DPPC:MPOx chimeric advanced Drug Delivery nano Systems (chi-aDDnSs): Physicochemical and structural characterization, stability and drug release studies

International Journal of Pharmaceutics 450 (2013) 1–10

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical Nanotechnology

DPPC:MPOx chimeric advanced Drug Delivery nano Systems (chi-aDDnSs): Physicochemical and structural characterization, stability and drug release studies Natassa Pippa a,b , Maria Merkouraki a , Stergios Pispas b , Costas Demetzos a,∗ a Department of Pharmaceutical Technology, Faculty of Pharmacy, Panepistimioupolis Zografou 15771, National and Kapodistrian University of Athens, Athens, Greece b Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, 11635, Athens, Greece

a r t i c l e

i n f o

Article history: Received 13 February 2013 Received in revised form 21 March 2013 Accepted 28 March 2013 Available online 22 April 2013 Keywords: Gradient block copolymer Chimeric aDDnSs Self-assembly Fractal dimension Polymersomes Drug release

a b s t r a c t Chimeric advanced Drug Delivery nano Systems (chi-aDDnSs) could be defined as mixed nanosystems composed of different biomaterials that can be organized into new nanostructures that can offer advantages as drug carriers. In this work, we report on the self assembly behavior and on stability studies of chi-aDDnSs consisting of DPPC (dipalmitoylphosphatidylcholine) and poly(2-methyl-2-oxazoline)-gradpoly(2-phenyl-2-oxazoline) (MPOx) gradient copolymer in Phosphate Buffer Saline (PBS). Light scattering techniques were used in order to extract information on their physicochemical and structural characteristics (i.e. -potential, Polydispersity Index (PD.I.), size/shape and morphology), while their stability was also studied as a function of gradient block copolymer content, as well as temperature. The colloidal stability of the chimeric nanovectors and their thermoresponsive behavior indicates that these nanosystems could be considered as sterically stabilized nanocontainers. DPPC:MPOx chimeric advanced Drug Delivery nano Systems were found to be effective nanocontainers for the incorporation of indomethacin (IND). The combination of gradient block copolymers with phospholipids for the development of novel chimeric nanovectors is reported for the first time and appears very promising, mostly due to the fact that the MPOx acts as a modulator for the release rate of the IND. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Soft Nanotechnology is a relatively young scientific and technological discipline (Hamley, 2003; Nayak and Lyon, 2005; Whitesides and Lipomi, 2009). Pharmaceutical nanotechnology has brought a wide variety of new possibilities into biomedical discovery and clinical practice because nano-scaled carriers have revolutionalized drug delivery (Hughes, 2005; Ravichandron, 2009; Mishra et al., 2010; Souza et al., 2010). The importance of nanotechnology in drug delivery is based on its ability to manipulate supramolecular self-assembled structures in order to produce devices with programmed functions (Whitesides and Grzybowski, 2007). Over the last years liposomes have been proved as the archetypical nanoscale vectors and represent one of the most thoroughly studied categories of colloidal nanocarries (Bangham et al., 1965; Gregoriadis et al., 1974). On the other hand, polymersomes are a

Abbreviations: DPPC, dipalmitoylphosphatidylcholine; df , mass fractal; MPOx, poly(2-methyl-2-oxazoline)-grad-poly(2-phenyl-2-oxazoline); PBS, phosphate buffer saline; aDDnSs, advanced Drug Delivery nano Systems. ∗ Corresponding author. Tel.: +30 2107274596; fax: +30 2107274027. E-mail address: [email protected] (C. Demetzos). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.03.052

class of artificial vesicles made from synthetic amphiphilic block copolymers. Polymersomes have relatively thick (3–4 nm) and robust membranes formed by amphiphilic block copolymers with relatively high molecular weight (Discher and Eisenberg, 2002; Le Meins et al., 2011; Meng and Zhong, 2011; Lee and Feijen, 2012; Thompson et al., 2012). From a biophysical point of view polymersomes, as well as liposomes, can be considered as interesting cell membrane mimics (Le Meins et al., 2011). Their closed bilayer structure is a first step toward compartmentalization, which is one of the key architectural requirements to reproduce the natural environment of living cells. Interestingly, membrane proteins can be incorporated into such bio-mimetic membranes by reconstitution methods, leading to so-called proteopolymersomes (Nallani et al., 2011). Additionally, the application of polymers in medicine as components of drug nanocarriers are considered essential for producing and developing new formulations against several human diseases. Amphiphilic block copolymers and vesicle forming surfactants have attracted major scientific interest in recent years due to their intriguing self-assembly behavior in aqueous media, which results in a plethora of nanoassemblies and their potential applications in drug delivery (Pispas and Sarantopoulou, 2007; Pispas, 2011a,b). Lipopolymers self-assembled into biocompatible

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nanostructured multifunctional biomaterials offer many potential and attractive applications in drug, protein and nucleotide delivery, nanomedicine and diagnostics, too (Liu et al., 2007; Shi et al., 2010; Angelova et al., 2011). Amphiphilic block copolymers are able to form a range of different nanoparticulate morphologies for pharmaceutical applications (Letchford and Burt, 2007). A large number of studies indicate that it is possible to achieve absorption or penetration of block copolymers onto preformed liposomes or self-assembled superstructures in aqueous media (Stoenescu et al., 2004; Leiske et al., 2011; Nam et al., 2011). The interactions and the insertion mechanism between lipids and block copolymers are of paramount importance due to their pharmaceutical applications (Firestone and Seifert, 2005; Ruysschaert et al., 2005; Amado et al., 2009; Antunes et al., 2009; Kita-Tokarczyk et al., 2009; Leiske et al., 2011; Nam et al., 2011). Furthermore, Modulatory Controlled Release Drug Delivery nano Systems (MCRDDnSs) are considered as suitable selfassembled nanocarriers that can be composed of more than one biomaterial, i.e. lipids producing liposomes and polymers, dendrimers or dendritic structures which can be mixed in order to produce new and effective delivery systems with unique biophysical properties and controlled release profile. Such DDnSs with a Modulatory Controlled Release profile, are denoted as advanced Modulatory Controlled Release Drug Delivery Systems and could be categorized as hybridic or chimeric based on the nature of the mixing elements that could be the same or different, respectively (Demetzos, 2010a,b; Gardikis et al., 2011; Du et al., 2012; Kaditi et al., 2012). These nanovectors can be characterized as mixed nanosystems due to the combination of different in nature materials and are used for biomimetic delivery. The interest in such systems stems from the possibilities for basic understanding of biological behavioral motifs, since biological systems also extensively use mixed materials in order to create “smart” self-assembled nanostructures, (Demetzos, 2010a,b). Although the Pluronics, composed of poly(ethylene oxide) and poly(propylene oxide) blocks are the most widely studied amphiphilic block copolymers, poly(2-oxazoline)s may present important advantages for clinical applications (Adams and Schubert, 2007; Hoogenboom, 2009; Knop et al., 2010; Barz et al., 2011). Additionally, poly(2-oxazoline)s and their copolymer are characterized as bioinspired materials due to the pseudopeptide nature of the oxazoline segments, while poly(2methyl-2-oxazoline) is proposed as an alternative to PEG in terms of biocompatibility and stealth properties (Kempe et al., 2009; Schlaad et al., 2010; Barz et al., 2011; Lambermont-Thijs et al., 2011, Bauer et al., 2013). Furthermore, polymers of the poly(2-oxazoline) family, as modifiers of the liposomal surface, are efficient in conveying long-circulating and stealth properties to liposomes in mice (Woodle et al., 1994; Zalipsky et al., 1996). Controlling the morphology of nanoparticles is of key importance for exploiting their functionality and their properties in several emerging technologies (Henry, 2005). The morphology of nanoparticles is critical to their biological interactions, like protein binding (Semple et al., 1998). The polymers induce significant morphological perturbations when included in lipid mixtures used for preparation of liposomal bilayers. According to the recent literature, the demixing of lipid-rich and polymer-rich membrane domains within the same vesicle bilayer was demonstrated and these morphological and structural differences were discussed as the possible resultant interdomain interactions within the mixed liposomal membrane (Nam et al., 2011). The morphology and the shape of these systems are directly related to their colloidal behavior (Pippa et al., 2012a,b). Validated assays are important for detecting and quantifying nanopharmaceuticals, Drug Delivery Systems or biologically active drug products, and how biophysical

characteristics and structure may impact product quality in clinical use (Chen et al., 2007). The goal of this study is to design and develop novel chimeric advanced Drug Delivery nano Systems (chi-aDDnSs) composed of dipalmitoylphosphatidylcholine (DPPC) and poly(2-methyl-2oxazoline)-grad-poly(2-phenyl-2-oxazoline (MPOx) at different molar ratios. Prime interest is focused on the determination of the physicochemical and structural characteristics (i.e. -potential, Polydispersity Index (PD.I.), size/shape and morphology) of the produced chimeric nanostructures composed of different concentrations of DPPC:MPOx in PBS as well as of the effect of the temperature on their size/shape, PD.I., distribution and their fractal dimension. We also studied the chimeric nanoassemblies formed by the incorporation of indomethacin (IND) and the drug release profile of this lipophilic drug which is correlated with their structural characteristics. 2. Materials and methods 2.1. Materials The phospholipid used for liposomal formulations was 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). It was purchased from Avanti Polar Lipids Inc., (Albaster, AL, USA) and used without further purification. Chloroform and all other reagents used were of analytical grade and purchased from Sigma–Aldrich Chemical Co. Indomethacin was supplied by Fluka and was used as received. The MPOx amphiphilic gradient block copolymer was prepared via cationic polymerization (Milonaki et al., 2012). In this copolymer phenyl-oxazoline segments comprise the hydrophobic components and methyl-oxazoline segments the hydrophilic ones. The copolymer is considered biocompatible due to the pseudopeptide nature of the oxazoline segments. Poly(2-methyl-2-oxazoline) is proposed as an alternative to PEG in terms of biocompatibility and stealth properties. Molecular weight and molecular weight distribution of the MPOx copolymer was determined by size exclusion chromatography (SEC) using a Waters system, with a Waters 1515 isocratic pump, a set of three ␮-Styragel mixed bed columns, hav˚ a Waters 2414 refractive index ing a porosity range of 102 –106 A, detector (at 40 ◦ C) and controlled through Breeze software. CHCl3 was the mobile phase used, at a flow rate of 1.0 mL/min at 25 ◦ C. The set-up was calibrated with polystyrene standards having weight average molecular weight in the range 1200–900,000 g/mol. Average composition of the copolymer was determined by 1 H-NMR spectroscopy in CDCl3 , using a Bruker AC 300 spectrometer in CDCl3 at 30 ◦ C. The gradient copolymer MPOx was characterized by SEC and 1 H-NMR and it was found to have the following molecular characteristics: Mw = 3300, Mw /Mn = 1.26, 39 wt% PhOxz (hydrophobic component). The structures of the components of chimeric nanostructures are presented in Fig. 1. The neat amphiphilic gradient copolymer was found to form polymersomes in aqueous media (Milonaki et al., 2012). 2.2. Preparation of chimeric aDDnSs Different liposomal formulations have been prepared using the thin-film hydration method, composed of DPPC:MPOx (9:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 0:9 molar ratios). Briefly, appropriate amounts of DPPC:MPOx mixtures were dissolved in chloroform/methanol (9:1 v/v) and then transferred into a round flask connected to a rotary evaporator (Rotavapor R-114, Buchi, Switzerland). Vacuum was applied (vacuum 1.0 × 10−2 mbar) and the phospholipid thin film was formed by slow removal of the solvent at 50 ◦ C. The mixed phospholipid film was maintained under

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Fig. 1. Chemical structures of (a) DPPC lipid and (b) the gradient block copolymer MPOx, employed in this study. (c) The molecular architecture of the gradient block copolymer MPOx.

vacuum for at least 24 h in a desiccator to remove traces of solvent and subsequently it was hydrated in Phosphate Buffer Saline (PBS) (pH = 7.40 and I = 0.154 M), by slowly stirring for 1 h in a water bath above the phase transition of lipids (41◦ C for DPPC). The resultant multilamellar vesicles (MLVs) were subjected to two, 3 min and 3 min sonication cycles (amplitude 70, cycle 0.7) interrupted by a 3 min resting period, in water bath, using a probe sonicator (UP 200S, Dr. Hielsher GmbH, Berlin, Germany). The resultant small unilamellar vesicles (SUVs) were allowed to anneal for 30 min. The liposomal formulation with drug were prepared by dissolving IND in the initial lipid/copolymer mixture resulting in the following molar ratios: DPPC:MPOx:IND 9:1:1, DPPC:MPOx:IND 1:9:1, DPPC:MPOx:IND 5:5:1, and MPOx:IND 9:1. The effect of IND incorporation in the chimeric liposomal preparations was evaluated by measuring the size, size distribution, fractal dimension and potential of the liposomes. The mean hydrodynamic diameter was used for the characterization of the nanoassemblies immediately after preparation (t = 0 d).

2.3. IND incorporation efficiency Chimeric nanostructures incorporating IND were frozen at −80 ◦ C overnight and were subjected to lyophilization in order to reconstitute by chloroform and calculate the incorporation efficiency. The lyophilization was achieved using a freeze drier (TELESTARQ7 Cryodos-50, Spain) under the following conditions: condenser temperature from −50 ◦ C, vacuum 8.2 × 10−2 mbar) (Armstrong et al., 2002). The lyophilized liposomal suspensions were stored at 4 ◦ C. Freeze-dried liposomes were reconstituted by chloroform to the original volume of the preparation under gentle agitation. Each sample was allowed to anneal for 30 min followed by vortexing, and a relaxation period of 15 min. The percentage of IND incorporated into chimeric nanocarriers was estimated by spectrophotometry (Stat Fax® 4200, Microplate Reader, NEOGEN® Corporation). IND concentration was estimated

with the aid of the following IND calibration curve in chloroform: IND concentration (mg/ml) =

absorbance − 0.0099 0.2741

(R2 = 0.9965)

(1)

The absorbance was measured at 492 nm. Non incorporated IND was separated from liposomal formulations on a Sephadex G75column. Incorporation efficiency (IE) was calculated by using the following equation: %IE =

IND (after column) × 100 IND (initial)

(2)

2.4. In vitro IND release studies The release profile of IND from DPPC:MPOx (9:1:1, 1:9:1 and 5:5:1 molar ratio) chimeric nanovectors and MPOx polymersomes (MPOx:IND 9:1 molar ratio) was studied in PBS at 37 ◦ C. Nanovectors incorporating IND (1 ml of each sample) were placed in dialysis sacks (molecular weight cut off 12,000; Sigma–Aldrich). Dialysis sacks were inserted in 10 mL (PBS) in shaking water bath set at 37 ◦ C. Aliquots of samples were taken from the external solution at specific time intervals and that volume was replaced with fresh release medium in order to maintain sink conditions. The amount of IND released at various times, up to 10 h, was determined using spectrophotometry (Stat Fax® 4200, Microplate Reader, NEOGEN® Corporation) at max = 492 nm with the aid of the calibration curve of the equation (1). 2.5. Dynamic and static light scattering The hydrodynamic radius (Rh ) of chimeric nanoassemblies and the Polydispersity Index (PD.I.) were measured by dynamic light scattering (DLS) and the fractal dimension was determined by static light scattering (SLS). Mean values and standard deviations were

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calculated from three independent samples. For dynamic and static light scattering measurements, an AVL/CGS-3 Compact Goniometer System (ALV GmbH, Germany) was used, equipped with a cylindrical JDS Uniphase 22 mV He-Ne laser, operating at 632.8 nm, and an Avalanche photodiode detector. The system was interfaced with an ALV/LSE-5003 electronics unit, for stepper motor drive and limit switch control, and an ALV-5000/EPP multi-tau digital correlator. Autocorrelation functions were analyzed by the cumulants method and the CONTIN software. For evaluating the temperature stability of the systems the cell temperature was varied from 25 ◦ C to 50 ◦ C (temperature higher than the phase transition of DPPC), using a temperature controlled circulating bath (model 9102 from Polyscience, USA). Heating and cooling cycles were performed, with equilibration of the systems at intermediate temperatures. Apparent hydrodynamic radii, Rh , at finite concentrations, were calculated by aid of Stokes–Einstein equation:

viscosity of water. The function f( ˛) depends on particle shape. While if ˛ > 1: f ( ˛) = 1.5 +

9 75 + 2( ˛) 2( ˛)2

(7)

the above function refers to chimeric dispersions of the present study. 2.7. Statistical analysis Results are shown as mean value ± standard deviation (S.D.) of three independent measurements. Statistical analysis was performed using Student’s t-test and multiple comparisons were done using one-way ANOVA. P-values <0.05 were considered statistically significant. All statistical analyses were performed using “EXCELL”. 3. Results and discussion

kB T Rh = 6n0 D

(3)

where kB is the Boltzmann constant, 0 is the viscosity of water at temperature T, and D is the diffusion coefficient at a fixed concentration. The polydispersity of the particle sizes was given as the 2 / 2 (PD.I.) from the cumulants method, where  is the average relaxation rate, and 2 is its second moment. Light scattering has been used widely in the study of the fractal dimensions of aggregates. In static light scattering, a beam of light is directed into a sample and the scattered intensity is measured as a function of the magnitude of the scattering vector q, with: q=

4n0 sin 0

   2

(4)

where n0 is the refractive index of the dispersion medium,  is the scattering angle and 0 is the wavelength of the incident light. Measurements were made at the angular range of 30–150◦ (i.e. the range of the wave vector was 0.01 < q < 0.03 cm−1 ). The general relation for the angular dependence of the scattered intensity, I(q) is: I(q)∼q−df

(5)

where df is the fractal dimension of the liposomes or aggregates with 1 ≤ df ≤ 3 (df = 3 corresponds to the limit of a completely compact Euclidean sphere where less compact structures are characterized by lower df values). The above equation is the classical result used to determine the mass fractal dimension from the negative slope of the linear region of a log-log plot of I vs. q. 2.6. Electrophoretic mobility–microelectrophoresis The zeta potential (-potential) plays an important role in colloidal stability of nanoparticles and can be readily measured by the technique of microelectrophoresis (Delgado et al., 2007). The zeta potential of chimeric nanostructures was measured using Zetasizer 3000HAS, Malvern Instruments, Malvern, UK. 50 ␮l of the dispersions was 30-fold diluted in dispersion medium and -potential was measured at room temperature at 633 nm. The zeta potentials were calculated from electrophoretic mobilities, E , by using the Henry correction of the Smoluchowski equation: =

3E n 1 2ε0 εr f ( a)

(6)

where ε0 is the permittivity of the vacuum, εr is the relative permittivity, ˛ is the particle radius, is the Debye length, and n is the

3.1. The physicochemical and morphological characteristics of DPPC:MPOx chimeric nanostructures Physicochemical and structural characteristics of DPPC:MPOx chimeric nanostructures in PBS are presented in Table 1. It should be noted that PBS was chosen as dispersion medium because the pH (∼7.4) and the ionic strength of PBS resembles the conditions met within the human body. The nanostructures formed by the combined assembly of DPPC and MPOx copolymer are polydisperse in size. Nanoassemblies incorporating MPOx and conventional DPPC liposomes did not exhibit the same physicochemical characteristics (size and size distribution) (Table 1) in PBS. Additionally, for the majority of chimeric nanovectors, the incorporation of MPOx leads to nanoassemblies (tentatively assigned as mixed/chimeric vesicles) of larger size in PBS. On the other hand, it should be pointed out that the DPPC:MPOx (8:2molar ratio) in PBS exhibit smaller Rh than all formulations. Furthermore, the chimeric liposomal nanovectors have no consistent tendency regarding size distribution (Table 1). The two components have substantially different molecular weights, hydrophilic/hydrophobic ratio, self-assembly properties and biocompatibility/biodegradability characteristics. These differences in chemical structure and functionality support, to our opinion, this phenomenology of quite different physicochemical and morphological characteristics for the combined nanoassemblies. Zeta potential is another important parameter that gives an indication concerning the surface charge of liposomal nanovectors. The -potential of DPPC liposomes was found near zero, because of the absence of net charge on the liposome surface. The structural and the morphological changes during the formation process of chimeric liposomal nanovectors was caused only a small change of zeta potential values from DPPC liposomes (Table 1), probably because of the different screening effects as a result of the ionic strength (I = 0.154 M) of PBS. On the other hand, the potential of the MPOx vesicles presented more negative values (Table 1). The fractal dimension (df ) (represents the parameter of the quantification of the morphology of nanostructures) was found near to 2.5 for conventional DPPC liposomes and for MPOx polymersomes. A decrease of df values was observed for DPPC:MPOx chimeric liposomes in PBS, especially at 7:3 and 6:4 molar ratios (Table 1). The morphology (df ) of chimeric liposomal systems in PBS presents statistically significant difference as the molar ratio of MPOx changed (Table 1). The fractal dimension (df ) was found closer to 2 for DPPC:MPOx nanocarriers in PBS at the higher contents of the polymeric component (DPPC:MPOx 3:7, 2:8 molar ratios). These observations indicate that the polymeric chains induce some morphological perturbations when included in lipid

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Table 1 The physicochemical and morphological characteristics of chimeric DPPC:MPOx nanoassemblies. Rh (nm)a

DPPC liposomes DPPC:MPOx (9:1) DPPC:MPOx (8:2) DPPC:MPOx (7:3) DPPC:MPOx (6:4) DPPC:MPOx (5:5) DPPC:MPOx (4:6) DPPC:MPOx (3:7) DPPC:MPOx (2:8) DPPC:MPOx (1:9) MPOx polymersomes

95.9 96.6 139.6 152.9 141.7 116.2 98.5 127.4 80.1 270.2 344.1

± ± ± ± ± ± ± ± ± ± ±

PD.I.a 7.6 7.9 20.5 6.2 4.3 2.1 1.4 8.1 6.8 11.7 12.6

0.50 0.59 0.54 0.52 0.58 0.47 0.41 0.52 0.55 0.50 0.50

-potential (mV) ± ± ± ± ± ± ± ± ± ± ±

0.01 0.02 0.04 0.05 0.04 0.02 0.03 0.05 0.03 0.02 0.04

± ± ± ± ± ± ± ± ± ± ±

+0.7 +5.1 +8.7 +6.4 +2.8 -5.8 +2.7 -0.8 +10.9 +5.2 -17.1

0.1 2.3 1.6 1.6 1.2 1.2 1.4 0.6 2.1 0.5 1.4

df b 2.55 2.32 2.45 2.08 2.17 2.21 2.27 2.07 2.28 2.18 2.49

± ± ± ± ± ± ± ± ± ± ±

0.1 0.0 0.1 0.1 0.1 0.0 0.1 0.1 0.1 0.0 0.1

Determined by the cumulants method. Determined by static light scattering.

physicochemical characteristics at least for three weeks (Fig. 1(c)). In the case of polymersomes, the molar mass and polydispersity of the hydrophilic block play an important role on the vesicle size and size distribution (Lee and Feijen, 2012). According to the literature, it has been shown that the curvature energy of the vesicles is lowered by segregation of the hydrophilic chains, the inner leaflet being enriched with the shorter ones while the outer leaflet is enriched with the longer ones (Le Meins et al., 2011).

Rh (nm)

(a) 450 350

9:1

250

8:2

150

7:3 6:4

50 1

0

3

5

7

10 15 20 28

5:5

t (days)

(b) 1000 800 Rh (nm)

mixtures used for the preparation of liposomal membrane bilayers (Firestone and Seifert, 2005; Li et al., 2012). The biomolecular sculpture of MPOx gradient block copolymer must play a significant role for this phenomenology. The gradient copolymer chain is expected to have several entry and exit points in the lipid membrane, in contrast to lipid-hydrophilic polymer conjugates and amphiphilic diblock copolymers, where the hydrophobic part is incorporated into the lipid membrane and the hydrophilic polymer chain is anchored on the membrane (Scheme 1). This gradient copolymer chain arrangement may have several consequences in the structure and properties of the membrane itself and the chimeric nanostructure as a whole, as the current results indicate. In the Scheme 1, the MPOx molecules are shown anchored on the outer surface of chimeric nanovectors. It is most probable that the polymeric molecules can also anchor on the inner surface. In our opinion, the MPOx molecules, which are anchored on the outer surface of nanocarriers, are responsible for steric stabilization of the produced nanovectors and for their interactions with the environment. The physical stability over time of all chimeric formulations was assessed by measuring the size and size distribution for a period of 28 days. All chimeric formulations in PBS, apart from DPPC:MPOx (9:1 and 1:9), were found to retain their original physicochemical characteristics (size and size distribution) at least for two weeks after their preparation (Fig. 2(a) and (b)). This demonstrates the effect of gradient copolymer content in the chimeric nanostructures at physiological conditions. The presence of increasing amounts of MPOx gradient block copolymer stabilizes the assemblies to greater extend compared to neat DPPC liposomes. The observed colloidal stability indicates that steric repulsion should be responsible for keeping the chimeric nanovectors far enough to avoid van der Waals attraction, due to the absence of electrostatic repulsion, which was predicted from classical DLVO theory (Derjaguin and Landau, 1941; Verwey and Overbeek, 1948). These observations indicate that these chimeric nanostructures are also sterically stabilized nanovectors, despite the different molecular architecture of the gradient copolymer in comparison to lipid-polymer conjugates and amphiphilic diblock copolymers. Aggregation of neat DPPC liposomes was observed because liposomal dispersions are not thermodynamically stable, while MPOx polymersomes were found to retain their original

1:9

600

2:8

400

3:7

200

4:6 5:5

0 0

1

3

5

7 10 15 20 28 t (days)

(c) 1000 800

Rh (nm)

a b

Sample

600 400

0:9

200

9:0

0 0

1

3

5

7 10 15 20 28

t (days)

Scheme 1. The biomolecular sculpture of the DPPC/MPOx chimeric membrane.

Fig. 2. Stability assessment of chimeric DPPC:MPOx formulations (a) and (b), and for neat DPPC and MPOx vesicles in PBS (c). Mean of three independent experiments run in triplicate, SD < 10%.

N. Pippa et al. / International Journal of Pharmaceutics 450 (2013) 1–10

400 350 300 250 200 150 100 50

(a)

0.65 9:0 9:1 8:2 7:3 6:4

20 25 30 35 40 45 50 55

5:5

9:1

0.45

8:2

0.35

7:3 6:4 20 25 30 35 40 45 50 55

5:5

Temperature (C)

(c) 9:0 9:1 8:2 7:3 6:4 20 25 30 35 40 45 50 55

5:5

Temperature (C)

I (kHz)

df

9:0

0.25

Temperature (C)

3 2.8 2.6 2.4 2.2 2 1.8 1.6

(b)

0.55 PD.I.

Rh (nm)

6

21000 18000 15000 12000 9000 6000 3000 0

(d) 9:0 9:1 8:2 7:3 6:4 20 25 30 35 40 45 50 55

5:5

Temperature (C)

Fig. 3. (a) Rh , (b) PD.I., (c) df and (d) I vs. temperature for chimeric nanostructures with larger DPPC content in PBS (the concentration of chimeric nanostructures is constant at 5 × 10−2 mg/ml).

3.2. DPPC:MPOx chimeric nanostructures as thermoresponsive nanoassemblies The physical and chemical properties of some polymersomes’ membranes are changeable in response to external stimuli (Amstad et al., 2012). Various polymers, which are responsive to pH, temperature, redox conditions, light, magnetic field, ionic strength and concentration of glucose, have been used to form polymersomes for programmed drug delivery (Amstad et al., 2012). Polymers, which are sensitive to various stimuli (i.e. temperature, pH, etc.) can be encapsulated with drugs or proteins in polymersomes and this may change the morphology of the interior of polymersomes (Li et al., 2012; Marguet et al., 2012). Thermoresponsive polymer-modified liposomal nanocarriers and thermoresponsive polymersomes are considered to be a promising approach to site-specific delivery of drugs and to triggered drug release (Kono et al., 1999a,b; Kono, 2001; Amstad et al., 2012). Having this in mind we investigated the temperature dependence of the structural parameters of chimeric nanocarriers in the process of heating in the two aqueous dispersion media (Figs. 3 and 4). These types of experiments may also simulate the response of chimeric nanocarriers after injection to a fever situation or inflammatory conditions, although the temperature range investigated is wider than normal and abnormal body temperatures. The hydrodynamic radii (Rh ) of DPPC liposomes in PBS decreased in the process of heating up to 50 ◦ C (Fig. 3(a)). On the other hand, the hydrodynamic radii (Rh ) of chimeric liposomal nanostructures remained unaffected during the process of the heating. The population of chimeric nanocarriers becomes more heterogeneous in the process of heating up to 50 ◦ C (polydispersity increases) (Fig. 3(b)). The fractal dimension (df ) values increased during the heating process only for DPPC:MPOx (5:5 molar ratio) chimeric nanocarriers. Rh increased for chimeric polymeric nanostructures DPPC:MPOx 1:9 molar ratio in PBS in the process of heating up to 50 ◦ C (Fig. 4(a)), while the df values remained unaffected (Fig. 4(c)). On the other hand, at the highest temperature

the population of chimeric polymeric nanocarriers became more homogeneous also in PBS (Fig. 4(b)). The fractal dimension offers a quantification of the changes on nanoassemblies’ morphology under different thermal conditions, detecting differences that the hydrodynamic size characteristics alone do not reveal, especially for the nanosystems with the higher content of MPOx (Fig. 4(c)). The mass of the chimeric nanocarriers also decreased (Figs. 3(d) and 4(d)) in the temperature range 25–50 ◦ C, as it is indicated by the decrease in the scattered intensity values (analogous to the mass of the nanoassemblies), especially for chimeric polymersomes. For neat DPPC liposomes, the mass remained virtually unchanged in the temperature range 25–45 ◦ C (temperature higher than the main transition temperature of DPPC lipids, Tm = 41 ◦ C for DPPC lipids), because the scattering intensity from the liposomal dispersion remains almost constant (within experimental error) (Figs. 3(d) and 4(d)). This observation indicates that the chimeric nanostructures are changing their morphological characteristics under increased temperature conditions and can be considered as thermoresponsive nanoassemblies. The main influence of temperature seems to be a decrease in mass of the nanoassemblies, probably through some partial dissociation of the nanostructures. This process should be related to the main transition temperature of DPPC lipids and which may be further modulated by the presence of the polymer segments within the lipid membrane. Heating also seems to result in less compact structures as the decrease in df indicates in some cases. Morphological changes of the nanovectors are expected to influence the release profile of incorporated drugs. 3.3. IND incorporation in DPPC:MPOx chimeric nanovectors and drug release Indomethacin (IND) belongs to the class of nonsteroidal antiinflammatory drugs (NSAIDs), which are highly effective in the treatment of a wide variety of illnesses and in the prevention of

N. Pippa et al. / International Journal of Pharmaceutics 450 (2013) 1–10

1050 950 850 750 650 550 450 350 250 150 50

(a)

0.6 0:9 2:8 3:7

(b) 0:9

0.5 PD.I.

Rh (nm)

1:9

1:9 0.4

2:8 3:7

0.3

4:6 20 25 30 35 40 45 50 55

5:5

4:6

0.2 20 25 30 35 40 45 50 55

3 2.8 2.6 2.4 2.2 2 1.8 1.6

5:5

Temperature ( C)

Temperature ( C)

(c) 0:9 1:9 2:8 3:7 4:6 5:5

20 25 30 35 40 45 50 55

I (kHz)

df

7

(d)

5000 4500 4000 3500 3000 2500 2000 1500 1000

0:9 1:9 2:8 3:7 4:6 20 25 30 35 40 45 50 55

5:5

Temperature ( C)

Temperature ( C)

Fig. 4. (a) Rh , (b) PD.I., (c) df and (d) I vs. temperature for chimeric nanostructures with larger MPOx in PBS (the concentration of chimeric nanostructures is constant at 5 × 10−2 mg/ml).

human cancer (Xu, 2002; Soh and Weinstein, 2003; Rao and Reddy, 2004). On the other hand, the long term use of NSAIDs results in gastrointestinal toxicity (Zhou et al., 2010). In view of the required decreased adverse drug reactions of IND formulations, the encapsulation within nanostructures seems to be an attractive approach from the pharmaceutical nanotechnology point of view (Chen et al., 2007; Jaafar-Maalej et al., 2011; Milonaki et al., 2012; Lim et al., 2012; Sugihara et al., 2012). Having established a picture regarding the self-assembly behavior of the different chimeric nanocarriers in PBS, chimeric liposomes and polymersomes, it was decided to investigate the possibilities for encapsulation of IND within these nanovectors in order to ameliorate the ADME (Absorption biodistribution Metabolism and Excretion) profile of this lipophilic drug. Chimeric nanocarrier size and size distribution, -potential values and df after IND incorporation are presented in Table 2. The incorporation of indomethacin led to a increase in the size of chimeric nanocarriers and of MPOx polymersomes (Tables 1 and 2). The -potential values did not present any significant difference after the incorporation of IND for the chimeric nanovectors (Table 2). On the other hand, a shift of -potential to positive values was observed for MPOx nanovectors (Tables 1 and 2). The morphological characteristics of chimeric nanocarriers changed significantly after the incorporation of IND, especially for MPOx nanoparticles. Most probably IND having some amphiphilic character alters the spatiotemporal liposomal phase behavior as well as the solvation properties of the nanosystems. The present observations provide additional information on the chimeric nanocarrier/drug interactions (Lúcio et al., 2008; Nunes et al.,

2011). The drug incorporation efficiency was 17.3% and 11.5% for DPPC:MPOx:IND chimeric nanoassemblies 9:1:1 and 1:9:1 molar ratios in PBS, respectively (Table 2). The incorporation efficiency of chimeric nanocarriers for IND was increased by the decrease of MPOx content. The DPPC/MPOx chimeric nanocarriers seem to be effective nanocontainers for the encapsulation of IND, especially those having the lowest molar ratio of the gradient block copolymer. This should be correlated to the highest hydrophobicity of the DPPC component. Manipulation of the biophysical properties of aDDnSs, especially for chimeric systems, provides improved control over the pharmacokinetics (PK) and pharmacodynamics (PD) of the encapsulated drugs relative to free drugs (Moghimi and Szebeni, 2003; Allen et al., 2006). The concept of fractal geometry can be applied to describe the complexity of the heterogeneous nature of drug processes in the human body and the dissolution of bioactive compounds (Dokoumetzidis et al., 2004, 2008; Pereira, 2010; Dokoumetzidis and Macheras, 2011). It should be pointed out that drug release from the polymersomes is governed by the diffusion of the drug through the membrane (Christian et al., 2009; Lee and Feijen, 2012). The driving force for the drug release is a concentration gradient of the drug between polymersomes and surrounding medium (Lee and Feijen, 2012). According to the literature, when the drug is diffusing from the core of the polymersomes to the surrounding medium the release rate is a function of the square root of time and for the polymersomes in polymersomes the release data can be fitted by the experimental law established by Peppas and Ritger and the size

Table 2 The physicochemical and morphological characteristics and incorporation efficiency of DPPC:MPOx:IND nanoassemblies in PBS. Composition

Rh (nm)

DPPC:MPOx:IND (9:1:1 molar ratio) DPPC:MPOx:IND (5:5:1 molar ratio) DPPC:MPOx:IND (1:9:1 molar ratio) MPOx:IND (9:1 molar ratio)

146.1 206.8 301.4 458.2

± ± ± ±

PD.I. 10.2 12.4 13.8 14.5

0.52 0.42 0.49 0.45

df ± ± ± ±

0.05 0.02 0.04 0.02

2.18 2.01 1.99 1.91

-potential (mV) ± ± ± ±

0.1 0.0 0.1 0.1

+5.9 +6.6 +6.2 +9.1

± ± ± ±

1.6 2.6 2.8 1.9

% Incorporation Efficiency 17.3 10.6 11.5 27.8

± ± ± ±

0.7 1.1 1.6 1.8

8

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(a) 120 % drug release

100 80

DPPC:MPOx:IND 9:1:1

60

DPPC:MPOx:IND 5:5:1

40

DPPC:MPOx:IND 1:9:1 MPOx:IND 9:1

20 0 0

1

2

3

4

5

6

7

8

9

10

T (hours)

(b) 120 % drug release

100 80 DPPC:MPOx:IND 9:1:1

60

DPPC:MPOx:IND 5:5:1

40

DPPC:MPOx:IND 1:9:1

20

MPOx:IND 9:1

0 0

1

2

3

4

5

6

7

8

9

10

T (hours)

(c) 120 % drug release

100 80

60

DPPC:MPOx:IND 9:1:1 DPPC:MPOx:IND 5:5:1

40

DPPC:MPOx:IND 1:9:1

20

MPOx:IND 9:1

0 0

1

2

3

4

5

6

7

8

9

10

T (hours) Fig. 5. Cumulative drug release from DPPC:MPOx: IND 9:1:1, 5:5:1, 1:9:1 and MPOx:IND 9:1 molar ratio at (a) 25 ◦ C, (b) 37 ◦ C and (c) 45 ◦ C. Mean of three independent experiments run in triplicate, SD < 10%.

distribution of the polymersomes will also play a role in the overall release rate (Christian et al., 2009; Lee and Feijen, 2012; Marguet et al., 2012). The in vitro release of the IND from the chimeric nanovectors and polymersomes at three different temperatures (25 ◦ C, 37 ◦ C and 45 ◦ C) is presented in Fig. 5. It is observed that the in vitro release of the drug from the prepared chimeric nanostructures is quite fast only for the mixed nanovectors prepared with the lowest ratio of gradient block copolymer (DPPC:MPOx:IND 9:1:1) (Fig. 5) at 25 ◦ C and at 37 ◦ C. The combination of gradient block copolymers with phospholipids for the development of a novel chimeric nanovector appears very promising, mostly due to the fact that the MPOx acts as a modulator for the release rate of the IND. This phenomenology could serve as a control factor for the preparation and development of chimeric formulations with the desired release profile, modulating the release rate of the IND, improving its therapeutic index and finally decreasing any unwanted side effects. On the other hand, polymersomes can be easily loaded with hydrophilic molecules in the internal cavity, as well as hydrophobic ones in the membrane, allowing for dual functioning therapy. They present a low passive permeability to low-molecular-weight solutes, allowing a controlled release by external triggers such as temperature, pH and light. It is observed that the in vitro release of the drug from the prepared polymersomes MPOx:IND (9:1 molar ratio) is quite slow in comparison with the chimeric nanocontainers (Fig. 5). This kinetic behavior has been validated in simulation studies of drug release in fractal polymeric matrices. In our opinion, it seems likely that the spatiotemporal variation in membrane

permeability of the dynamically swelling MPOx polymer is close enough to the percolation threshold for non-classical diffusion effects to impinge on release kinetics (Dokoumetzidis et al., 2004, 2008; Pereira, 2010; Dokoumetzidis and Macheras, 2011). The design of a Modulatory Controlled Release System at the nano scale level (MCRnS) is an issue that needs biomaterials which can modulate the release of the drug in a zero-kinetic order. However, innovative therapies require innovative delivery systems by controlling the physicochemical properties of the biomaterials utilized, as well as their interactions between them and with the encapsulated drug (Demetzos, 2010a,b). Furthermore, the in vitro release of the drug from the prepared chimeric nanostructures is faster only for all the chimeric nanovectors prepared at 45 ◦ C, temperature higher than Tm (=41 ◦ C) of DPPC lipids (Fig. 5(c)) in comparison to the other two lower temperatures (Fig. 5(a) and (b)). In other words, for the chimeric nanovectors, the drug release rate increased with increasing temperature. According to the literature, a conformational change of C C single bonds in the alkyl chains of the DPPC lipids leads to an increase in the total volume occupied by the hydrocarbon chains in the membrane, and therefore increases the permeability of the mixed bilayer membrane. At Tm (41 ◦ C for DPPC lipids) permeability is additionally increased as result of the coexistence of membrane areas in both liquid crystalline phases (Gaber et al., 1995; Hossann et al., 2007, 2012). The increase of the membrane permeability is most probably the reason for the increased release rates for chimeric liposomes at 45 ◦ C. However, the presence of MPOx chains modulates the behavior since the chimeric nanovectors show different temperature dependent release rate, influences by the DPPC/MPOx ratio. On the other hand, the IND release rate for MPOx polymersomes remained unaffected by the increase of temperature (Fig. 5.) (Christian et al., 2009). In this research direction, the combination of gradient block copolymers with liposomes for the development of a novel chimeric nanovector appears to be very promising, mostly due to the fact that the MPOx acts as a modulator for the release rate of IND. Although the exact mechanism of release modulation is still under investigation, the present studies shed light on the physicochemical interactions between the components of chimeric nanosystems comprised by different self-assembled materials.

4. Conclusions Chimeric nanocarriers consisted of DPPC and MPOx gradient block copolymers were successfully prepared in PBS. These chimeric nanosystems can be utilized as advanced Drug Delivery nano Systems. The physicochemical and structural behavior of these chimeric nanoassemblies was found to depend on the molar ratio between the components (i.e. DPPC and MPOx) and the temperature. The incorporation of MPOx gradient block copolymer into liposomal bilayer increases the physicochemical stability of the initial vesicles, especially for the higher MPOx molar ratio, while their structural variability remains unaffected in the case of the hydrodynamic radii (Rh ) (size/shape) while in the case of the fractal dimension (df ) (morphology) decreased in the process of heating up to 50 ◦ C. This observation indicates that these chimeric nanostructures are changing their structural characteristics under increased temperature conditions and can be considered as thermoresponsive nanoassemblies. IND was successfully incorporated into different in molar ratio of the components DPPC and MPOx chimeric formulations and it was found that the structural characteristics of these thermosensitive and sterically stabilized nanocarriers did present significant differences after the incorporation IND while their physicochemical characteristics remained unaffected. The in vitro release of IND from the prepared chimeric

N. Pippa et al. / International Journal of Pharmaceutics 450 (2013) 1–10

nanostructures is quite fast only for the mixed nanovectors prepared with the lowest ratio of gradient block copolymer. For the chimeric nanovectors, the drug release rate increased with increasing temperature, while for MPOx polymersomes the IND release rate did not change significantly. The results were very promising in terms of drug encapsulation and release rate, factors that can alter the therapeutic profile of a drug with low therapeutic index such as IND. The present studies show that there are a significant number of parameters that can be used in order to alter the morphology of chi-aDDnSs and this is advantageous to the design and the development of chimeric nanocarriers for drug delivery. The results presented highlight the existing potential for controlling the colloidal behavior and the morphology of chimeric nanocontainers, in order to achieve programmed in vitro drug release. In conclusion, the composition and the fractal sculpture of the chimeric systems play a key role on their self-assembly properties, which could be a road map for designing chi-aDDnSs with knowledge of their in vitro drug release profile.

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