International Journal of Pharmaceutics 473 (2014) 80–86
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Preparation, development and in vitro release evaluation of amphotericin B–loaded amphiphilic block copolymer vectors Natassa Pippa a,b , Maria Mariaki a , Stergios Pispas b , Costas Demetzos a, * a Department of Pharmaceutical Technology, Faculty of Pharmacy, National and Kapodistrian University of Athens, Panepistimioupolis Zografou, 15771 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
A B S T R A C T
Article history: Received 26 May 2014 Received in revised form 30 June 2014 Accepted 1 July 2014 Available online 3 July 2014
The aim of this work is to design and develop a suitable polymeric formulation incorporating amphotericin B (Ampho B) in order to overcome its water insolubility problem. To this end, we have chosen the poly(isoprene-b-ethylene oxide) amphiphilic block copolymer (IEO) family. We investigate the self assembly behavior and the stability kinetics of IEO copolymer based nanostructures formed in HPLC grade water and in phosphate buffer saline (PBS). The IEO block copolymer samples investigated have different molecular weights and compositions. A gamut of light scattering techniques (static, dynamic and electrophoretic) were used in order to extract information on the size, z-potential and morphological characteristics of the structures formed, as a function of the molar ratio of incorporated lipophilic drug Ampho B. The amphiphilic character and the colloidal stability of the particular polymeric drug vectors indicate that these nanostructures can be utilized as effective containers for the particular hydrophobic drug. The incorporation of Ampho B led to alteration of the physicochemical and morphological characteristics of the pure polymeric carriers. It is observed that the in vitro release of Ampho B from the prepared vectors IEO-b:Ampho B was quite slow, while the IEO-a carriers did not release Ampho B. ã 2014 Published by Elsevier B.V.
Keywords: Amphiphilic block copolymer Amphotericin B Self-assembly Morphology Drug release
1. Introduction The main goal of advanced drug delivery nano systems is the improvement of the therapeutic index and the reduced toxicity and side effects of the encapsulated active substance (Rowland et al., 2012; Crommelin and Florence, 2013; Demetzos and Pippa, 2014). Amphiphilic block copolymers have been in use as pharmaceutical excipients in different forms for a long time and their application is experiencing rapid growth in pharmaceutics and in nanomedicine (Adams et al., 2003; Uchegbu, 2006; Cabral et al., 2011; Xiong et al., 2012). The rapid development of amphiphilic block copolymers applications in the pharmaceutical sciences is primarily due to the chemical flexibility of their structure, which provides an opportunity for the design of versatile drug nanocarriers.
Abbreviations: IEO, poly(isoprene-b-ethylene oxide) copolymer; Ampho B, amphotericin B; df, mass fractal; Rg/Rh, radius of gyration/hydrodynamic radius; PBS, phosphate buffer saline; DLVO theory, Derjaguin, Landau, Verwey and Overbeek theory. * Corresponding author. Tel.: +30 2107274596; fax: +30 2107274027. E-mail address: [email protected] (C. Demetzos). http://dx.doi.org/10.1016/j.ijpharm.2014.07.001 0378-5173/ ã 2014 Published by Elsevier B.V.
Micelles are nanoparticulate colloidal systems that exist in equilibrium with the molecules or ions in aqueous solution from which they are formed. Micelles are characterized as selfassembled nanostructures in a liquid and composed of amphiphilic soft materials in general amphiphilic di- or tri-block copolymers made of solvophilic and solvophobic blocks (Torchilin, 2004, 2007). It should be noted that the micelle formation and stability are concentration-dependent. The utility and applications of polymeric micelles formed through the multimolecular assembly of block copolymers as novel core–shell type colloidal carriers for drug, peptide, protein and gene delivery, controlled release and targeting has been discussed (Kataoka et al., 2001; Gaucher et al., 2005; Mahmud et al., 2007). In Pharmaceutical Nanotechnology, polymersomes are a new class of artificial vesicular systems, tiny hollow spherical carriers that enclose an aqueous medium. Polymersomes are structured using amphiphilic block copolymers to form the vesicle membrane and are used in order to encapsulate drugs, peptides/proteins and nucleic acids (Discher and Eisenberg, 2002; Le Meins et al., 2011; Meng and Zhong, 2011; Lee and Feijen, 2012; Thompson et al., 2012; Kowalczuk et al., 2014). The polymersome membrane provides a biophysical barrier that isolates the incorporated bioactive molecule from external
N. Pippa et al. / International Journal of Pharmaceutics 473 (2014) 80–86
medium, such as those found in blood stream (Rösler et al., 2001; Adams et al., 2003; Taubert et al., 2004; Pippa et al., 2013). Amphotericin B (Ampho B) is a macrocyclic polyene antifungal bioactive compound and is used in clinical application intravenously for systemic fungal infections. This antifungal agent is commercially available as liposomal formulation, cholesteryl sulfate complex and as a lipid complex. The first formulation has been already developed to improve the tolerability for the patient and to reduce toxicity, and to alter the pharmacokinetic profile compared to plain Ampho B (Moen et al., 2009; Hamill, 2013). To reduce the side effects of Ampho B, several lipid- and polymer-based formulations were designed and developed compared to those are marketed (Choi et al., 2008; Italia et al., 2009; Jung et al., 2009; Wasan et al., 2009; Sheikh et al., 2010; Van de Ven et al., 2012). Additionally, Ampho B belongs to Class IV (low solubility, low permeability) of the Biopharmaceutical Classification System (BSC) and to B of ABG system (Amidon et al., 1995; Fagerholm, 2007; Macheras and Karalis, 2014). In this investigation, we designed and prepared polymeric micellar nanoparticles composed of poly(isoprene-b-ethylene oxide) copolymers (IEO) and antimicrobial antiprotozal agent, amphotericin B (Ampho B), was incorporated into these polymeric carriers. Ampho B-incorporating polymeric vectors of IEO copolymers with different composition of the hydrophobic polyisoprene block were characterized and the drug release was evaluated in vitro. The polyisoprene (PI) block has great chemical similarities with the hydrophobic part of Ampho B as can be seen in Fig. 1. So it is expected that the PI cores of IEO copolymer micelles in aqueous media may have a significant ability to encapsulate the desired drug. The PEO hydrophilic block provides the well known solubility, biocompatibility and stealth properties to the nanostructures. 2. Materials and methods 2.1. Materials The IEO block copolymers were prepared via anionic polymerization using procedures that have been reported earlier (Pispas, 2006). The chemical structures and the molecular characteristics of copolymer samples are presented in Fig. 1 and Table 1,
81
Table 1 Molecular characteristics of the block copolymers. Sample
Mwa
Mw/Mna
%wt PIb
IEO-a IEO-b
16,400 33,000
1.08 1.02
46 28
a b
By SEC in THF using polystyrene standards. By 1H NMR in CDCl3.
respectively. Ampho B was supplied by Fluka and was used as received. The dialysis membranes with a molecular weight cutoff (MWCO) of 12,000 g/mol were purchased from Spectra/ProTM Membranes. Methanol and dimethyl sulfoxide (DMSO) were of HPLC grade and used without further purification. 2.2. Preparation of polymeric carriers Polymeric carriers were prepared using a solvent evaporation method. Briefly, appropriate amounts of IEO-a/-b:Ampho B (9:0, 9:0.1, 9:0.2 and 9:0.3 molar ratios) mixtures were dissolved in DMSO:methanol (1:1 v/v). DMSO samples were evaporated to dryness on a HT-4X series Genevac centrifugal evaporation system equipped with Lyo-speed. Evaporation was performed during 3 h, in a pre-heated chamber (40 C), with low rotor speed (approximately 350 g) and 5 mbar pressure. Vacuum was applied and a thin film was formed by removal of the solvent. The film was maintained under vacuum for at least 24 h in a desiccator to remove traces of solvent and subsequently it was hydrated in HPLC grade water and phosphate buffer saline (PBS) (pH 7.40 and I = 0.154 M), by slowly stirring for 1 h in a water bath at 50 C. The resultant structures were subjected to two, 3 min and 2 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 nanostructures were allowed to anneal for 30 min. The effect of Ampho B incorporation in the chimeric preparations was evaluated by measuring the size, size distribution, fractal dimension and z-potential of the resulting nanostructures. The mean hydrodynamic diameter was used for the characterization of the nanoassemblies immediately after preparation (t = 0 day), as well as for monitoring their physical stability over time (t = 21 days). 2.2.1. Ampho B incorporation efficiency Polymeric structures incorporating Ampho B were frozen at 80 C overnight and were subjected to lyophilization in order to be reconstituted 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 8.2 102 mbar). The lyophilized polymeric suspensions were stored at 4 C. Freezedried nanostructures 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 Ampho B incorporated into polymeric nanocarriers was estimated by spectrophotometry (Stat Fax1 4200, Microplate Reader, NEOGEN1 Corporation). The absorbance was measured at 405 nm. Non incorporated Ampho B was separated from chimeric formulations on a Sephadex G75 column. Incorporation efficiency (IE) was calculated by using the following equation: % IE ¼
Fig. 1. Chemical structures of (a) PI-b-PEO (IEO) block copolymers and (b) amphotericin B, employed in this study.
Ampho B ðafter columnÞ 100 Ampho B ðinitialÞ
(1)
Ampho B concentration was estimated with the aid of the following Ampho B calibration curve in chloroform:
82
N. Pippa et al. / International Journal of Pharmaceutics 473 (2014) 80–86
Ampho B concentration ðmg=mlÞ absorbance 0:0142 2 ðR ¼ 0:9928Þ ¼ 1:1565
(2)
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. 2.5. Electrophoretic mobility–microelectrophoresis
2.3. In vitro Ampho B release The release profile of Ampho B from IEO:Ampho B (9:0.1, 9:0.2 and 9:0.3 molar ratio) polymeric nanovectors was studied in PBS at 37 C. Block copolymer nanovectors incorporating Ampho B (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 Ampho B released at various times, up to 20 h, was determined using spectrophotometry (Stat Fax1 4200, Microplate Reader, NEOGEN1 Corporation) at lmax = 405 nm with the aid of the calibration curve of Eq. (2). 2.4. Dynamic and static light scattering The hydrodynamic diameter (Dh) of polymeric carriers and the polydispersity index (PDI) were measured by dynamic light scattering (DLS) and the fractal dimension was determined by static light scattering (SLS). Mean values and standard deviations were 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 multitau digital correlator. Autocorrelation functions were analyzed by the cumulants method and the CONTIN software. Heating and cooling cycles were performed, with equilibration of the systems at intermediate temperatures. Apparent hydrodynamic diameters, Dh, at finite concentrations were calculated by aid of Stokes–Einstein equation: Dh ¼
kB T 6ph0 D
(3)
where kB is the Boltzmann constant, h0 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 m2/’2 (PDI) from the cumulants method, where G is the average relaxation rate, and m2 is its second moment. Light scattering has been widely used 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: 4ph0 u sin (4) q¼ l0 2 where h0 is the refractive index of the dispersion medium, u is the scattering angle and l0 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 cm1). The general relation for the angular dependence of the scattered intensity, I(q) is: IðqÞ qdf
(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
The zeta potential (z-potential) values play an important role in the colloidal stability of colloidal polymeric particles. The z-potential of polymeric carriers was measured by the technique of microelectrophoresis, using Zetasizer 3000HAS, Malvern Instruments, Malvern, UK. 50 ml of the dispersions was 30-fold diluted in dispersion medium and z-potential was measured at room temperature at 633 nm. The z-potentials were calculated from electrophoretic mobilities, mE, by using the Henry correction of the Smoluchowski equation:
z¼
3mE h 1 2e0 er f ðkaÞ
(6)
where e0 is the permittivity of the vacuum, er is the relative permittivity, a is the particle radius, k is the Debye length, and h is the viscosity of water. The function f(ka) depends on particle shape. While if ka > 1: f ðkaÞ ¼ 1:5 þ
9 75 þ 2ðkaÞ 2ðkaÞ2
(7)
The above function refers to polymeric dispersions of the present study. 2.6. Statistical analysis Results are shown as mean value standard deviation (SD) 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 3.1. Physicochemical characteristics of polymeric carriers Physicochemical and structural characteristics of IEO-a/-b nanostructures with and without Ampho B in two aqueous media (HPLC grade water and PBS) are presented in Table 2. The mean hydrodynamic diameter of empty IEO-a drug containers was found Dh = 210 nm and the population of particles is polydisperse in HPLC grade water. On the other hand, the mean hydrodynamic diameter of empty IEO-a in PBS was found Dh = 120 nm and the population of polymeric nanoparticles was quite monodisperse (PDI = 0.35). The z-potential values of IEO-a/b assemblies in aqueous media were found slightly negative (8 mV) because of the absence of net charges on polymeric structures’ surface. The fractal dimension (df) was found near to 2.00 and the ratio of the radius of the gyration to the hydrodynamic radius, Rg/Rh, was found equal to 0.81, indicating spherical particles in the two aqueous media. The mean hydrodynamic diameter of empty IEO-b nanocontainers in the two aqueous media was found around 100–120 nm, having z-potential near zero and somewhat lower polydispersity, especially in HPLC grade water in comparison to the polymeric structures composed by IEO-a (PDI 0.20) (Table 2). The morphological characteristics, as quantified by df, did not present significant differences between the prepared polymeric nanovectors. From the results at hand, and taking into account the molecular characteristics of the copolymers and their hydrophobic/hydrophilic ratio, it can be concluded that samples IEO-a and IEO-b form
N. Pippa et al. / International Journal of Pharmaceutics 473 (2014) 80–86
83
Table 2 Physicochemical and morphological characteristics of polymeric carriers. PDI
z-potential (mV)
df
Rg/Rh
% EE
210.3 19.9 122.9 4.6 164.7 2.1
0.51 0.10 0.35 0.30 0.58 0.08
8.6 5.5 8.3 3.0 14.6 0.3
2.00 1.87 2.35
0.71 0.73 0.93
– – 91.1
271.3 10.3
0.58 0.01
15.6 1.5
2.34
0.84
93.0
HPLC grade water
1402.0 45.6
0.51 0.01
12.4 2.1
2.56
1.69
97.0
PBS
1151.4 84.0
0.49 0.00
17.2 1.3
2.45
1.64
96.5
HPLC grade water
1498.2 220.5
0.47 0.00
16.4 0.6
2.38
1.50
88.0
PBS
766.2 68.7
0.51 0.00
15.8 1.6
2.56
1.42
88.6
HPLC grade water PBS HPLC grade water
120.2 2.5 100.0 0.3 32.0 1.3
0.23 0.02 0.20 0.03 0.18 0.03
4.7 2.1 1.4 0.2 5.4 0.1
2.10 1.97 2.14
0.68 0.72 1.65
– – 74.8
PBS
54.4 2.3
0.23 0.05
8.2 0.4
2.21
1.40
78.0
HPLC grade water
38.2 2.5
0.35 0.00
10.8 1.5
1.76
1.33
74.5
PBS
59.3 4.3
0.33 0.03
12.2 1.2
1.75
1.03
67.4
HPLC grade water
180.4 4.8
0.43 0.07
18.7 1.6
2.18
1.30
66.3
PBS
523.5 3.5
0.49 0.09
21.8 5.8
2.46
0.72
69.7
Composition
Dispersion medium
IEO-a IEO-a IEO-a:Ampho B (9:0.1 molar ratio) IEO-a:Ampho B (9:0.1 molar ratio) IEO-a:Ampho B (9:0.2 molar ratio) IEO-a: Ampho B (9:0.2 molar ratio) IEO-a:Ampho B (9:0.3 molar ratio) IEO-a:Ampho B (9:0.3 molar ratio) IEO-b IEO-b IEO-b:Ampho B (9:0.1 molar ratio) IEO-b:Ampho B (9:0.1 molar ratio) IEO-b:Ampho B (9:0.2 molar ratio) IEO-b:Ampho B (9:0.2 molar ratio) IEO-b:Ampho B (9:0.3 molar ratio) IEO-b:Ampho B (9:0.3 molar ratio)
HPLC grade water PBS HPLC grade water PBS
Dh (nm)
spherical micellar like aggregates (Table 2). Their composition helps to this direction of self-assembly in aqueous solution. Sample IEO-b most probably forms micelles, as the small size of the assemblies and the Rg/Rh (close to unity) ratio indicate (Table 2) (Burchard, 1983). For IEO-a in both dispersion media, the Rg/Rh values are close to 0.775 indicating hard uniform sphere morphology (Table 2) (Burchard, 1983). The large polydispersity of the nanoassemblies may be attributed to the structure of the copolymer that poses some spatial constrains in the arrangement of macromolecules in better defined structures. It is also interesting to note that although the particular preparation protocol, via intermediate thin film formation followed by hydration, in certain cases of amphiphilic block copolymers leads to the preparation of polymersomes, the particular copolymers do not form vesicles (also based on the determined Rg/Rh and df values for the polymeric aggregates). Taking into account, the large dimensions of the aggregates of copolymer IEO-a in both media, compared to the dimensions of the single copolymer chains, and the Rg/Rh values most probably the structures observed in these systems are spherical compound micelles and not vesicles. This may be due to the amphiphilic character of the formed nanostructures, also based on the amphiphilicity of the components and their weight fraction. It seems that this is an effect of the dispersion medium as has been discussed previously.
As presented in Section 2, Ampho B was incorporated within IEO in three different proportions (copolymer:Ampho B = 9:0.1, 9:0.2 and 9:0.3 molar ratio). Polymeric carrier’s size and size distribution, z-potential values and df after Ampho B incorporation are presented in Table 2. The incorporation of Ampho B at the lowest molar ratio led to a small decrease in the size of IEO-a nanocarriers because the hydrodynamic radius of IEO-a:Ampho B nanoparticles was found equal to 164 nm in HPLC grade water (Table 2) and that indicates that this lipophilic drug alters the structural characteristics of the copolymer/drug mixed assemblies, by modulating the organization of the polymeric components. On the other hand, the incorporation of Ampho B led to a significant increase in the size of IEO-a nanocarriers at 9:0.2 and 9:0.3 molar ratios (Table 2). This may be an indication of an increase of the already high hydrophobic character of the IEO-a assemblies due to the presence of hydrophobic drug molecules in the aggregate. A shift of z-potential to more negative values was observed for IEO-a vectors (Table 2). The population of polymeric carriers remained polydisperse in the two aqueous dispersion media, while the df values remained statistically unaltered
3.2. Amphotericin B incorporation into IEO nanostructures The hydrophobic drug amphotericin B (Ampho B) was successfully incorporated within the copolymer structures, following the aforementioned encapsulation protocol, and the properties of the mixed aggregates were studied in comparison to the initial pure copolymer assemblies (Scheme 1). The chemical structure, the amphiphilic character and the biocompatibility of IEO block copolymers make them good candidates for incorporating Ampho B, because this antifungal drug consists of polyene hydrophobic groups similar to the PI block of IEO copolymers (Fig. 1).
Scheme 1. Schematic representation of IEO block copolymer nanocarriers incorporating Ampho B.
84
N. Pippa et al. / International Journal of Pharmaceutics 473 (2014) 80–86
(Table 2) in both dispersion media in 9:0.1 and 9:0.3 molar ratios. The morphology of polymeric IEO-a carriers changed significantly after the incorporation of Ampho B in different molar ratios, as indicated by df and Rg/Rh values (Table 2). Namely, for IEO-a: Ampho B (9:0.1 molar ratio) the Rg/Rh ratio indicates vesicle like morphology (values close to unity) (Table 2). The Rg/Rh ratio increased as the ratio of the incorporated drug increased. This observation indicates an open/loose conformation in these cases or more elongated structures. On the contrary, the size of IEO-b nanocontainers decreased significantly when Ampho B was incorporated at 9:0.1 and 9:0.2 molar ratios (Table 2), while the Rg/Rh ratio increased significantly (Table 2). This can be attributed to the partial amphiphilic character of Ampho B due to the presence of a COOH group, an NH2 group and multiple OH groups. The polymeric nanoparticles containing Ampho B also had lower polydispersity (Table 2). On the other hand, the IEO-b nanocontainers increased significantly when Ampho B was incorporated at 9:0.3 molar ratio, especially in PBS (Table 2). The morphological characteristics of nanocarriers changed significantly after the incorporation of Ampho B, as can be implied from the df values, and the Rg/Rh values which, together with the size changes, indicate a more compact structure for the mixed copolymer/drug aggregates (Table 2). Furthermore, the drug incorporation efficiency was estimated to 90%, for IEO-a containers and 70% for IEO-b. This difference in incorporation efficiency should be attributed to the higher hydrophobic content of IEO-a. The incorporation efficiency was increased significantly for IEO-a copolymer assemblies at the lower molar ratios of Ampho B (Table 2). These observations may be a result of the interplay between the copolymer composition and the amphiphilic character of the drug.
3.3. Stability studies Temporal colloidal stability of the initially formed structures incorporating Ampho B in two dispersion media was also investigated. Generally, size and size distribution control is of a great importance in preparing formulations, because particle size can modulate the biological stability, the ADME profile of encapsulated drug and the capture mechanism by macrophages. The physical stability over time of all formulations without and with incorporated antifungal drug was assessed by measuring the size for a period of 21 days. All formulations composed by IEO-a were found to retain significantly their original physicochemical characteristics (hydrodynamic radius) at least for the time period they have been investigated in the two aqueous dispersion media (Fig. 2). On the other hand, the polymeric assemblies composed by IEO-b were physicochemical stable only in PBS. The incorporation of Ampho B into polymeric carriers stabilized the drug vectors in the two aqueous dispersion media at 9:0.1 and 9:0.2 molar ratios (Fig. 3). Aggregation of the initial polymeric particles was observed for pure IEO-b assemblies (Fig. 3a) and for IEO-b: Ampho B 9:0.3 molar ratio polymeric carriers in HPLC grade water. Taking into consideration the ionic strength of PBS one can assume that the above observations may be due to differences in the solvation state of hydrophilic polymeric chains when ions are present in the solution. According to the extended DLVO theory, another explanation can be based on the changes in the hydration force of water, due to the presence of ions in PBS, as well as the, at least partially, coordination of these ions to the hydrophilic polymeric chains, something that would make them more hydrophilic, improving the hydration characteristics of the polymeric chains, leading to the stabilization of drug carriers (Derjaguin and Landau, 1941; Verwey and Overbeek, 1948; Okhi and Ohshima, 1999; Okhi and Arnold, 2000).
a. 300
a. 1800 1600
250
1400
200
1000
9:0.0
800
9:0.1
600
9:0.2
400
9:0.3
200 0
0
1
5
7
10
15
Dh (nm)
Dh (nm)
1200
9:0.2 9:0.3
50 0
21
0
1
5
7
10
15
21
t (days)
b. 1800
b. 1800
1600
1600
1400
1400
1200 1000
9:0.0
800
9:0.1
600 400
1200 1000
9:0.0
800
9:0.1
9:0.2
600
9:0.2
9:0.3
400
9:0.3
200
Dh (nm)
Dh (nm)
9:0.1
100
t (days)
0
9:0.0
150
200
0
1
5
7
9
15
21
t (days) Fig. 2. Stability assessment of IEO-a:Ampho B (9:0, 9:0.1, 9:0.2 and 9:0.3 molar ratios) formulations in (a) HPLC grade water and (b) PBS. Mean of three independent experiments run in triplicate, SD < 10%.
0
0
1
5
7
10
15
21
t (days) Fig. 3. Stability assessment of IEO-b:Ampho B (9:0, 9:0.1, 9:0.2 and 9:0.3 molar ratios) formulations in (a) HPLC grade water and (b) PBS. Mean of three independent experiments run in triplicate, SD < 10%.
N. Pippa et al. / International Journal of Pharmaceutics 473 (2014) 80–86
Acknowledgements
100 90
The authors express their thanks to Prof. A.L. Skaltsounis and to Dr. K. Vougogiannopoulou (Faculty of Pharmacy, Department of Pharmacognosy and Natural Product Chemistry, University of Athens) for their help in the lyophilization of the samples.
80
% drug release
70 60 50
9:0.1
40
9:0.2
30
9:0.3
20 10 0
85
0
2
4
6
8
10
12
14
16
18
20
T (hours)
Fig. 4. Cumulative drug release from IEO-b:Ampho B nanoassemblies (9:0.1, 9:0.2 and 9:0.3 molar ratios). Mean of three independent experiments run in triplicate, SD < 10%.
3.4. In vitro release of Ampho B The in vitro release of the Ampho B from the polymeric carriers in different molar ratio of the bioactive antifungal agent is presented in Fig. 4. It should be noted that the IEO-a polymeric carriers did not release the Ampho B. For the IEO-a drug carriers, the in vitro release of Ampho B is faster from the polymeric particles with the higher ratio of the incorporated antifungal agent (Fig. 4). Namely, the in vitro release of the Ampho B is faster from the polymeric carriers with the higher ratio of this antifungal drug (Fig. 4). The polymeric vectors with the lower ratio of the incorporated Ampho B released only the 60% of the incorporated drug (Siepmann and Peppas, 2001; Macheras and Illiadis, 2006; Rinaki et al., 2003). These differences may also point to a different spatial incorporation of drug molecules within the two block copolymer nanocarriers. From the point of view of kinetics, this release behavior of Ampho B from polymeric vectors has been validated in simulation and in vitro studies of drug release in fractal polymeric matrices (Pereira 2010; Dokoumetzidis and Macheras, 2011; Pippa et al., 2014). 4. Conclusions Drug nanocarriers based on poly(isoprene-b-ethylene oxide) (PI-b-PEO) block copolymers have been designed and developed. We investigated the self assembly behavior and the stability kinetics of IEO copolymer nanostructures formed in aqueous media. The physicochemical behavior of the nanoassemblies, as well as their drug encapsulation and drug release properties, were found to depend on the molecular structure of the copolymer, the surrounding aqueous medium and the molar ratio of incorporated Ampho B. The presented results elucidate several aspects concerning the self-assembly processes in pure copolymers systems, and those incorporating a hydrophobic drug, and can be utilized as guidelines for the production of new drug delivery systems composed of such copolymers. In conclusion, the composition of the block copolymer itself, as well as the physicochemical characteristics of the drug and the copolymer/drug interactions and weight ratios, play a key role in the self assembly behavior of drug carriers, as well as in the encapsulation efficiency and release of a lipophilic drug and should be taken into account in the preparation of polymeric formulations incorporating insoluble drugs in terms of their physical stability, release kinetics and consequently of their effectiveness.
References Adams, M.L., Lavasanifar, A., Kwon, G.S., 2003. Amphiphilic block copolymers for drug delivery. J. Pharm. Sci. 92, 1343–1455. Amidon, G.L., Lennernas, H., Shah, V.P., Crison, J.R., 1995. A theoretical basis for biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm. Res. 12, 413–420. Burchard, W., 1983. Static and dynamic light scattering from branched polymers and biopolymers. Adv. Polym. Sci. 48, 1–124. Cabral, H., Nishiyama, N., Kataoka, K., 2011. Supramolecular nanodevices: from design validation to threnostic nanomedicine. Acc. Chem. Res. 44, 999–1008. Choi, K.C., Bang, J.Y., Kim, P.I., Kim, C., Song, C.E., 2008. Amphotericin B-incorporated polymeric micelles composed of poly(D,L-lactide-co-glycolide)/dextran graft copolymer. Int. J. Pharm. 355, 224–230. Crommelin, D.J., Florence, A.T., 2013. Towards more effective advanced drug delibery systems. Int. J. Pharm. 451, 496–511. Demetzos, C., Pippa, N., 2014. Advanced drug delivery nanosystems (aDDnSs): a mini-review. Drug Deliv. 21, 250–257. Derjaguin, B.V., Landau, L.D., 1941. Theory of the stability of stronglycharged lyophobic sols and of adhesion of strongly charged particles in solution of electrolytes. Acta Physicochim. URRS 14, 633–662. Discher, D.E., Eisenberg, A., 2002. Polymer vesicles. Science 297, 967–973. Dokoumetzidis, A., Macheras, P., 2011. The changing face of the rate concept in biopharmaceutical sciences: from classical to fractal and finally to fractional. Pharm. Res. 28, 1229–1232. Fagerholm, U., 2007. Evaluation and suggested improvements of the biopharmaceutics classification system (BCS). J. Pharm. Pharmacol. 59, 751–757. Gaucher, G., Dufresne, M.H., Sant, V.P., Kang, N., Maysinger, D., Leroux, J.C., 2005. Block copolymer micelles: preparation, characterization and application in drug delivery. J. Control. Release 109, 1690188. Hamill, R.J., 2013. Amphotericin B formulations: a comperative review of efficacy and toxicity. Drugs 73, 919–934. Italia, J.L., Yahya, M.M., Singh, D., Ravi Kumar, M.N., 2009. Biodegradable nanoparticles improve oral bioavailability of amphotericin B and show reduced nephrotoxicity compared to intravenous Fungizone. Pharm. Res. 26, 1324–1331. Jung, S.H., Lim, D.H., Jung, S.H., Lee, J.E., Jeong, K.S., Seong, H., Shin, B.C., 2009. Amphotericin B-entrapping lipid nanoparticles and their in vitro and in vivo characteristics. Eur. J. Pharm. Sci. 37, 313–320. Kataoka, K., Harada, A., Nagasaki, Y., 2001. Block copolymer micelles for drug delivery: design, characterization and biological significance. Adv. Drug Deliv. Rev. 47, 113–131. Kowalczuk, A., Trzcinska, R., Trzebicka, B., Müller, A.H.E., Dwora, A., Tsvetanov, C.B., 2014. Loading of polymer nanocarriers: factors, mechanisms and applications. Prog. Polym. Sci. 39, 43–86. Le Meins, J.F., Sandre, O., Lecommandoux, S., 2011. Recent trends in the tuning of polymersomes membrane properties. Eur. Phys. J. E Soft Matter 34, 1–16. Lee, J.S., Feijen, J., 2012. Polymersomes for drug delivery: design, formation and characterization. J. Control. Release 161, 473–483. Macheras, P., Illiadis, A., 2006. Modelling in Biopharmaceutics, Pharmacokinetics and Pharmacodynamics: Homogeneous and Heterogeneous Approaches. Springer, New York, pp. 174–178. Macheras, P., Karalis, V., 2014. A non-binary biopharmaceutical classification of drugs: the ABG system. Int. J. Pharm. 464, 85–90. Mahmud, A., Xiong, X.B., Aliabadi, H.M., Lavasanifar, A., 2007. Polymeric micelles for drug targeting. J. Drug Target. 15, 553–584. Meng, F., Zhong, Z., 2011. Polymersomes spanning from nano- to microscales: advanced vehicles for controlled drug delivery and robust vesicles for virus and cell mimicking. J. Phys. Chem. Lett. 2, 1533–1539. Moen, M.D., Lyseng-Williamson, K.A., Scott, L.J., 2009. Liposomal amphotericin B: a review of its use as empirical therapy in febrile neutropenia and the treatment of invasive fungal infections. Drugs 69, 361–392. Okhi, S., Ohshima, H., 1999. Interaction and aggregation of lipid vesicles (DLVO theory versus modified DLVO theory). Colloids Surf. B: Biointerfaces 14, 27–45. Okhi, S., Arnold, K., 2000. A mechanism for ion-induced lipid fusion. Colloids Surf. B: Biointerfaces 18, 83–97. Pereira, L.M., 2010. Fractal pharmacokinetics. Comput. Math. Methods Med. 11, 161–184. Pippa, N., Merkouraki, M., Pispas, S., Demetzos, C., 2013. DPPC:MPOx chimeric advanced drug delivery nanosystems (chi-aDDnSs): physicochemical and structural characterization, stability and drug release studies. Int. J. Pharm. 450, 1–10. Pippa, N., Dokoumetzidis, A., Pispas, S., Demetzos, C., 2014. The interplay between the rate of release from polymer grafted liposomes and their fractal morphology. Int. J. Pharm. 465, 63–69.
86
N. Pippa et al. / International Journal of Pharmaceutics 473 (2014) 80–86
Pispas, S., 2006. Double hydrophilic block copolymers of sodium(2-sulfamate-3carboxylate)isoprene and ethylene oxide. J. Polym. Sci. Part A: Polym. Chem. 44, 606–613. Rinaki, E., Valsami, G., Macheras, P., 2003. The power law can describe the ‘entire’ drug release curve from HPMC-based matrix tablets: a hypothesis. Int. J. Pharm. 255, 199–207. Rösler, A., Vandermeulen, G.W., Klock, H.A., 2001. Advanced drug delivery devices via self-assembly of amphiphilic block copolymers. Adv. Drug Deliv. Res. 53, 95–108. Rowland, M., Noe, C.R., Smith, D.A., Tucker, G.T., Crommelin, D.J., Peck, C.C., Rocci Jr, M.L., Basançon, L., Shah, V.P., 2012. Impact of the pharmaceutical sciences on health care: a reflection over the past 50 years. J. Pharm. Sci. 101, 4075–4099. Sheikh, S., Ali, S.M., Ahmad, M.U., Ahmad, A., Musthaq, M., Paithankar, M., Mandal, J., Saptarishi, D., Sehgal, A., Maheshwari, K., Ahmad, I., 2010. Nanosomal amphotericin B is an efficacious alternative to ambisome for fungal therapy. Int. J. Pharm. 397, 103–108. Siepmann, J., Peppas, N.A., 2001. Modeling of drug release from delivery systems based on hydroxypropyl methylcellulose (HPMC). Adv. Drug Deliv. Rev. 48, 139–157. Taubert, A., Napoli, A., Meier, W., 2004. Self-assembly of reactive amphiphilic block copolymers as mimetics for biological membranes. Curr. Opin. Chem. Biol. 8, 598–603.
Thompson, K.L., Chambon, P., Versber, R., Armes, S.P., 2012. Can polymersomes form colloidosomes? J. Am. Chem. Soc. 134, 12450–12453. Torchilin, V.P., 2004. Targeted polymeric micelles for delivery of poorly soluble drugs. Cell. Mol. Life Sci. 61, 2549–2559. Torchilin, V.P., 2007. Micellar nanocarriers: pharmaceutical perspectives. Pharm. Res. 21, 1–15. Uchegbu, I.F., 2006. Pharmaceutical nanotechnology: polymeric vesicles for drug and gene delivery. Expert Opin. Drug Deliv. 3, 629–640. Van de Ven, H., Paulussen, C., Feijens, P.B., Matheeussen, A., Rombaut, P., Kayaert, P., Van den Mooter, G., Weyenberg, W., Cos, P., Maes, L., Ludwig, A., 2012. PLGA nanoparticles and nanosuspensions with amphotericin B: potent in vitro and in vivo alternatives to Fungizone and AmBisome. J. Control. Release 161, 795–803. Verwey, E.J.B., Overbeek, J.Th.G., 1948. Theory of the Stability of Lyophobic Colloids. Elsevier, Amsterdan. Wasan, E.K., Bartlett, K., Gershkovich, P., Sivak, O., Banno, B., Wong, Z., Gagnon, J., Gates, B., Leon, C.G., Wasan, K.M., 2009. Development and characterization of oral lipid-based amphotericin B formulations with enhanced drug solubility, stability and antifungal activity in rats infected with Apsergillus fumigatus of Candida albicans. Int. J. Pharm. 372, 76–84. Xiong, X.B., Binkhathlan, Z., Molavi, O., Lavasanifar, A., 2012. Amphiphilic block copolymers: preparation and application in nanodrug and gene delivery. Acta Biomater. 8, 2017–2033.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Preparation, development and in vitro release evaluation of amphotericin B–loaded amphiphilic block copolymer vectors Natassa Pippa a,b , Maria Mariaki a , Stergios Pispas b , Costas Demetzos a, * a Department of Pharmaceutical Technology, Faculty of Pharmacy, National and Kapodistrian University of Athens, Panepistimioupolis Zografou, 15771 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
A B S T R A C T
Article history: Received 26 May 2014 Received in revised form 30 June 2014 Accepted 1 July 2014 Available online 3 July 2014
The aim of this work is to design and develop a suitable polymeric formulation incorporating amphotericin B (Ampho B) in order to overcome its water insolubility problem. To this end, we have chosen the poly(isoprene-b-ethylene oxide) amphiphilic block copolymer (IEO) family. We investigate the self assembly behavior and the stability kinetics of IEO copolymer based nanostructures formed in HPLC grade water and in phosphate buffer saline (PBS). The IEO block copolymer samples investigated have different molecular weights and compositions. A gamut of light scattering techniques (static, dynamic and electrophoretic) were used in order to extract information on the size, z-potential and morphological characteristics of the structures formed, as a function of the molar ratio of incorporated lipophilic drug Ampho B. The amphiphilic character and the colloidal stability of the particular polymeric drug vectors indicate that these nanostructures can be utilized as effective containers for the particular hydrophobic drug. The incorporation of Ampho B led to alteration of the physicochemical and morphological characteristics of the pure polymeric carriers. It is observed that the in vitro release of Ampho B from the prepared vectors IEO-b:Ampho B was quite slow, while the IEO-a carriers did not release Ampho B. ã 2014 Published by Elsevier B.V.
Keywords: Amphiphilic block copolymer Amphotericin B Self-assembly Morphology Drug release
1. Introduction The main goal of advanced drug delivery nano systems is the improvement of the therapeutic index and the reduced toxicity and side effects of the encapsulated active substance (Rowland et al., 2012; Crommelin and Florence, 2013; Demetzos and Pippa, 2014). Amphiphilic block copolymers have been in use as pharmaceutical excipients in different forms for a long time and their application is experiencing rapid growth in pharmaceutics and in nanomedicine (Adams et al., 2003; Uchegbu, 2006; Cabral et al., 2011; Xiong et al., 2012). The rapid development of amphiphilic block copolymers applications in the pharmaceutical sciences is primarily due to the chemical flexibility of their structure, which provides an opportunity for the design of versatile drug nanocarriers.
Abbreviations: IEO, poly(isoprene-b-ethylene oxide) copolymer; Ampho B, amphotericin B; df, mass fractal; Rg/Rh, radius of gyration/hydrodynamic radius; PBS, phosphate buffer saline; DLVO theory, Derjaguin, Landau, Verwey and Overbeek theory. * Corresponding author. Tel.: +30 2107274596; fax: +30 2107274027. E-mail address: [email protected] (C. Demetzos). http://dx.doi.org/10.1016/j.ijpharm.2014.07.001 0378-5173/ ã 2014 Published by Elsevier B.V.
Micelles are nanoparticulate colloidal systems that exist in equilibrium with the molecules or ions in aqueous solution from which they are formed. Micelles are characterized as selfassembled nanostructures in a liquid and composed of amphiphilic soft materials in general amphiphilic di- or tri-block copolymers made of solvophilic and solvophobic blocks (Torchilin, 2004, 2007). It should be noted that the micelle formation and stability are concentration-dependent. The utility and applications of polymeric micelles formed through the multimolecular assembly of block copolymers as novel core–shell type colloidal carriers for drug, peptide, protein and gene delivery, controlled release and targeting has been discussed (Kataoka et al., 2001; Gaucher et al., 2005; Mahmud et al., 2007). In Pharmaceutical Nanotechnology, polymersomes are a new class of artificial vesicular systems, tiny hollow spherical carriers that enclose an aqueous medium. Polymersomes are structured using amphiphilic block copolymers to form the vesicle membrane and are used in order to encapsulate drugs, peptides/proteins and nucleic acids (Discher and Eisenberg, 2002; Le Meins et al., 2011; Meng and Zhong, 2011; Lee and Feijen, 2012; Thompson et al., 2012; Kowalczuk et al., 2014). The polymersome membrane provides a biophysical barrier that isolates the incorporated bioactive molecule from external
N. Pippa et al. / International Journal of Pharmaceutics 473 (2014) 80–86
medium, such as those found in blood stream (Rösler et al., 2001; Adams et al., 2003; Taubert et al., 2004; Pippa et al., 2013). Amphotericin B (Ampho B) is a macrocyclic polyene antifungal bioactive compound and is used in clinical application intravenously for systemic fungal infections. This antifungal agent is commercially available as liposomal formulation, cholesteryl sulfate complex and as a lipid complex. The first formulation has been already developed to improve the tolerability for the patient and to reduce toxicity, and to alter the pharmacokinetic profile compared to plain Ampho B (Moen et al., 2009; Hamill, 2013). To reduce the side effects of Ampho B, several lipid- and polymer-based formulations were designed and developed compared to those are marketed (Choi et al., 2008; Italia et al., 2009; Jung et al., 2009; Wasan et al., 2009; Sheikh et al., 2010; Van de Ven et al., 2012). Additionally, Ampho B belongs to Class IV (low solubility, low permeability) of the Biopharmaceutical Classification System (BSC) and to B of ABG system (Amidon et al., 1995; Fagerholm, 2007; Macheras and Karalis, 2014). In this investigation, we designed and prepared polymeric micellar nanoparticles composed of poly(isoprene-b-ethylene oxide) copolymers (IEO) and antimicrobial antiprotozal agent, amphotericin B (Ampho B), was incorporated into these polymeric carriers. Ampho B-incorporating polymeric vectors of IEO copolymers with different composition of the hydrophobic polyisoprene block were characterized and the drug release was evaluated in vitro. The polyisoprene (PI) block has great chemical similarities with the hydrophobic part of Ampho B as can be seen in Fig. 1. So it is expected that the PI cores of IEO copolymer micelles in aqueous media may have a significant ability to encapsulate the desired drug. The PEO hydrophilic block provides the well known solubility, biocompatibility and stealth properties to the nanostructures. 2. Materials and methods 2.1. Materials The IEO block copolymers were prepared via anionic polymerization using procedures that have been reported earlier (Pispas, 2006). The chemical structures and the molecular characteristics of copolymer samples are presented in Fig. 1 and Table 1,
81
Table 1 Molecular characteristics of the block copolymers. Sample
Mwa
Mw/Mna
%wt PIb
IEO-a IEO-b
16,400 33,000
1.08 1.02
46 28
a b
By SEC in THF using polystyrene standards. By 1H NMR in CDCl3.
respectively. Ampho B was supplied by Fluka and was used as received. The dialysis membranes with a molecular weight cutoff (MWCO) of 12,000 g/mol were purchased from Spectra/ProTM Membranes. Methanol and dimethyl sulfoxide (DMSO) were of HPLC grade and used without further purification. 2.2. Preparation of polymeric carriers Polymeric carriers were prepared using a solvent evaporation method. Briefly, appropriate amounts of IEO-a/-b:Ampho B (9:0, 9:0.1, 9:0.2 and 9:0.3 molar ratios) mixtures were dissolved in DMSO:methanol (1:1 v/v). DMSO samples were evaporated to dryness on a HT-4X series Genevac centrifugal evaporation system equipped with Lyo-speed. Evaporation was performed during 3 h, in a pre-heated chamber (40 C), with low rotor speed (approximately 350 g) and 5 mbar pressure. Vacuum was applied and a thin film was formed by removal of the solvent. The film was maintained under vacuum for at least 24 h in a desiccator to remove traces of solvent and subsequently it was hydrated in HPLC grade water and phosphate buffer saline (PBS) (pH 7.40 and I = 0.154 M), by slowly stirring for 1 h in a water bath at 50 C. The resultant structures were subjected to two, 3 min and 2 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 nanostructures were allowed to anneal for 30 min. The effect of Ampho B incorporation in the chimeric preparations was evaluated by measuring the size, size distribution, fractal dimension and z-potential of the resulting nanostructures. The mean hydrodynamic diameter was used for the characterization of the nanoassemblies immediately after preparation (t = 0 day), as well as for monitoring their physical stability over time (t = 21 days). 2.2.1. Ampho B incorporation efficiency Polymeric structures incorporating Ampho B were frozen at 80 C overnight and were subjected to lyophilization in order to be reconstituted 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 8.2 102 mbar). The lyophilized polymeric suspensions were stored at 4 C. Freezedried nanostructures 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 Ampho B incorporated into polymeric nanocarriers was estimated by spectrophotometry (Stat Fax1 4200, Microplate Reader, NEOGEN1 Corporation). The absorbance was measured at 405 nm. Non incorporated Ampho B was separated from chimeric formulations on a Sephadex G75 column. Incorporation efficiency (IE) was calculated by using the following equation: % IE ¼
Fig. 1. Chemical structures of (a) PI-b-PEO (IEO) block copolymers and (b) amphotericin B, employed in this study.
Ampho B ðafter columnÞ 100 Ampho B ðinitialÞ
(1)
Ampho B concentration was estimated with the aid of the following Ampho B calibration curve in chloroform:
82
N. Pippa et al. / International Journal of Pharmaceutics 473 (2014) 80–86
Ampho B concentration ðmg=mlÞ absorbance 0:0142 2 ðR ¼ 0:9928Þ ¼ 1:1565
(2)
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. 2.5. Electrophoretic mobility–microelectrophoresis
2.3. In vitro Ampho B release The release profile of Ampho B from IEO:Ampho B (9:0.1, 9:0.2 and 9:0.3 molar ratio) polymeric nanovectors was studied in PBS at 37 C. Block copolymer nanovectors incorporating Ampho B (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 Ampho B released at various times, up to 20 h, was determined using spectrophotometry (Stat Fax1 4200, Microplate Reader, NEOGEN1 Corporation) at lmax = 405 nm with the aid of the calibration curve of Eq. (2). 2.4. Dynamic and static light scattering The hydrodynamic diameter (Dh) of polymeric carriers and the polydispersity index (PDI) were measured by dynamic light scattering (DLS) and the fractal dimension was determined by static light scattering (SLS). Mean values and standard deviations were 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 multitau digital correlator. Autocorrelation functions were analyzed by the cumulants method and the CONTIN software. Heating and cooling cycles were performed, with equilibration of the systems at intermediate temperatures. Apparent hydrodynamic diameters, Dh, at finite concentrations were calculated by aid of Stokes–Einstein equation: Dh ¼
kB T 6ph0 D
(3)
where kB is the Boltzmann constant, h0 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 m2/’2 (PDI) from the cumulants method, where G is the average relaxation rate, and m2 is its second moment. Light scattering has been widely used 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: 4ph0 u sin (4) q¼ l0 2 where h0 is the refractive index of the dispersion medium, u is the scattering angle and l0 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 cm1). The general relation for the angular dependence of the scattered intensity, I(q) is: IðqÞ qdf
(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
The zeta potential (z-potential) values play an important role in the colloidal stability of colloidal polymeric particles. The z-potential of polymeric carriers was measured by the technique of microelectrophoresis, using Zetasizer 3000HAS, Malvern Instruments, Malvern, UK. 50 ml of the dispersions was 30-fold diluted in dispersion medium and z-potential was measured at room temperature at 633 nm. The z-potentials were calculated from electrophoretic mobilities, mE, by using the Henry correction of the Smoluchowski equation:
z¼
3mE h 1 2e0 er f ðkaÞ
(6)
where e0 is the permittivity of the vacuum, er is the relative permittivity, a is the particle radius, k is the Debye length, and h is the viscosity of water. The function f(ka) depends on particle shape. While if ka > 1: f ðkaÞ ¼ 1:5 þ
9 75 þ 2ðkaÞ 2ðkaÞ2
(7)
The above function refers to polymeric dispersions of the present study. 2.6. Statistical analysis Results are shown as mean value standard deviation (SD) 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 3.1. Physicochemical characteristics of polymeric carriers Physicochemical and structural characteristics of IEO-a/-b nanostructures with and without Ampho B in two aqueous media (HPLC grade water and PBS) are presented in Table 2. The mean hydrodynamic diameter of empty IEO-a drug containers was found Dh = 210 nm and the population of particles is polydisperse in HPLC grade water. On the other hand, the mean hydrodynamic diameter of empty IEO-a in PBS was found Dh = 120 nm and the population of polymeric nanoparticles was quite monodisperse (PDI = 0.35). The z-potential values of IEO-a/b assemblies in aqueous media were found slightly negative (8 mV) because of the absence of net charges on polymeric structures’ surface. The fractal dimension (df) was found near to 2.00 and the ratio of the radius of the gyration to the hydrodynamic radius, Rg/Rh, was found equal to 0.81, indicating spherical particles in the two aqueous media. The mean hydrodynamic diameter of empty IEO-b nanocontainers in the two aqueous media was found around 100–120 nm, having z-potential near zero and somewhat lower polydispersity, especially in HPLC grade water in comparison to the polymeric structures composed by IEO-a (PDI 0.20) (Table 2). The morphological characteristics, as quantified by df, did not present significant differences between the prepared polymeric nanovectors. From the results at hand, and taking into account the molecular characteristics of the copolymers and their hydrophobic/hydrophilic ratio, it can be concluded that samples IEO-a and IEO-b form
N. Pippa et al. / International Journal of Pharmaceutics 473 (2014) 80–86
83
Table 2 Physicochemical and morphological characteristics of polymeric carriers. PDI
z-potential (mV)
df
Rg/Rh
% EE
210.3 19.9 122.9 4.6 164.7 2.1
0.51 0.10 0.35 0.30 0.58 0.08
8.6 5.5 8.3 3.0 14.6 0.3
2.00 1.87 2.35
0.71 0.73 0.93
– – 91.1
271.3 10.3
0.58 0.01
15.6 1.5
2.34
0.84
93.0
HPLC grade water
1402.0 45.6
0.51 0.01
12.4 2.1
2.56
1.69
97.0
PBS
1151.4 84.0
0.49 0.00
17.2 1.3
2.45
1.64
96.5
HPLC grade water
1498.2 220.5
0.47 0.00
16.4 0.6
2.38
1.50
88.0
PBS
766.2 68.7
0.51 0.00
15.8 1.6
2.56
1.42
88.6
HPLC grade water PBS HPLC grade water
120.2 2.5 100.0 0.3 32.0 1.3
0.23 0.02 0.20 0.03 0.18 0.03
4.7 2.1 1.4 0.2 5.4 0.1
2.10 1.97 2.14
0.68 0.72 1.65
– – 74.8
PBS
54.4 2.3
0.23 0.05
8.2 0.4
2.21
1.40
78.0
HPLC grade water
38.2 2.5
0.35 0.00
10.8 1.5
1.76
1.33
74.5
PBS
59.3 4.3
0.33 0.03
12.2 1.2
1.75
1.03
67.4
HPLC grade water
180.4 4.8
0.43 0.07
18.7 1.6
2.18
1.30
66.3
PBS
523.5 3.5
0.49 0.09
21.8 5.8
2.46
0.72
69.7
Composition
Dispersion medium
IEO-a IEO-a IEO-a:Ampho B (9:0.1 molar ratio) IEO-a:Ampho B (9:0.1 molar ratio) IEO-a:Ampho B (9:0.2 molar ratio) IEO-a: Ampho B (9:0.2 molar ratio) IEO-a:Ampho B (9:0.3 molar ratio) IEO-a:Ampho B (9:0.3 molar ratio) IEO-b IEO-b IEO-b:Ampho B (9:0.1 molar ratio) IEO-b:Ampho B (9:0.1 molar ratio) IEO-b:Ampho B (9:0.2 molar ratio) IEO-b:Ampho B (9:0.2 molar ratio) IEO-b:Ampho B (9:0.3 molar ratio) IEO-b:Ampho B (9:0.3 molar ratio)
HPLC grade water PBS HPLC grade water PBS
Dh (nm)
spherical micellar like aggregates (Table 2). Their composition helps to this direction of self-assembly in aqueous solution. Sample IEO-b most probably forms micelles, as the small size of the assemblies and the Rg/Rh (close to unity) ratio indicate (Table 2) (Burchard, 1983). For IEO-a in both dispersion media, the Rg/Rh values are close to 0.775 indicating hard uniform sphere morphology (Table 2) (Burchard, 1983). The large polydispersity of the nanoassemblies may be attributed to the structure of the copolymer that poses some spatial constrains in the arrangement of macromolecules in better defined structures. It is also interesting to note that although the particular preparation protocol, via intermediate thin film formation followed by hydration, in certain cases of amphiphilic block copolymers leads to the preparation of polymersomes, the particular copolymers do not form vesicles (also based on the determined Rg/Rh and df values for the polymeric aggregates). Taking into account, the large dimensions of the aggregates of copolymer IEO-a in both media, compared to the dimensions of the single copolymer chains, and the Rg/Rh values most probably the structures observed in these systems are spherical compound micelles and not vesicles. This may be due to the amphiphilic character of the formed nanostructures, also based on the amphiphilicity of the components and their weight fraction. It seems that this is an effect of the dispersion medium as has been discussed previously.
As presented in Section 2, Ampho B was incorporated within IEO in three different proportions (copolymer:Ampho B = 9:0.1, 9:0.2 and 9:0.3 molar ratio). Polymeric carrier’s size and size distribution, z-potential values and df after Ampho B incorporation are presented in Table 2. The incorporation of Ampho B at the lowest molar ratio led to a small decrease in the size of IEO-a nanocarriers because the hydrodynamic radius of IEO-a:Ampho B nanoparticles was found equal to 164 nm in HPLC grade water (Table 2) and that indicates that this lipophilic drug alters the structural characteristics of the copolymer/drug mixed assemblies, by modulating the organization of the polymeric components. On the other hand, the incorporation of Ampho B led to a significant increase in the size of IEO-a nanocarriers at 9:0.2 and 9:0.3 molar ratios (Table 2). This may be an indication of an increase of the already high hydrophobic character of the IEO-a assemblies due to the presence of hydrophobic drug molecules in the aggregate. A shift of z-potential to more negative values was observed for IEO-a vectors (Table 2). The population of polymeric carriers remained polydisperse in the two aqueous dispersion media, while the df values remained statistically unaltered
3.2. Amphotericin B incorporation into IEO nanostructures The hydrophobic drug amphotericin B (Ampho B) was successfully incorporated within the copolymer structures, following the aforementioned encapsulation protocol, and the properties of the mixed aggregates were studied in comparison to the initial pure copolymer assemblies (Scheme 1). The chemical structure, the amphiphilic character and the biocompatibility of IEO block copolymers make them good candidates for incorporating Ampho B, because this antifungal drug consists of polyene hydrophobic groups similar to the PI block of IEO copolymers (Fig. 1).
Scheme 1. Schematic representation of IEO block copolymer nanocarriers incorporating Ampho B.
84
N. Pippa et al. / International Journal of Pharmaceutics 473 (2014) 80–86
(Table 2) in both dispersion media in 9:0.1 and 9:0.3 molar ratios. The morphology of polymeric IEO-a carriers changed significantly after the incorporation of Ampho B in different molar ratios, as indicated by df and Rg/Rh values (Table 2). Namely, for IEO-a: Ampho B (9:0.1 molar ratio) the Rg/Rh ratio indicates vesicle like morphology (values close to unity) (Table 2). The Rg/Rh ratio increased as the ratio of the incorporated drug increased. This observation indicates an open/loose conformation in these cases or more elongated structures. On the contrary, the size of IEO-b nanocontainers decreased significantly when Ampho B was incorporated at 9:0.1 and 9:0.2 molar ratios (Table 2), while the Rg/Rh ratio increased significantly (Table 2). This can be attributed to the partial amphiphilic character of Ampho B due to the presence of a COOH group, an NH2 group and multiple OH groups. The polymeric nanoparticles containing Ampho B also had lower polydispersity (Table 2). On the other hand, the IEO-b nanocontainers increased significantly when Ampho B was incorporated at 9:0.3 molar ratio, especially in PBS (Table 2). The morphological characteristics of nanocarriers changed significantly after the incorporation of Ampho B, as can be implied from the df values, and the Rg/Rh values which, together with the size changes, indicate a more compact structure for the mixed copolymer/drug aggregates (Table 2). Furthermore, the drug incorporation efficiency was estimated to 90%, for IEO-a containers and 70% for IEO-b. This difference in incorporation efficiency should be attributed to the higher hydrophobic content of IEO-a. The incorporation efficiency was increased significantly for IEO-a copolymer assemblies at the lower molar ratios of Ampho B (Table 2). These observations may be a result of the interplay between the copolymer composition and the amphiphilic character of the drug.
3.3. Stability studies Temporal colloidal stability of the initially formed structures incorporating Ampho B in two dispersion media was also investigated. Generally, size and size distribution control is of a great importance in preparing formulations, because particle size can modulate the biological stability, the ADME profile of encapsulated drug and the capture mechanism by macrophages. The physical stability over time of all formulations without and with incorporated antifungal drug was assessed by measuring the size for a period of 21 days. All formulations composed by IEO-a were found to retain significantly their original physicochemical characteristics (hydrodynamic radius) at least for the time period they have been investigated in the two aqueous dispersion media (Fig. 2). On the other hand, the polymeric assemblies composed by IEO-b were physicochemical stable only in PBS. The incorporation of Ampho B into polymeric carriers stabilized the drug vectors in the two aqueous dispersion media at 9:0.1 and 9:0.2 molar ratios (Fig. 3). Aggregation of the initial polymeric particles was observed for pure IEO-b assemblies (Fig. 3a) and for IEO-b: Ampho B 9:0.3 molar ratio polymeric carriers in HPLC grade water. Taking into consideration the ionic strength of PBS one can assume that the above observations may be due to differences in the solvation state of hydrophilic polymeric chains when ions are present in the solution. According to the extended DLVO theory, another explanation can be based on the changes in the hydration force of water, due to the presence of ions in PBS, as well as the, at least partially, coordination of these ions to the hydrophilic polymeric chains, something that would make them more hydrophilic, improving the hydration characteristics of the polymeric chains, leading to the stabilization of drug carriers (Derjaguin and Landau, 1941; Verwey and Overbeek, 1948; Okhi and Ohshima, 1999; Okhi and Arnold, 2000).
a. 300
a. 1800 1600
250
1400
200
1000
9:0.0
800
9:0.1
600
9:0.2
400
9:0.3
200 0
0
1
5
7
10
15
Dh (nm)
Dh (nm)
1200
9:0.2 9:0.3
50 0
21
0
1
5
7
10
15
21
t (days)
b. 1800
b. 1800
1600
1600
1400
1400
1200 1000
9:0.0
800
9:0.1
600 400
1200 1000
9:0.0
800
9:0.1
9:0.2
600
9:0.2
9:0.3
400
9:0.3
200
Dh (nm)
Dh (nm)
9:0.1
100
t (days)
0
9:0.0
150
200
0
1
5
7
9
15
21
t (days) Fig. 2. Stability assessment of IEO-a:Ampho B (9:0, 9:0.1, 9:0.2 and 9:0.3 molar ratios) formulations in (a) HPLC grade water and (b) PBS. Mean of three independent experiments run in triplicate, SD < 10%.
0
0
1
5
7
10
15
21
t (days) Fig. 3. Stability assessment of IEO-b:Ampho B (9:0, 9:0.1, 9:0.2 and 9:0.3 molar ratios) formulations in (a) HPLC grade water and (b) PBS. Mean of three independent experiments run in triplicate, SD < 10%.
N. Pippa et al. / International Journal of Pharmaceutics 473 (2014) 80–86
Acknowledgements
100 90
The authors express their thanks to Prof. A.L. Skaltsounis and to Dr. K. Vougogiannopoulou (Faculty of Pharmacy, Department of Pharmacognosy and Natural Product Chemistry, University of Athens) for their help in the lyophilization of the samples.
80
% drug release
70 60 50
9:0.1
40
9:0.2
30
9:0.3
20 10 0
85
0
2
4
6
8
10
12
14
16
18
20
T (hours)
Fig. 4. Cumulative drug release from IEO-b:Ampho B nanoassemblies (9:0.1, 9:0.2 and 9:0.3 molar ratios). Mean of three independent experiments run in triplicate, SD < 10%.
3.4. In vitro release of Ampho B The in vitro release of the Ampho B from the polymeric carriers in different molar ratio of the bioactive antifungal agent is presented in Fig. 4. It should be noted that the IEO-a polymeric carriers did not release the Ampho B. For the IEO-a drug carriers, the in vitro release of Ampho B is faster from the polymeric particles with the higher ratio of the incorporated antifungal agent (Fig. 4). Namely, the in vitro release of the Ampho B is faster from the polymeric carriers with the higher ratio of this antifungal drug (Fig. 4). The polymeric vectors with the lower ratio of the incorporated Ampho B released only the 60% of the incorporated drug (Siepmann and Peppas, 2001; Macheras and Illiadis, 2006; Rinaki et al., 2003). These differences may also point to a different spatial incorporation of drug molecules within the two block copolymer nanocarriers. From the point of view of kinetics, this release behavior of Ampho B from polymeric vectors has been validated in simulation and in vitro studies of drug release in fractal polymeric matrices (Pereira 2010; Dokoumetzidis and Macheras, 2011; Pippa et al., 2014). 4. Conclusions Drug nanocarriers based on poly(isoprene-b-ethylene oxide) (PI-b-PEO) block copolymers have been designed and developed. We investigated the self assembly behavior and the stability kinetics of IEO copolymer nanostructures formed in aqueous media. The physicochemical behavior of the nanoassemblies, as well as their drug encapsulation and drug release properties, were found to depend on the molecular structure of the copolymer, the surrounding aqueous medium and the molar ratio of incorporated Ampho B. The presented results elucidate several aspects concerning the self-assembly processes in pure copolymers systems, and those incorporating a hydrophobic drug, and can be utilized as guidelines for the production of new drug delivery systems composed of such copolymers. In conclusion, the composition of the block copolymer itself, as well as the physicochemical characteristics of the drug and the copolymer/drug interactions and weight ratios, play a key role in the self assembly behavior of drug carriers, as well as in the encapsulation efficiency and release of a lipophilic drug and should be taken into account in the preparation of polymeric formulations incorporating insoluble drugs in terms of their physical stability, release kinetics and consequently of their effectiveness.
References Adams, M.L., Lavasanifar, A., Kwon, G.S., 2003. Amphiphilic block copolymers for drug delivery. J. Pharm. Sci. 92, 1343–1455. Amidon, G.L., Lennernas, H., Shah, V.P., Crison, J.R., 1995. A theoretical basis for biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm. Res. 12, 413–420. Burchard, W., 1983. Static and dynamic light scattering from branched polymers and biopolymers. Adv. Polym. Sci. 48, 1–124. Cabral, H., Nishiyama, N., Kataoka, K., 2011. Supramolecular nanodevices: from design validation to threnostic nanomedicine. Acc. Chem. Res. 44, 999–1008. Choi, K.C., Bang, J.Y., Kim, P.I., Kim, C., Song, C.E., 2008. Amphotericin B-incorporated polymeric micelles composed of poly(D,L-lactide-co-glycolide)/dextran graft copolymer. Int. J. Pharm. 355, 224–230. Crommelin, D.J., Florence, A.T., 2013. Towards more effective advanced drug delibery systems. Int. J. Pharm. 451, 496–511. Demetzos, C., Pippa, N., 2014. Advanced drug delivery nanosystems (aDDnSs): a mini-review. Drug Deliv. 21, 250–257. Derjaguin, B.V., Landau, L.D., 1941. Theory of the stability of stronglycharged lyophobic sols and of adhesion of strongly charged particles in solution of electrolytes. Acta Physicochim. URRS 14, 633–662. Discher, D.E., Eisenberg, A., 2002. Polymer vesicles. Science 297, 967–973. Dokoumetzidis, A., Macheras, P., 2011. The changing face of the rate concept in biopharmaceutical sciences: from classical to fractal and finally to fractional. Pharm. Res. 28, 1229–1232. Fagerholm, U., 2007. Evaluation and suggested improvements of the biopharmaceutics classification system (BCS). J. Pharm. Pharmacol. 59, 751–757. Gaucher, G., Dufresne, M.H., Sant, V.P., Kang, N., Maysinger, D., Leroux, J.C., 2005. Block copolymer micelles: preparation, characterization and application in drug delivery. J. Control. Release 109, 1690188. Hamill, R.J., 2013. Amphotericin B formulations: a comperative review of efficacy and toxicity. Drugs 73, 919–934. Italia, J.L., Yahya, M.M., Singh, D., Ravi Kumar, M.N., 2009. Biodegradable nanoparticles improve oral bioavailability of amphotericin B and show reduced nephrotoxicity compared to intravenous Fungizone. Pharm. Res. 26, 1324–1331. Jung, S.H., Lim, D.H., Jung, S.H., Lee, J.E., Jeong, K.S., Seong, H., Shin, B.C., 2009. Amphotericin B-entrapping lipid nanoparticles and their in vitro and in vivo characteristics. Eur. J. Pharm. Sci. 37, 313–320. Kataoka, K., Harada, A., Nagasaki, Y., 2001. Block copolymer micelles for drug delivery: design, characterization and biological significance. Adv. Drug Deliv. Rev. 47, 113–131. Kowalczuk, A., Trzcinska, R., Trzebicka, B., Müller, A.H.E., Dwora, A., Tsvetanov, C.B., 2014. Loading of polymer nanocarriers: factors, mechanisms and applications. Prog. Polym. Sci. 39, 43–86. Le Meins, J.F., Sandre, O., Lecommandoux, S., 2011. Recent trends in the tuning of polymersomes membrane properties. Eur. Phys. J. E Soft Matter 34, 1–16. Lee, J.S., Feijen, J., 2012. Polymersomes for drug delivery: design, formation and characterization. J. Control. Release 161, 473–483. Macheras, P., Illiadis, A., 2006. Modelling in Biopharmaceutics, Pharmacokinetics and Pharmacodynamics: Homogeneous and Heterogeneous Approaches. Springer, New York, pp. 174–178. Macheras, P., Karalis, V., 2014. A non-binary biopharmaceutical classification of drugs: the ABG system. Int. J. Pharm. 464, 85–90. Mahmud, A., Xiong, X.B., Aliabadi, H.M., Lavasanifar, A., 2007. Polymeric micelles for drug targeting. J. Drug Target. 15, 553–584. Meng, F., Zhong, Z., 2011. Polymersomes spanning from nano- to microscales: advanced vehicles for controlled drug delivery and robust vesicles for virus and cell mimicking. J. Phys. Chem. Lett. 2, 1533–1539. Moen, M.D., Lyseng-Williamson, K.A., Scott, L.J., 2009. Liposomal amphotericin B: a review of its use as empirical therapy in febrile neutropenia and the treatment of invasive fungal infections. Drugs 69, 361–392. Okhi, S., Ohshima, H., 1999. Interaction and aggregation of lipid vesicles (DLVO theory versus modified DLVO theory). Colloids Surf. B: Biointerfaces 14, 27–45. Okhi, S., Arnold, K., 2000. A mechanism for ion-induced lipid fusion. Colloids Surf. B: Biointerfaces 18, 83–97. Pereira, L.M., 2010. Fractal pharmacokinetics. Comput. Math. Methods Med. 11, 161–184. Pippa, N., Merkouraki, M., Pispas, S., Demetzos, C., 2013. DPPC:MPOx chimeric advanced drug delivery nanosystems (chi-aDDnSs): physicochemical and structural characterization, stability and drug release studies. Int. J. Pharm. 450, 1–10. Pippa, N., Dokoumetzidis, A., Pispas, S., Demetzos, C., 2014. The interplay between the rate of release from polymer grafted liposomes and their fractal morphology. Int. J. Pharm. 465, 63–69.
86
N. Pippa et al. / International Journal of Pharmaceutics 473 (2014) 80–86
Pispas, S., 2006. Double hydrophilic block copolymers of sodium(2-sulfamate-3carboxylate)isoprene and ethylene oxide. J. Polym. Sci. Part A: Polym. Chem. 44, 606–613. Rinaki, E., Valsami, G., Macheras, P., 2003. The power law can describe the ‘entire’ drug release curve from HPMC-based matrix tablets: a hypothesis. Int. J. Pharm. 255, 199–207. Rösler, A., Vandermeulen, G.W., Klock, H.A., 2001. Advanced drug delivery devices via self-assembly of amphiphilic block copolymers. Adv. Drug Deliv. Res. 53, 95–108. Rowland, M., Noe, C.R., Smith, D.A., Tucker, G.T., Crommelin, D.J., Peck, C.C., Rocci Jr, M.L., Basançon, L., Shah, V.P., 2012. Impact of the pharmaceutical sciences on health care: a reflection over the past 50 years. J. Pharm. Sci. 101, 4075–4099. Sheikh, S., Ali, S.M., Ahmad, M.U., Ahmad, A., Musthaq, M., Paithankar, M., Mandal, J., Saptarishi, D., Sehgal, A., Maheshwari, K., Ahmad, I., 2010. Nanosomal amphotericin B is an efficacious alternative to ambisome for fungal therapy. Int. J. Pharm. 397, 103–108. Siepmann, J., Peppas, N.A., 2001. Modeling of drug release from delivery systems based on hydroxypropyl methylcellulose (HPMC). Adv. Drug Deliv. Rev. 48, 139–157. Taubert, A., Napoli, A., Meier, W., 2004. Self-assembly of reactive amphiphilic block copolymers as mimetics for biological membranes. Curr. Opin. Chem. Biol. 8, 598–603.
Thompson, K.L., Chambon, P., Versber, R., Armes, S.P., 2012. Can polymersomes form colloidosomes? J. Am. Chem. Soc. 134, 12450–12453. Torchilin, V.P., 2004. Targeted polymeric micelles for delivery of poorly soluble drugs. Cell. Mol. Life Sci. 61, 2549–2559. Torchilin, V.P., 2007. Micellar nanocarriers: pharmaceutical perspectives. Pharm. Res. 21, 1–15. Uchegbu, I.F., 2006. Pharmaceutical nanotechnology: polymeric vesicles for drug and gene delivery. Expert Opin. Drug Deliv. 3, 629–640. Van de Ven, H., Paulussen, C., Feijens, P.B., Matheeussen, A., Rombaut, P., Kayaert, P., Van den Mooter, G., Weyenberg, W., Cos, P., Maes, L., Ludwig, A., 2012. PLGA nanoparticles and nanosuspensions with amphotericin B: potent in vitro and in vivo alternatives to Fungizone and AmBisome. J. Control. Release 161, 795–803. Verwey, E.J.B., Overbeek, J.Th.G., 1948. Theory of the Stability of Lyophobic Colloids. Elsevier, Amsterdan. Wasan, E.K., Bartlett, K., Gershkovich, P., Sivak, O., Banno, B., Wong, Z., Gagnon, J., Gates, B., Leon, C.G., Wasan, K.M., 2009. Development and characterization of oral lipid-based amphotericin B formulations with enhanced drug solubility, stability and antifungal activity in rats infected with Apsergillus fumigatus of Candida albicans. Int. J. Pharm. 372, 76–84. Xiong, X.B., Binkhathlan, Z., Molavi, O., Lavasanifar, A., 2012. Amphiphilic block copolymers: preparation and application in nanodrug and gene delivery. Acta Biomater. 8, 2017–2033.
