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Studying the colloidal behavior of chimeric liposomes by cryo-TEM, micro-differential scanning calorimetry and high-resolution ultrasound spectroscopy
Colloids and Surfaces A 555 (2018) 539–547
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Colloids and Surfaces A journal homepage: www.elsevier.com/locate/colsurfa
Studying the colloidal behavior of chimeric liposomes by cryo-TEM, microdifferential scanning calorimetry and high-resolution ultrasound spectroscopy
T
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Natassa Pippaa,b, Diego Romano Perinellic, Stergios Pispasb, Giulia Bonacucinac, , ⁎ Costas Demetzosa, , Aleksander Forysd, Barbara Trzebickad a
Department of Pharmaceutical Technology, Faculty of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, Athens, Greece School of Pharmacy, University of Camerino, 62032 Camerino, MC, Italy d Centre of Polymer and Carbon Materials, Polish Academy of Sciences, Zabrze, Poland b c
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Nanovesicles Copolymers Chimeric systems Thermal analysis High-resolution ultrasound spectroscopy cryo-TEM
The investigation of colloidal properties of nanosystems represents a fundamental issue for the development of nanotechnology-based medicines. The aim of this study is to combine various techniques in order to characterize more comprehensively chimeric nanovesicles composed of block or gradient block copolymers with different architectures and compositions. Several chimeric systems were prepared and the impact of the block [poly(εcaprolactone)−poly(ethylene oxide); PEO-b-PCL] and gradient block [poly(2-methyl-2-oxazoline)-grad-poly(2phenyl-2-oxazoline); MPOx] copolymers on the physicochemical and morphological characteristics of conventional 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes was examined. Light scattering techniques and cryo-TEM were used for the physicochemical and morphological characterization of the prepared systems. The size and the morphology were strongly related to the architecture and the composition of the polymeric compounds. Micro differential scanning calorimetry and high-resolution ultrasound spectroscopy were used for investigating the interactions between the DPPC lipids and the polymeric guest. An increase in the
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Corresponding authors. E-mail addresses: [email protected] (G. Bonacucina), [email protected] (C. Demetzos).
https://doi.org/10.1016/j.colsurfa.2018.07.025 Received 4 June 2018; Received in revised form 16 July 2018; Accepted 17 July 2018 Available online 20 July 2018 0927-7757/ © 2018 Elsevier B.V. All rights reserved.
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main transition temperature was observed for the prepared chimeric systems in comparison to DPPC liposomes. In conclusion, a detailed characterization of the colloidal behavior of chimeric liposomes can benefit from the combination of the aforementioned techniques that operate synergistically, giving information on their physicochemical and morphological characteristics as well as on their thermotropic behavior.
1. Introduction
characterized as core-shell structures comprising polymer cores and lipid/lipid-PEG shells [4,7,8,15]. They exhibit unique properties such as colloidal stability, biocompatibility, high loading efficiency, different thermotropic behavior in comparison to pure liposomal membrane, vesicular morphology, etc. [4,7,8,16]. They also have been used as model of cellular membranes due to the macromolecular sculpture of the polymer-grafted liposomal membrane [17]. Furthermore, a gamut of techniques has been already utilized in order to investigate in depth the structure and the solution behavior of the aforementioned colloidal systems. Light scattering techniques are used for the elucidation of size and zeta potential of the colloidal particles [4,18]. Static light scattering offers also the possibility of studying the morphology of colloidal particles using fractal geometry [5]. Thermal analysis techniques (i.e. differential scanning calorimetry, microcalorimetry etc.) are also used for studying the microstructure and the loading capacity of PEGylated and polymer grafted liposomes [18,19]. Additionally, cryo-TEM is a very useful technique to visualize the morphology of colloidal systems [20]. Acoustic spectroscopy is useful to analyze the highly structured colloidal dispersion and gives information about the materials during pre-formulation and formulation studies [21]. The aim of this study is to combine different techniques in order to study chimeric liposomes composed of block or gradient block copolymers with different architectures and compositions. We prepared different chimeric systems in order to examine the impact of the block and gradient block copolymers on the physicochemical and morphological characteristics of conventional 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) liposomes. The block copolymers PEO-b-PCL consist of two blocks, one hydrophilic (PEO) and one hydrophobic part (PCL). In the MPOx copolymers, the gradual compositional change along the length of the polymer, which is related to the hydrophilic and hydrophobic units, is presented in Scheme 1. The investigated block copolymers were selected for different reasons including their difference in structure and architecture, high biocompatibility, the ability to entry in lipid membranes (as shown in our previous investigations) and the steric stabilizing effects in DPPC liposomes [4–6]. Light scattering techniques and cryo-TEM were used for the physicochemical and morphological characterization of the prepared systems. Micro
With its accelerated development in the past three decades, pharmaceutical nanotechnology is an innovative field for the design and the preparation of drug delivery systems. These drug delivery systems are nanocarriers which belong to the class of soft colloidal nanomaterials with unique properties e.g. stimuli-responsiveness [1,2]. A new class of smart nanostructured platform with applications in drug delivery and targeting is represented by advanced chimeric drug delivery systems, including polymer-grafted liposomes [3–8]. The interactions of lipids and polymers play a key role on the morphology of the resulting chimeric nanosystems i.e. weak polymer-lipid attraction (Coulombic and hydrogen interactions) and hydrophobic interactions [3,7]. Poly-ε-caprolactone (PCL) is a biodegradable polymer with several applications in drug delivery and tissue engineering [9]. Poly(ethylene glycol) (PEG) is a biocompatible and hydrophilic polymer, well-known for its low immunogenicity and “stealth” properties. The synthesis of PEO-b-PCL copolymers is generally performed via ring opening polymerization as described in the literature [10]. The nanoparticles based on PEO-b-PCL have been used as systems for vaccines, genes, waterinsoluble active ingredients, cancer passive and active targeting, stimuli-responsive applications e.g. controlled drug release. In all cases, due to their biocompatible and biodegradable nature, they improve the therapeutic index and the effectiveness of the encapsulated agent [9–11]. Additionally, poly(2-oxazolines) are water soluble and biocompatible polymers. Several investigations showed the wide variety of their applications in pharmaceutical nanotechnology. Drug and protein conjugates of poly(2-oxazoline) increase the half-life and the biological stability of the active substance. A very important class of them is the thermoresponsive poly(2-oxazoline)s with many applications in cancer chemotherapy characterized them as smart bio-inspired polymers due to their solution properties and thermo-responsiveness [12,13]. Synthesis of poly(2-methyl-2-oxazoline)-grad-poly(2-phenyl-2-oxazoline) copolymers (MPOx) is achieved via cationic polymerization [14]. Recently, systems composed of lipids and polymers have been described in the literature as controlled released drug nanocarriers. These systems are generally referred as mixed, hybrid or chimeric and can be
Scheme 1. Chemical structures of (a) DPPC lipid, (b) the block copolymer PEO-b-PCL, (c) the gradient block copolymer MPOx. Macromolecular architecture of (d) PEO-b-PCL (hydrophilic component - PEO: purple line and hydrophobic component- PCL: orange line) and (e) MPOx (hydrophilic component (MeOxz): blue line and hydrophobic component (PhOxz): red line) employed in this study. The boxes with dashed lines show the entry/ exit points into/from the liposomal membrane (For interpretation of the references to colour in this Scheme legend, the reader is referred to the web version of this article).
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2.5. Cryogenic transmission Electron microscopy (cryo-TEM)
differential scanning calorimetry and high-resolution ultrasound spectroscopy were used for investigating the interactions between the DPPC lipids and the polymeric guest. To best of our knowledge, this is the first report in the literature, in which all the aforementioned techniques have been combined for a detailed investigation on the effect of block copolymers on the morphology and thermal behavior of colloidal mixed lipid/copolymer nanovesicles.
Cryogenic Transmission Electron Microscopy images were obtained using a Tecnai F20 TWIN microscope (FEI Company, USA) equipped with field emission gun, operating at an acceleration voltage of 200 kV. Images were recorded on the Eagle 4k HS camera (FEI Company, USA) and processed with TIA software (FEI Company, USA). Specimen preparation was done by vitrification of the aqueous solutions on grids with holey carbon film (Quantifoil R 2/2; Quantifoil Micro Tools GmbH, Germany). Prior to use, the grids were activated for 15 s in oxygen plasma using a Femto plasma cleaner (Diener Electronic, Germany). Cryo-TEM samples were prepared by applying a droplet (3 μL) of the solution to the grid, blotting with filter paper and rapid freezing in liquid ethane using a fully automated blotting device Vitrobot Mark IV (FEI Company, USA). After preparation, the vitrified specimens were kept under liquid nitrogen until they were inserted into a cryo-TEM-holder Gatan 626 (Gatan Inc., USA) and analyzed in the TEM at −178 °C.
2. Materials and methods 2.1. Materials The phospholipid used for liposomal formulations was 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 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 (St. Louis, MO, USA).
2.6. Micro-DSC analyses
2.2. Synthesis and chemical characterization of block copolymers
mDSC analyses were carried out using a microcalorimeter (mDSC III, Setaram, France). Samples were loaded inside the Hastelloy cells (700 μL) and subjected to the following thermal programme: isotherm at 5 °C for 20 min; heating ramp from 5 °C to 80 °C at 1 °C/min; cooling ramp from 80 °C to 5 °C at 1 °C/min. The peak temperature (Tm, °C), onset temperature (Tonset, °C) and enthalpy (ΔH, J/g of solution) were calculated from the peak and the area of the transition by the tangent method using the software of the instrument (Setsoft2000, Setaram, France). Measurements were performed in triplicate.
The PEO-b-PCL block copolymer was prepared by ring opening polymerization of ε-caprolactone monomer using a monohydroxy terminated PEO (Mn = 5000) as the macroinitiator and stannous octoate as the catalyst [4]. The MPOx amphiphilic gradient block copolymers were prepared via cationic polymerization [14]. In this copolymer, phenyl-oxazoline segments comprise the hydrophobic components and methyl-oxazoline segments the hydrophilic ones. The copolymers are considered biocompatible due to the pseudopeptide nature of the oxazoline segments. Poly(2-methyl-2-oxazoline) has been proposed as an alternative to PEG in terms of biocompatibility and stealth properties. The details related to synthesis and chemical characterization (NMR and SEC chromatography) of the block and gradient block copolymers are summarized in our previous publications [4,14]. The structures and the molecular characteristics of copolymer samples are presented in Scheme 1 and in Table 1, respectively.
2.7. High-resolution ultrasound spectroscopy High-resolution ultrasound spectroscopy is used to characterize concentrated colloidal dispersions without dilution, in such a way as to be able to study highly structured nanosystems [22]. Ultrasonic attenuation and ultrasonic sound speed were monitored as a function of temperature using a HR-US 102 high-resolution spectrometer (Ultrasonic Scientific, Ireland). Around 2 mL of sample and reference (HPLC grade water) were filled in the ultrasonic cells and subjected to the same thermal programmes as described for mDSC analyses. Sample transitions were calculated by the peak value from the attenuation profile or from the first derivate of the signal in the case of sound speed. Measurements were performed in triplicate.
2.3. Preparation of pure and chimeric structures Different chimeric formulations and pure liposomes have been prepared using the thin-film hydration method as described in our previous publications [4–6]. The formulations studied here are presented in Table 2. The concentration of colloidal dispersion (phospholipids and polymers) is 10 mg/mL. Appropriate amounts of DPPC:PEOb-PCL and 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 and the mixed phospholipids/block copolymer thin film was formed by slow removal of the solvent at 45–50 °C. The mixed film was maintained under vacuum for at least 24 h in a desiccator to remove traces of solvent and, subsequently, hydrated in HPLC-grade water by slowly stirring for 1 h in a water bath above the phase transition temperature of lipids (41 °C for DPPC). The resultant nanostructures (apparently multilamellar vesicles, MLVs) were subjected to two 5 min sonication cycles (amplitude 70, cycle 0.7) interrupted by a 5 min resting period, in a water bath, using a probe sonicator (UP 200S, dr. Hielsher GmbH, Berlin, Germany). The resultant nanostructures were allowed annealing for 30 min. DPPC and phospholipid/copolymer chimeric liposomes were analysed immediately after their preparation.
3. Results and discussion 3.1. The impact of architecture and composition of the polymeric guest on size and morphology of chimeric liposomes We prepared different chimeric systems in order to examine the impact of the block or gradient block copolymers, as well as their composition, on the physicochemical and morphological characteristics of copolymer/lipid mixed liposomes in comparison to conventional DPPC liposomes. Table 2 reports the hydrodynamic diameter (Dh), Table 1 Molecular characteristics of block and gradient block copolymers utilized.
2.4. Light scattering techniques The protocols used for the physicochemical characterization of the nanostructures by dynamic and electrophoretic light scattering in aqueous media are described in our previous papers [4–6].
Sample
Mwa
Mw/Mna
%wt (hydrophobic component)b
PEO-b-PCL 1 PEO-b-PCL 2 MPOx 1 MPOx 2
10,600 7,700 5,200 3,300
1.43 1.18 1.14 1.26
53 30 28 39
a b
541
by SEC in CHCl3 using polystyrene standards. by1H-NMR in CDCl3.
(PCL) (PCL) (PhOxz) (PhOxz)
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Table 2 Physicochemical characteristics of DPPC liposomes and chimeric lipid/polymer formulations. Values are reported as mean of three independent experiments run in triplicate. Composition
DPPC DPPC:PEO-b-PCL 1 9_1 DPPC:PEO-b-PCL 2 9_1 DPPC:MPOx 1 9_1 DPPC:MPOx 1 9_3 DPPC:MPOx 2 5_5 DPPC:MPOx 2 9_1 a b
Lipid/Polymer (molar ratio)
9:1 9:1 9:1 9:3 5:5 9:1
Dh (nm)a
PDIb
240.3 ± 4.8 68.7 ± 0.1 68.9 ± 0.1 92.9 ± 0.9 99.3 ± 0.6 143.3 ± 0.6 80.0 ± 2.1
0.65 0.17 0.26 0.26 0.27 0.33 0.54
ζ-potential (mV)
± ± ± ± ± ± ±
0.03 0.01 0.01 0.02 0.02 0.08 0.01
+7.2 +2.8 −9.3 +1.5 −3.5 −5.8 +5.1
± ± ± ± ± ± ±
0.1 0.5 0.1 0.2 0.1 1.2 2.3
Hydrodynamic Diameter. Polydispersity Index.
Fig. 1. Cryo TEM images and size distributions from DLS of the chimeric lipid/copolymer systems DPPC:PEO-b-PCL 1 9_1 (A) and DPPC:PEO-b-PCL 2 9_1 (B).
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Fig. 2. Cryo TEM images and size distributions from DLS of the chimeric lipid/copolymer systems DPPC:MPOx1 9_1 (A) and DPPC:MPOx1 9_3 (B).
polydispersity index (PDI) and ζ-potential of pure DPPC liposomes and mixed DPPC:PEO-b-PCL 1 and DPPC:PEO-b-PCL 2 (9:1 molar ratio), DPPC:MPOx 1 (9:1 and 9:3 molar ratios) and DPPC:MPOx 2 (5:5 and 9:1 molar ratios) systems. Size distribution graphs and cryo-TEM images are presented in Figs. 1–3. The size (Dh) of pure DPPC liposomes, used as control, are around 240 nm with high polydispersity and low ζ-potential due to the zwitterionic nature of the phospholipids used (Table 2). The presence of copolymers reduced the size of the chimeric nanostructures in a copolymer architecture dependent manner. It has been observed, that the reduction of size is higher for PEO-b-PCL grafted liposomes in comparison to MPOx grafted liposomes. This finding is strongly dependent on the number of entry points of copolymer chains into the lipid bilayer and the consequent packing of phospholipids in the presence of the polymeric guest. Indeed, for MPOx the gradient of co-monomer sequence exhibits several entry points into DPPC bilayers, because of the hydrophobic and hydrophilic repetition within the copolymer chain. On the other hand, the block copolymer PEO-b-PCL exhibits only one entry point into the bilayer (the junction
point between the two blocks). Additionally, for the PEO-b-PCL block copolymers the hydrophilic part is more elongated in comparison to methyl-oxazoline sequences within the MPOx gradient block copolymer and induces stronger steric intra-vesicular repulsions, leading to a smaller hydrodynamic diameter for PEO-b-PCL in comparison to the MPOx ones (Table 2) [13,23,24]. In details, the size of chimeric DPPC:PEO-b-PCL 1 and 2 is comparable and around 70 nm according to DLS measurements (Table 2 and Fig. 1). In this case, the different % ratio of the hydrophobic component in PEO-b-PCL copolymer did not affect the size of the resulted nanocarriers (i.e. Dh = 68.7 ± 0.1 nm for DPPC:PEO-b-PCL 1 and Dh = 68.9 ± 0.1 nm for DPPC:PEO-b-PCL 2). Nanovesicles of diameters between 50 and 200 nm, which correspond to DPPC:PEO-bPEO 1 and DPPC:PEO-b-PEO 2 chimeric liposomes, are observed in cryo-TEM images (Fig. 1). Generally, using this technique, we observed vesicles larger than those measured by DLS. In our opinion, the strong steric interactions due to the PEO-hydrophilic chains produce vesicles with smaller hydrodynamic diameter in the disperse liquid state (as 543
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Fig. 3. Cryo TEM images and size distributions from DLS of the chimeric lipid/copolymer systems DPPC:MPOx2 9_1 (A) and DPPC:MPOx2 5_5 (B).
On the contrary, the size for chimeric liposomes formed by MPOx gradient copolymers was found to be dependent on the percentage of the hydrophobic component. Indeed, for DPPC:MPOx1 and DPPC:MPOx2 (9:1 molar ratio), the size resulted to be 10 nm smaller for liposomes in which MPOx participates with the lower ratio of the hydrophobic component (i.e. copolymer MPOx1, 28% hydrophobic component). Similarly, for DPPC:MPOx1, the size and size distribution of the prepared chimeric liposomes increased slightly with the increase of the molar ratio of the polymeric guest (Table 2). From cryo-TEM images, self-assembled vesicular objects are observed of diameter between 50–100 nm for DPPC:MPOx1 (9:1 molar ratio) (Fig. 2A1) and 50200 nm for DPPC:MPOx 1 (9:3 molar ratio) due to a more polydisperse population (Fig. 2B1) [27–29]. The increase of MPOx2 molar ratio caused a greater size of the prepared systems (Table 2) (i.e. Dh = 143.3 ± 0.6 nm for DPPC:MPOx2 (5:5 molar ratio) and Dh = 80.0 ± 2.1 nm for DPPC:MPOx2 (9:1 molar ratio). The population of DPPC:MPOx2 (9:1
measured by DLS), while the effects from solvent freezing may affect vesicles size in cryo-TEM images. The thickness of the mixed liposomal membrane is equal to 4–5 nm, a little bit higher than the thickness of pure liposomes’ membrane (∼3–4 nm) and lower than the thickness of pure PEO-b-PCL polymersomes’ membrane (∼8-12 nm) [7,25,26]. Moreover, spherical objects with diameter between 15–40 nm can be observed, recognized as pure PEO-b-PCL micelles formed from the copolymer chains that have not been incorporated in DPPC lipid bilayers during the formation of chimeric liposomes [27–29]. Some large vesicles around 200 nm of diameter have some smaller vesicles encapsulated in the interior. According to the literature, these systems can be characterized as “pregnant vesicles”, where smaller vesicles are enclosed within larger chimeric liposomes [30]. In Figs. 2A1 and 3B1, the black, branched objects with strong contrast are a contamination from liquid ethane. According to recent literature, ethane could remain in the samples after the vitrifying procedure on a film of cubic nanoparticles [20]. 544
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Additionally, for DPPC:MPOx1 systems, the peak transition temperature increased by 7 °C for the two molar ratios in comparison to pure DPPC liposomes. For these systems, the thermotropic peaks are broader than those obtained from DPPC: PEO-b-PCL 1 and 2. It should be pointed out that for all the MPOx grafted liposomes the peaks are not sharp but rather wide (Fig. 4A). In other words, new metastable phases were created at 50 °C for all the MPOx chimeric liposomes. The reduction of the enthalpy is much higher for the MPOx 2 i.e. for the copolymer having the higher amount of the hydrophobic component (PhOx). This means that the cooperativity between the two biomaterials (i.e. lipid and gradient block copolymer) is limited, leading the DPPC lipid to feel the MPOx 2 as an “impurity” for the liquid crystalline dance of the phospholipid molecules. For the PEO-b-PCL block copolymers, the cooperativity is higher because the macromolecular structure of block copolymers and phospholipids are the same (i.e. the hydrophilic PEO chain corresponds to phospholipid’s head group and the hydrophobic PCL chain to the lipid alkyl chains). This observation may also be a manifestation of the effects of the number of entry points in each case. The DSC cooling scans are illustrated in Fig. 4B. A small hysteresis in the cooling curves at the main transition temperature is observed, a common phenomenon well established in the literature [32]. For the DPPC:MPOx2 (9:1 molar ratio), a new peak at 46 °C has appeared, very close to the main transition temperature at 52 °C. Additionally, for DPPC:MPOx1 (9:3 and 9:1 molar ratio), a shoulder has appeared two degrees before the main transition temperature at 40 °C (Fig. 4B). These highly reproducible metastable phases have been also reported in our previous publications with DSC experiments [19]. These metastable phases can be used in order to make correlations with the release mechanism from the above chimeric drug delivery systems (i.e. faster release rate of slightly amphiphilic drugs like indomethacin) from DPPC:MPOx1 (9:1 molar ratio) chimeric liposomes in comparison to DPPC:MPOx2 (9:1 molar ratio) [19,35]. Furthermore, in this investigation we observed some differences between the thermotropic characteristics of the prepared systems in comparison to those studied in our previous studies. These differences are strongly associated with the different concentration i.e. we used 40 mg/mL colloidal dispersion for DSC experiments while for mDSC studies the colloidal dispersion is 4 timer lower (10 mg/mL). The colloidal dispersion affects several physicochemical and thermotropic parameters of the nanosystems during the self-assembly process. In all cases, we should highlight that the observed Tm corresponds to the Tm of the whole systems (lipid and guest polymer) and not only to the DPPC (lipid). Therefore, Tm of chimeric nanovesicles is higher than the main transition temperature of the pure lipid liposomes, possibly because of restrictions to the mobility of the lipids due to the presence of
molar ratio) chimeric liposomes is quite polydisperse (PDI = 0.54 ± 0.01) (Table 2) due to the presence of two populations, the first one is around 50 nm and the second one around 400 nm, as shown in the size distribution graph (Fig. 3A2). Cryo-TEM images are in accordance with DLS measurements since vesicles of a diameter 50–100 nm and vesicles of diameter 300–400 nm are observed. For DPPC:MPOx2 (9:1 molar ratio) and DPPC:MPOx2 (5:5 molar ratio) spherical objects of diameter 15–50 nm are also evident, which can be identified as MPOx 2 micelles or micellar aggregates. The membrane thickness is around 5 nm for the DPPC:MPOx1 and DPPC:MPOx2 chimeric liposomes (Fig. 2A1 and B1) [7,25,26]. Finally, in all cryo-TEM images, there are objects in the form of strands (sometimes forming lamella). According to data reported in the literature, by using thin-film hydration method, as preparation protocol for polymer/lipid chimeric liposomes, some structures like worm-like micelles, discs and pan-like micelles are generally observed by cryo-TEM images [7,8,26,31]. 3.2. The impact of architecture and the composition of the polymeric guest on the thermotropic properties of DPPC liposomes We also studied how the presence of block or gradient block copolymer chains influence the thermotropic behavior of the DPPC lipid bilayers. For this purpose, we used microcalorimetry analysis (mDSC) and high-resolution ultrasonic spectroscopy (HR-US) [32–34]. Fig. 4 shows the mDSC traces of the samples related to the heating and the cooling scans, while Table 3 reports the thermodynamic parameters (peak temperature, onset temperature and enthalpy) related to the main and pre-transition of all the systems calculated from the heating ramp. The DPPC liposomes’ thermodynamic parameters are in accordance to the literature [32]. The presence of PEO-b-PCL 1 and 2 block copolymers led to a significant increase of the main transition temperature (consequently the onset temperature increased, which corresponds to the temperature at which the thermal phenomenon starts). The increase is about 14 °C for DPPC:PEO-b-PCL 1 and 15 °C for DPPC:PEO-b-PCL 1 (Table 3). The main transition enthalpy decreased significantly by the incorporation of PEO-b-PCL into DPPC liposomes (Table 3). The presence of hydrophobic PCL chain into DPPC liposomes caused reduction of the energy needed for the melting of the phospholipids. The reduction is higher for DPPC-b-PEO 1 because the hydrophobic chain is longer (53 wt% hydrophobic component). The MPOx gradient block copolymers, as mentioned before, exhibit several entry and exit points into the DPPC liposomal membrane and interact by this way in several points with the phospholipid head groups. For this reason, the pre-transition enthalpy is decreased because the mobility of the phospholipid head groups is lower, due to the presence of the MPOx gradient block copolymer chains/segments in their vicinity.
Fig. 4. mDSC traces of DPPC and chimeric lipid/copolymer systems obtained from the first heating (A) and cooling scans (B) at a temperature rate of 1 °C/min. 545
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Table 3 Thermodynamic parameters (peak temperature, °C; onset temperature, °C; and enthalpy, J/g of solution) related to the main and pre-transition of DPPC and chimeric lipid/copolymer systems as calculated from the first heating ramp by microcalorimetry. Values are reported as mean ± SD of measurements performed in triplicate. Main transition
DPPCa DPPC:PEO-b-PCL 1 9_1 DPPC:PEO-b-PCL 2 9_1 DPPC:MPOx1 9_1 DPPC:MPOx1 9_3 DPPC:MPOx2 5_5 DPPC:MPOx2 9_1 a
Pre-transition
Onset (°C)
Peak (°C)
40.62 50.68 52.18 43.16 42.73 45.66 48.44
41.31 54.76 55.14 50.27 50.31 50.96 53.12
± ± ± ± ± ± ±
0.23 0.06 0.07 0.04 0.02 0.98 0.72
± ± ± ± ± ± ±
0.49 0.01 0.04 0.01 0.03 0.04 0.18
Enthalpy (J/g of solution)
Onset (°C)
Peak (°C)
0.587 0.165 0.191 0.114 0.119 0.032 0.018
33.47 40.90 32.04 35.49 34.48 30.26 38.68
36.62 43.48 34.99 37.35 36.38 35.44 39.55
± ± ± ± ± ± ±
0.021 0.002 0.001 0.004 0.011 0.030 0.032
± ± ± ± ± ± ±
0.13 0.39 0.84 0.47 0.53 0.98 1.39
± ± ± ± ± ± ±
Enthalpy (J/g of solution) 0.00 0.04 0.06 0.25 0.91 1.96 2.02
0.016 0.015 0.017 0.005 0.004 0.007 0.002
± ± ± ± ± ± ±
0.002 0.001 0.001 0.001 0.001 0.001 0.000
For DPPC the thermodynamic parameters were calculated from deconvolution analysis.
the copolymer chains. Thermotropic properties of DPPC liposomes and the chimeric DPPC/copolymer vesicles were also studied by high-resolution ultrasonic spectroscopy. It is a technique able to detect the variation of ultrasound parameters as attenuation and sound speed as a function of temperature. The variation of these parameters over temperature was found to be sensitive to detect the main transition of liposomes composed of phospholipids and PEGylated-phospholipids [18,33]. During the main transition, phospholipids in the lipid bilayer change their physical state from an ordered gel phase into a less ordered liquid phase, thereby affecting the total volume compressibility, which, in turn, influences attenuation and sound speed parameter [21]. Fig. 5a shows the variation of attenuation measured over temperature for DPPC liposomes and all analyzed chimeric systems. Phase transition can be detected since attenuation in the vicinity of the temperature at which the transition occurs deviates from the baseline. Particularly, attenuation increases up to reaching a maximum, and then, decreases, returning to the baseline values. The maximum value of attenuation can be considered as the transition temperature. The increase of attenuation at a temperature close to the transition is associated to the larger degree of inhomogeneity of the lipid bilayer during the conversion from the gel to the liquid phase. Thermal transition of phospholipids in liposomal membranes can be also detected by sound speed (Fig. 5). This parameter refers to the variation of the velocity of the ultrasound wave through the materials, which is dependent on the physical properties of the material itself. Sound speed is markedly affected by temperature (sound speed decreases linearly by increasing temperature) but also by temperature-dependent transitions, which can be highlighted as a stepwise decrease in sound speed signal. Phase transition temperatures calculated from sound speed and
Table 4 Transition temperature of DPPC and chimeric lipid/copolymer samples as calculated from the attenuation and sound speed profile obtained from high-resolution ultrasound spectroscopy analysis during the first heating scan. Values are reported as mean ± SD of measurements performed in triplicate. Transition temperature (°C)
DPPC DPPC:PEO-b-PCL 1 9_1 DPPC:PEO-b-PCL 2 9_1 DPPC:MPO 1 9_1 DPPC:MPO 1 9_3 DPPC:MPO 2 5_5 DPPC:MPO 2 9_1
Attenuation
Sound speed
41.27 51.72 52.22 52.46 45.81 50.42 50.41
42.76 50.81 51.71 50.79 46.09 49.61 48.80
± ± ± ± ± ± ±
0.61 0.26 0.10 0.32 0.27 0.58 0.50
± ± ± ± ± ± ±
0.42 0.34 0.20 0.39 0.21 0.43 0.38
attenuation for DPPC liposomes and phospholipids/copolymers chimeric systems are reported in Table 4. These values were found to be comparable to those determined by microcalorimetry as previously observed for other thermal transitions of different colloidal systems [18,34]. 4. Conclusions The aim of this investigation was to combine different techniques in order to characterize more comprehensively chimeric nanovesicles composed of lipids and block or gradient block copolymers with a different architectures and compositions. For this reason, we used DLS and cryo-TEM to examine the impact of block or gradient block copolymers in DPPC based liposomes in terms of size and morphology, while
Fig. 5. Attenuation (1/m) and sound speed (m/s) profiles of DPPC and chimeric lipid/copolymer systems recorded during the heating ramp from high-resolution ultrasonic spectroscopy. 546
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mDSC and ultrasound spectroscopy were employed to investigate the thermotropic behavior of chimeric liposomes. Particularly, these techniques resulted to be effective in studying the case of chimeric liposomes since they highlighted the effect exerted by the presence of the copolymer on both the self-organization and thermal transitions of the mixed nanovesicles. Indeed, the presence of block and gradient copolymers reduced the size of the chimeric nanostructures in a copolymer architecture dependent manner in comparison to DPPC pure liposomes. Moreover, their inclusion in DPPC bilayers has markedly influenced the thermotropic behavior of chimeric vesicles, determining a strong increase (around 9–14 °C) in the main transition of liposomal membrane and a marked decrease of the enthalpy associated to this transition. In conclusion, a detailed characterization of the colloidal behavior of chimeric liposomes can benefit from a gamut of techniques that operate synergistically, thereby providing information on their complex nature at the physicochemical, morphological and thermotropic level. The combination of these techniques could be extended in the future to other drug delivery systems i.e. lipid nanoparticles, micelles etc. and/or to other chimeric liposomes. In this way, a better understanding of the structural characteristics, influencing their thermal behavior, can be achieved. This information could be helpful for the design of studies aimed at investigating the formulation and performances of more effective drug loaded nanosystems.
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Colloids and Surfaces A journal homepage: www.elsevier.com/locate/colsurfa
Studying the colloidal behavior of chimeric liposomes by cryo-TEM, microdifferential scanning calorimetry and high-resolution ultrasound spectroscopy
T
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Natassa Pippaa,b, Diego Romano Perinellic, Stergios Pispasb, Giulia Bonacucinac, , ⁎ Costas Demetzosa, , Aleksander Forysd, Barbara Trzebickad a
Department of Pharmaceutical Technology, Faculty of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, Athens, Greece School of Pharmacy, University of Camerino, 62032 Camerino, MC, Italy d Centre of Polymer and Carbon Materials, Polish Academy of Sciences, Zabrze, Poland b c
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Nanovesicles Copolymers Chimeric systems Thermal analysis High-resolution ultrasound spectroscopy cryo-TEM
The investigation of colloidal properties of nanosystems represents a fundamental issue for the development of nanotechnology-based medicines. The aim of this study is to combine various techniques in order to characterize more comprehensively chimeric nanovesicles composed of block or gradient block copolymers with different architectures and compositions. Several chimeric systems were prepared and the impact of the block [poly(εcaprolactone)−poly(ethylene oxide); PEO-b-PCL] and gradient block [poly(2-methyl-2-oxazoline)-grad-poly(2phenyl-2-oxazoline); MPOx] copolymers on the physicochemical and morphological characteristics of conventional 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes was examined. Light scattering techniques and cryo-TEM were used for the physicochemical and morphological characterization of the prepared systems. The size and the morphology were strongly related to the architecture and the composition of the polymeric compounds. Micro differential scanning calorimetry and high-resolution ultrasound spectroscopy were used for investigating the interactions between the DPPC lipids and the polymeric guest. An increase in the
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Corresponding authors. E-mail addresses: [email protected] (G. Bonacucina), [email protected] (C. Demetzos).
https://doi.org/10.1016/j.colsurfa.2018.07.025 Received 4 June 2018; Received in revised form 16 July 2018; Accepted 17 July 2018 Available online 20 July 2018 0927-7757/ © 2018 Elsevier B.V. All rights reserved.
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main transition temperature was observed for the prepared chimeric systems in comparison to DPPC liposomes. In conclusion, a detailed characterization of the colloidal behavior of chimeric liposomes can benefit from the combination of the aforementioned techniques that operate synergistically, giving information on their physicochemical and morphological characteristics as well as on their thermotropic behavior.
1. Introduction
characterized as core-shell structures comprising polymer cores and lipid/lipid-PEG shells [4,7,8,15]. They exhibit unique properties such as colloidal stability, biocompatibility, high loading efficiency, different thermotropic behavior in comparison to pure liposomal membrane, vesicular morphology, etc. [4,7,8,16]. They also have been used as model of cellular membranes due to the macromolecular sculpture of the polymer-grafted liposomal membrane [17]. Furthermore, a gamut of techniques has been already utilized in order to investigate in depth the structure and the solution behavior of the aforementioned colloidal systems. Light scattering techniques are used for the elucidation of size and zeta potential of the colloidal particles [4,18]. Static light scattering offers also the possibility of studying the morphology of colloidal particles using fractal geometry [5]. Thermal analysis techniques (i.e. differential scanning calorimetry, microcalorimetry etc.) are also used for studying the microstructure and the loading capacity of PEGylated and polymer grafted liposomes [18,19]. Additionally, cryo-TEM is a very useful technique to visualize the morphology of colloidal systems [20]. Acoustic spectroscopy is useful to analyze the highly structured colloidal dispersion and gives information about the materials during pre-formulation and formulation studies [21]. The aim of this study is to combine different techniques in order to study chimeric liposomes composed of block or gradient block copolymers with different architectures and compositions. We prepared different chimeric systems in order to examine the impact of the block and gradient block copolymers on the physicochemical and morphological characteristics of conventional 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) liposomes. The block copolymers PEO-b-PCL consist of two blocks, one hydrophilic (PEO) and one hydrophobic part (PCL). In the MPOx copolymers, the gradual compositional change along the length of the polymer, which is related to the hydrophilic and hydrophobic units, is presented in Scheme 1. The investigated block copolymers were selected for different reasons including their difference in structure and architecture, high biocompatibility, the ability to entry in lipid membranes (as shown in our previous investigations) and the steric stabilizing effects in DPPC liposomes [4–6]. Light scattering techniques and cryo-TEM were used for the physicochemical and morphological characterization of the prepared systems. Micro
With its accelerated development in the past three decades, pharmaceutical nanotechnology is an innovative field for the design and the preparation of drug delivery systems. These drug delivery systems are nanocarriers which belong to the class of soft colloidal nanomaterials with unique properties e.g. stimuli-responsiveness [1,2]. A new class of smart nanostructured platform with applications in drug delivery and targeting is represented by advanced chimeric drug delivery systems, including polymer-grafted liposomes [3–8]. The interactions of lipids and polymers play a key role on the morphology of the resulting chimeric nanosystems i.e. weak polymer-lipid attraction (Coulombic and hydrogen interactions) and hydrophobic interactions [3,7]. Poly-ε-caprolactone (PCL) is a biodegradable polymer with several applications in drug delivery and tissue engineering [9]. Poly(ethylene glycol) (PEG) is a biocompatible and hydrophilic polymer, well-known for its low immunogenicity and “stealth” properties. The synthesis of PEO-b-PCL copolymers is generally performed via ring opening polymerization as described in the literature [10]. The nanoparticles based on PEO-b-PCL have been used as systems for vaccines, genes, waterinsoluble active ingredients, cancer passive and active targeting, stimuli-responsive applications e.g. controlled drug release. In all cases, due to their biocompatible and biodegradable nature, they improve the therapeutic index and the effectiveness of the encapsulated agent [9–11]. Additionally, poly(2-oxazolines) are water soluble and biocompatible polymers. Several investigations showed the wide variety of their applications in pharmaceutical nanotechnology. Drug and protein conjugates of poly(2-oxazoline) increase the half-life and the biological stability of the active substance. A very important class of them is the thermoresponsive poly(2-oxazoline)s with many applications in cancer chemotherapy characterized them as smart bio-inspired polymers due to their solution properties and thermo-responsiveness [12,13]. Synthesis of poly(2-methyl-2-oxazoline)-grad-poly(2-phenyl-2-oxazoline) copolymers (MPOx) is achieved via cationic polymerization [14]. Recently, systems composed of lipids and polymers have been described in the literature as controlled released drug nanocarriers. These systems are generally referred as mixed, hybrid or chimeric and can be
Scheme 1. Chemical structures of (a) DPPC lipid, (b) the block copolymer PEO-b-PCL, (c) the gradient block copolymer MPOx. Macromolecular architecture of (d) PEO-b-PCL (hydrophilic component - PEO: purple line and hydrophobic component- PCL: orange line) and (e) MPOx (hydrophilic component (MeOxz): blue line and hydrophobic component (PhOxz): red line) employed in this study. The boxes with dashed lines show the entry/ exit points into/from the liposomal membrane (For interpretation of the references to colour in this Scheme legend, the reader is referred to the web version of this article).
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2.5. Cryogenic transmission Electron microscopy (cryo-TEM)
differential scanning calorimetry and high-resolution ultrasound spectroscopy were used for investigating the interactions between the DPPC lipids and the polymeric guest. To best of our knowledge, this is the first report in the literature, in which all the aforementioned techniques have been combined for a detailed investigation on the effect of block copolymers on the morphology and thermal behavior of colloidal mixed lipid/copolymer nanovesicles.
Cryogenic Transmission Electron Microscopy images were obtained using a Tecnai F20 TWIN microscope (FEI Company, USA) equipped with field emission gun, operating at an acceleration voltage of 200 kV. Images were recorded on the Eagle 4k HS camera (FEI Company, USA) and processed with TIA software (FEI Company, USA). Specimen preparation was done by vitrification of the aqueous solutions on grids with holey carbon film (Quantifoil R 2/2; Quantifoil Micro Tools GmbH, Germany). Prior to use, the grids were activated for 15 s in oxygen plasma using a Femto plasma cleaner (Diener Electronic, Germany). Cryo-TEM samples were prepared by applying a droplet (3 μL) of the solution to the grid, blotting with filter paper and rapid freezing in liquid ethane using a fully automated blotting device Vitrobot Mark IV (FEI Company, USA). After preparation, the vitrified specimens were kept under liquid nitrogen until they were inserted into a cryo-TEM-holder Gatan 626 (Gatan Inc., USA) and analyzed in the TEM at −178 °C.
2. Materials and methods 2.1. Materials The phospholipid used for liposomal formulations was 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 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 (St. Louis, MO, USA).
2.6. Micro-DSC analyses
2.2. Synthesis and chemical characterization of block copolymers
mDSC analyses were carried out using a microcalorimeter (mDSC III, Setaram, France). Samples were loaded inside the Hastelloy cells (700 μL) and subjected to the following thermal programme: isotherm at 5 °C for 20 min; heating ramp from 5 °C to 80 °C at 1 °C/min; cooling ramp from 80 °C to 5 °C at 1 °C/min. The peak temperature (Tm, °C), onset temperature (Tonset, °C) and enthalpy (ΔH, J/g of solution) were calculated from the peak and the area of the transition by the tangent method using the software of the instrument (Setsoft2000, Setaram, France). Measurements were performed in triplicate.
The PEO-b-PCL block copolymer was prepared by ring opening polymerization of ε-caprolactone monomer using a monohydroxy terminated PEO (Mn = 5000) as the macroinitiator and stannous octoate as the catalyst [4]. The MPOx amphiphilic gradient block copolymers were prepared via cationic polymerization [14]. In this copolymer, phenyl-oxazoline segments comprise the hydrophobic components and methyl-oxazoline segments the hydrophilic ones. The copolymers are considered biocompatible due to the pseudopeptide nature of the oxazoline segments. Poly(2-methyl-2-oxazoline) has been proposed as an alternative to PEG in terms of biocompatibility and stealth properties. The details related to synthesis and chemical characterization (NMR and SEC chromatography) of the block and gradient block copolymers are summarized in our previous publications [4,14]. The structures and the molecular characteristics of copolymer samples are presented in Scheme 1 and in Table 1, respectively.
2.7. High-resolution ultrasound spectroscopy High-resolution ultrasound spectroscopy is used to characterize concentrated colloidal dispersions without dilution, in such a way as to be able to study highly structured nanosystems [22]. Ultrasonic attenuation and ultrasonic sound speed were monitored as a function of temperature using a HR-US 102 high-resolution spectrometer (Ultrasonic Scientific, Ireland). Around 2 mL of sample and reference (HPLC grade water) were filled in the ultrasonic cells and subjected to the same thermal programmes as described for mDSC analyses. Sample transitions were calculated by the peak value from the attenuation profile or from the first derivate of the signal in the case of sound speed. Measurements were performed in triplicate.
2.3. Preparation of pure and chimeric structures Different chimeric formulations and pure liposomes have been prepared using the thin-film hydration method as described in our previous publications [4–6]. The formulations studied here are presented in Table 2. The concentration of colloidal dispersion (phospholipids and polymers) is 10 mg/mL. Appropriate amounts of DPPC:PEOb-PCL and 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 and the mixed phospholipids/block copolymer thin film was formed by slow removal of the solvent at 45–50 °C. The mixed film was maintained under vacuum for at least 24 h in a desiccator to remove traces of solvent and, subsequently, hydrated in HPLC-grade water by slowly stirring for 1 h in a water bath above the phase transition temperature of lipids (41 °C for DPPC). The resultant nanostructures (apparently multilamellar vesicles, MLVs) were subjected to two 5 min sonication cycles (amplitude 70, cycle 0.7) interrupted by a 5 min resting period, in a water bath, using a probe sonicator (UP 200S, dr. Hielsher GmbH, Berlin, Germany). The resultant nanostructures were allowed annealing for 30 min. DPPC and phospholipid/copolymer chimeric liposomes were analysed immediately after their preparation.
3. Results and discussion 3.1. The impact of architecture and composition of the polymeric guest on size and morphology of chimeric liposomes We prepared different chimeric systems in order to examine the impact of the block or gradient block copolymers, as well as their composition, on the physicochemical and morphological characteristics of copolymer/lipid mixed liposomes in comparison to conventional DPPC liposomes. Table 2 reports the hydrodynamic diameter (Dh), Table 1 Molecular characteristics of block and gradient block copolymers utilized.
2.4. Light scattering techniques The protocols used for the physicochemical characterization of the nanostructures by dynamic and electrophoretic light scattering in aqueous media are described in our previous papers [4–6].
Sample
Mwa
Mw/Mna
%wt (hydrophobic component)b
PEO-b-PCL 1 PEO-b-PCL 2 MPOx 1 MPOx 2
10,600 7,700 5,200 3,300
1.43 1.18 1.14 1.26
53 30 28 39
a b
541
by SEC in CHCl3 using polystyrene standards. by1H-NMR in CDCl3.
(PCL) (PCL) (PhOxz) (PhOxz)
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Table 2 Physicochemical characteristics of DPPC liposomes and chimeric lipid/polymer formulations. Values are reported as mean of three independent experiments run in triplicate. Composition
DPPC DPPC:PEO-b-PCL 1 9_1 DPPC:PEO-b-PCL 2 9_1 DPPC:MPOx 1 9_1 DPPC:MPOx 1 9_3 DPPC:MPOx 2 5_5 DPPC:MPOx 2 9_1 a b
Lipid/Polymer (molar ratio)
9:1 9:1 9:1 9:3 5:5 9:1
Dh (nm)a
PDIb
240.3 ± 4.8 68.7 ± 0.1 68.9 ± 0.1 92.9 ± 0.9 99.3 ± 0.6 143.3 ± 0.6 80.0 ± 2.1
0.65 0.17 0.26 0.26 0.27 0.33 0.54
ζ-potential (mV)
± ± ± ± ± ± ±
0.03 0.01 0.01 0.02 0.02 0.08 0.01
+7.2 +2.8 −9.3 +1.5 −3.5 −5.8 +5.1
± ± ± ± ± ± ±
0.1 0.5 0.1 0.2 0.1 1.2 2.3
Hydrodynamic Diameter. Polydispersity Index.
Fig. 1. Cryo TEM images and size distributions from DLS of the chimeric lipid/copolymer systems DPPC:PEO-b-PCL 1 9_1 (A) and DPPC:PEO-b-PCL 2 9_1 (B).
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Fig. 2. Cryo TEM images and size distributions from DLS of the chimeric lipid/copolymer systems DPPC:MPOx1 9_1 (A) and DPPC:MPOx1 9_3 (B).
polydispersity index (PDI) and ζ-potential of pure DPPC liposomes and mixed DPPC:PEO-b-PCL 1 and DPPC:PEO-b-PCL 2 (9:1 molar ratio), DPPC:MPOx 1 (9:1 and 9:3 molar ratios) and DPPC:MPOx 2 (5:5 and 9:1 molar ratios) systems. Size distribution graphs and cryo-TEM images are presented in Figs. 1–3. The size (Dh) of pure DPPC liposomes, used as control, are around 240 nm with high polydispersity and low ζ-potential due to the zwitterionic nature of the phospholipids used (Table 2). The presence of copolymers reduced the size of the chimeric nanostructures in a copolymer architecture dependent manner. It has been observed, that the reduction of size is higher for PEO-b-PCL grafted liposomes in comparison to MPOx grafted liposomes. This finding is strongly dependent on the number of entry points of copolymer chains into the lipid bilayer and the consequent packing of phospholipids in the presence of the polymeric guest. Indeed, for MPOx the gradient of co-monomer sequence exhibits several entry points into DPPC bilayers, because of the hydrophobic and hydrophilic repetition within the copolymer chain. On the other hand, the block copolymer PEO-b-PCL exhibits only one entry point into the bilayer (the junction
point between the two blocks). Additionally, for the PEO-b-PCL block copolymers the hydrophilic part is more elongated in comparison to methyl-oxazoline sequences within the MPOx gradient block copolymer and induces stronger steric intra-vesicular repulsions, leading to a smaller hydrodynamic diameter for PEO-b-PCL in comparison to the MPOx ones (Table 2) [13,23,24]. In details, the size of chimeric DPPC:PEO-b-PCL 1 and 2 is comparable and around 70 nm according to DLS measurements (Table 2 and Fig. 1). In this case, the different % ratio of the hydrophobic component in PEO-b-PCL copolymer did not affect the size of the resulted nanocarriers (i.e. Dh = 68.7 ± 0.1 nm for DPPC:PEO-b-PCL 1 and Dh = 68.9 ± 0.1 nm for DPPC:PEO-b-PCL 2). Nanovesicles of diameters between 50 and 200 nm, which correspond to DPPC:PEO-bPEO 1 and DPPC:PEO-b-PEO 2 chimeric liposomes, are observed in cryo-TEM images (Fig. 1). Generally, using this technique, we observed vesicles larger than those measured by DLS. In our opinion, the strong steric interactions due to the PEO-hydrophilic chains produce vesicles with smaller hydrodynamic diameter in the disperse liquid state (as 543
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Fig. 3. Cryo TEM images and size distributions from DLS of the chimeric lipid/copolymer systems DPPC:MPOx2 9_1 (A) and DPPC:MPOx2 5_5 (B).
On the contrary, the size for chimeric liposomes formed by MPOx gradient copolymers was found to be dependent on the percentage of the hydrophobic component. Indeed, for DPPC:MPOx1 and DPPC:MPOx2 (9:1 molar ratio), the size resulted to be 10 nm smaller for liposomes in which MPOx participates with the lower ratio of the hydrophobic component (i.e. copolymer MPOx1, 28% hydrophobic component). Similarly, for DPPC:MPOx1, the size and size distribution of the prepared chimeric liposomes increased slightly with the increase of the molar ratio of the polymeric guest (Table 2). From cryo-TEM images, self-assembled vesicular objects are observed of diameter between 50–100 nm for DPPC:MPOx1 (9:1 molar ratio) (Fig. 2A1) and 50200 nm for DPPC:MPOx 1 (9:3 molar ratio) due to a more polydisperse population (Fig. 2B1) [27–29]. The increase of MPOx2 molar ratio caused a greater size of the prepared systems (Table 2) (i.e. Dh = 143.3 ± 0.6 nm for DPPC:MPOx2 (5:5 molar ratio) and Dh = 80.0 ± 2.1 nm for DPPC:MPOx2 (9:1 molar ratio). The population of DPPC:MPOx2 (9:1
measured by DLS), while the effects from solvent freezing may affect vesicles size in cryo-TEM images. The thickness of the mixed liposomal membrane is equal to 4–5 nm, a little bit higher than the thickness of pure liposomes’ membrane (∼3–4 nm) and lower than the thickness of pure PEO-b-PCL polymersomes’ membrane (∼8-12 nm) [7,25,26]. Moreover, spherical objects with diameter between 15–40 nm can be observed, recognized as pure PEO-b-PCL micelles formed from the copolymer chains that have not been incorporated in DPPC lipid bilayers during the formation of chimeric liposomes [27–29]. Some large vesicles around 200 nm of diameter have some smaller vesicles encapsulated in the interior. According to the literature, these systems can be characterized as “pregnant vesicles”, where smaller vesicles are enclosed within larger chimeric liposomes [30]. In Figs. 2A1 and 3B1, the black, branched objects with strong contrast are a contamination from liquid ethane. According to recent literature, ethane could remain in the samples after the vitrifying procedure on a film of cubic nanoparticles [20]. 544
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Additionally, for DPPC:MPOx1 systems, the peak transition temperature increased by 7 °C for the two molar ratios in comparison to pure DPPC liposomes. For these systems, the thermotropic peaks are broader than those obtained from DPPC: PEO-b-PCL 1 and 2. It should be pointed out that for all the MPOx grafted liposomes the peaks are not sharp but rather wide (Fig. 4A). In other words, new metastable phases were created at 50 °C for all the MPOx chimeric liposomes. The reduction of the enthalpy is much higher for the MPOx 2 i.e. for the copolymer having the higher amount of the hydrophobic component (PhOx). This means that the cooperativity between the two biomaterials (i.e. lipid and gradient block copolymer) is limited, leading the DPPC lipid to feel the MPOx 2 as an “impurity” for the liquid crystalline dance of the phospholipid molecules. For the PEO-b-PCL block copolymers, the cooperativity is higher because the macromolecular structure of block copolymers and phospholipids are the same (i.e. the hydrophilic PEO chain corresponds to phospholipid’s head group and the hydrophobic PCL chain to the lipid alkyl chains). This observation may also be a manifestation of the effects of the number of entry points in each case. The DSC cooling scans are illustrated in Fig. 4B. A small hysteresis in the cooling curves at the main transition temperature is observed, a common phenomenon well established in the literature [32]. For the DPPC:MPOx2 (9:1 molar ratio), a new peak at 46 °C has appeared, very close to the main transition temperature at 52 °C. Additionally, for DPPC:MPOx1 (9:3 and 9:1 molar ratio), a shoulder has appeared two degrees before the main transition temperature at 40 °C (Fig. 4B). These highly reproducible metastable phases have been also reported in our previous publications with DSC experiments [19]. These metastable phases can be used in order to make correlations with the release mechanism from the above chimeric drug delivery systems (i.e. faster release rate of slightly amphiphilic drugs like indomethacin) from DPPC:MPOx1 (9:1 molar ratio) chimeric liposomes in comparison to DPPC:MPOx2 (9:1 molar ratio) [19,35]. Furthermore, in this investigation we observed some differences between the thermotropic characteristics of the prepared systems in comparison to those studied in our previous studies. These differences are strongly associated with the different concentration i.e. we used 40 mg/mL colloidal dispersion for DSC experiments while for mDSC studies the colloidal dispersion is 4 timer lower (10 mg/mL). The colloidal dispersion affects several physicochemical and thermotropic parameters of the nanosystems during the self-assembly process. In all cases, we should highlight that the observed Tm corresponds to the Tm of the whole systems (lipid and guest polymer) and not only to the DPPC (lipid). Therefore, Tm of chimeric nanovesicles is higher than the main transition temperature of the pure lipid liposomes, possibly because of restrictions to the mobility of the lipids due to the presence of
molar ratio) chimeric liposomes is quite polydisperse (PDI = 0.54 ± 0.01) (Table 2) due to the presence of two populations, the first one is around 50 nm and the second one around 400 nm, as shown in the size distribution graph (Fig. 3A2). Cryo-TEM images are in accordance with DLS measurements since vesicles of a diameter 50–100 nm and vesicles of diameter 300–400 nm are observed. For DPPC:MPOx2 (9:1 molar ratio) and DPPC:MPOx2 (5:5 molar ratio) spherical objects of diameter 15–50 nm are also evident, which can be identified as MPOx 2 micelles or micellar aggregates. The membrane thickness is around 5 nm for the DPPC:MPOx1 and DPPC:MPOx2 chimeric liposomes (Fig. 2A1 and B1) [7,25,26]. Finally, in all cryo-TEM images, there are objects in the form of strands (sometimes forming lamella). According to data reported in the literature, by using thin-film hydration method, as preparation protocol for polymer/lipid chimeric liposomes, some structures like worm-like micelles, discs and pan-like micelles are generally observed by cryo-TEM images [7,8,26,31]. 3.2. The impact of architecture and the composition of the polymeric guest on the thermotropic properties of DPPC liposomes We also studied how the presence of block or gradient block copolymer chains influence the thermotropic behavior of the DPPC lipid bilayers. For this purpose, we used microcalorimetry analysis (mDSC) and high-resolution ultrasonic spectroscopy (HR-US) [32–34]. Fig. 4 shows the mDSC traces of the samples related to the heating and the cooling scans, while Table 3 reports the thermodynamic parameters (peak temperature, onset temperature and enthalpy) related to the main and pre-transition of all the systems calculated from the heating ramp. The DPPC liposomes’ thermodynamic parameters are in accordance to the literature [32]. The presence of PEO-b-PCL 1 and 2 block copolymers led to a significant increase of the main transition temperature (consequently the onset temperature increased, which corresponds to the temperature at which the thermal phenomenon starts). The increase is about 14 °C for DPPC:PEO-b-PCL 1 and 15 °C for DPPC:PEO-b-PCL 1 (Table 3). The main transition enthalpy decreased significantly by the incorporation of PEO-b-PCL into DPPC liposomes (Table 3). The presence of hydrophobic PCL chain into DPPC liposomes caused reduction of the energy needed for the melting of the phospholipids. The reduction is higher for DPPC-b-PEO 1 because the hydrophobic chain is longer (53 wt% hydrophobic component). The MPOx gradient block copolymers, as mentioned before, exhibit several entry and exit points into the DPPC liposomal membrane and interact by this way in several points with the phospholipid head groups. For this reason, the pre-transition enthalpy is decreased because the mobility of the phospholipid head groups is lower, due to the presence of the MPOx gradient block copolymer chains/segments in their vicinity.
Fig. 4. mDSC traces of DPPC and chimeric lipid/copolymer systems obtained from the first heating (A) and cooling scans (B) at a temperature rate of 1 °C/min. 545
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Table 3 Thermodynamic parameters (peak temperature, °C; onset temperature, °C; and enthalpy, J/g of solution) related to the main and pre-transition of DPPC and chimeric lipid/copolymer systems as calculated from the first heating ramp by microcalorimetry. Values are reported as mean ± SD of measurements performed in triplicate. Main transition
DPPCa DPPC:PEO-b-PCL 1 9_1 DPPC:PEO-b-PCL 2 9_1 DPPC:MPOx1 9_1 DPPC:MPOx1 9_3 DPPC:MPOx2 5_5 DPPC:MPOx2 9_1 a
Pre-transition
Onset (°C)
Peak (°C)
40.62 50.68 52.18 43.16 42.73 45.66 48.44
41.31 54.76 55.14 50.27 50.31 50.96 53.12
± ± ± ± ± ± ±
0.23 0.06 0.07 0.04 0.02 0.98 0.72
± ± ± ± ± ± ±
0.49 0.01 0.04 0.01 0.03 0.04 0.18
Enthalpy (J/g of solution)
Onset (°C)
Peak (°C)
0.587 0.165 0.191 0.114 0.119 0.032 0.018
33.47 40.90 32.04 35.49 34.48 30.26 38.68
36.62 43.48 34.99 37.35 36.38 35.44 39.55
± ± ± ± ± ± ±
0.021 0.002 0.001 0.004 0.011 0.030 0.032
± ± ± ± ± ± ±
0.13 0.39 0.84 0.47 0.53 0.98 1.39
± ± ± ± ± ± ±
Enthalpy (J/g of solution) 0.00 0.04 0.06 0.25 0.91 1.96 2.02
0.016 0.015 0.017 0.005 0.004 0.007 0.002
± ± ± ± ± ± ±
0.002 0.001 0.001 0.001 0.001 0.001 0.000
For DPPC the thermodynamic parameters were calculated from deconvolution analysis.
the copolymer chains. Thermotropic properties of DPPC liposomes and the chimeric DPPC/copolymer vesicles were also studied by high-resolution ultrasonic spectroscopy. It is a technique able to detect the variation of ultrasound parameters as attenuation and sound speed as a function of temperature. The variation of these parameters over temperature was found to be sensitive to detect the main transition of liposomes composed of phospholipids and PEGylated-phospholipids [18,33]. During the main transition, phospholipids in the lipid bilayer change their physical state from an ordered gel phase into a less ordered liquid phase, thereby affecting the total volume compressibility, which, in turn, influences attenuation and sound speed parameter [21]. Fig. 5a shows the variation of attenuation measured over temperature for DPPC liposomes and all analyzed chimeric systems. Phase transition can be detected since attenuation in the vicinity of the temperature at which the transition occurs deviates from the baseline. Particularly, attenuation increases up to reaching a maximum, and then, decreases, returning to the baseline values. The maximum value of attenuation can be considered as the transition temperature. The increase of attenuation at a temperature close to the transition is associated to the larger degree of inhomogeneity of the lipid bilayer during the conversion from the gel to the liquid phase. Thermal transition of phospholipids in liposomal membranes can be also detected by sound speed (Fig. 5). This parameter refers to the variation of the velocity of the ultrasound wave through the materials, which is dependent on the physical properties of the material itself. Sound speed is markedly affected by temperature (sound speed decreases linearly by increasing temperature) but also by temperature-dependent transitions, which can be highlighted as a stepwise decrease in sound speed signal. Phase transition temperatures calculated from sound speed and
Table 4 Transition temperature of DPPC and chimeric lipid/copolymer samples as calculated from the attenuation and sound speed profile obtained from high-resolution ultrasound spectroscopy analysis during the first heating scan. Values are reported as mean ± SD of measurements performed in triplicate. Transition temperature (°C)
DPPC DPPC:PEO-b-PCL 1 9_1 DPPC:PEO-b-PCL 2 9_1 DPPC:MPO 1 9_1 DPPC:MPO 1 9_3 DPPC:MPO 2 5_5 DPPC:MPO 2 9_1
Attenuation
Sound speed
41.27 51.72 52.22 52.46 45.81 50.42 50.41
42.76 50.81 51.71 50.79 46.09 49.61 48.80
± ± ± ± ± ± ±
0.61 0.26 0.10 0.32 0.27 0.58 0.50
± ± ± ± ± ± ±
0.42 0.34 0.20 0.39 0.21 0.43 0.38
attenuation for DPPC liposomes and phospholipids/copolymers chimeric systems are reported in Table 4. These values were found to be comparable to those determined by microcalorimetry as previously observed for other thermal transitions of different colloidal systems [18,34]. 4. Conclusions The aim of this investigation was to combine different techniques in order to characterize more comprehensively chimeric nanovesicles composed of lipids and block or gradient block copolymers with a different architectures and compositions. For this reason, we used DLS and cryo-TEM to examine the impact of block or gradient block copolymers in DPPC based liposomes in terms of size and morphology, while
Fig. 5. Attenuation (1/m) and sound speed (m/s) profiles of DPPC and chimeric lipid/copolymer systems recorded during the heating ramp from high-resolution ultrasonic spectroscopy. 546
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mDSC and ultrasound spectroscopy were employed to investigate the thermotropic behavior of chimeric liposomes. Particularly, these techniques resulted to be effective in studying the case of chimeric liposomes since they highlighted the effect exerted by the presence of the copolymer on both the self-organization and thermal transitions of the mixed nanovesicles. Indeed, the presence of block and gradient copolymers reduced the size of the chimeric nanostructures in a copolymer architecture dependent manner in comparison to DPPC pure liposomes. Moreover, their inclusion in DPPC bilayers has markedly influenced the thermotropic behavior of chimeric vesicles, determining a strong increase (around 9–14 °C) in the main transition of liposomal membrane and a marked decrease of the enthalpy associated to this transition. In conclusion, a detailed characterization of the colloidal behavior of chimeric liposomes can benefit from a gamut of techniques that operate synergistically, thereby providing information on their complex nature at the physicochemical, morphological and thermotropic level. The combination of these techniques could be extended in the future to other drug delivery systems i.e. lipid nanoparticles, micelles etc. and/or to other chimeric liposomes. In this way, a better understanding of the structural characteristics, influencing their thermal behavior, can be achieved. This information could be helpful for the design of studies aimed at investigating the formulation and performances of more effective drug loaded nanosystems.
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