitro release as a function of pH. Free and formulated curcumin were further investigated for cytotoxicity, apoptosis-induction and caspase-8, and 9 activation in chemosensitive HL-60 and its resistant sublines HL-60/Dox and HL-60/CDDP. Formulated curcumin was superior cytotoxic and apoptogenic agent vs. the free drug. The mechanistic assay demonstrated that the potent proapoptotic effects of pH-sensitive liposomal curcumin presumably mediated via recruitment of both extrinsic and intrinsic apoptotic pathways in both HL-60 and HL-60/CDDP cells. Key words: pH-sensitive hybrid liposomes, curcumin, inclusion complex, polyoxyethylated tert-buthylcalix[4]arenes, apoptosis, poly(isoprene-b-acrylic acid)

Introduction Curcumin is the active principle of the spice turmeric, produced by the rhizome of Curcuma longa (Zingiberaceae) (Singh and Khar, 2006), which is widely used in traditional eastern medicine as a hepatoprotective, anti-infectious and antiinflammatory remedy (Shehzad et al., 2010). A compelling body of recent evidence 2

has shown that curcumin is endowed by pleiotropic antineoplastic effects, due to modulation of NFκB and other cell signaling pathways, implicated in cell survival, apoptosis and angiogenesis (Bansal et al., 2011; Shehzad et al., 2010; Sou, 2012). Curcumin has been also documented to exert chemopreventive and drug-resistance modulating activities (Ilieva et al., 2014; Shehzad et al., 2010; Singh and Khar, 2006). Despite these promising pharmacological properties curcumin is characterized by unfavorable biopharmaceutical and pharmacokinetic properties that have generally prevented its advancing to the clinical setting (Bansal et al., 2011). Curcumin is characterized by an exceptionally low aqueous solubility (11 ng/ml), and by marked chemical instability (t1/2 in buffer solution at pH 7.4 at 37°C is less than 10 min) (Priyadarsini, 2009; Wang et al., 1997; Zhongfa et al., 2012). It is well appreciated that the cytotoxic and apoptogenic properties of curcumin are concentrationdependent and require continuous exposure of cancer cells to 5-50 μM of curcumin (Ilieva et al., 2014; Sou, 2012). Unfortunately, even applied at extensive oral doses of several grams daily, curcumin has a very low bioavailability. The pharmacokinetic studies typically report low nanomolar concentrations of the drug (Bansal et al., 2011; Sharma et al., 2004; Sou, 2012) and the highest published plasma concentration is 1.7 μM, after 8 g daily dosing (Cheng et al., 2001). Moreover, curcumin is promptly cleared from the circulation and extensively metabolized and thus is incapable of reaching therapeutic levels in target tissues outside the gastrointestinal tract (Bansal et al., 2011; Garcea et al., 2005; Garcea et al., 2004; Sou, 2012). Being highly lipophilic and hence unsuitable for intravenous application, curcumin is a good candidate for incorporation into nanoparticulate drug delivery systems that could make parenteral application feasible (Bansal et al., 2011; Sou, 2012). Curcumin has been formulated in 3

liposomes (Barui et al., 2014; Berginc et al., 2014; Chen et al., 2012; Drakalska et al., 2014; Gosangari and Watkin, 2012; Karewicz et al., 2013; Li et al., 2012; Pandelidou et al., 2011; Tang et al., 2013), nanoemulsions (Ganta and Amiji, 2009), solid lipid nanoparticles (Gota et al., 2010), nanosuspensions (Gao et al., 2010), polymeric nanoparticles (Feng et al., 2012; Khalil et al., 2013; Misra and Sahoo, 2011), etc. Based on their advantageous properties, liposomes are considered among the most promising nano-carriers for curcumin (Kurzrock et al., 2004; Sou, 2012), but nevertheless the practical elaboration of liposomal curcumin faces certain setbacks. Being hydrophobic, curcumin is entirely localized in the liposomal membranes, which could compromise the bilayer integrity when the drug concentration exceeds certain level (Barry et al., 2009; Hung et al., 2008). This in turn is a prerequisite for the low entrapment capacity of curcumin in liposomes. A feasible strategy for increasing the loading efficiency is based on hybrid liposomal platforms, whereby curcumin is entrapped as water soluble complexes with macrocyclic cavitands, which allows the drug to occupy both the phospholipid membranes and the central aqueous cavity of liposomes (Dhule et al., 2012; Matloob et al., 2014; Rahman et al., 2012). Recently we have reported a series of polyoxyethylated tert-butyl calix[4]arenes whose selfassembly in aqueous medium leads to formation of spherical nanosized aggregates with high solubilization capacity for curcumin and size, suitable of systemic delivery (Momekova et al., 2012). Eventually a hybrid liposomal system was developed, whereby inclusion complexes of curcumin with these polyoxyethylated tert-butyl calix[4]arenes were entrapped inside lipid vesicles with a high loading capacity (Drakalska et al., 2014). Considering the fact that the main mechanisms for cellular internalization of liposomes are endocytosis and membrane fusion, another problem that has to be 4

addressed is the potential endosomal sequestration and degradation of the carrier with the entrapped curcumin. Due to the hydrophobicity of curcumin and its ability to form hydrogen bonds with cellular membrane components, there is a tendency for sequestration of the drug within membrane compartments, leading to low intracellular levels. A rational approach for optimizing the intracellular delivery is the loading of curcumin in pH-sensitive liposomes, which have been well appreciated to escape endosomal sequestration and increase the intracellular availability of their cargo (Chu et al., 1990; Schroit et al., 1986). On these grounds, the present investigation is focused upon the elaboration, characterization and cytotoxicity evaluation of hybrid, pH-sensitive liposomes based on dipalmitoylphosphathydilcholine:cholesterol (DPPC:CHOL), for targeted delivery and release of curcumin, using polyoxyethylated calix[4]arene and a pH-sensitive block poly(isoprene-b-acrylic acid) (pI-pAA) copolymer.

2. Materials and Methods 2.1 Materials Polyoxyethylated tert-buthylcalix[4]arene (BEC-X) (Mw=32300 g/mol total degree of polymerization of PEG moieties of 180) (Figure 1a) was synthesized by anionic polymerization as described elsewhere (Momekova et al., 2012). The рН-sensitive block copolymer poly(isoprene-b-acrylic acid) (pI-pAA) (Mw = 12000 g/mol) (Figure 1b) was synthesized by combination of anionic polymerization and a post polymerization hydrolysis reaction scheme. In brief, a poly(isoprene-b-tertbutylacrylate) precursor copolymer was synthesized by anionic polymerization of isoprene in benzene using n-BuLi as the initiator, followed by the polymerization of tert-butylacrylate in a THF/benzene mixture, after capping the polyisoprenyl-lithium 5

active chain ends with 1,1-diphenylethylene, and in the presence of LiCl. After isolation of the poly(isoprene-b-tert-butylacrylate) copolymer the tert-butyl groups were removed by acid hydrolysis resulting in the desired poly(isoprene-b-acrylic acid) copolymer. Dipalmithoylphosphathydilcholine (DPPC), cholesterol (CHOL), curcumin (95 % purity), SpectraPor 7 dialysis membranes (MWCO 10000), Lglutamine, RPMI-1640 cell medium and fetal calf serum (FCS) were purchased from Sigma–Aldrich (USA).

2.2. Preparation of curcumin:BEC-X inclusion complexes Curcumin:BEC-X inclusion complexes were prepared by a solvent evaporation method as described earlier (Drakalska et al., 2014). In brief, BEC-X (6 mg/ml) and curcumin (1mg/ml) were dissolved in absolute ethanol and evaporated to dryness using a Buchi rotation-type vacuum evaporator. To avoid the formation of supramolecular aggregates during hydration step the concentrations of BEC-X was kept below its critical aggregation concentration of 7.7 mg/ml corresponding to 0.24 μmol/ml (Momekova et al., 2012). Then the dried films were hydrated with deionized water and were left for 2h at 50°C and thereafter in dark at room temperature for 24 h under constant agitation to attain equilibrium. Afterwards, the samples were centrifuged at 5000 rpm for 10 minutes to allow sedimentation of undissolved curcumin. The transparent yellow supernatants containing the curcumin:BEC-X inclusion complexes were collected, analyzed for curcumin content using a validated UV-VIS spectrophotometric method and used as a hydration medium for preparation of hybrid liposomal systems.

6

2.3. Preparation of conventional and pH-sensitive hybrid liposomes. All liposomal formulations were prepared by a modified thin film hydration method and extrusion. Briefly, phospholipids DPPC:CHOL (2:1 mol ratio; total lipid concentration 10 μmol/ml) were dissolved in chloroform. For the preparation of hybrid pH-sensitive liposomes, the pH-sensitive copolymer pI-pAA was added as a membrane component in concentrations from 2.5 to 7.5 mol % in respect to total lipid. The pI hydrophobic block is expected to be incorporated within the lipid membrane of the liposomes, whereas the pAA block is tethered on the membrane sides well solvated in the aqueous medium. Thereafter, the organic solvent was evaporated on a rotation type vacuum evaporator (Buchi, Germany). Traces of chloroform were removed under vacuum overnight. Then the thin lipid films were hydrated according to the aim of the study with isotonic solution of sodium chloride or aqueous solution of calcein of concentration 100 mM, at which the fluorescence of the latter is self-quenching. During the hydration step, liposomal suspensions were subjected to 11 consecutive freeze/thaw cycles (-140°C/50°C). Afterwards, the liposomal formulations were extruded 15 times through polycarbonate membrane filters of 100 nm pore size using 10 mL thermobarrel extruder (Lipex, Nortern Lipids INC). Curcumin loaded pH-sensitive, hybrid liposomes were prepared using the same method with the only difference that curcumin was incorporated as a free drug into the phospholipid membrane (0.1:1 drug to phospholipids molar ratio) and also encapsulated in aqueous core of the vesicles in the form of water soluble curcumin:BEC-X inclusion complexes, used as a hydration medium. The 7

concentration of curcumin into the inclusion complex was 128 μg/ml. The nonentrapped curcumin was removed by passing the liposomal suspension through a Sephadex G50 column (Pharmacia, Uppsala, Sweden), equilibrated with isotonic solution of sodium chloride. The encapsulation efficiency (EE) was calculated according to the equation: EE (%) = (Cl/Ct) × 100, where Cl – curcumin in liposomes, Ct – total curcumin used for the preparation of liposomes. Drug loading capacity (DL) was calculated according to the equation: DL= Cl (mol)/CTL (mol) where Cl (mol) – entrapped curcumin and CTL - total lipid (mol) The loss of DPPC and curcumin determined at the end of the preparation process (after the extrusion and gel filtration) were 9.42 % and 11.2 % respectively.

2.4. Dynamic light scattering (DLS) and zeta-potential The mean size, size distribution patterns and zeta-potential of liposomal formulations either empty or loaded with curcumin were determined using ZetaSizer NanoZS (Malvern Instruments Ltd., Spring Lane South, Malvern, UK.), equipped with a 633 nm laser. The parameters were evaluated from measurements at the scattering angle of 173° (back scattering detection) at 25°C. The hydrodynamic diameters dh were calculated using the Stokes–Einstein equation: dh= kT/3πηD

8

where kT is the thermal energy factor; ηis the temperature dependent viscosity of the solvent and D is the diffusion coefficient. Each measurement was performed in triplicate.

2.5. Cryogenic-transmission electron microscopy (Cryo-TEM) A droplet of the tested liposomal suspensions was placed onto a copper grid, coated with perforated polymer film and was frozen at -140°С in liquid ethane. The samples were analyzed using a Zeiss EM 902A electron microscope. The working temperature during the Cryo-TEM measurements was maintained below 180 К. The lipid concentration of the samples was 3 mM.

2.6. Leakage assays. 50 μL of 100 mM calcein-loaded plain or pI-pAA surface modified liposomes, separated from the non-encapsulated dye, were added to 2ml of buffered solutions of pH ranging from 4.5 to 7. The fluorescence of calcein at concentration of 80 mM and above is self-quenched. As the polar dye leaks out from the pores of liposomal membranes, it becomes diluted and the fluorescense intensity increases. After incubation of 15 minutes at 37°C, the fluorescence was assessed at 520 nm after excitation at 490 nm, before and after the addition of 100 μl of a 10 wt.% solution of Triton X-100 using a Hitachi F-7000 spectrofluorimeter. The percentage of calcein leakage was calculated according to the following equation: Cal leakage (%) = (IpH – I 7.4) / (It − I7.4) × 100

9

where IpH is the corrected intensity at acidic pH before the addition of Triton X-100, I7.4 is the fluorescense at pH 7.4 and It is the total fluorescense intensity evaluated after the destruction of liposomes.

10

2.8. In vitro curcumin release The curcumin release from conventional and hybrid pH-sensitive liposomes was investigated as a function of pH using membrane dialysis at 37°C. After 1 hour of incubation in isotonic buffered solutions (рН 4.5-7), acceptor phase samples were investigated for curcumin concentration using UV-Vis spectroscopy at 427 nm. Curcumin release from hybrid pH-sensitive liposomes was also tested as a function of time after 24 hour of incubation in phosphate buffer (pH 7) at 37 °C. At predermined time intervals, samples from the acceptor phase were withdrawn and curcumin content was determined by UV-Vis spectroscopy at 427 nm.

2.9. Cell lines and culture conditions Cell lines and culture conditions. The cytotoxic activity of free and formulated curcumin was assessed against the acute myelocyte leukemia-derived HL-60 cell line, and its multidrug-resistant (HL60/DOX) and cisplatin-resistant (HL-60/CDDP) sublines. HL-60 cells and HL60/DOX were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ GmbH, Braunschweig, Germany). HL-60/CDDP was established in the authors’ lab by continuous selection in growth medium with gradually increasing concentrations of cisplatin. The cells were grown in controlled environment – cell culture flasks at 37oC in an incubator 'BB 16-Function Line' Heraeus (Kendro, Hanau, Germany) with humidified atmosphere and 5% CO2. The growth medium was 90% RPMI-1640 + 10% FBS. HL-60/CDDP were cultivated in the presence of 25 μM cisplatin, and HL-60/DOX were grown in medium containing 0.2 μM doxorubicin in order to maintain their drug resistance phenotype. Three days prior the bioassays, cell 11

were re-cultured in drug-free medium in order to avoid possible synergistic interactions with the tested active treatments.

2.10. Cytotoxicity assessment (MTT-dye reduction assay). The cellular viability after exposure to free curcumin or its formulations was assessed using the standard MTT-dye reduction assay as previously described (Mosmann, 1983) with slight modifications (Konstantinov et al., 1999). The method is based on the reduction of the yellow tetrazolium salt MTT to a violet MTT-formazan via the mitochondrial succinate dehydrogenase in viable cells. In brief, exponentially proliferating cells were seeded in 96-well flat-bottomed microplates (100 μl/well) at a density of 1×105 cells per ml and after 24h incubation at 37°C, they were exposed to various concentrations of the tested curcumin formulations (see below) or cisplatin, used as a reference antineoplastic agent for 72h. For each concentration a set of at least 8 wells were used. After the exposure period, 10 μl MTT solution (10 mg/ml in PBS) aliquots were added to each well. Thereafter, the microplates were incubated for 4h at 37°C and the MTT-formazan crystals formed were dissolved through addition of 100 μl/well 5% formic acid-acidified 2-propanol). The MTT-formazan absorption was recorded using a LabeximLMR-1 microplate reader at 580 nm. Cell survival fractions were calculated as percentage of the untreated control. In addition, IC50 values were derived from the concentration-response curves (see below).

Bioassay data processing and statistics The cell survival data were normalized as percentage of the untreated control (set as 100% viability) and were fitted to sigmoidal dose response curves and the corresponding IC50 values (concentrations causing 50% suppression of cellular 12

viability) were calculated using non-linear regression analysis (GraphPad Prizm Software for PC). The statistical processing of biological data included the paired Student’s t-test whereby values of p ≤ 0.05 were considered as statistically significant. The MTT-bioassay derived IC50 values of free curcumin were divided by those derived with the corresponding formulations to yield the modulation indices (MI). In addition, the resistance indices as a relative merit for the level of resistance in HL60/CDDP were determined as the ratio between the IC50 in the multi-drug resistant HL-60/CDDP and the corresponding IC50 in the sensitive parent line HL-60.

2.11. Apoptosis assay The apoptotic DNA fragmentation was examined using a commercially available ‘Cell-death detection’ ELISA kit (Roche Applied Science). This assay allows semiquantitative determination of the characteristic for the apoptotic process histoneassociated mono- and oligonucleosomal DNA-fragments using ‘sandwich’ ELISA. Exponentially proliferating HL-60 or HL-60/CDDP cells were exposed to equieffective concentrations (1/2 IC50 and IC50) of the tested formulations for 24 h and thereafter cytosolic fractions of 1×104 cells per group (treated or untreated) served as antigen source in a sandwich ELISA, utilizing a primary anti-histone antibodycoated microplate and a secondary peroxidase-conjugated anti-DNA antibody. The spectrophotometric immunoassay for histone-associated DNA fragments was performed according to the manufacturers’ instructions at 405 nm, using a microprocessor-controlled microplate reader (Labexim LMR-1). The results are expressed as the oligonucleosome enrichment factor (representing a ratio between the absorption in the treated vs. the untreated control samples). The assay was run in quadruplicate. 13

2.12. Western immunoblot analysis HL-60, and HL-60/CDDP cells were exposed to equieffective concentrations of curcumin formulations (1/2 IC50 and IC50) for 24 h. Thereafter, samples (2x106 cells) were lysed on ice with buffer containing 100 mM Tris-HCl with pH 8.0 (Sigma), 4 % SDS (Sigma), 20 % Glycerol (Sigma), 200 mM DTT (dithiotriethol, Sigma) supplemented with complete protease inhibitor cocktail tablets (Roche). The cell lysates were boiled for 10 min and then centrifuged at 13,000 rpm for 10 min at 4oC. Aliquots of 10 µl were taken from lysates before adding DTT, diluted five times in distilled water and quantified for protein concentration using Piece Protein Assay (Pierce, USA). Lysates were subjected to denaturing polyacrylamide gel electrophoresis on 4–20% Precise Protein Gels (Thermo Scientific, Rockford, IL, USA). The separated proteins were transferred onto polyvinylidene-difluoride membranes. After transfer, the blot was blocked with 5 % (w/v) skimmed milk in PBS, and incubated for 2 h with the following primary antibodies: procaspase 8 (sc-6136), procaspase 9 (sc-17784) and β-actin (sc-8432). Immunoblots were developed using a HRP-conjugated anti-mouse or anti-goat antibody. Investigated proteins were visualized after 45 min incubation with the secondary antibody and washing. The chemiluminescent reaction was detected by an ECL system, according to the manufacturer’s instruction (Amersham, USA). Densitometric analysis of the blots was performed using the Quantity One 4.6.9 software (BioRad, Hercules, CA, USA).

14

3. Results 3.1.

Size and size distribution patterns of unloaded liposomes

The main physicochemical parameters of the conventional and hybrid pH-sensitive liposomes – diameter, size distribution patterns and ζ-potential were determined by dynamic light scattering, using a Nano-ZS instrument. The data are summarized in Table 1. Table 1. Size, size distribution and zeta-potential of conventional and hybrid, рН sensitive liposomes based on DPPC:CHOL (2:1 molar ratio) at preparation and after 1month storage at 4°C. Formulation

DPPC:Chol

Diameter (nm) At

After

At

After

preparation

storage

preparation

storage

0.096 ±

0.123 ±

0.002

0.005

127.4 ± 2.6 131.5 ± 0.084

± 0.115

124.0 ± 4.2 120.0± 3.2

DPPC:Chol:pI-pAA 2,5 mol %

DPPC:Chol:pI-pAA 5 mol % DPPC:Chol:pI-pAA 7,5 mol %

Polydispersity index

1.14

0.002

137.2 ± 3.5 136.8 ± 0.12 ±0.008

ζ – potential (mV) - 22.0 ±4.2

± - 28.1 ± 5.5

0.082 0.23 ±0.074

- 34.0 ± 2.3

4.1 139.0 ± 2.8 142.7 ± 0.163 3.3

0.082

± 0.168

± - 40.8 ±3.8

0.052

The incorporation of the copolymer in growing concentrations of 2.5 to 7.5 mol % is accompanied by a slight increase of the mean vesicle size by ca 13 nm, as compared to the plain liposomes, which is attributed to the formation of polymer layers around the phospholipid membranes by the hydrophilic poly(acrylic acid) chains of the 15

copolymer. The formation of the steric stabilizing coating is corroborated also by the clear decrease in the particle ζ–potential from -22 mV of the plain liposomes to -40.8 mV of the liposomes of the highest copolymer content. The presence of polymer coating imparting a well pronounced negative ζ-potential is a prerequisite for increased colloid stability of the vesicles. An interesting finding from the DLS measurements is the very low polydispersity index of the systems (lower or close to 0.1), which indicates firmly monomodal size distribution patterns (Table 1). Only for the liposomes of the highest copolymer content, the polydispersity index rises to 0.163, still indicating a fairly narrow size distribution. After one month of storage at 4 °C no significant changes in mean diameter or polydispersity index were observed, which confirms the physical stability of the prepared vesicles (Table 1).

3.2. Structure and lamellarity of hybrid pH-sensitive liposomes Another main parameter with a crucial influence on the biopharmaceutical and pharmacokinetic behavior of liposomes as drug carriers is their morphology. It was investigated as a function of copolymer content. Liposomes were visualized and structurally characterized using cryogenic transmission electron microscopy (cryoТЕМ), which allows assessment of the structure and lamellarity of the vesicles. Representative cryo-TEM photomicrographs are depicted in Figure 2. The chosen preparation allows the formation of well-defined unilamellar liposomes (Figure 2). In general, the incorporation of the pH-sensitive copolymer imparts steric stabilization of the liposomes. Thus, in contrast to the plain liposomes, the modified vesicles were separated and did not tend to aggregate (Figure 2 b and c). At copolymer concentrations of 2.5 mol % the only observed objects were well-separated unilamellar liposomes. The first signs for membrane destabilization were detected at 5 16

mol %, as evidenced by the appearance of small spherical micellar objects (Figure 2b), whereas the further increase of the copolymer concentrations to 7.5 mol% was associated with a significant increase of the micelle fraction. Therefore, as the optimum in terms of morphology of the liposomes was selected 2.5 mol% concentration of a pH-sensitive copolymer.

3.3. pH-induced calcein leakage For imparting of рН-sensitivity the membranes of the DPPC:CHOL liposomes were grafted with the рН-responsive block copolymer pI-pAA. This copolymer contains a pAA block which is pH-responsive, being fully deprotonated and negatively charged in alkaline milieu, and fully protonated and less negatively charged in a medium with pH below 4.5. This protonation-deprotonation process is accompanied by a conformational change of the pAA block from coil to more extended chain conformation, respectively, due to strong repulsion between negatively charged COO- side groups. A series of DPPC:CHOL liposomes were prepared, modified with increasing molar fractions of the copolymer. The effect of the copolymer on membrane integrity was monitored by the leakage of a fluorescent marker as a function of copolymer content and the pH of the medium. Calcein was chosen as a fluorescent probe because its fluorescence is not dependent on changes of the medium pH. Liposomes were investigated in the pH range of 4.5-7.0 in order to evaluate the stability of the carrier at the physiological pH of the blood (рН 7.4), on one hand, and to estimate the possibility for selective release of encapsulated cargo in a medium with slightly acidic pH, corresponding to the conditions in the tumor environment (рН 6.5-6.8), as well as in cellular endosomes (рН 4.5-5), on the other. The latter comprise the most important cellular compartment, whereby the foreign particulate matter (incl. 17

nanomedicines) are sequestrated following endocytosis. The fluorescent dye leakage as a function of copolymer content and medium pH is depicted on Figure 3. As evident from the presented data, the plain liposomes are devoid of pH-dependent release of their cargo. Even at the lowest pH (4.5) the quantity of released calcein is less than 15%. In a dissimilar fashion, the copolymer-decorated liposomes showed a marked trend towards a pH-dependent calcein leakage. At the most acidic conditions (рН 4.5), the liposomes modified with 2.5 or 5 mol% pI-pAA release 60% and 65% of the entrapped calcein, respectively. It is worth mentioning that this pH value coincides with the рКа of the pAA block in the copolymer. Under these circumstances, the pAA chains undergo transition to a protonated state, abruptly lower their solubility and, accordingly, tend to isolate from the aqueous phase, thus causing significant rearrangements in the bilayer membrane such as formation of large openings/pores and stabilization of the transient ones, through which the fluorescent dye leaks out. These findings indicate that the inclusion of the pH-responsive block copolymer in the membranes of DPPC:CHOL liposomes is a feasible approach towards imparting pHsensitivity in conventional liposomes and, hence, escaping endosomal sequestration and degradation of the labile curcumin.

3.4. Characterization of curcumin loaded pH-sensitive, hybrid liposomes. 3.4.1. Determination of encapsulation efficiency and drug loading capacity In the presented pH-sensitive hybrid platforms, curcumin is loaded by both incorporation into the phospholipid membranes and by taking up water solubleinclusion complexes (Curc:BEC-X) in the aqueous core of the liposomes. The results showed high encapsulation efficiency (85%) for the pH-sensitive system, which is comparable to those of the plain vesicles and well correlate with our previous study 18

with curcumin loaded hybrid non-pH-sensitive liposomes (Drakalska et al., 2014). These findings implied that the membrane grafting with 2.5 mol% pI-pAA was not detrimental for liposomal capability for accommodation of the active ingredient (Table 2). Table 2. Encapsulation efficiency and loading capacity of conventional and pHsensitive hybrid liposomes. Formulation

DPPC:Chol:Curcumin

Encapsulation efficiency (%) 98

Drug loading capacity 0.09

Ammount of entrapped curcumin (µg/ml) 240 ±1.4

85

0.17

364 ±2.3

DPPC:Chol:рI-pAA(2.5 mol%): Curc:BEC-X

Albeit, the presented data indicate that all tested formulation are characterized by high encapsulating efficiency (exceeding 85%). In contrast to the conventional liposomes, the hybrid pH-sensitive systems showed a twice higher loading capacity, as evident from the almost doubled curcumin-to-phospholipid ratio (0.09 to 0.17). The incorporation of the hydrophilic inclusion complexes of curcumin (Curc:BEC-X) in the aqueous cavity of the vesicles allows to attain significant increase of the content of loaded drug with the same amount of phospholipid, without concomitant signs for disruption of the membrane integrity.

3.4.2. In vitro curcumin release as a function of pH The curcumin release profiles from the hybrid pH-sensitive vesicles were recorded as a function of the pH of the medium, at 37°С over 1 h. They were compared to those of the analogous hybrid non-pH-sensitive liposomes (DPPC:Chol:Curc:BEC-X). The results are depicted on Figure 4a. 19

In line with the calcein leakage data, the non-modified systems did not show a pHdependent curcumin release behavior. Even at the lowest pH value, the amount of released curcumin is below 15% after 1 h incubation. In contrast, the hybrid pI-pAAmodified vesicles demonstrated clear increase of the released curcumin with decreasing pH, whereby at рН 4.5 it exceeded 30%. These findings indicate that the curcumin loading (as free drug into the bilayer and as inclusion complexes inside the central cavity) is not associated with compromising their pH-responsiveness. A very important prerequisite of liposomes as drug delivery systems is their ability to retain the encapsulated compound until they reach target tissues. In this line, the pHsensitivity is useless if the liposomes are not able to retain their cargo in the blood. To evaluate the extent to which the membrane permeability was affected by the incorporation of 2.5mol% of pI-pAA, leakage assay in phosphate buffer (pH 7) upon 24 hours of incubation at 37°C was carried out (Fig. 4b). As evident from the presented data, the ability of pH-sensitive hybrid liposomes to retain curcumin was not affected by the incorporation of the tested copolymer. The released drug at the end of experimental period was less than 40 % and was similar to the amount the drug released from the counterpart hybrid, non pH-sensitive liposomes. These findings give us reason to consider the presented pH-sensitive hybrid liposomes as a feasible platform for controlled intracellular delivery of curcumin.

3.5. Cytotoxicity assessment of free and formulated curcumin A comparative evaluation of the cytotoxic effect of pH-sensitive curcumin loaded system vs. free drug (DMSO solution) in the chemosensitive acute myeloid leukemiaderived HL-60 cell line and its two resistant derivatives, namely HL-60/Dox and HL60/CDDP was performed. For the sake of comprehensiveness, the anti-cancer 20

cytotoxicity bioassay was also performed for alternative formulations specifically, conventional curcumin-loaded DPPC:CHOL liposomes and the two previously described carriers, that are the inclusion complexes of curcumin (Curc:BEC-X) and the non-pH-sensitive hybrid system, loaded with the latter complexes (Lipo:BECX:Curcumin) (Drakalska et al., 2014). The growth inhibitory concentration-response curves are presented on Figure 5 and the corresponding equieffective IC50 values are summarized in Table 3. In order to evaluate the alteration of the cytotoxicity of formulated vs. free curcumin, the corresponding modulation indices were calculated as the ratios between the IC50 value of free drug and the IC50 value of the respective formulation. In addition, resistance indices were calculated as a quantitative merit for the relative responsiveness in HL-60/Dox and HL-60/CDDP vs. HL-60 and are summarized in Table 4. Evident from the presented bioassay data, the formulated curcumin was superior in terms of cytotoxic activity as compared to the free drug. The concentration-response curves were shifted to the lower concentrations and respectively the IC50 values were lower as compared to the effects of curcumin, applied as a DMSO-solution diluted in RPMI-1640 medium. The evaluation of the modulation indices indicates that the pHsensitive hybrid platform, based on DPPC:CHOL:pI-pAA:Curc:BEC-X proved to ensure the most prominent augmentation of cytotoxicity of its cargo with modulation indices ranging ca. 5-19 (Table 3). Noteworthy, these phenomena were more prominent in the resistant HL-60 variants. Thus, while free curcumin was less effective in HL-60/Dox and HL-60/CDDP, relative to the chemosensitive HL-60 with resistance indices of 1.5 and 1.9 its formulations showed lower resistance indices, especially in the cisplatin-resistant derivative cell line. This modulation of the resistance patterns was most pronounced with the pH-sensitive liposomal carrier. For 21

instance, in HL-60/CDDP this formulation was 19 times more potent than free curcumin, and moreover, it proved to completely bypass the resistance phenotype with a resistance index of less than 1, indicating collateral sensitivity (i.e. increased responsiveness in the resistant vs. the sensitive cell line) (Table 4).

Table 3. Equieffective concentrations of tested curcumin formulations, vs. the free drug and the reference anticancer drug cisplatin. IC50 μM (MI*) DPPC:CHO

DPPC:CHOL:

Curc:BE

L:

pI-

C-X

Curc:BEC-

pAA:Curc:BE

X

C-X

Liposom Cell line Cisplati Curcumi al n

n

Curcumi n

HL-60

8.3 ± 0.8

9.1 ±1.1

3.8 ±1.2

4.7 ±1.1

2.9 ±0.4

1.7 ±0.09

(2.4)

(1.9)

(3.1)

(5.3)

HL-

12.2 ±

13.5 ±

5.1 ±1.6

6.2 ±0.7

3.4 ±1.8

2.1 ±0.21

60/DOX

1.2

1.7

(2.6)

(2.2)

(3.97)

(6.4)

144.4 ±

26.7 ±

4.4 ±1.5

5.9 ±1.4

3.2 ±1.1

1.4 ±0.1

9.8

2.1

(6.1)

(4.5)

(8.3)

(19.1)

HL60/CDD P

*MI (modulation indices) = IC50 (free curcumin) / IC50 (encapsulated curcumin)

22

Table 4. Resistance indices of free and loaded curcumin Resistance indices (RI*)

Resistan t HL-60 derivati ve HL60/DOX

Liposom

DPPC:CHO

DPPC:CHOL:

Cisplati Curcumi al

Curc:BE

L:

pI-

n

C-X

Curc:BEC-

pAA:Curc:BE

X

C-X

n

Curcumi n

1.5

1.5

1.3

1.3

1.2

1.2

17.4

2.9

1.15

1.25

1.1

0.8

HL60/CDD P *resistance indices = IC50 (resistant HL-60 variant) / IC50 (chemosensitive HL-60) In order to elucidate the mechanistic aspect of the observed cytotoxicity, we studied the ability of curcumin and its formulations to induce apoptosis by quantifying the degree of genomic DNA fragmentation. The latter is a consequence of the action of specific nucleases, which degrade the higher order chromatin structure during the apoptotic process to histone-associated mono- and oligonucleosome fragments. To meet this objective, exponentially growing HL-60 and HL-60/CDDP cells were exposed to equieffective concentrations of curcumin formulations for 24 h and thereafter the cytosolic enrichment with histone-associated DNA fragments was established using a commercially available “Cell death detection” ЕLISA-kit. Data presented in Figure 6 show that exposure to the tested free and encapsulated curcumin is consistent with a concentration-dependent increase in the DNA-fragmentation intensity in treated cells, showing that its cytotoxicity is at least partly mediated by induction of apoptosis. In line with the MTT-assay data, in HL-60 cells the pH-sensitive hybrid formulation proved to induce the most pronounced induction of apoptosis, whereby it was more 23

potent versus the equieffective concentrations of free curcumin. The other tested formulations also caused concentration-dependent DNA-fragmentation, albeit less intensively as compared to the free drug. Noteworthy, they were applied at lower concentrations based on the differences in the corresponding IC50 values (Figure 6 left). Interestingly, the apoptogenic effects of the tested formulations were more pronounced in the cisplatin-resistant HL-60/CDDP as compared to the chemosensitive parent cell line (Figure 6 right). In this setting, all liposomal formulations were more potent than free curcumin. The calixarene complex showed similar apoptogenic activity as compared to non-encapsulated curcumin. In compliance to the findings with HL-60 again the hybrid pH-sensitive formulation was the most potent inducer of apoptosis, albeit applied at lower levels, based on its quite lower IC50 values. As the apoptotic process is associated with cascade activation of the caspase proteases, we sought to evaluate the level of DNA-fragmentation following coincubation of the cell lines with the non-selective pan-caspase inhibitor BocAsp(OMe)-fluoromethyl ketone (PCI). Evident form the presented data (see Figure 6), the co-administration of the pan-caspase inhibitor greatly ameliorated the apoptogenic effects of curcumin, but nevertheless it did not completely abrogate it. These finding implied for the involvement of caspase-dependent (mainly) and independent cell signaling in the apoptogenic effects of free and encapsulated curcumin. Noteworthy, even in the setting of total caspase inhibition, the pH-sensitive liposomal curcumin was the most potent inducer of apoptosis. To delineate the involvement of the extrinsic and intrinsic apoptotic pathway signaling in the apoptogenic effects of free and entrapped curcumin, further Westernblot analysis for caspase activation was performed. We mimicked the apoptosis assay 24

exposure protocol and treated HL-60 and HL-60/CDDP with equieffective concentrations of free curcumin or its formulations for 24 h. Figure 7 shows the expression of the apoptotic markers, following exposure to free curcumin and its formulations. Activation of caspase-9 (indicative for the recruitment of the intrinsic apoptotic pathway) was indicated by the disappearance of a 46-kDaprocaspase band. Likewise, processing of the caspase-8, involved in the extrinsic apoptotic pathway signaling was indicated by the disappearance of the corresponding 55-kDa procaspase-9 band. Evident form the presented Western-blots and the corresponding β-actin-normalized relative expression data, at equitoxic concentrations the tested curcumin formulations proved to elicit differential effects on the caspase signaling cascades. In the chemosensitive parent cell line HL-60, the pH-sensitive liposomal formulation and free curcumin were the most potent activators of caspase 9 (Figure 7 upper panel). There was some disappearance of the procaspase band also at the higher concentration of plain liposomal curcumin, whereas all of the other tested formulations failed to induce caspase-9 activation. Interestingly, in HL-60 cells only the pH-sensitive hybrid liposomal platform caused disappearance of the procaspase-8 band, indicative for recruitment of the extrinsic apoptotic signaling pathway. In the cisplatin-resistant HL-60/CDDP variant, there was more pronounced concentration-dependent activation of caspase 9 following exposure to the different curcumin formulations. Most marked procaspase 9 band disappearance was detected after exposure to the pH-sensitive hybrid liposomal formulation and free curcumin (Figure 7 bottom panel). Regarding the recruitment of the extrinsic pathway, while free curcumin caused only minimal activation of caspase 8, the tested nanosized formulations caused 25

disappearance of the procaspase band, especially at higher concentrations. Evident from the β-actin-normalized data for procaspase band density, the hybrid liposomal carriers were the most potent activators of caspase 8.

Discussion The present contribution presents the development of a hybrid delivery system for curcumin based on DPPC:CHOL liposomes embarking calixarene-encapsulated drug whose membranes are grafted with poly(isoprene-b-acrylic acid) copolymer to confer pH-responsiveness and ability for triggered drug release. The chosen composition and pH-sensitive copolymer have been shown to confer beneficial modulation of the properties of encapsulated curcumin and to address several issues, namely: i) preparing liposomes with physicochemical properties suitable for parenteral application, ii) attaining optimal drug-to-lipid loading capacity, iii) conferring aciditytriggerable destabilization and release properties. The polyisoprene block is mimicking the prenyl functionalities, which are important membrane anchoring moieties of signaling molecules in mammalian cells (Alvarado and Giles, 2007; Gelb et al., 2006). While liposomes have been repeatedly reported as suitable carriers for curcumin (Bansal et al., 2011; Kurzrock et al., 2004; Sou, 2012), the design of liposomal platforms for this agent faces several concerns to consider. Although curcumin is feasibly incorporated in membrane bilayers of liposomes, this quite limits the amount of drug to be incorporated, since it is only confined to the membrane domain of the vesicles, while the much more bulky central aqueous reservoirs remain unloaded, due to the hydrophobicity of the compound. Moreover, curcumin itself has been shown to destabilize the integrity of the phospholipid bilayer, which further limits the maximal 26

quantity of the active ingredient to be incorporated (Barry et al., 2009; Hung et al., 2008). Among the investigated conventional liposomal carriers of curcumin, maximal entrapment capacity has been described with dimyristoyl phosphatidylcholine (DMPC) (Bansal et al., 2011; Sou et al., 2008). Unfortunately, this phospholipid has a relatively low gel-to-liquid crystalline phase transition temperature (Tc) of 25°C, which makes such liposomes leaky and unstable at physiological conditions. In contrast, dipalmitoyl phosphatidylcholine (DPPC) is a phospholipid with a Tc of 42°C, which renders it more suitable for elaboration of liposomal carriers. Unfortunately, DPPC-based liposomes have been found to accommodate much less efficiently curcumin. Further complexity to the issue is added by the influence of cholesterol, which is an almost inevitable component of liposomal membranes, but has been found to decrease the capacity of DPPC liposomes to accommodate curcumin, because it competitively hampers the formation of hydrogen bonds between the drug and the phospholipid molecules (Sou, 2012). In order to address this stability vs. entrapment capacity dilemma, we have developed a hybrid DPPC-based liposomal system, whereby curcumin is introduced as water soluble complexes with a previously described PEO-modified calix[4]arene. Evident form the presented data, increased water solubility of curcumin allows the drug to be accommodated also in the central aqueous cavity of liposomes, leading to an increase of the loading capacity by a factor of 1.7. Apparently, the pH-responsive polymer at 2.5 mol% was not detrimental neither for the drug loading efficacy, nor for the structural integrity of the liposomes. The Cryo-TEM and DLS characterization of the DPPC:CHOL:pI-pAA:BEC-X:Curcumin shows that the chosen preparatory method allows the formation of well defined, spherical vesicles with a mean diameter of 145 27

nm and monomodal size-distribution patterns. Moreover, the high negative charge imparted by the grafted copolymer conditions superior colloidal stability as a further advantageous characteristic of this formulation as a drug delivery system. A central objective of the study was to impart pH-responsiveness of the vesicles in order to allow triggered destabilization in acidic media and hence ability to escape endosomal sequestration and inactivation of the cargo. Evident from the calcein leakage experiments, pI-pAA at 2.5mol%, which have been shown to be inert towards membrane and colloidal stability of the liposomes, confers marked pH-responsiveness of the vesicles, in contrast to the non-modified liposomes. This is an additional beneficial feature of the presented formulation because the molecular targets of curcumin are intracellular and thus pH-sensitive fusogenic liposomes are expected to beneficially increase its intracellular delivery and hence augment its biological activities. An important issue to address in this contribution was whether encapsulation of curcumin inclusion complexes in a pH-responsive hybrid liposomal system would translate into an enhancement of its antineoplastic activity in comparison to free drug, its polyoxyethylated calix[4]arene complexes or plain liposomes. Considering the importance of multidrug resistance phenomena as impediment of anticancer chemotherapy and the potential role of curcumin as resistance-reversal agent, we evaluated its cytotoxicity in cisplatin-resistant, multi-drug resistant and chemosensitive (parent) variant of the acute myeloid leukemia cell line HL-60. Free curcumin was a potent cytotoxic agent in HL-60, but the responsiveness of HL60/Dox, and especially of HL-60/CDDP was lower. These findings are in line with previous investigations of the drug in HL-60/Dox, and could be ascribed to active

28

unilateral efflux of the drug via the xenobiotic efflux pump overexpressed in this cell line (Alaikov et al., 2007; Ilieva et al., 2014). The MTT-bioassay data unambiguously showed that the pH-sensitive liposomal formulation of curcumin proved to significantly outclass the free drug in terms of relative potency; it inhibited the viability and proliferation of chemosensitive and especially of the resistant cell lines at low micromolar concentrations, with IC50 values 5 times lower in HL-60, ca. 6 times in HL-60/Dox and almost 20-fold lower in the cisplatin resistant HL-60/CDDP, relative to the non-formulated agent. The other nanocarriers, namely, the BEC-X inclusion complexes, plain liposomes, and the hybrid-non-pH-sensitive liposomes also caused shifting of the dose-response curves to lower concentrations and conversely augmentation of the cytotoxic activity, but less efficiently than DPPC:CHOL:pI-pAA:BEC-X:Curcumin. These findings well parallel numerous preceding reports showing that by virtue of enhancing solubility, stability, and cellular uptake of the cargo, the liposomal curcumin is multifold more effective than equimolar non-formulated curcumin (Apiratikul et al., 2013; Bansal et al., 2011; Rahman et al., 2012). The established high efficacy of formulated curcumin in resistant cells is of special interest, since this compound has been shown to restore the responsiveness to anticancer drugs and is considered as a potential multidrug-resistance reversal agent (Li et al., 2013; Li et al., 2011; Misra and Sahoo, 2011; Rao et al., 2014; Roy and Mukherjee, 2014; Sreekanth et al., 2011; Sreenivasan et al., 2012). The apoptosis assay firmly demonstrated that liposomal curcumin is a potent inducer of apoptotic DNA-fragmentation in HL-60, in compliance to preceding reports (Alaikov et al., 2007; Liao et al., 2008; Tan et al., 2006) and especially in HL-60/CDDP cells which proved to be more sensitive to the apoptogenic effects of equieffective levels of 29

curcumin. Noteworthy, the pH-sensitive platform was the most potent formulation in this respect although applied at lowest concentrations (based on the IC50 values from the MTT-bioassay). Non-selective inhibition of caspase activities reduced the levels of DNA-fragmentation, but nevertheless it did not completely hamper the apoptogenic activity of the tested curcumin formulations. These findings well corroborate the reported involvement of both caspase-dependent (Anto et al., 2002; Chang et al., 2014; Gogada et al., 2011; Kuo et al., 2011; Li et al., 2014; Sikora et al., 2006; Singh et al., 2009; Wu et al., 2005; Wu et al., 2010)) and independent mechanisms (Hilchie et al., 2010; Lu et al., 2009; Piwocka et al., 2002; Piwocka et al., 2001; Thayyullathil et al., 2008) to underlie the proapoptotic effects of the polyphenol. In order to further delineate the mechanistic peculiarities of formulated and free curcumin, we mimicked the apoptosis assay protocol in HL-60 and HL-60/CDDP cells to evaluate the recruitment of the intrinsic mitochondrial (caspase-9-dependent) and extrinsic (caspase-8-dependent) apoptotic signaling pathways. The findings clearly showed differential responses in the chemosenstive and the cisplatin-resistant cell lines. While in the parent HL-60 there was recruitment of caspase-9 especially after exposure to free curcumin or the pH-sensitive system, only the latter induced some disappearance of the procaspase-8 band. These data are indicative for activation of the mitochondrial, intrinsic-apoptotic signaling pathway, in unison with preceding reports (Kuo et al., 2011; Liao et al., 2008), albeit the recruitment of the extrinsic pathway by the pH-sensitive system is in line with published data for free curcumin as well (Bush et al., 2001; Ilieva et al., 2014). Interestingly, in HL-60/CDDP formulated curcumin evoked stronger activation of caspase-9 and especially of caspase-8, as compared to the effects in the parent cell line. The recruitment of the extrinsic pathway was considerable after treatment with 30

the plain hybrid system and the pH-sensitive platform, leading to more than 70% disappearance of the procaspase bands. Taken together, the pharmacological-bioassay data firmly indicated that the formulation of curcumin in pH-sensitive hybrid liposomes, loaded with inclusion complexes of the drug is a feasible approach towards augmentation of its cytotoxic and apoptogenic effects. Noteworthy, the resistant cell sub-line HL-60/CDDP not only demonstrated higher sensitivity to DPPC:CHOL:pI-pAA:BEC-X:Curcumin, but also more marked apoptotic DNA-fragmentation and activation of both caspase-9 and caspase-8. These findings are of special interest since curcumin has been reportedly effective in restoring the responsiveness of resistant cells to cisplatin (Chen et al., 2014; Roy and Mukherjee, 2014; Ye et al., 2012) and even more has been shown to reduce the adverse clastogenic effects of this drug (Alaikov et al., 2007), which makes this plant polyphenol an advantageous candidate for combination regimens with the platinum drug.

Conclusion Hybrid pH-sensitive liposomes (DPPC:CHOL:pI-pAA:Curc:BEC-X) were elaborated as a novel drug delivery platform for curcumin. The liposomal system is characterized with high curcumin loading capacity due to encapsulation of the drug both into phospholipid membrane as well as into aqueous liposomal cavity in form of inclusion complex with polyoxyethylated tert-buthylcalix[4]arene. The bioassay data showed that formulated curcumin was superior in terms of cytotoxic activity as compared to the free drug. The pH-sensitive liposomal formulation inhibited the viability and proliferation of chemosensitive and especially of the resistant cell lines at lower micromolar concentrations as compared to free drug and curcumin formulated in 31

hybrid, but non-pH-sensitive liposomes. These findings give us reason to conclude that the presented pH-sensitive liposomal system of curcumin is a feasible platform to ensure augmentation of its cytotoxic and apoptogenic properties with a concomitant anticipated beneficial modulation of the pharmacokinetic behavior, based on the well known generic properties of liposomes as drug delivery systems.

Acknowledgements: Financial support from DFNI B01/25 2012) is gratefully acknowledged. We are grateful to Dr. Jonny Eriksson for performing the cryo-TEM analysis.

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Figure captions:

Figure 1. Schematic representation of the structure of: a) polyoxyethylated tertbuthylcalix[4]arene and b).poly(isoprene-b-acrylic acid)

Figure 2. Cryo-TEM images of DРPC:CHOL (2:1): plain (a) and surface modified with 5 (b) and 7.5 (c) mol % of pI–pAA. Bars = 200 nm. Arrow shows small micellar objects.

Figure 3. Calcein release from DPPC:CHOL (2:1) liposomes: plain (■) and modified with 2.5 mol.% (♦) and 5 mol.% (▲) polyisoprene-b-polyacrylic acid

Figure 4. Curcumin release profiles from hybrid non pH-sensitive DPPC:CHOL:Curc:BEC-X (■) and from рН-sensitive hybrid (DPPC:CHOL:pIpAA:Curc:BEC-X(•) liposomes modified with 2.5 mol% pI-pAA as a function of: pH (a) and time at 37°C in phosphate buffer (pH 7) (b).

Figure 5. The growth inhibitory concentration-response curves as determined by the MTT-dye reduction assay after 72 hours continous expousure. Each data point represents the arithmetic mean ±SD of 8 separate experimints.

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Figure 6. Apoptotic DNA-fragmentation of HL-60 (left) and HL-60/CDDP cells (right) after exposure to either free curcumin or its formulations, with or without cotreatment with the pan-caspase inhibitor Boc-Asp(OMe)-fluoromethyl ketone (PCI), as assessed by the commercially available ‘Cell death detection’ ELISA kit. Each test was run in quadruplicate.

Figure 7. Western blot analysis of caspase-8 and 9 activation following 24 h exposure of HL-60 and HL-60/CDDP to curcumin and its formulations, as evidenced by the disappearance of the corresponding 55 kDa and 46 kDa procaspase bands. The numbers indicate the percentage decrease of the actin normalized expression levels; N.D. denotes bands with no decrease relative to the untreated control.

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Grraph hical absttracct

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